Heart Physiology and Pathophysiology

HEART PHYSIOLOGY AND PATHOPHYSIOLOGY F O U R T H E D I T I O N

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HEART PHYSIOLOGY AND PATHOPHYSIOLOGY F O U R T H E D I T I O N

Edited by

NICHOLAS SPERELAKIS (Editor-in-Chief) University of Cincinnati Medical Center and Sperelakis Research and Consulting (www.SperelakisConsulting.com) Cincinnati, Ohio

YOSHIHISA KURACHI Department of Pharmacology II Graduate School of Medicine Osaka University Osaka, Japan

ANDRE TERZIC Division of Cardiovascular Diseases and Department of Internal Medicine, and Department of Molecular Pharmacology and Experimental Therapeutics Mayo Clinic and Foundation Rochester, Minnesota

MICHAEL V. COHEN Departments of Medicine and Physiology University of South Alabama College of Medicine Mobile, Alabama

ACADEMIC PRESS A Harcourt Science and Technology Company

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Front cover images: (Left) Three-dimensional (NMR) structure of CaM bound to a synthetic peptide corresponding to the CaM-binding domain (residues 796815) of smooth muscle MLCK. (Right) Three-dimensional (X-ray crystallographic) structure of free CaM with four bound Ca2⫹ ions. For more information, see color Figure 4 in Chapter 32. (Background) Model of calcium-dependent inactivation of the L-type 움1C channel. For more information, see Figure 5 in Chapter 13.

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Dedication

The fourth edition of Heart Physiology and Pathophysiology is dedicated to Planet Earth and its animal and plant inhabitants. The destruction of the environment and toxic pollution of the air, water, and soil are still occurring at an alarming rate. Our wilderness, forests, parks, and farmland are under increasing pressure. There is an urgent need for global population stabilization and global human rights. Our main salvation may be those numerous national and international nonprofit organizations that are dedicated to halting the ruthless destruction of our planet and the inhumane treatment of animals and humans. These organizations are concerned about the environment, wildlife, forests, farmland, overpopulation, animal rights and welfare, and human rights. Such deserving organizations are in desperate need of support from all of us. We urge all readers of this book to express their serious and urgent concern for the well-being of Planet Earth and all of its inhabitants, and to help educate the public and governments worldwide.

Diagram provided by courtesy of Richard S. Babb.

‘‘The Foundation of Every State is Education of its Youth.’’ Diogenes

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Contents Contributors xiii Foreword xix Preface xxi

8. Gap-Junction Channels and Healing-Over of Injury 149 DAVID C. SPRAY, SYLVIA O. SUADICANI, MONIQUE J. VINK, AND MIDUTURU SRINIVAS P A R T

I

P A R T

II

PUMPING ACTION AND ELECTRICAL ACTIVITY OF THE HEART

CELLULAR ELECTROPHYSIOLOGY OF HEART AND VASCULAR SMOOTH MUSCLE

1. Sequence of Cardiac Activation and Ventricular Mechanics 3 JAMES M. DOWNEY AND GERD HEUSCH

9. Electrogenesis of the Resting Potential 175

2. Coronary Circulation and Hemodynamics 19

NICHOLAS SPERELAKIS, MASANORI SUNAGAWA, AND MARIKO NAKAMURA

JOS A. E. SPAAN, JAN J. PIEK, AND MARIA SIEBES

10. Cardiac Action Potentials 199

3. Neurohumoral Control of Cardiac Function 45

GORDON M. WAHLER

JEFFREY L. ARDELL

11. Electrophysiology of Vascular Smooth Muscle 213

4. Control of Cardiac Output and its Alterations during Exercise and in Heart Failure 61

JANE A. MADDEN AND NANCY J. RUSCH

JAMES M. DOWNEY AND GERD HEUSCH

12. Sodium Channels 229

5. Ultrastructure of Cardiac Muscle and Blood Vessels 71

KATSUSHIGE ONO AND MAKOTO ARITA

MICHAEL S. FORBES

13. Voltage-Dependent Calcium Channels 247

6. Excitability and Impulse Propagation 99

´ AND FRANZ HOFMANN LUBICA LACINOVA

MORTON F. ARNSDORF AND JONATHAN C. MAKIELSKI

14. Voltage-Dependent K⫹ Channels 259

7. Electrocardiogram and Cardiac Excitation 133

HAROLD C. STRAUSS, MICHAEL J. MORALES, SHIMIN WANG, MULUGU V. BRAHMAJOTHI, AND DONALD L. CAMPBELL

YORAM RUDY

vii

viii

Contents

15. Inwardly-Rectifying K⫹ Channels in the Heart 281

24. Transport in Nucleus 437 CARMEN M. PEREZ-TERZIC, A. MARQUIS GACY, AND ANDRE TERZIC

MASAYUKI TANEMOTO, AKIKAZU FUJITA, AND YOSHIHISA KURACHI

⫹

16. Voltage and Calcium-Activated K Channels of Coronary Smooth Muscle 309

25. Sarcoplasmic Reticulum Ca2⫹ Transport 447 ISTVAN EDES, GUOXIANG CHU, AND EVANGELIA G. KRANIAS

JURE MARIJIC AND LIGIA TORO

17. Ion Channels in Vascular Smooth Muscle 327

26. Calcium Release from Cardiac Sarcoplasmic Reticulum 461 GERHARD MEISSNER

JUN YAMAZAKI AND KENJI KITAMURA

18. Cardiac Pacemaker Currents 357 D. DIFRANCESCO, A. MORONI, M. BARUSCOTTI, AND ERIC A. ACCILI

P A R T

IV VASCULAR ENDOTHELIUM

19. Chloride Channels in Heart 373 ROBERT D. HARVEY AND JOSEPH R. HUME

27. Function of Vascular Endothelium 473 STEPHANIE H. WILSON AND AMIR LERMAN

20. Regulation of Cardiac Ion Channels by Phosphorylation, Ca2⫹, Cytoskeleton, and Stretch 389 MASAYASU HIRAOKA, YUJI HIRANO, SEIKO KAWANO, AND TETSUSHI FURUKAWA

28. Ion Channels in Vascular Endothelium 481 BERND NILIUS AND GUY DROOGMANS

P A R T

P A R T

III

V

PUMPS AND EXCHANGERS

EXCITATION–CONTRACTION COUPLING AND PHARMACOMECHANICAL COUPLING

21. Cardiac Na⫹/K⫹ Pump 407 JOSEPH R. STIMERS

22. Cardiac Na⫹ –Ca2⫹ Exchanger: Pathophysiology and Pharmacology 417

29. Electromechanical and Pharmacomechanical Coupling in Vascular Smooth Muscle Cells 501

JUNKO KIMURA

GUY DROOGMANS, BERND NILIUS, HUMBERT DE SMEDT, JAN B. PARYS, AND LUDWIG MISSIAEN

23. Na⫹/H⫹ Exchanger and pH Regulation 427

30. Mechanisms Regulating Cardiac Myofilament Response to Calcium 519

M. PUCE´AT

R. JOHN SOLARO

ix

Contents

31. Vascular Smooth Muscle Contraction 527

39. Diadenosine Polyphosphate Signaling in the Heart 693

GARY J. KARGACIN AND MICHAEL P. WALSH

ALEKSANDAR JOVANOVIC, SOFIJA JOVANOVIC, AND ANDRE TERZIC

P A R T

VI METABOLISM AND ENERGETICS

P A R T

VIII DEVELOPMENTAL CHANGES AND AGING

32. Myocardial Energy Metabolism 543 PAUL F. KANTOR, GARY D. LOPASCHUK, AND LIONEL H. OPIE

40. Cardiac Development and Regulation of Cardiac Transcription 705

33. Metabolism and Energetics of Vascular Smooth Muscle 571

FRE´DE´RIC CHARRON AND MONA NEMER

CHRISTOPHER D. HARDIN, TARA J. ALLEN, AND RICHARD J. PAUL

41. Developmental Changes of Ion Channels 719 HISASHI YOKOSHIKI AND NORITSUGU TOHSE

P A R T

VII SIGNALING SYSTEMS 34. Adrenergic Receptors in the Cardiovascular System 599 JON W. LOMASNEY AND LEE F. ALLEN

42. Aging of the Cardiovascular System 737 EDWARD G. LAKATTA, YING-YING ZHOU, RUI-PING XIAO, AND MARVIN BOLUYT

43. Changes in Autonomic Responsiveness during Development 761 RICHARD B. ROBINSON, MICHAEL R. ROSEN, AND SUSAN F. STEINBERG

35. Cardiac Action of Angiotensin II 609 MASAO ENDOH

P A R T

IX 36. ATP and Adenosine Signal Transductions 633 AMIR PELLEG, GUY VASSORT, AND JOHN A. AUCHAMPACH

MECHANISM OF ACTION OF CARDIOACTIVE DRUGS

37. Kinase Signaling in the Cardiovascular System 657

44. Inotropic Mechanism in Cardiac Muscle 779

JUN-ICHI ABE, CHEN YAN, JAMES SURAPISITCHAT, AND BRADFORD C. BERK

DONALD M. BERS

38. Calcium Signaling 679

45. Mechanisms of Action of Calcium Antagonists 789

DEREK TERRAR, STEVAN RAKOVIC, AND ANTONY GALIONE

HELMUT A. TRITTHART

x

Contents

46. Cyclic Nucleotides and Protein Phosphorylation in Vascular Smooth Muscle Relaxation 805 GIOVANNI M. PITARI, DONALD H. MAURICE, BRIAN M. BENNETT, AND SCOTT A. WALDMAN

47. K⫹ Channel Openers 829

55. Diabetic Vascular Disease 1011 WILLIAM G. MAYHAN

56. Angiogenesis and Coronary Collateral Circulation 1031 WOLFGANG SCHAPER AND JUTTA SCHAPER

ARSHAD JAHANGIR, WIN-KUANG SHEN, AND ANDRE TERZIC

48. Mode of Action of Antiarrhythmic Drugs 837

57. Molecular Pathophysiology of Cardiomyopathies 1045 ALI J. MARIAN AND ROBERT ROBERTS

AUGUSTUS O. GRANT AND VIJAY S. CHAUHAN

P A R T

X PATHOPHYSIOLOGY 49. Cellular Mechanisms of Cardioprotection 853 MASAFUMI KITAKAZE AND MASATSUGU HORI

50. Ischemic Preconditioning: Description, Mechanism, and Significance 867

58. Signal Transduction of Cardiac Myocyte Hypertrophy 1065 HIROKI AOKI AND SEIGO IZUMO

59. Electrophysiological Changes in Hypertrophy 1087 VALENTINO PIACENTINO III AND STEVEN R. HOUSER

60. Molecular Basis of Inherited Long QT Syndromes and Cardiac Arrhythmias 1097 DENIS ESCANDE, MILOU D. DRICI, AND JACQUES BARHANIN

MICHAEL V. COHEN AND JAMES M. DOWNEY

51. Cardioplegia and Surgical Ischemia 887

61. Molecular Mechanisms of Atrial Fibrillation 1107

D. J. CHAMBERS AND D. J. HEARSE

DAVID R. VAN WAGONER AND JEANNE M. NERBONNE

52. Apoptosis 927

62. Lipids Released during Ischemia and Arrhythmogenesis 1125

ARMIN HAUNSTETTER AND SEIGO IZUMO

GARY D. LOPASCHUK

53. Calcium Overload in Ischemia/ Reperfusion Injury 949

63. Ion Channels in the Heart 1137

NARANJAN S. DHALLA, RANA M. TEMSAH, THOMAS NETTICADAN, AND MANJOT S. SANDHU

ROBERT S. KASS, HUGUES ABRIEL, AND ILARIA RIVOLTA

54. Coronary Atherosclerosis and Restenosis 967

64. Cardiac Arrhythmias: Reentry and Triggered Activity 1153

SHMUEL BANAI, ADI KURGAN, AND S. DAVID GERTZ

CHARLES ANTZELEVITCH AND ALEXANDER BURASHNIKOV

xi

Contents

65. Myocardial Reperfusion Injury— Role of Free Radicals and Mediators of Inflammation 1181

67. Regulation of Gene Expression by Hypoxia 1225 ANDREW P. LEVY

BENEDICT R. LUCCHESI

68. Gene Transfer in Cardiovascular Therapy 1233 66. Cardiac Toxicology 1211 ROSITA J. RODRIGUEZ AND DANIEL ACOSTA, JR.

IFTIKHAR J. KULLO AND ROBERT D. SIMARI

Index 1245

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Contributors The numbers in parentheses indicate the pages on which the authors’ contributions begin.

M. Baruscotti (357) Department of Physiology and General Biochemistry, University of Milan, 20133 Milan, Italy

Jun-ichi Abe (657) Cardiovascular Research Center, University of Rochester Medical Center, Rochester, New York 14642

Brian M. Bennett (805) Departments of Pharmacology and Toxicology, Faculty of Medicine, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Hugues Abriel (1137) Department of Pharmacology, Columbia University, New York, New York 10032

Bradford C. Berk (657) Cardiovascular Research Center, University of Rochester Medical Center, Rochester, New York 14642

Eric A. Accili (357) School of Kinesiology, Faculty of Applied Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6

Donald M. Bers (779) Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153

Daniel Acosta, Jr. (1211) College of Pharmacy, University of Cincinnati, Cincinnati, Ohio 45267

Marvin Boluyt (737) Department of Movement Science, Laboratory of Molecular Kinesiology, University of Michigan, Ann Arbor, Michigan 48109

Lee F. Allen (599) Department of Early Clinical Research, Pfizer, Inc., Groton, Connecticut 06340

Mulugu V. Brahmajothi (259) Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214

Tara J. Allen (571) Department of Physiology, University of Missouri, Columbia, Missouri 65212

Eugene Braunwald (xix) Department of Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Charles Antzelevitch (1153) Masonic Medical Research Laboratory, Utica, New York 13501 Hiroki Aoki (1065) Cardiovascular Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Alexander Burashnikov (1153) Masonic Medical Research Laboratory, Utica, New York 13501

Jeffrey L. Ardell (45) Department of Pharmacology, James H. Quillen College of Medicine, Johnson City, Tennessee 37614

Donald L. Campbell (259) Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214

Makoto Arita (229) Department of Physiology, Oita Medical University, Oita 879-5593, Japan

D. J. Chambers (887) Cardiac Surgical Research/ Cardiothoracic Surgery, Guy’s and St. Thomas’ NHS Trust, The Rayne Institute, King’s College, St. Thomas’ Hospital, London SE1 7EH, England

Morton F. Arnsdorf (99) Department of Medicine, Section of Cardiology, University of Chicago, Chicago, Illinois 60637

Fre´de´ric Charron (705) Laboratory of Cardiac Growth and Differentiation, Montreal Clinical Research Institute, and Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada H2W 1R7

John A. Auchampach (633) Department of Medicine, University of Louisville, Louisville, Kentucky 40292 Shmuel Banai (967) Department of Cardiology, Bikur Cholim Hospital, and Department of Anatomy and Cell Biology, The Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel

Vijay S. Chauhan (837) Duke University Medical Center, Durham, North Carolina 27710; and University of Western Ontario, Ontario, Canada Guoxiang Chu (447) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Jacques Barhanin (1097) Institut de Pharmacologie Mole´culaire et Cellulaire, Sophia Antipolis, 06560 Valbonne, France

xiii

xiv

Contributors

Michael V. Cohen (867) Departments of Medicine and Physiology, University of South Alabama, College of Medicine, Mobile, Alabama 36688

S. David Gertz (967) Department of Anatomy and Cell Biology, The Hebrew University, Hadassah Medical School, Jersualem 91120, Israel

Humbert De Smedt (501) Department of Physiology, Catholic University of Leuven, B-3000 Leuven, Belgium

Augustus O. Grant (837) Duke University Medical Center, Durham, North Carolina 27710; and University of Western Ontario, Ontario, Canada

Naranjan S. Dhalla (949) Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R2H 2A6

Christopher D. Hardin (571) Department of Physiology, University of Missouri, Columbia, Missouri 65212

D. DiFrancesco (357) Department of Physiology and General Biochemistry, University of Milan, 20133 Milan, Italy

Armin Haunstetter (927) Department of Cardiology, University of Heidelberg, Heidelberg, Germany

James M. Downey (3, 61, 867) Department of Physiology, University of South Alabama, College of Medicine, Mobile, Alabama 36688 Milou D. Drici (1097) Institut de Pharmacologie Mole´culaire et Cellulaire, Sophia Antipolis, 06560 Valbonne, France Guy Droogmans (481, 501) Department of Physiology, Catholic University of Leuven, B-3000 Leuven, Belgium Istvan Edes (447) Department of Heart and Lung Diseases, University Medical School, HU-4032 Debrecen, Hungary Masao Endoh (609) Department of Pharmacology, Yamagata University School of Medicine, Yamagata 990-9585, Japan Denis Escande (1097) Physiopathologie et Pharmacologie Cellulaires et Mole´culaires, Hoˆpital G. R. Laennec, 44035 Nantes, France Michael S. Forbes (71) ‘‘Merry Oaks,’’ Troy, Virginia 22974 Akikazu Fujita (281) Department of Veterinary Pharmacology, Faculty of Agriculture, University of Osaka Prefecture, Osaka 599-8531, Japan Tetsushi Furukawa (389) Department of Physiology I, Akita University School of Medicine, Akita 0108543, Japan A. Marquis Gacy (437) Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Antony Galione (679) Department of Pharmacology, Oxford University, Oxford OX1 3QT, England

Robert D. Harvey (373) Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

D. J. Hearse (887) Cardiovascular Research, King’s Centre for Cardiovascular Biology and Medicine, The Rayne Institute, King’s College, St. Thomas’ Hospital, London SE1 7EH, England Gerd Heusch (3, 61) Department of Pathophysiology, University of Essen, 45147 Essen, Germany Yuji Hirano (389) Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan Masayasu Hiraoka (389) Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan Franz Hofmann (247) Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, D80802 Mu¨nchen, Germany Masatsugu Hori (853) Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita 565-0871, Japan Steven R. Houser (1087) Molecular and Cellular Cardiology Laboratory, Cardiovascular Research Group, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Joseph R. Hume (373) Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Seigo Izumo (927, 1065) Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 Arshad Jahangir (829) Division of Cardiovascular Diseases and Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Aleksandar Jovanovic (693) Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland

Contributors

xv

Sofija Jovanovic (693) Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland

Gary D. Lopaschuk (543, 1125) Cardiovascular Research Group, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

Paul F. Kantor (543) McMaster University, Hamilton, Canada

Benedict R. Lucchesi (1181) Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109

Gary J. Kargacin (527) Smooth Muscle Research Group, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada T2N 4N1 Robert S. Kass (1137) Department of Pharmacology, Columbia University, New York, New York 10032

Jane A. Madden (213) Department of Neurology, Medical College of Wisconsin, Zablocki Veterans Administration Medical Center, Milwaukee, Wisconsin 53295

Seiko Kawano (389) Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan

Jonathan C. Makielski (99) Departments of Medicine and Physiology, University of Wisconsin, Madison, Wisconsin 53792

Junko Kimura (417) Department of Pharmacology, Fukushima Medical University, School of Medicine, Fukushima 960-1295, Japan

Ali J. Marian (1045) Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

Masafumi Kitakaze (853) Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita 565-0871, Japan

Jure Marijic (309) Department of Anesthesiology, University of California, Los Angeles School of Medicine, Los Angeles, California 90095

Kenji Kitamura (327) Department of Pharmacology, Fukuoka Dental College, Fukuoka 814-0193, Japan

Donald H. Maurice (805) Departments of Pathology and Pharmacology and Toxicology, Faculty of Medicine, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Evangelia G. Kranias (447) Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Iftikhar J. Kullo (1233) Mayo Clinic and Foundation, Rochester, Minnesota 55905 Yoshihisa Kurachi (281) Department of Pharmacology II, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan

William G. Mayhan (1011) Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198 Gerhard Meissner (461) Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Adi Kurgan (967) Department of Surgery B, Shaare Zedek Hospital, Jerusalem 91031, Israel

Ludwig Missiaen (501) Department of Physiology, Catholic University of Leuven, B-3000 Leuven, Belgium

Lubica Lacinova´ (247) Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, D80802 Mu¨nchen, Germany

Michael J. Morales (259) Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214

Edward G. Lakatta (737) Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224 Amir Lerman (473) Mayo Clinic and Foundation, Rochester, Minnesota 55905 Andrew P. Levy (1225) Technion Faculty of Medicine, Bat Galim 31096, Israel Jon W. Lomasney (599) Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611

A. Moroni (357) Department of Physiology and General Biochemistry, University of Milan, 20133 Milan, Italy Mariko Nakamura (175) Department of Physiology I, School of Medicine, University of the Ryukyus, Okinawa 903-0125, Japan Mona Nemer (705) Laboratory of Cardiac Growth and Differentiation, Montreal Clinical Research Institute, and Department of Medicine, Division of Experimental Medicine, McGill University, and Department of Pharmacology, University of Montreal, Montreal, Quebec, Canada H2W 1R7

xvi

Contributors

Jeanne M. Nerbonne (1107) Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Robert Roberts (1045) Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

Thomas Netticadan (949) Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R2H 2A6

Richard B. Robinson (761) Departments of Pharmacology, Medicine, and Pediatrics, Columbia University, New York, New York 10032

Bernd Nilius (481, 501) Department of Physiology, Catholic University of Leuven, B-3000 Leuven, Belgium Katsushige Ono (229) Department of Physiology, Oita Medical University, Oita 879-5593, Japan Lionel H. Opie (543) University of Cape Town, RSA 7925 Cape Town, South Africa Jan B. Parys (501) Department of Physiology, Catholic University of Leuven, B-3000 Leuven, Belgium Richard J. Paul (571) Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Amir Pelleg (633) Departments of Medicine and Pharmacology, MCP Hahnemann University, Philadelphia, Pennsylvania 19102 Carmen M. Perez-Terzic (437) Division of Cardiovascular Diseases and Department of Internal Medicine, Department of Molecular Pharmacology and Experimental Therapeutics, and Department of Physical Medicine and Rehabilitation, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Valentino Piacentino III (1087) Molecular and Cellular Cardiology Laboratory, Cardiovascular Research Group, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Jan J. Piek (19) Department of Cardiology, CRIA, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands Giovanni M. Pitari (805) Departments of Medicine and Biochemistry and Molecular Pharmacology, Division of Clinical Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 M. Puce´at (427) Research Center of Macromolecular Biochemistry, CNRS UPR 1086, Cedex 5, France Stevan Rakovic (679) Department of Pharmacology, Oxford University, Oxford OX1 3QT, England Ilaria Rivolta (1137) Department of Pharmacology, Columbia University, New York, New York 10032

Rosita J. Rodriguez (1211) College of Pharmacy, Oregon State University, Corvallis, Oregon 97331 Michael R. Rosen (761) Departments of Pharmacology, Medicine, and Pediatrics, Columbia University, New York, New York 10032 Yoram Rudy (133) Cardiac Bioelectricity Research and Training Center, Case Western Reserve University, Cleveland, Ohio 44106 Nancy J. Rusch (213) Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Manjot S. Sandhu (949) Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R2H 2A6 Jutta Schaper (1031) Department of Experimental Cardiology, Max Planck Institute, D-61231 Bad Nauheim, Germany Wolfgang Schaper (1031) Department of Experimental Cardiology, Max Planck Institute, D-61231 Bad Nauheim, Germany Win-Kuang Shen (829) Division of Cardiovascular Diseases and Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Maria Siebes (19) Department of Cardiology, CRIA, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands Robert D. Simari (1233) Mayo Clinic and Foundation, Rochester, Minnesota 55905 R. John Solaro (519) Department of Physiology and Biophysics, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612 Jos A. E. Spaan (19) Department of Medical Physics, CRIA, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands Nicholas Sperelakis (175) Department of Molecular and Cellular Physiology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267

Contributors

David C. Spray (149) Departments of Neuroscience and Medicine (Molecular Cardiology), Albert Einstein College of Medicine, Bronx, New York 10461 Miduturu Srinivas (149) Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461 Susan F. Steinberg (761) Departments of Pharmacology, Medicine, and Pediatrics, Columbia University, New York, New York 10032 Joseph R. Stimers (407) Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Harold C. Strauss (259) Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214 Sylvia O. Suadicani (149) Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461; and Universidade Sao Judas Tadeu, Sao Paulo, Brazil Masanori Sunagawa (175) Department of Physiology I, School of Medicine, University of the Ryukyus, Okinawa 903-0125, Japan James Surapisitchat (657) Cardiovascular Research Center, University of Rochester Medical Center, Rochester, New York 14642 Masayuki Tanemoto (281) Department of Pharmacology II, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan Rana M. Temsah (949) Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R2H 2A6

xvii California, Los Angeles School of Medicine, Los Angeles, California 90095

Helmut A. Tritthart (789) Institut fu¨r Medizinische Physik und Biophysik, Karl-Franzens-Universita¨t Graz, A-8010 Graz, Austria David R. Van Wagoner (1107) Department of Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 Guy Vassort (633) INSERM U-390 Physiopathologie Cardiovasculaire, FR-34095 Montpellier, France Monique J. Vink (149) Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461 Gordon M. Wahler (199) Department of Physiology, Midwestern University, Downers Grove, Illinois 60515 Scott A. Waldman (805) Departments of Medicine and Biochemistry and Molecular Pharmacology, Division of Clinical Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Michael P. Walsh (527) Smooth Muscle Research Group, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada T2N 4N1 Shimin Wang (259) Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214 Stephanie H. Wilson (473) Mayo Clinic and Foundation, Rochester, Minnesota 55905 Rui-Ping Xiao (737) Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224

Derek Terrar (679) Department of Pharmacology, Oxford University, Oxford OX1 3QT, England

Jun Yamazaki (327) Department of Pharmacology, Fukuoka Dental College, Fukuoka 814-0193, Japan

Andre Terzic (437, 693, 829) Division of Cardiovascular Diseases and Department of Internal Medicine, and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota 55905

Chen Yan (657) Cardiovascular Research Center, University of Rochester Medical Center, Rochester, New York 14642

Noritsuga Tohse (719) Department of Physiology, Sapporo Medical University School of Medicine, Sapporo 060, Japan Ligia Toro (309) Departments of Anesthesiology and Molecular & Medical Pharmacology, University of

Hisashi Yokoshiki (719) Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan Ying-Ying Zhou (737) Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224

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Foreword When we examine the fourth edition of Heart Physiology and Pathophysiology we can be reassured that physiology’s place as the ‘‘Queen of the Biological Sciences’’ remains secure. Even more than its three illustrious predecessors, this edition provides a contemporary and comprehensive view of cardiovascular function, beginning with ultrastructure, events at the cell membrane, the processes of contraction and relaxation, the integrated function of the system, and the key mechanisms responsible for its derangement. Both the heart and the vascular system are considered, and the similarities and differences in structure and function of cardiac myocytes and vascular smooth muscle cells are clarified. The endothelium has long been thought of as a relatively inert casing separating the blood from the vascular wall, but its important roles in vascular smooth muscle function and coagulation receive appropriate attention. The mechanisms of action of the major classes of cardio-

vascular drugs are also clearly presented. In the final analysis, cellular function is controlled by gene expression, and important new information on the molecular bases of cardiac and vascular injury and dysfunction is provided. The talented editors, Drs. Sperelakis, Kurachi, Terzic, and Cohen, should be congratulated for selecting a stellar team of authors and for coordinating their efforts in preparing this important text. The fourth edition of Heart Physiology and Pathophysiology is certain to be of intense interest and immense value not only to the broad community of cardiovascular scientists and their trainees but also to cardiologists concerned with the why and wherefore of normal and abnormal cardiovascular function. Eugene Braunwald, M.D. Boston, Massachusetts

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Preface The first three editions of Physiology and Pathophysiology of the Heart were successful as advanced reference books for heart researchers and academic cardiologists. The books had developed a solid reputation as being the leaders in the field of cardiovascular science. The first and second editions were translated into Russian. The book was very comprehensive and authoritative. The various chapters were written by leading investigators in their respective specialty areas of cardiovascular science. Although there were some other focused books on heart function, our book was very broad based, covering an array of topics concerning heart function and malfunction. This was the background for planning of a fourth edition. However, it was suggested that the book be remodeled to make it more of a textbook, as well as a reference book, not only for cardiovascular researchers and academic cardiologists, but also for cardiology residents, practicing cardiologists, and graduate students interested in cardiovascular science. To help in this major endeavor, three well-known cardiovascular scientists were recruited to serve as co-editors and to provide valuable input about topics that should be covered and the experts who should be invited to write chapters on those topics. The editorial work was divided among the four editors. The editors asked each contributor to make a concise and didactic presentation of the assigned topic. Since the book would become a textbook, we requested that the contributors limit the bibliography to about 50 references, citing only key research reports, reviews, and books. We also asked that their chapter be well illustrated with clear figures. To help achieve our goal of getting the attention of the cardiology resident and practitioner, we have placed much more emphasis on the relevant pathophysiology and mechanism of drug action. This includes coverage of cardioprotection, ischemia/reperfu-

sion injury, ischemic preconditioning, calcium overload, atherosclerosis, diabetic vascular disease, angiogenesis, cardioplegia, heart failure, cardiomyopathies, hypertrophy, apoptosis, arrhythmias, gene expression, gene therapy, free radical damage, and toxicology. This book provides the foundation for the basic science of heart function and dysfunction, and attempts to bridge the gap between basic cardiovascular science and clinical science. The fourth edition has been greatly reorganized, including the section headings. A number of previous chapters have been dropped, and a large number of new chapters have been added. There are now 68 chapters compared to 59 chapters in the third edition. Discussions of vascular smooth muscle, endothelial cells, and coronary circulation are integrated and presented in a logical sequence alongside discussion of cardiac muscle. The publisher of the book is now Academic Press. We wanted to keep the price of the book in a range that students, residents, and researchers could afford. The name of the book was changed to Heart Physiology and Pathophysiology to reflect the new publisher and the new orientation toward a textbook. The chapters were written by a distinguished group of experts and outstanding scientists from around the world. It has been our pleasure and honor to work with them in the preparation of the fourth edition. We hope that the reader will find the book useful, clear, and comprehensive. Finally, Dr. Sperelakis wishes to acknowledge the expert technical help of Emily May and Lori Asbury in the preparation of this book. Nicholas Sperelakis Yoshihisa Kurachi Andre Terzic Michael V. Cohen

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1 Sequence of Cardiac Activation and Ventricular Mechanics JAMES M. DOWNEY

GERD HEUSCH

Department of Physiology University of South Alabama Mobile, Alabama 36688

Department of Pathophysiology University of Essen Essen, Germany

I. INTRODUCTION

basis of the force-generating apparatus. Similarly, both contract in response to an action potential on the sarcolemmal membrane. The electrophysiology of the two muscles differs dramatically, however. In skeletal muscle an action potential at the end plate of a motor neuron causes the skeletal muscle cell to depolarize through release of a transmitter substance, acetylcholine. Thus the skeletal muscle is termed neurogenic, as it contracts only in response to a neural action potential. The skeletal muscle cells are insulated from each other and are, therefore, not affected by the activation of neighboring cells. Skeletal muscle contracts in an all-or-none fashion, and the force generated for any given length will be the same for every twitch. The action potential, however, is so short in skeletal muscle that a significant amount of force can only be generated by stimulating the fiber repeatedly with a train of neural discharges (temporal summation). Cardiac muscle differs in three important respects. First, the cardiac action potential is not initiated by neural activity. Instead, specialized muscle tissue in the heart itself spontaneously initiates the action potential, making the heart’s contraction myogenic (originating within the muscle). Because of gap junctions between adjacent cardiac muscle cells, electrical activation spreads directly from muscle cell to muscle cell. Second, the duration of the cardiac action potential is much longer than that in skeletal muscle and lasts for almost a third of a second. As a result, a single action potential maintains tension development throughout systole. Neural and humoral influences have only a modulatory effect on heart rate. The third difference is that the contraction is not an all-or-none phenomenon. Skeletal muscle grades the strength of contraction through tem-

The primary function of the heart is to circulate blood through the body. It does this by acting as a mechanical pump. While on the surface this function seems simple, investigators have devoted an enormous amount of time and resources to study this pumping process. The ultimate goal is to achieve a complete understanding of how the molecular processes in the individual cells of the heart result in the gross pumping of blood. To reach that goal we must understand how the cells are activated, how tension development and shortening occur on the cellular level, and how the geometry of the fibers, which shorten in a single direction, is coupled to pressure development in the lumen and the accompanying expulsion of volume from it. Unfortunately, our understanding of each of these points remains incomplete. This book has divided the physiology of the heart into many discrete parts. Each chapter focuses on its assigned part and reviews what is known about it in detail. This chapter is designed to serve as an introduction to the basic physiology of the beating heart. The overview provided by this introductory chapter will set the stage for the following in-depth chapters.

II. CARDIAC MUSCLE IS SIMILAR TO OTHER MUSCLE TYPES Cardiac muscle, like skeletal muscle, is a striated muscle and much of the mechanism of contraction of the two muscle types is similar. Both use the proteins actin and myosin arranged in a highly organized lattice as the

Heart Physiology and Pathophysiology, Fourth Edition

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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I. Pumping Action and Electrical Activity of the Heart

poral and spatial summation. Because the heart’s contraction is in response to a single action potential transmitted to all fibers, the cardiac muscle cells have evolved a sophisticated system by which the force of contraction can be modulated from beat to beat.

III. ACTIVATION OF THE HEART A. Excitation Originates within the Pacemaker Several types of cardiac muscle fibers exist within the heart. It is often useful to classify these types broadly as either contractile or conductile. Contractile cells are the cells of the working myocardium and constitute the bulk of the muscle cells that make up the atria and the ventricles. An action potential in any one of these cells leads to vigorous force development and/or mechanical shortening. Conductile cells are specialized muscle cells that are involved with the initiation or propagation of action potentials rather than direct generation of force. The conducting cells are principally concentrated in the structures indicated in Fig. 1. Of critical importance is the sinoatrial (SA) node. The SA node lies in the right atrium near the entrance of the superior vena cava. SA nodal cells generate spontaneous action potentials and act as the normal pacemaker of the heart. An action potential in a cardiac muscle cell will, through its gap junctions, stimulate neighboring cells to generate an action potential such that each action potential originating in the SA node will be propagated over the whole heart. Because the SA node is located in the atria, action potentials will first be propagated over the atria, making them the first structures in the heart to contract.

Action potentials spreading across the atria eventually reach another conducting structure known as the atrioventricular (AV) node. The AV node is located in the proximal part of the interventricular septum between the origin of the coronary sinus and the septal leaflet of the tricuspid valve. The AV node serves two important functions. The first is to relay the wave of depolarization from the atria to the ventricles. A sheet of connective tissue associated with the valves separates the atria from the ventricles, and the AV node is normally the only conductive link between the atria and the ventricles. The second function of the AV node is to delay the spread of excitation from the atria to the ventricles. AV node cells are specialized to conduct very slowly from cell to cell. This delay permits the atrial contraction to fill the ventricles with blood before the latter begin to contract. Fibers of the AV node give rise to fibers of the AV bundle (common bundle or bundle of His), which in turn divides into the left bundle branch and the right bundle branch. These branches then divide into an extensive network of Purkinje fibers. Purkinje fibers are conductile cells that conduct action potentials very rapidly. They are interwoven among the contractile cells of the ventricles and serve to quickly spread the wave of excitation throughout the ventricles. If conduction over the ventricles were slow, the heart would contract in a peristaltic wave from base to apex, which would be very inefficient at displacing blood out of the ventricles. The rapidly conducting Purkinje fibers, however, cause the ventricular cardiomyocytes to contract almost simultaneously. It is important to emphasize that all of these conducting structures (i.e., SA node, AV node, Purkinje network) are composed of specialized cardiac muscle cells (Noble, 1978).

B. Cell-to-Cell Conduction Occurs through Gap Junctions

FIGURE 1 Structure of the conduction system in the heart. See text for details. Modified with permission from A. M. Katz, ‘‘Physiology of the Heart,’’ Raven Press, New York (1992).

The heart is made up of millions of individual rodshaped cells, each arranged such that their long axes are oriented parallel to a plane tangent to the heart’s surface. The myocytes are arranged in the heart in a staggered pattern much like bricks in a wall. The region where adjacent myocytes adjoin is termed the intercalated disc and would be analogous to the mortar between the bricks (see Fig. 2). The discs run transversely or perpendicular to the cells’ long axis where the ends of two adjacent myocytes abut. The disc then turns 90⬚ and runs longitudinally between the myocytes until another end abutment begins. The transverse aspect of the intercalated disc is filled with structures called desmosomes. The desmosomes make strong mechanical attachments between the cells, as the cardiomyocytes will shorten

1. Cardiac Activation and Ventricular Mechanics

FIGURE 2 An electron micrograph of ventricular muscle. The A, I, and Z bands are clearly seen, as are mitochondria (M). The thin line that meanders through the field is an intercalated disk separating two adjacent cells. Details show gap junctions (GJ) and desmosomes (D) on the intercalated disk. Reproduced with permission from S. R. Goodman, ‘‘Medical Cell Biology,’’ Lippincott, Philadelphia (1994).

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in the direction of their long axis. The longitudinally oriented region of the intercalated disc is rich in lowresistance connections between the cells called gap junctions. Small pores in the center of each gap junction allow ions and even small peptides to flow from one cell to another along their electrochemical gradients. An action potential in one muscle cell is propagated to adjacent muscle cells via direct electrotonic propagation across the gap junctions. Because of the gap junctions, every cell in the heart is electrically coupled to the next and, thus, the heart can behave as a single motor unit. Ventricular muscle conducts electrical impulses at about 1 m/sec. Theoretically, an ion inside an SA nodal cell could travel throughout the heart via the gap junctions. The ion could visit every cell without ever having to enter the extracellular space. Purkinje cells contain less contractile proteins than contractile cells and are specialized for rapid propagation. The rate of transmission of an action potential from one end of a cell to the other increases in proportion to the cell’s size. The conduction velocity is aided by the large diameter of the Purkinje cells. A Purkinje fiber also has a high density of gap junctions between its cells and it will conduct action potentials four times faster than a ventricular myocyte (4 m/sec). Because of their large size, Purkinje cells are a popular cell type to study in single cell electrophysiological investigations (Cohen et al., 1981). Cells of the AV and SA nodes, like those in the Purkinje fibers, also have a reduced quantity of contractile proteins. Nodal cells are much smaller than either contractile cells or Purkinje cells, which results in a low propagation velocity of only 0.05 m/sec. AV nodal cells also have a reduced density of gap junctions to further

depress their conduction velocity. In addition, nodal cells lack fast sodium channels, which causes the upsweep of the action potential to occur much more slowly. The reduced rate of rise of the action potential voltage also slows the rate of conduction. The low propagation velocity of AV nodal cells accounts for the delay between atrial and ventricular contraction as was noted earlier. While all of these factors act to reduce conduction velocity in the AV node, they also reduce the safety factor in this tissue. The safety factor refers to the ratio of the current actually injected into a neighboring cell to the threshold amount required to initiate an action potential. As this ratio diminishes and approaches one, the probability of conduction failure increases. As a result, conduction block in the AV node is a common clinical occurrence. Ultrastructural features of a typical cardiac muscle cell are illustrated in Fig. 3. At first glance, the ultrastructure appears much like that in a skeletal muscle cell. Common features include the characteristic A, I, and Z bands where the actin and myosin filaments overlap, a T tubule system, and sarcoplasmic reticulum (SR). Because the pattern of banding is repetitive, it is convenient to refer to the unit from one Z band to the next as a sarcomere, the basic unit of the contractile apparatus. There are some subtle differences, however, between cardiac muscle and skeletal muscle ultrastructure. One major difference lies in the T tubules (the T stands for ‘‘transverse’’ tubules because they are transversely oriented or perpendicular to the long axis of the cell). They are centered on the Z band with only one tubule per sarcomere, unlike the case in skeletal muscle where there are two tubules per sarcomere located at A–I junctions. In addition, the SR is less developed in the cardiac muscle

FIGURE 3 Ultrastructure of a contractile cell. A contractile cell in the heart is very similar to a skeletal muscle cell in its basic organization. Modified with permission from A. M. Katz, ‘‘Physiology of the Heart,’’ Raven Press, New York (1992).

1. Cardiac Activation and Ventricular Mechanics

cell. This feature has important physiological consequences, as will be discussed later. The SR in cardiac contractile cells consists of two types of structures: (a) the sarcotubular network, making up the bulk of the SR, in close proximity to the contractile machinery; and (b) the subsarcolemmal cisternae, which abut the T tubules.

C. Cardiac Muscle Experiences an Action Potential Figure 4 shows an action potential typical of what would be recorded from a contractile cell in the ventri-

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cle. The action potential causes calcium to enter the cell and to be released from the SR into the cytosol, which in turn activates the actin and myosin filaments. Note that the action potential is about 300 msec in duration, whereas an action potential in a nerve or skeletal muscle cell lasts only about 1 msec (Cranefield, 1977). The plateau phase of the action potential prolongs the active state, preventing the heart from relaxing before the ventricular contents are ejected. The rapid phase of depolarization is termed phase 0. The small initial period of positive potential is called phase 1. Phases 0 and 1 are the result of opening of fast sodium channels (Cohen et al., 1981) and the simultaneous closure of potassium channels. This causes the cell to approach the equilibrium potential for sodium. Figure 4 indicates that sodium conductance becomes high at that time. Phase 1 is followed by a long period, the plateau phase or phase 2, during which the membrane remains depolarized. Note that phase 2 is largely maintained by calcium entry through L-type calcium channels. After the plateau phase, there is a phase of repolarization, during which the membrane potential returns to its resting level. The repolarization phase, termed phase 3, results from the reopening of potassium channels. The resting potential between beats is referred to as phase 4, and at that time the cell is close to the equilibrium potential for potassium.

D. Excitation–Contraction Coupling Is Accomplished by Calcium Ions

FIGURE 4 Simplified diagram of the time course of some of the permeability changes that contribute to the cardiac action potential. Modified with permission from A. M. Katz, ‘‘Physiology of the Heart,’’ Raven Press, New York (1992).

Cardiac muscle cells, like skeletal muscle cells, have a highly developed SR system. Excitation–contraction coupling in cardiac muscle cells is similar to that in skeletal muscle cells. Specifically, an action potential travels down the T tubules and causes the release of Ca2⫹ from the SR, which in turn activates the contractile machinery by attaching to troponin C-binding sites located on actin filaments. During the cardiac action potential there is a sustained increase in Ca2⫹ conductance, and all Ca2⫹ ions moving into the cell at that time contribute to mechanical activation. Figure 5 shows a diagram of how calcium activates the cardiac muscle cell to contract. There are three pools of Ca2⫹ that are important to the cardiac muscle cell: the extracellular fluid, the SR, and the cytoplasm. Only Ca2⫹ in the latter compartment is able to bind with the troponin-binding sites and initiate contraction. During an action potential, Ca2⫹ entry through the sarcolemma increases the concentration of Ca2⫹ in the cytoplasm. Because the amount of Ca2⫹ entering is relatively small, however, it accounts for only a fraction of the activation of the contractile proteins. Through mechanisms that are not fully understood, it appears that this relatively

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the cell. As shown in Fig. 5, a Na⫹ –Ca2⫹ exchange system in the sarcolemma is primarily responsible for removing calcium from the cytosol. The exchanger is not a pump and can function only in concert with the membrane Na⫹ –K⫹ pump, which itself is dependent on ATP as a source of energy. The exchanger will pass three Na⫹ ions in one direction for one Ca2⫹ ion in the other direction, but because of its status as an exchanger rather than a pump, it will only do so when the concentration gradients for both ions are favorable. Because the Na⫹ –K⫹ pump maintains a strong transmembrane gradient for sodium, any free Ca2⫹ that appears in the cytosol will favorably be exchanged for three Na⫹ ions from the extracellular fluid. There are also true calcium pumps in the sarcolemma, which are not shown in Fig. 5; they account for only a small percentage of the calcium flux. FIGURE 5 A simplified model of calcium fluxes in the excitation– contraction coupling process in cardiac muscle cells. From L. R. Johnson, ‘‘Essential Medical Physiology,’’ 2nd Ed., Lippincott-Raven, Philadelphia (1998), used with permission.

small amount of Ca2⫹ entering the cell during the action potential triggers the release of sequestered Ca2⫹ within the SR. The importance of this facilitation is evidenced by the fact that heart muscle will not contract when the influx of Ca2⫹ across the sarcolemma is prevented, even though adequate stores of Ca2⫹ are still present in the SR. Once Ca2⫹ reaches the cytosol it is free to bind to troponin C located on the thin actin filaments. This binding, through a complex series of chemical actions, results in modification of the myosin molecule such that it will form crossbridges with the actin filaments and begin the contraction process.

E. Relaxation Is Accomplished by Removing Ca2⫹ from the Cytosol In cardiac muscle, as in skeletal muscle, there is a Ca2⫹ pump in the SR, often referred to as sarcoendoplasmic reticulum calcium ATPase (SERCA). The Ca2⫹ pump in Fig. 5 removes Ca2⫹ from the cytoplasmic pool and pumps it back into the SR. When enough Ca2⫹ is removed from the cytosol, the muscle relaxes. The Ca2⫹ pump restores the concentration of Ca2⫹ in the intracellular pool (i.e., SR) to its preaction potential level. With each action potential some extracellular Ca2⫹, which is the ultimate source of SR Ca2⫹ stores, moves into the cell. If all of the cytosolic Ca2⫹ were pumped into the SR, then after only a short period of time the ability of the SR to hold all of the Ca2⫹ would be exceeded. Clearly, a mechanism is needed to remove Ca2⫹ from

F. The Strength of Contraction Can Be Modulated in Cardiac Muscle The strength of contraction generated by cardiac muscle cells is dependent on the cells’ contractility, sometimes called the inotropic state, which in turn is related to the Ca2⫹ fluxes described earlier. An increased contractility means that for any given length the muscle is capable of generating a greater force or shortening to a greater extent. It is important to note that the amount of Ca2⫹ released from the SR with each action potential is not sufficient to fully occupy all of the troponin C-binding sites. Therefore, any manipulation that leads to an enhanced cytosolic Ca2⫹ during systole will result in a more complete activation of the filaments and thus stronger muscle contraction. The Ca2⫹ fluxes can, in turn, be altered by a variety of physiological control systems, such as the sympathetic nerves working through the 웁 adrenergic receptors. Details of these systems are discussed in detail in Chapters 3, 34, and 44.

G. Pacemaker Cells Control Heart Rate The SA node is the normal pacemaker of the heart. Action potentials in the SA node are somewhat different from action potentials in contractile cells. The first and most obvious difference is that these action potentials lack a rapid phase 0 depolarization, as cells in the SA node lack the fast voltage-dependent Na⫹ channels. Another difference between contractile and nodal cells is that phase 4 resting potentials are unstable. The resting potential starts from its minimum value of about ⫺60 mV and then depolarizes slowly until it reaches the threshold for a regenerative action potential. Upon completion of the action potential, the cell again begins to depolarize and another action potential is initiated.

1. Cardiac Activation and Ventricular Mechanics

This process occurs about 60 to 100 times each minute, resulting in the spontaneous heart beat. Cells in the AV node also have pacemaker potentials, but they are slower to reach threshold (only about 40 beats/min) and thus the SA node dominates with its faster rhythm. Purkinje fibers also have intrinsic pacemaker activity. An isolated strip of Purkinje fibers will spontaneously generate action potentials with a frequency of about 25 to 40 action potentials/min, a rate slower than that of either SA or AV nodal cells. The AV node and Purkinje fibers are called latent pacemakers because they will assume the pacemaker role should the signals from the more proximate and dominant pacemaker be interrupted. Thus the AV node takes over when there is SA nodal arrest, and the pacemaker moves to Purkinje fibers when conduction below the AV node is interrupted, blocking transmission of the impulse generated by higher pacemakers. The firing rate of the SA node can be modulated by both sympathetic and parasympathetic nervous systems. In addition, circulating hormones such as catecholamines and thyroid hormones also affect the rate.

IV. CONTRACTION OF THE HEART Section II reviewed how the heart is activated. This section examines how the heart pumps blood. The pumping action of the heart occurs when cardiac fibers contract, causing the walls of the hollow ventricle to move inward, raising the pressure of the contained blood and expelling it. The blood then flows along its pressure gradients through the structures of the cardiovascular system. Because of the geometry of the muscle fibers in the ventricular wall, a direct relationship exists between linear tension development in an individual fiber and pressure development in the ventricle. For the most part there has been good agreement between studies of isolated muscle strips such as small animal papillary muscles and whole heart mechanics. For that reason it is useful to first examine the behavior of the muscle strip.

A. The Length–Tension Relationship The length–tension relationship is the cornerstone of muscle mechanics. Figure 6 shows a plot of tension vs length for a strip of cardiac muscle. There are three curves on the graph. The first is the passive tension curve collected for muscle in the noncontracting (relaxed) state. Note that muscle is freely extensible with little increase in tension at shorter lengths, but then becomes quite stiff as it is stretched further. This increased tension is thought to derive from the connective tissue

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FIGURE 6 A length–tension curve for a strip of cardiac muscle. The active tension curve is derived by subtracting the passive tension from the total tension at each length. Lmax occurs at the peak of the active tension curve.

surrounding the myocytes as well as from cytoskeletal elements within the myocytes. It is often termed parallel elastic tension because it is in parallel with actual contractile elements. The uppermost curve represents the total tension obtained when the muscle is contracting. This is termed total because it represents the sum of tension from both contractile elements and parallel elastic elements. The third curve is not actually measured, but is rather derived from the first two by subtraction. The resulting active tension curve in theory should depict the tension developed by the contractile elements. Two things are obvious. Active tension development is maximal at an intermediate muscle length termed Lmax . Second, active tension approaches zero at either end of the curve. Physiologically the heart operates with the muscle fibers at a length below Lmax . Note that in that range increasing the resting length of the muscle results in a more forceful contraction. There has been a concerted search for the determinants of the length–tension curve. Gordon et al. (1966) demonstrated that the length–tension relationship, at least in part, derives from the geometric overlap of actin and myosin filaments. Indeed fiber arrangement does explain the decline in active tension at lengths beyond Lmax . At these long lengths the overlap diminishes until at zero active tension there is no overlap. The ascending limb of the curve is much more interesting to the physiologist, as it is in this region that the heart operates. Unfortunately, it is also more difficult to explain. At one time the ascending limb was assumed to result from the geometry of thick and thin filament overlap that might be altering the distance between actin and myosin filaments. However, from studies of skinned muscle fibers that have had their sarcolemma function-

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ally removed with detergents, it was demonstrated that a length-dependent calcium sensitivity largely accounted for the length–tension curve. The concentration of Ca2⫹ required to produce 50% maximal tension (EC50) actually increases as the fibers shorten (Hofmann and Fuchs, 1988). The molecular mechanism is not understood, but may in part be related to closer proximity of actin to myosin when the fibers are stretched (Solaro and Van Eyk, 1996). It has been convenient to classify muscle contractions as either isotonic or isometric. If both ends of the muscle are firmly fixed the length cannot change and the contraction is termed isometric; only tension can be developed. The curves in Fig. 6 were generated in isometric muscles by varying the muscle length between beats and measuring tensions during contraction and relaxation. However, if the muscle contracts against a finite load then it will shorten whenever its ability to develop force exceeds the load against which it is contracting. If the load remains constant throughout the contraction (as often occurs in the laboratory) the contraction is termed isotonic. Look at the example in Fig. 7. The muscle strip at its starting length is under a resting tension termed the preload. When caused to contract the muscle tenses from point A to point B in an isometric (no change in length) fashion. At that length the muscle is capable of developing much more force (D) than the load (afterload) it is being asked to contract against. Therefore, at point B the muscle begins to shorten in an isotonic fashion toward point C. The length–tension relationship is useful in determining how much a muscle will shorten. Figure 7 reveals that the muscle strip will shorten until its length reaches a point where the total developed tension has a value equal to the load it is shortening against (C). At this point further shortening

is impossible as the muscle would be incapable of supporting this afterload at a shorter length.

B. The Frank–Starling Relationship The length–tension relationship can be extrapolated to the whole heart. The German physiologist Otto Frank was the first investigator to systematically examine how stroke volume is controlled. His research demonstrated that the amount of ventricular pressure actively generated by a frog’s contracting heart is dependent on the volume of fluid within the ventricle at the end of diastole. A fluid-filled balloon was placed within the ventricle of a beating frog’s heart and the pressure inside the balloon was measured with a catheter. The balloon was then filled to different volumes and ventricular pressure was recorded at each volume both during diastole and systole. Plotting diastolic and peak systolic pressures against volume yielded a pressure–volume curve that is remarkably similar to the length–tension curve seen for a muscle strip. Studying the canine heart, the British physiologist Ernest Starling extended Frank’s concept to demonstrate that this length dependency ensures that the heart will pump a volume of blood equal to that which it receives. Because of the pioneering work of these two physiologists, we now refer to the concept that the volume expelled and/or force of ejection during systole is proportional to the length of the muscle fibers or degree of cardiac filling at end diastole as the Frank– Starling law. What they saw was simply a reflection of the length–tension relationship described earlier, but in a whole heart. We can apply the physiologic principles learned from a strip of cardiac muscle to the whole heart by changing the tension and length axes used for the former to pressure and volume within the ventricle.

C. Pressure–Volume Relationships in the Ventricle

FIGURE 7 The path followed by an isotonic contraction plotted on a length–tension curve. See text for details.

The relationship between pressure and volume for a distensible container is its compliance. As the compliance of a structure increases, the more its volume will change for a given change in filling pressure. It is useful to think of pressure–volume curves for the ventricle in terms of compliance as well. Figure 8 reveals that during diastole the heart is very compliant, as a small increment in filling pressure will cause a large increment in ventricular volume. Note that during systole the slope of the pressure–volume curve increases dramatically, implying that the ventricle is much less compliant. In the example described earlier it was shown how the end systolic length should in theory be predicted from the length–tension curve. However, when Frank tried to

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FIGURE 8 The heart describes an ejection loop with every beat. LVEDV is determined by the venous filling pressure (preload), and LVESV is determined by the aortic pressure (afterload) and systolic compliance curve. The difference between LVESV and LVEDV is stroke volume.

calculate how much blood the frog heart would eject, he found that the relationship was quite complex because the frog heart begins to relax before ejection is complete (Frank, 1959). It was assumed that the mammalian heart would behave in a similar fashion until the studies of Kichi Sagawa in the 1970s. Sagawa found that the mammalian heart, unlike the frog heart, does completely eject before it begins to relax and, as a result, the length–tension curve accurately predicts the end systolic length (Sagawa, 1978). Based on these assumptions the ejection loop has emerged as a simple yet powerful method of analyzing the whole heart’s mechanics. Figure 8 depicts a pressure–volume curve for the heart. The ventricle fills passively with blood during diastole. The filling pressure for the left ventricle is the left ventricular end diastolic pressure (LVEDP), which is largely determined by pulmonary venous pressure. LVEDP typically will be between 3 and 7 mm Hg and is analogous to the preload in Fig. 7. In fact, cardiologists often use the terms LVEDP and preload interchangeably. LVEDP determines the left ventricular end diastolic volume (LVEDV) and hence the resting length of the ventricular muscle fibers. When the heart contracts it behaves as if its compliance was greatly decreased, i.e., the ventricle holds less volume for any given pressure than it did during diastole. Thus, with each beat the heart moves from the diastolic compliance curve to the systolic compliance curve. The decreasing compliance raises pressure in the left ventricle, resulting in closure of the mitral valve.

After mitral valve closure the pressure rises in the ventricle isovolumetrically. The constant-volume condition is depicted by the vertical line drawn between points A and B in Fig. 8. The ventricle in Fig. 8 would be capable of developing a pressure much higher than aortic pressure, but as soon as the ventricular pressure exceeds the pressure in the aorta the aortic valve opens and the ventricle begins to eject blood into the aorta. At point B in Fig. 8 the mode of muscle contraction changes abruptly from isometric (A to B) to isotonic. Shortening against the aortic pressure, the heart moves horizontally from point B to C. Just as the filling pressure is often referred to as the preload for the ventricle, the aortic pressure is often called the afterload. Because of the Frank–Starling relationship the ability of the ventricle to develop pressure is diminished as the ventricular volume decreases during ejection, and at point C a stable equilibrium is reached. The heart then stays at point C, the left ventricular end systolic volume (LVESV), until the action potential subsides and the heart begins to relax. Regardless of how the contraction began, it will always end with LVESV positioned on the systolic compliance curve (Weber et al., 1976). At the end of ejection the aortic valve closes and the heart relaxes isovolumetrically, causing the pressure–volume curve to move vertically from point C to D where it rejoins the diastolic compliance curve. As pressure in the ventricle falls below atrial pressure, the mitral valve opens and the ventricle refills with pulmonary venous blood, causing the loop to return to the starting point A and close. In the example shown in Fig. 8, stroke volume is calculated as the difference between points B and C. The fraction of the total blood content that the heart ejects during systole is called the ejection fraction and is calculated as the stroke volume divided by the LVEDV. The shape of systolic and diastolic compliance curves will vary from heart to heart, but if the curves are known for any heart then its output can be predicted accurately.

D. Contractility Two factors determine the force of contraction in cardiac muscle. The first, the length of the muscle, has already been discussed in detail. The second is termed contractility. As was explained earlier, insufficient calcium is released into the cytosol to cover all of the troponin C calcium-binding sites during each action potential. As a result the strength of contraction is less than maximal. There are many mechanisms within the cardiomyocyte by which the calcium concentration can be modulated such as occurs when adrenergic 웁 receptors are stimulated. If the amount of calcium entering

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I. Pumping Action and Electrical Activity of the Heart

the cytosol is increased, the force generated for any given muscle length is also increased, causing an upward shift of the systolic pressure–volume curve shown in Fig. 9. This is termed an increase in contractility. The effect of an increase in contractility on the ejection loop is to move LVESV to the left (toward a smaller end systolic volume). If filling pressure, which is determined by the venous pressure, is unchanged, then the end diastolic volume will be unchanged. The reduced end systolic volume will result in an increased stroke volume, as indicated by the shaded area in Fig. 9. Conversely, decreasing contractility tends to decrease stroke volume.

E. Preload The end diastolic volume is determined primarily by venous filling pressure. Because the ventricle is very compliant, small changes in venous filling pressure have a major effect on end diastolic volume and thus stroke volume. This relationship makes venous filling pressure a primary determinant of cardiac output, which is discussed in further detail in Chapter 4. Because of its pivotal role in controlling cardiac output, the pulmonary venous pressure is of great importance clinically and is usually monitored closely in the critically ill patient. Figure 10 shows that an increase in venous pressure increases stroke volume, as indicated by the shaded area, entirely by changing end diastolic volume and has no effect on end systolic volume. Because the healthy heart is operating at near-optimal end diastolic fiber length and volume, only small increases in end diastolic filling will actually increase cardiac output (Braunwald et al., 1976). This dependence of cardiac output on dia-

FIGURE 9 The effect of an increase in contractility on the ejection loop. Note that stroke volume increases because LVESV decreases, as indicated by the shaded area.

FIGURE 10 The effect of an increase in filling pressure on the ejection loop. Note that stroke volume increases because LVEDV increases, as indicated by the shaded area.

stolic filling results in a beat-to-beat adaptation of right and left ventricular stroke volumes during the variable right- and left-sided pre- and afterloads seen during the various phases of ventilation.

F. Afterload It is easy to see in Fig. 11 that LVESV is determined by aortic pressure. As aortic pressure falls, the equilibrium point will occur at a smaller end systolic volume. The resulting decrease in LVESV will increase stroke volume by the amount indicated by the shaded area in Fig. 11. Thus, a rise in aortic pressure tends to decrease stroke volume, whereas a falling aortic pressure tends

FIGURE 11 The effect of a decrease in aortic pressure on the ejection loop. Note that stroke volume increases because LVESV falls, as indicated by the shaded area.

1. Cardiac Activation and Ventricular Mechanics

13

to increase stroke volume. Note that as was seen with contractility, changes in aortic pressure affect stroke volume by changing only the end systolic volume. In general, aortic pressure is highly regulated and thus is not used by the body to control cardiac output as are venous pressure and contractility. Nevertheless, when changes in aortic pressure do occur, such as acutely in stress situations or more chronically in hypertension, stroke volume and thus cardiac output will be affected.

G. Diastolic Compliance While the body has the ability to change the heart’s contractility from moment to moment, no such control system exists for changing the diastolic compliance curve. Because myocytes of the normal heart are fully relaxed, their stiffness is determined wholly by the structures that make up their parallel elastic elements. However, diastolic compliance can be altered by disease. When the heart becomes hypertrophied, as often occurs with persistent hypertension, the walls of the ventricle become very thick as the muscle cells increase their diameter and connective tissue is deposited within the heart wall. The thickened wall reduces compliance and reduces filling during diastole.

H. The Auxotonic Contraction For the sake of clarity we have simplified the ejection loops by keeping the aortic pressure constant during ejection. This produced an isotonic contraction. In actuality, isotonic contractions are rare in the body, as afterload usually does change during the contraction in most muscles, and the heart is no exception. Figure 12 reveals that blood pressure actually rises and falls through ejection, starting at point A, the diastolic pressure, rising to point B, the peak systolic pressure, and finally falling to C, the left ventricular end systolic pressure (LVESP). A contraction against a varying afterload is termed an auxotonic beat. The diagrams that we have developed earlier, therefore, would have to be modified slightly as in Fig. 12 to take into account the auxotonic conditions in a real heart.

I. Assessment of Contractility The contractile state of the ventricle is of much interest to the physician as many disease states cause abnormal contractility. In the hyperthyroid patient, contractility may be elevated to the point where the heart may hypertrophy because of the hyperkinetic state. Conversely, in the patient with cardiac failure, contractility may be reduced to a point where an adequate cardiac output cannot be achieved. Because of the heart’s inac-

FIGURE 12 The ejection loops in Figs. 8–11 have been simplified by assuming ejection is isotonic. A more accurate representation of the ejection loop shown here accounts for the continuously changing afterload throughout systole. Points A, B, and C of the blood pressure tracing in the inset are indicated on the ejection loop. From L. R. Johnson, ‘‘Essential Medical Physiology,’’ 2nd Ed., Lippincott-Raven, Philadelphia (1998), used with permission.

cessible position in the chest, it has been surprisingly difficult to measure the heart’s contractility in the clinical setting. Over the years several indices of cardiac contractility have been introduced and the student should have some understanding of the major ones. 1. The Ventricular Function Curve Pressure–volume loops revl that the heart’s stroke volume, which can be measured in the clinical or laboratory setting, is determined in part by the heart’s contractility. However, two other factors, filling pressure and aortic pressure, also affect stroke volume. This problem was first addressed by Sarnoff and Berglund in 1954. To isolate the effect of filling pressure they proposed a simple plot of stroke volume as a function of end diastolic pressure. If one examines the ejection loop in Fig. 10 it can easily be seen that if contractility and aortic pressure are held constant, then a simple relationship exists between stroke volume and filling pressure. That relationship is depicted in Fig 13 (top). Similarly, it can easily be appreciated that decreasing the contractility of the heart will shift that line downward, as indicated in Fig. 13. The problem with this approach is that the stroke volume filling pressure curve is also affected by aortic pressure. As shown in Fig. 13 (top), increasing aortic pressure also shifts the curve downward. Thus, it is not possible to tell if a shift in the stroke volume filling pressure curve resulted from a change in contractility or aortic pressure. The ambiguity was cleverly elimi-

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I. Pumping Action and Electrical Activity of the Heart

FIGURE 13 Evolution of the ventricular function curve. When filling pressure is plotted against stroke volume (top), the curve can be shifted by changes in either contractility or aortic pressure. If stroke work is chosen for the vertical axis (bottom), only changes in contractility can shift the curve.

nated by plotting stroke work rather than stroke volume on the vertical axis. In a fluid system, physical work is the product of the volume pumped and the pressure it is pumped against: stroke volume times aortic pressure in this case. Stroke work for the ventricle is relatively independent of the aortic pressure because the aortic pressure and stroke volume are reciprocally related. As a result their product (stroke work) remains constant over a wide range of pressures. The stroke work filling pressure plot shown in Fig. 13 (bottom) usually called the ventricular function curve, is only affected by changes in contractility. In practice the filling pressure is changed over several beats by the rapid infusion of intravenous fluids or by suddenly lifting the patient’s legs. Stroke volume and aortic pressure are monitored, and from these data the plot is made. Any factor that shifts that plot is said to have changed contractility. The shortcomings are that very invasive procedures are required to obtain such data, thus limiting clinical application.

and the contraction becomes isometric. Increasing the resting length of the muscle (preload) rotates the curve counterclockwise with Vmax acting as the pivot point. As expected, P0 increases, and at any load (except zero) the contraction is faster. Brutsaert and Sonnenblick (1969) discovered that changes in contractility tended to shift the curve upward along its entire length, including Vmax . That prompted them to propose that Vmax could be used as an independent index of contractility. However, there is a problem when one actually attempts to estimate Vmax for a patient’s heart. Because the initial phase of systole occurs with zero load on the contractile elements, the cardiac fibers in theory should shorten briefly at Vmax at the start of every beat. The velocity of shortening can be assessed by measuring the rate at which ventricular pressure rises during the isovolumetric period of contraction. Geometric analysis predicts that the velocity of fiber shortening in the isovolumetric phase is approximated by dP/dt/P (Grossman et al., 1971). Regardless of the method, it is necessary to extrapolate the shortening velocity–load curve back to zero load to determine Vmax (dotted lines in Fig. 14). While it is feasible to make such a measurement from a catheter placed in a patient’s ventricle, the prognostic value of these measurements has been disappointing. A simpler approach has been the measurement of the rate of pressure development (dP/dt). The recording of ventricular pressure from a catheter can be differentiated electronically so that a continuous tracing of ventricular dP/dt can be monitored. Unfortunately, a rise in preload, in this case ventricular filling pressure, also increases the rate of shortening. However, a rise in con-

2. Velocity-Related Indices of Contractility The length–tension curve tells us nothing about the temporal relationships in the contracting muscle. For that information we can look at the force–velocity curve shown in Fig. 14. The velocity of shortening is plotted against the load that the muscle is shortening against. A hyperboloid relationship is seen. At zero load the velocity of shortening is maximal (Vmax). As the load increases the velocity of shortening decreases until a load is reached where no shortening is possible, P0 ,

FIGURE 14 The force–velocity curve for isotonically contracting cardiac muscle. As the afterload decreases, the muscle shortens faster. Extrapolation to zero load (dotted lines) yields a maximum velocity of shortening, Vmax . Changing the preload causes the curves to rotate about Vmax . Only an increase in contractility causes a change in Vmax . P0 indicates the point where the velocity of shortening is reduced to zero and represents the isometric tension for that muscle length.

1. Cardiac Activation and Ventricular Mechanics

15

tractility usually evokes a fall in venous pressure. Thus an increase in dP/dt can unambiguously be interpreted as an increase in contractility if the end diastolic pressure is either unchanged or falls. The major shortcomings of dP/dt as a marker of contractility are that it is imprecise, it is affected by changes in preload and afterload, and its measurement requires insertion of a high-fidelity ventricular catheter. It is often used in animal experiments where a simple index of contractility is to be monitored continuously. 3. The Ejection Fraction The ejection fraction, as mentioned earlier, is calculated by dividing the stroke volume by the end diastolic volume. It is literally the fraction of the end diastolic ventricular volume that is ejected with each beat. The ejection fraction can be measured by a variety of methods in the human heart and, as a result, has become the primary clinical index of contractility. These methods now include ultrasound, nuclear medicine scans, and Xray angiography. While the ejection fraction is strongly influenced by contractility, the ejection loop analysis reveals that it is also influenced by both preload and afterload. Nevertheless, because of its ease of measurement, the ejection fraction remains useful. Normally, the ejection fraction should be about 0.6 for a healthy heart. Ejection fractions below 0.5 suggest disease and below 0.3 are associated with high mortality. 4. The End Systolic Volume–Pressure Relationship The best assessment of contractility would be derived from an actual plot of the systolic pressure–volume curves for that ventricle. As the heart contracts its compliance curve gets steeper until it reaches a point of maximum slope, which is referred to as the maximum elastance, Emax . Because it can be shown that the heart is at Emax at the end of ejection, Emax can be measured by determining the relationship between left ventricular end systolic volume and aortic pressure (Suga et al., 1973). It is now possible to obtain a continuous ventricular volume recording with a conductance catheter (Baan et al., 1984). By having a computer plot the ventricular volume signal against ventricular pressure, an ejection loop for each beat can be displayed. If aortic pressure is varied over several beats, the pressure and volume at the end of systole can be determined for each of those beats, as shown in Fig. 15. Connecting these end systolic points results in graphic representation of the relationship between LVESV and LVESP over a range of afterloads, and the slope of that curve is Emax . As estimated from the end systolic pressure–volume relationship, Emax is clearly the most accurate measurement

FIGURE 15 Calculation of the end systolic pressure–volume relationship. Ventricular volume is displayed on the horizontal axis and ventricular pressure on the vertical axis. Each loop depicts data from a single beat. Filling pressure is elevated for several beats by injecting saline into the left atrium so that several different ejection loops are traced. A line is then fit to the end systolic points. When contractility was elevated with epinephrine, the systolic compliance curve (Emax) became steeper. Reprinted from Suga et al. (1973).

of contractility available today. Unfortunately, the method still requires relatively invasive instrumentation, which limits its clinical utility.

J. The Systolic Pressure–Volume Area Predicts Oxygen Consumption of the Heart Most of the heart’s energy is derived from oxidative metabolism of fatty acids and, to a lesser extent, carbohydrates. As a result there is a linear relationship between the heart’s energy production and its oxygen consumption. Because most of the heart’s energy expenditure is related to its mechanical work, it is not surprising that a close correlation exists between the oxygen consumption of the heart and its mechanical activity. The external physical work (pressure times volume) done by the heart is exactly represented by the area within the ejection loop. That area, however, correlates very poorly with the heart’s oxygen requirements. If, however, an internal work component, as shown in Fig. 16A, is added to the external work, then the sum correlates well with the heart’s energy demands (Suga et al., 1983). This systolic pressure–volume area index leads to some interesting and surprising predictions. For example, it can easily be shown that raising the aortic pressure decreases stroke volume and may even decrease external work but always increases oxygen demand. This analysis explains that high blood pressure is so stressful to the heart because of the increased

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I. Pumping Action and Electrical Activity of the Heart

FIGURE 16 (A) The oxygen consumption of the heart per beat is determined very accurately by the area encompassed by the ejection loop (external work) plus the triangle between systolic and diastolic compliance curves (internal work). Note how increasing the afterload increases the oxygen demand greatly because of increased internal work, even though the external work is hardly altered. (B) For any given state of contractility, the oxygen consumption of the heart is related linearly to the PVA. If the contractile state is elevated, then the line is shifted upward (C).

internal work and, therefore, increased sum of internal and external components. Conversely, reducing aortic pressure increases cardiac output and decreases oxygen demand. Figure 16B reveals that there is a basal oxygen consumption that is unrelated to the pressure– volume work area. If contractility is increased (increased Emax), basal oxygen consumption will also be increased, causing a parallel upward shift of the relationship between pressure–volume area and oxygen consumption (Fig. 16C). It is not fully understood why the systolic pressure–volume area is such an accurate predictor of the heart’s oxygen consumption, but it is hoped that as the energetics of contraction become better appreciated, the underlying mechanism may become obvious. The physical work of the heart consists of internal work (deformation), external work (pressure–volume area), and kinetic work (acceleration). The pressure– volume area does not consider the time course of the

cardiac cycle. Therefore, all energy spent for the acceleration of blood during ejection is not accounted for in the pressure–volume diagram; normally, less than 10% of energy is spent for the acceleration of blood during ejection, but this term may increase at high heart rates, e.g., during intense exercise (Antoni, 1996).

K. Practical Considerations We now know that the concept of a single quantity called contractility is an unrealistic one. As discussed in later chapters in this book, the heart has a variety of mechanisms whereby it can vary its strength of contraction. Varying the calcium concentration in the cell does not account for all of the changes. For example, we know that contractile proteins can be phosphorylated by several kinases with resultant alteration of the performance of those proteins. The force–velocity curve is

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1. Cardiac Activation and Ventricular Mechanics

quite revealing. At one end of the curve is P0 , which is determined logically by the number of crossbridges between the actin and the myosin filaments participating in the contraction. It is easy to correlate P0 with Ca2⫹ concentration, as that will in turn determine the number of myosin attachment sites that will be activated. The other end of the curve, Vmax , is more enigmatic. Because of the zero loading condition, Vmax should be independent of the number of crossbridges, but rather sensitive to the rate at which each of them can be made and broken. Factors that control that rate are not understood, but certainly will not be a simple function of the number of troponin-binding sites occupied by Ca2⫹. Another complicating factor is the ever-changing active state. In a tetanized skeletal muscle the intracellular calcium concentration is constant during stimulation. In cardiac muscle it is changing continuously during a contraction. Thus, although the heart’s myofibers should theoretically pass through Vmax at the onset of every beat, the intracellular Ca2⫹ concentration would still be quite low that early in the cardiac cycle. As a result, Vmax measured at that time would likely grossly underestimate the contractile state evident during ejection when calcium levels are higher. This is further complicated by the fact that all of the ventricular myocytes are not stimulated simultaneously because slow cell-to-cell conduction in the ventricle results in a 50- to 100-msec delay in activation of the ventricular myocardium. The late phase of systole is similarly affected. Because a plateau of activation has never actually been identified for cardiac muscle, the end of ejection should occur in a declining state of activation. Attempts to demonstrate a disparity between pressure–volume curves obtained during isovolumetric conditions when peak pressure is plotted and also at end systole in the ejecting heart have proven futile, suggesting that this latter consideration is more of a theoretical problem than a practical one.

V. SUMMARY Electrical activation of the heart is conducted from cell to cell through gap junctions. The SA node serves as the pacemaker and the AV node controls conduction from the atria to the ventricles. Contraction is initiated by a rise in cytosolic calcium coming from both the sarcoplasmic reticulum and the interstitial fluid with each beat. Relaxation is effected by removing calcium from the cytosol by both a sarcolemmal calcium/sodium exchanger and by a sarcoplasmic calcium ATPase. Because insufficient calcium is present during systole to occupy all calcium binding sites on the troponin C mole-

cules, the heart controls the heart’s contractility by varying the cytosolic calcium concentration during systole. The force of contraction is determined not only by the contractility but also by the length of the muscle fibers. A cardiac cycle can be described by an ejection loop. Analysis of the ejection loop reveals that stroke volume can be varied by changing preload, afterload, or contractility. Contractility can be estimated by ventricular function curves, measurement of Vmax , dP/dt, ejection fraction, or the end systolic pressure–volume relation. Finally, the ejection loop can be used to derive a good index of myocardial oxygen consumption.

Bibliography Antoni, H. (1996). Functional properties of the heart. In ‘‘Comprehensive Human Physiology’’ (R. Greger and U. Windhorst, eds.). Springer, Berlin. Baan, J., van der Velde, E. T., de Bruin, H. G., Smeenk, G. J., Koops, J., van Dijk, A. D., Temmerman, D., Senden, J., and Buis B. (1984). Continuous measurement of left ventricular volume in animals and humans by conductance catheter. Circulation 70, 812–823. Braunwald, E., Ross, J., Jr., and Sonnenblick, E. H. (1976). ‘‘Mechanisms of Contraction of the Normal and Failing Heart,’’ 2nd Ed. Little, Brown and Company, Boston. Brutsaert, D. L., and Sonnenblick, E. H. (1969). Force-velocity-lengthtime relations of the contractile elements in heart muscle of the cat. Circ. Res. 24, 137–149. Cohen, C. J., Bean, B. P., Colatsky, T. J., and Tsien, R. W. (1981). Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. J. Gen. Physiol. 78, 383–411. Cranefield, P. F. (1977). Action potentials, afterpotentials, and arrhythmias. Circ. Res. 41, 415–423. Frank, O. (1959). On the dynamics of cardiac muscle (translated by C. B. Chapman and E. Wasserman). Am. Heart J. 58, 282– 378. Gordon, A. M., Huxley, A. F., and Julian, F. J. (1966). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. (Lond.) 184, 170–192. Grossman, W., Brooks, H., Meister, S., Sherman, H., and Dexter, L. (1971). New technique for determining instantaneous myocardial force-velocity relations in the intact heart. Circ. Res. 28, 290– 297. Hofmann, P. A., and Fuchs, F. (1988). Bound calcium and force development in skinned cardiac muscle bundles: effect of sarcomere length. J. Mol. Cell. Cardiol. 20, 667–677. Noble, D. (1978). ‘‘The Initiation of the Heartbeat.’’ Clarendon, Oxford. Sagawa, K. (1978). The ventricular pressure-volume diagram revisited. Circ. Res. 43, 677–687. Sarnoff, J. S., and Berglund, E. (1954). Ventricular function. I. Starling’s law of the heart studied by means of simultaneous left and right ventricular function curves in the dog. Circulation 9, 706–718. Solaro, R. J., and Van Eyk, J. (1996). Altered interactions among thin filament proteins modulate cardiac function. J. Mol. Cell. Cardiol. 28, 217–230. Suga, H., Hisano, R., Goto, Y., Yamado, O., and Igarashi, Y. (1983). Effect of positive inotropic agents on relation between oxygen

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consumption and systolic pressure volume area. Circ. Res. 53, 306–318. Suga, H., and Sagawa, K. (1974). Instantaneous pressure-volume relationships and their ratio in the excised and supported canine left ventricle. Circ. Res. 35, 117–126. Suga, H., Sagawa, K., and Shoukas, A. A. (1973). Load independence

of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio. Circ. Res. 32, 314–322. Weber, K. T., Janicki, J. S., and Hefner, L. L. (1976). Left ventricular force-length relations of isovolumic and ejecting contractions. Am. J. Physiol. 231, 337–343.

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2 Coronary Circulation and Hemodynamics JOS A. E. SPAAN,* JAN J. PIEK,† and MARIA SIEBES† Departments of *Medical Physics and †Cardiology, CRIA, Academic Medical Center University of Amsterdam, 1100 DE Amsterdam, The Netherlands

I. INTRODUCTION

ble part of the myocardium. The group of Hoffman (Domenech et al., 1969; Archie et al., 1974; Heymann et al., 1977) has been a driving force in this area of coronary research. Other new technologies have allowed the application of physiological insights gained from basic research to clinical decision making. Among others, Marcus (1983) and Gould (1998) played an important role in this field. More recently, we have seen the development of so-called smart guide wires. They incorporate a miniaturized pressure or Doppler flow velocity sensor at the tip of a flexible 0.014-inch diameter wire, which allows acute measurements of physiological signals in diseased coronary arteries of humans. At this moment, we have arrived in an era where clinical diagnosis and on the spot decision making regarding the treatment of coronary artery disease can be based on physiological principles. It is the purpose of this chapter to give an overview of coronary physiology that may be helpful in bridging the sometimes seemingly different worlds of basic scientists and clinicians.

The coronary circulation is designed to supply the heart with oxygen and nutrients in order to enable the heart to maintain the blood supply to the rest of the body. The metabolic needs of our body can vary widely and rapidly, which require an adequate adaptation of the heart and therefore coronary blood flow. Understanding the distribution and control of blood flow is important as an insufficient supply of blood flow to even a small area of heart tissue may result in mechanical dysfunction of the heart, irregularity of heart rhythm, infarction, or fibrillation. Coronary physiology has a long history. Scaramucci (1695) noted that coronary flow was pulsatile and that blood entering the heart muscle in diastole was stored and expelled in systole. In the first issue of the American Journal of Physiology, Porter (1898) noted that coronary flow could increase with work of the heart. With progress in instrumentation, insights into the mechanisms of coronary flow became more and more advanced. Pressure difference manometers were used in the middle of the century to evaluate the dynamics of coronary flow (Wiggers, 1954; Green et al., 1935; Gregg et al., 1972; Gregg, 1950). A great breakthrough in the analysis of coronary flow signals was achieved by the development of the electromagnetic flow meter by Gregg’s group, which for the first time allowed studies in the instrumented conscious dog. The next important step forward was the introduction of radioactive-labeled microspheres into coronary research (Heymann et al., 1977), which allowed differences between subepicardial and subendocardial perfusion to be analyzed and helped elucidate why the subendocardium is the most vulnera-

Heart Physiology and Pathophysiology, Fourth Edition

II. BASIC CHARACTERISTICS OF THE CORONARY CIRCULATION A. Anatomy The left and right coronary arteries are the first arteries to originate from the aorta, at the sinuses of Valsalva. The left main stem splits after a short distance into two major epicardial arteries, the left circumflex (LCX) and the left anterior descending artery (LAD), which course around and down the epicardial surface toward the apex.

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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I. Pumping Action and Electrical Activity of the Heart

The perfusion area of the LCX encompasses the lateral and posterior free wall of the left ventricle, and that of the LAD, the anterior free wall, and the upper part of the interventricular septum through one of its branches, the septal artery (SA), which runs almost completely intramurally. The right coronary artery runs over the epicardium of the free wall of the right heart. Its perfusion area is the free wall of the right ventricle and the right atrium, but up to a varying degree it also supplies a part of the posterior left ventricular free wall. The epicardial arteries branch into smaller penetrating arteries that perfuse the dense intramural vascular networks traversing the myocardial wall from the epicardium to the endocardium. A radiographed slice of myocardial tissue with vessels filled with Roentgen contrast is shown in Fig. 1.

Alongside the major epicardial arteries are epicardial veins, which join in the coronary sinus, a large venous structure that empties directly into the right atrium. Epicardial veins of the free wall of the right ventricle drain directly into the right atrium. A small percentage of venous blood drains directly into the ventricles via Thebesian veins. There are many anastomoses between epicardial veins. Occlusion of an epicardial vein easily directs venous flow into the direction of other epicardial veins. Such an anastomosis structure is quite useful. Because of the low pressures in epicardial veins, flow in them is obstructed easily. The heart also has a lymphatic system. Intramural lymph vessels drain into epicardial lymph vessels. Also the epicardial lymph system has many interconnections.

FIGURE 1 Radiograph of a myocardial cross section in which arteries have been filled with barium contrast. Note that transmural vessels originate from the outer layer of the wall (epicardium) and penetrate the myocardium where they further branch into smaller arteries and arterioles at the inner layer (endocardium). Radiograph provided by Professor W. Chilian, University of Wisconsin, Milwaukee, WI, used with permission.

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Lymph flow in a normal dog heart is very low, about 3 ml/hr/100 g tissue. It would take 7 hr to refresh the whole interstitial volume of the heart.

B. Pulsatile Coronary Arterial and Venous Flow Coronary arterial blood flow is phasic, which is caused by the aortic pressure wave and compression of the intramural coronary vessels by contraction of the cardiac muscle. This compression effect causes mean systolic arterial flow to be lower than mean diastolic flow, although the driving pressure in systole is higher. Figure 2 illustrates the characteristic features of pressure and flow waveforms in the left coronary artery of a dog. The systolic flow wave has an initial and a late dip, which are related to the rapid changes in systolic left ventricular pressure and correspond to the phases where the stiffness of the heart muscle is increasing and decreasing, respectively. During the relaxation phase, diastolic flow initially increases sharply above systolic levels and then decreases gradually with aortic pressure. Coronary venous flow is out of phase with coronary arterial flow. Venous flow is dominant in systole and is almost absent during the whole of diastole. The out-ofphase waveforms of arterial and venous flow demonstrate that intramural coronary blood volume is changing during each heartbeat. This volume change is brought about by the contraction of the heart muscle, squeezing the vessels running through it.

C. Intramyocardial Pump Model: General Concept The basic concept for interpretation of the phasic nature of coronary arterial and venous flow is the intra-

FIGURE 2 Blood flow in the left circumflex coronary artery of a dog, Qcor. Mean systolic flow is lower than mean diastolic flow, despite a higher aortic pressure, Pao, during systole. PLV is left ventricular pressure.

FIGURE 3 Intramyocardial pump model. The intramural arteries are represented by a ‘‘windkessel,’’ as is also used to explain the aortic pressure decay. Contraction compresses the windkessel and squeezes volume out, resulting in an increased venous outflow and a diminished arterial inflow. Furthermore, the windkessel causes a delay between inflow and outflow, also in prolonged diastoles as is demonstrated later, dV/dt refers to the rate of change of intramural volume, which equals the difference between inflow, Qin , and outflow, Qou , and is known as capacitive flow. Rin and Rout are inflow and outflow resistances, which are constant for the linear form of the model but dependent on volume, V, when the model is nonlinear.

myocardial pump model depicted in Fig. 3 (Spaan et al., 1981). Intramural intravascular volume is represented by a compliant compartment, which is compressed by an external force. This external force is related to a combination of left ventricular pressure and contractility, which are both time dependent. The compliance is called intramyocardial compliance and is filled and drained via resistances. It is difficult to assign the two conceptual resistances in the model to different anatomical parts of the coronary vascular network. Inlet resistance is dominated by resistance arteries and hence can vary due to coronary vascular control, which causes the diameter of these vessels to change. Outlet resistance certainly relates to intramural veins. The resistance of the capillary network has an influence on both resistances but mostly on outlet resistance. The model in Fig. 3 is conceptual and aims to elucidate some basic principles of the mechanics of coronary flow, which are summarized here, but discussed in more detail later in the chapter. 1. The squeezing of intramural vessels will increase systolic intravascular pressure, which in turn will result in an increased systolic venous outflow and decreased systolic coronary arterial inflow. The squeezing can result in an intramural intravascular pressure that exceeds coronary arterial input pressure. Normally occurring systolic forward flow will then change into retrograde flow. Systolic flow in the septal artery turns more easily into retrograde flow because it is embedded within contracting tissue.

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I. Pumping Action and Electrical Activity of the Heart

2. It takes time for the intramyocardial volume to change. Because of inlet and outlet resistances, intramural volume in systole will not reach a steady state. Systole is just too short to obtain this. 3. The change in intramural vascular volume is large enough to reduce vascular diameter and thereby increase vascular resistance in systole. 4. In diastole, the intramural vascular volume will restore itself by the fall of intravascular pressure resulting from the release of systolic squeezing. The amount of volume restoration will depend on the length of diastole. Normally, a diastole is not long enough to achieve a constant volume. 5. Because vascular volume is increasing in diastole, resistance is decreasing during diastole. 6. The elasticity of the walls of intramural vessels in interaction with surrounding myocardial tissue constitutes the so-called intramyocardial compliance. The model in Fig. 3 is too simple to describe all the characteristics of coronary flow in detail. It has been modified in several ways. For example, more units, such as the one shown in Fig. 3, have been put in series to describe the arterial, capillary, and venous parts of the circulation in more detail. The myocardium has been modeled by several layers with their inputs and outputs connected by resistances representing transmural arteries and veins, respectively, to predict endo-epicardial distribution of flow. Relationships between volume and resistance have been implemented in such models. Although coronary blood flow is phasic and the direction of flow varies with both vessel type and location in the myocardium, it is not necessary to take this fact into account at all levels of analysis of coronary flow. In general, one will use mean values of coronary flow and pressure to describe in global terms the distribution of blood flow across the ventricular wall, control of coronary blood flow, and hemodynamic consequences of an obstruction caused by coronary artery disease. However, one has to be aware of the fact that most of the elements are nonlinear and time dependent in their physical behavior, meaning that neglecting the timevarying nature of flow and pressure can result in erroneous conclusions.

this pressure will barely influence coronary flow when coronary oxygen consumption is constant. This characteristic is generally known as coronary autoregulation. Progressive dilation of resistance vessels serves to maintain coronary blood flow when the driving pressure is reduced because of an atherosclerotic obstruction or stenosis in one of the epicardial conduit vessels. However, when coronary arterial pressure is kept constant, coronary flow will vary with oxygen consumption. This phenomenon is referred to as metabolic flow adaptation. Other terms for this phenomenon are functional hyperemia or metabolic vasodilation. Autoregulation is best illustrated by the so-called autoregulation curves, as depicted in Fig. 4. In an autoregulation curve, flow is plotted versus pressure at constant oxygen consumption. An autoregulation curve exhibits a plateau phase, which is denoted as the autoregulatory range. The magnitude of flow within the range of this plateau depends on the oxygen consumption of the heart. Flow is higher at higher oxygen consumption. The pressure-flow relation without control (Fig. 4), i.e., at maximum vasodilation, delineates the low-pressure threshold above which regulation becomes apparent. This low-pressure boundary increases with oxygen consumption. It is believed that the upper pressure limit of the autoregulatory range is related to the condition of maximum tone development in resistance vessels. The pressure-flow relation at maximum vasodilatation plays an essential role in the physiological analysis of coronary stenosis severity. Metabolic flow adaptation is demonstrated in Fig. 5. It is important to note that these curves are incrementally linear, meaning that although the relationships are straight, they do not pass through the origin. Data dem-

III. BLOOD FLOW CONTROL A. Basic Characteristics of Coronary Blood Flow Control Under physiological circumstances, coronary blood flow is well adapted to the metabolic needs of the heart. Despite the fact that coronary arterial pressure is the driving pressure for coronary flow, limited changes in

FIGURE 4 Autoregulation curves at different levels of oxygen consumption. Note the parallel shift of the curves at different oxygen consumption values (see also Fig. 5). Data from P. Mosher et al., Circ. Res. 14, 250–259 (1964).

2. Coronary Circulation and Hemodynamics

FIGURE 5 Relation between coronary flow and oxygen consumption at constant perfusion pressure. Data were obtained in goats with a cannulated main stem and were corrected for the influences of perfusion pressure on consumption as discussed under the Gregg effect. Data from I. Vergroesen et al., Am. J. Physiol. 252, H545–H553 (1987).

onstrate that for a given myocardial oxygen consumption (MVO2), flow will be higher at a higher arterial pressure. This is only possible if the extraction of oxygen is lower at higher perfusion pressures. Hence, there is some room for adjustment of oxygen uptake by altering the level of oxygen extraction. This room, however, is quite limited in the coronary circulation, and the predominant means to increase oxygen uptake with increasing cardiac work at a constant hematocrit is to raise oxygen supply by increasing coronary flow.

B. Control of Tissue Oxygen Pressure Several factors have been identified that influence coronary blood flow, but the actual process of flow control has not yet been unraveled. We will address the interaction of several of the mechanisms involved, starting with the most likely candidate for a controlled variable, oxygen tissue pressure (PO2 ). That tissue PO2 is the controlled variable is based on the observation that coronary venous PO2 varies to a much smaller degree than flow for a variety of conditions, such as changes in heart rate and arterial pressure. Following the assumption that the control process aims to maintain myocardial PO2 at a certain level, the control of coronary flow can be derived from the oxygen control system. A simple model, depicted in Fig. 6, demonstrates this reasoning. The basic assumption in this model is that an increase in tissue PO2 results in vasoconstriction and a decrease in vasodilation. According to this concept, an increase in myocardial oxygen consumption causes a reduction in tissue PO2 that results in vasodilation, and

23

an increase in flow and oxygen delivery such that tissue PO2 rises again. In this way, metabolic adaptation can be explained. Conversely, a reduction in coronary arterial pressure will reduce oxygen supply and thereby tissue PO2 . The resulting vasodilation will then restore flow, which is exactly the action required for autoregulation. One can show that the simple oxygen control model basically describes an autoregulation curve and also the parallel shift of these curves with changing oxygen consumption. (Drake-Holland et al., 1984; Vergroesen et al., 1987; Broten and Feigl, 1992). Hence, the control of coronary blood flow can be derived from the control of tissue PO2 . Attempts have been made to quantify the quality of coronary autoregulation by, for example, relating the slope of the autoregulation curve to the slope of the pressure-flow relation at maximal vasodilation. However, one has to be very careful when studying autoregulation without knowledge of the oxygen consumption. In order to obtain an autoregulation curve, one has to vary coronary arterial pressure independently from oxygen consumption. A change in coronary arterial pressure is likely to induce a change in heart performance, thereby altering oxygen consumption. Moreover, the so-called Gregg effect causes an increase in oxygen consumption when arterial pressure is increased. This effect is stronger when the response of the tone in the resistance vessels to a change in arterial pressure is diminshed (see later). Hence, an autoregulation curve plateau can seemingly be more flat when oxygen consumption is decreasing with perfusion pressure or less flat if oxygen consumption is allowed to increase with pressure. When we accept that the control of coronary flow is the result of the control of tissue PO2 , the quality of control of this variable becomes a more relevant question than the quality of control of flow. Although coronary blood flow and venous oxygen pressure seem to be well controlled at the macroscopic

FIGURE 6 Tissue oxygen pressure model for the control of blood flow. It is assumed that tissue PO2 controls the arteriolar resistance by causing vasodilation when it becomes lower and vasoconstriction when it gets higher. Vc represents the volume of blood in the microcirculation forming a buffer for oxygen. MVO2 is rate of oxygen consumption.

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I. Pumping Action and Electrical Activity of the Heart

level in large coronary vessels, a different picture emerges from the distribution of these variables over smaller units in the myocardium. The smaller the unit into which tissue is cut for the analysis of microsphere distribution (see later for the technique), the larger the relative variance of this distribution becomes. As demonstrated by Bassingthwaighte et al. (1989), the halfwidth of the frequency distribution of the number of microspheres per tissue weight can be twice the mean value. The variance in flow distribution is caused by imperfections of the vascular tree and of the blood control system to distribute blood flow evenly. A similar picture arises when measuring the distribution of oxygen pressure in tissue or small veins where a wide distribution pattern can also be found (Weiss and Sinha, 1978). The heterogeneity in flow and oxygen distribution suggests that tissue PO2 is not well controlled. It seems that the tissue does not require a specific oxygen pressure, just a minimum level high enough for mitochondria to function. It has been demonstrated that isolated mitochondria can achieve maximal oxygen consumption at an oxygen pressure lower than 2 mm Hg. Hence, an intracellular mechanism must be responsible for controlling the oxygen consumption by mitochondria in the presence of a tissue PO2 level that is above the level needed for maximum consumption.

C. Oxygen Consumption and Evaluation of Vasodilation

flow will have to adapt but it cannot do so instantly. The simplest example of a dynamic response of coronary flow to an intervention is reactive hyperemia after a coronary occlusion of short duration. An example is provided in Fig. 7. In this experiment, the left anterior descending (LAD) artery was occluded for 15 sec and mean coronary flow responded to the occlusion with a flow overshoot four times above control. In the same experiment, coronary venous oxygen saturation has been measured and oxygen uptake has been calculated. After peak reactive hyperemic flow had been reached, coronary flow returned gradually back to control level. However, Fig. 7 demonstrates that the return of coronary flow to control is slower than the return of oxygen uptake. Hence, the relation between coronary blood flow and oxygen consumption in steady state does not hold under dynamic conditions. Reactive hyperemia has been studied extensively, and it has been shown that peak reactive hyperemic flow increases with occlusion duration up to a certain level, the maximal peak flow response. The integral of the flow response above control after an occlusion has been denoted flow overpayment. This overpayment increases with occlusion durations longer than that causing maximal peak reactive hyperemia. Adenosine plays a role in this phase of repayment, as the administration of adenosine deaminase diminishes this overpayment but not peak reactive hyperemic flow (Saito et al., 1981). The reactive hyperemic flow curves have also been char-

The interaction between the mechanisms responsible for autoregulation and metabolic adaptation means that one has to be cautious in evaluating the dilatory responses to other interventions and especially to drug administration. In fact, one should be careful in reporting on such mechanisms when coronary arterial pressure and myocardial oxygen consumption have not been measured. The administration of drugs may cause an increased oxygen demand, with a normal response of the oxygen supply. Such an increase of oxygen demand may result from an effect on cardiac function and the increase in flow as the consequence of that. Only when the increase of flow exceeds the increase to be expected on the basis of a concomitant change of oxygen demand can one refer to this drug as a vasodilator (Vergroesen et al., 1997).

IV. DYNAMICS OF CORONARY BLOOD FLOW CONTROL A. Reactive Hyperemia Under varying conditions, e.g., myocardial oxygen consumption or coronary arterial pressure, coronary

FIGURE 7 Reactive hyperemia induced by a 15-sec occlusion of the LAD of a dog. MVO2 was calculated after correction of the influence of unloading and recharging of the oxygen buffers in the microcirculation. Data from J. H. Ruiter et al., Am. J. Physiol. 4, H87–H94 (1978).

2. Coronary Circulation and Hemodynamics

acterized by the so-called repayment ratio, i.e., the ratio between flow overpayment and the product of control flow and the duration of occlusion. The idea behind this calculation was that the shortage of flow during occlusion has to be repaid after restoration of flow. The repayment ratio increased with the duration of occlusion. The terms oxygen debt and oxygen repayment have also been used in the literature. For a certain time it was thought that oxygen repayment ratios were also in the order of 200 to 400% (Coffman and Gregg, 1961). This would imply that not only depleted oxygen stores would be restored after occlusion but that metabolism would be less efficient in producing ATP in that postocclusion period. However, some of these studies used a continuous withdrawal technique for venous blood to measure oxygen uptake during the reactive hyperemic response. This withdrawal technique is not suited in dynamic conditions for measuring time-averaged oxygen extraction and resulted in a strong overestimation of oxygen consumption in the reactive hyperemic phase. With a more proper determination of dynamic changes of venous oxygen saturation, oxygen repayment was calculated to be in the order of 100%, hence just enough to replenish the oxygen stores of intramural hemoglobin in the blood and myoglobin in the tissue (Ruiter et al., 1978).

B. Transient Responses to Changes in Pressure and Heart Rate In the cannulated main stem preparation in anesthetized dogs and goats, experiments were performed to determine the rate of change of coronary resistance as induced by a change in heart rate, perfusion pressure, or flow. These experiments showed that times for half the relative change of resistance, T50 , varied between 5 and 10 sec. The relative change is defined as the change in resistance from control divided by the maximal difference in change. Hence, regardless of direction of change of coronary resistance, vasoconstriction or vasodilation, the index always starts at zero in controls and reaches ⫹1 at the end of the response. Figure 8 demonstrates the normalized response of coronary resistance to a change in HR, in which the responses to heart rate increase were pooled with those obtained at decreasing HR. However, the response to an increase in heart rate was on average 9% faster than the one to a decrease in HR. Perfusion pressure was constant and equaled 80 mm Hg. The index for each dog was calculated per beat. This curve represents the average response of about 20 interventions in five dogs. The responses were pooled. The response rate of coronary resistance to a change in heart rate can obviously not be faster than the rate

25

FIGURE 8 Response of resistance index to a change in heart rate at constant perfusion pressure of 80 mm Hg. Data from J. Dankelman et al., J. Physiol. Lond. 408, 295–312 (1989).

of change of oxygen consumption that is induced by this heart rate change. From dynamic measurements of oxygen consumption induced by a heart rate change, T50 was found to be in the order of 3.8 sec (Vergroesen and Spaan, 1988). This time constant agrees well with the rate of change by which mitochondria adapt to a change in the mechanical work load of the heart. Let us now interpret this figure for the case of a heart rate decrease. Coronary flow has to decrease in response to such an intervention, as resistance has to increase due to metabolic adaptation. However, the response is first in the opposite direction and it seems that resistance decreases at first. The duration of this effect is too long to be an epicardial compliance effect. The explanation for this behavior lies in the reduced compression effect on the coronary microcirculation. In fact, the initial vasodilation first compensates for that effect and the response therefore starts at a lower resistance value than the control value before the change in HR. Resistance index responses to changes in perfusion pressure are summarized in Fig. 9. Here we also see an opposite response immediately following the pressure step. Let us look at the case for the sudden pressure

FIGURE 9 Response of resistance index to a change in perfusion pressure at constant heart rate. Data from J. Dankelman et al., J. Physiol. Lond. 419, 703–715 (1989).

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I. Pumping Action and Electrical Activity of the Heart

reduction. Some blood will flow back because of capacitive discharge from epicardial and intramural vessels. In addition, the reduction in perfusion pressure will result in smaller vessel diameters at the same level of tone and, hence, an increase in resistance. The response to a pressure decrease then exhibits an overshoot and arrives at the new steady state much faster than the response to a pressure increase. The coronary circulation seems to have a mechanism that protects against underperfusion by a fast vasodilation. Although coronary flow control is mainly organized at the local level in the heart, the difference in the rates of coronary response is probably due to regulation via the central nervous system, as this differences in the rate of response disappears with chronic denervation of the heart (Vergroesen et al., 1999). The rate of response of the coronary circulation is also dependent on the mode of perfusion. When flow is kept constant during the heart beat, coronary pressure will change when heart rate or flow level is changed. Normalization of the coronary resistance change allows comparison between the two interventions. Whether in response to a change in heart rate or a change in perfusion, the coronary system was slower, i.e., T50 values were about 1.5 times longer at constant flow than at constant pressure perfusion. These differences are not the result of different control mechanisms, e.g., pressure versus flow-sensitive mechanisms. They can be explained by a change in feedback properties. At constant flow perfusion the feedback loop is cut open in the link between coronary resistance and flow since flow is not allowed to respond to changes in resistance. The rate of change in coronary flow has been measured in patients in the awake state and during anesthesia by applying a thermodilution catheter in the coronary sinus for coronary flow measurement. In the awake state, the half-time for flow adaptation to a change in heart rate was 5 sec, which is similar to what was found in anesthetized dogs. Under anesthesia this half-time doubled in the patients. The reason for this is as yet not understood (Van Wezel et al., 1996).

C. The Gregg Effect Gregg discovered that perfusion pressure affects myocardial oxygen consumption. In general, increased pressure is accompanied by an increase in contractility, which then provides a direct explanation for the increased oxygen consumption. The Gregg effect has also been explained by the so-called ‘‘garden hose’’ phenomenon. Like a garden hose, the intramural vessels become less deformable when the pressure difference between inside and outside is increased. The decrease in vascular deformability results in a stiffer myocardium, so that

more mechanical work is required to deform the heart muscle. This in turn causes an increase in oxygen consumption without a noticeable increase in contractility. Furthermore, a distending effect of perfusion pressure on the left ventricle may result in a larger end-diastolic volume, which would also result in more cardiac work. It has been argued that the interstitial space is larger at higher perfusion pressures and therefore the Ca2⫹ stores for contraction are bigger, causing stronger contractions and thus a higher oxygen consumption of the heart. In all these explanations the role of the resistance vessels seems to be very important, as has been shown by the group of Downey (Bai et al., 1994). They clearly demonstrated a correlation between intravascular and interstitial volume and oxygen consumption. In the presence of a high-quality autoregulation, i.e., a small change in flow in response to a large change in pressure, there was hardly a change in oxygen consumption with an increase in coronary pressure. One should be careful, however, in concluding that the Gregg effect is only present in hearts with poor autoregulation. When perfusion pressure is increased stepwise, there will also be a temporal increase of interstitial volume because of the transiently increased coronary flow. However, capillary pressure and thereby interstitial volume will be restored quickly, diminishing their effect on oxygen consumption. It is difficult to measure oxygen consumption dynamically from arterial-venous oxygen saturation differences because of the changing oxygen stores within the heart such as hemoglobin and myoglobin. However, blocking of ATP-sensitive potassium channels by the administration of glibenclamide slows down the rate of response of the coronary resistance vessels such that the rate of change of oxygen stores becomes small and transients in oxygen consumption can be measured. These dynamic measurements clearly demonstrated a transient increase in oxygen consumption in the period that coronary resistance was not yet adapted. Hence, the mechanism responsible for the Gregg effect is also functional in normal hearts and is responsible for part of the oxygen consumption of the myocardium. The mechanism for the Gregg effect acts as positive feedback in the control of flow. An increase in perfusion pressure will lead to an increase in oxygen consumption, which will result in vasodilation. This positive feedback has to be counteracted by the negative feedback stemming from autoregulation. Hence, the slope of the autoregulation curve, one way or the other, represents the balance between the positive and the negative feedback loop. If the negative feedback is not strong enough, the vascular bed could fully dilate even when the vessel’s smooth muscle is capable of developing tone.

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2. Coronary Circulation and Hemodynamics

V. MECHANISMS FOR CORONARY FLOW CONTROL The control of coronary flow, or, as argued earlier, the control of oxygen consumption, is the result of several interacting factors. These factors must all have their specific role in the system of flow control. It is not logical to control a variable by several mechanisms. A familiar example is the central heating system of a house. In general, there is a thermostat controlling the burners: turning them on when it is less warm than set by the thermostat or off when the temperature is above the preset value. An additional way for controlling the heat transfer to the rooms in the house is formed by thermostats on the radiators. When it is too warm the flow of hot water through the radiator is decreased and thereby the supply of heat is reduced. The basic rule for installation of a central heating system is not to have thermostatic valves on radiators in the same room where the central thermostat is located. Otherwise, when the thermostatic valve on the radiator is set at 18⬚C and the central thermostat at 20⬚C, the latter will never heat up above 18⬚C and hence will not turn off the burners. The rest of the house will then become very warm. Keeping the basic rule as just explained in mind, we have to try to define a principal role for each factor involved in the control of coronary blood flow. This section considers some of the main factors.

A. Adenosine Adenosine is the molecule that is the end product of dephosphorylation of ATP. It is always produced, also in healthy myocardium, but it is produced in excess under anoxic or ischemic conditions. For example, adenosine plays a role in reactive hyperemia and delays the recovery of flow back to its control value. Administration of adenosine deaminase reduces the overpayment of flow by 60%. It has also been hypothesized that adenosine is the link between metabolism and coronary flow under physiological conditions. Hence, the more energy that is used by the myocardial cells, the higher the ATP turnover will be and the more adenosine will be produced. An increase of MVO2 would then result in an increase of adenosine concentration in tissue and in blood, which could be responsible for the increase in flow matching the increase in MVO2 . Several studies did indeed identify an increased level of adenosine concentration in tissue fluid and in venous blood (Berne, 1980). However, later evidence argues against the adenosine hypothesis. Administration of adenosine deaminase to the arterial blood, enough to appear in pericardial fluid and cardiac lymph, decreased myocardial

adenosine concentrations, but hardly altered flow (Kroll and Feigl, 1985; Hanley et al., 1986). Essential for the adenosine hypothesis is the dose– response relation between adenosine concentration near smooth muscle and smooth muscle tone in resistance vessels. This adenosine concentration cannot be equated to concentrations either in cardiac lymph or in venous blood. Adenosine is easily metabolized into purines and is distributed over several intercellular compartments. A detailed mathematical model has been developed predicting the adenosine concentration at the smooth muscle level on the basis of venous adenosine concentration (Kroll et al., 1992). It has been shown in chronically instrumented dogs that the relevant adenosine concentration at the site of action remains below threshold levels even at high levels of exercise. These studies and the deanimase experiments make it unlikely that adenosine has a key role in normal physiological coronary flow control. Adenosine has a role in stimulating vasodilation when tissue becomes ischemic. Experiments using microspheres to occlude microvessels demonstrate such a role. In these experiments, where vasomotor tone was intact, the obstruction of small vessels resulted in an increase of flow in the major coronary artery but a decrease of peak reactive hyperemic flow (Hori et al., 1986). This effect could be blocked by theophylline, an adenosine-blocking drug. Apparently, adenosine is produced to levels above threshold in the small areas where perfusion is blocked, and then diffuses to neighboring resistance vessels which dilate. Higher perfusion of neighboring areas may then result in higher tissue oxygen pressure, which will facilitate oxygen shunting to the poorly perfused area.

B. Myogenic Response A change of the pressure difference across the wall of an artery or arteriole induces a change of vascular tone, which may result in a diameter response opposite to the pressure stimulus. Hence, an increase in luminal pressure will result in a decrease of diameter in a vessel with active tone instead of an increase in diameter as in a passive vessel. A typical myogenic response is demonstrated in Fig 10 which shows pressure steps imposed on the vessel (top) and response of the vessel when passive (middle). The diameter becomes larger with increasing pressure. However, when the vessel is active (bottom), the increase in pressure first results in an increase in diameter by which the vessel wall is stretched. Stretch sensitive receptors then induce vasoconstriction. Myogenic tone is needed for stabilization of the mechanics of the vascular wall. The law of Laplace dictates that in a cylindrical vessel the wall stress is higher at a

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I. Pumping Action and Electrical Activity of the Heart

is dependent on both pressure in the vessel and vessel diameter (VanBavel and Mulvany, 1994; VanBavel et al., 1998).

C. Flow-Dependent Vasodilation

FIGURE 10 Myogenic response of an isolated arteriole. In the passive vessel, diameter increases with pressure, whereas in active smooth muscle the diameter decreases CSA (cross-sectional area). Data from Wesselman et al., J. Vasc. Res. 33, 32–41 (1996).

smaller radius than at a larger radius at the same pressure. We know the effect of this law from inflating a balloon. Inflation is easier with increasing volume of the balloon. The passive pressure-diameter relation of vessels demonstrates a sigmoid curve: as in contrast to normal balloons, the vessels become stiffer at higher diameters. This is the result of the collagen matrix in the vessel walls, which prevents the vessels from overstretching. However, this collagen has hardly any effect on vessel wall stiffness when the diameter is determined by smooth muscle tone. The myogenic response guarantees that the vessel diameter can be maintained when pressure increases. The myogenic effect is stronger than needed for stabilization of the vascular wall in resistance vessels. The fact that the diameter can actually decrease upon an increase in pressure is consistent with autoregulation. As discussed earlier, coronary resistance has to increase when coronary arterial pressure increases in order to keep the increase in flow minimal when myocardial oxygen consumption remains constant. It has been demonstrated that the strength of myogenic tone depends on the size of the resistance vessel. The response is strongest for vessels with a diameter of 64 애m in the active condition and is less for larger vessels. It has been shown that the myogenic response can be sufficient to predict autoregulation curves at constant oxygen consumption. The myogenic response can be modulated by pulsations in pressure. Isolated vessels exhibiting myogenic tone diminish their tone when the magnitude of pressure pulsations is increased. Such a mechanism would favor subendocardial perfusion, since there the resistance vessels are under the effect of pulsating compression due to heart contraction. The definition of vascular tone is not straightforward and one should be careful not to derive changes in tone from changes in diameter alone. Tone should be defined as the percentage of active tension of the contractile apparatus in smooth muscle cells, since stress in the wall

Endothelial cells respond to shear stress by producing nitric oxide (NO), which is a potent vasodilator. Shear stress, ␴, is defined by

␴⫽애

dv dr

where 애 is viscosity of the blood at the wall, which will be close to plasma viscosity because of a plasma boundary layer. dv/dr is the velocity gradient at the vascular wall. When blood flow in a vessel increases, the shear stress will increase, more NO will be produced, and the vessel will dilate. When flow is kept at the higher level, the vessel diameter increase will reduce the shear stress until a new steady is obtained. Flow-dependent vasodilation by itself will not result in coronary regulatory properties. When, for example, the coronary arterial pressure is increased, flow will increase, resulting in vasodilation, which will result in an increase of flow and so on. The outcome of this chain of events would be full vasodilation.

D. Nervous Control in Coronary Flow Regulation Coronary flow regulation is mainly achieved at the local level. Both autoregulation and metabolic vasodilation can be found in isolated hearts as well. This is very important as it allows the transplantation of a heart, which is not innervated after implantation. In the normal heart, nervous control is predominantly parasympathetic (acetylcholine mediated) and results in vasodilation independent of myocardial metabolism. This mechanism is mediated by nitric oxide and is activated during baroreceptor and chemoreceptor reflexes (Feigl, 1998). Sympathetic innervation consists of 움 receptormediated vasoconstriction and 웁 receptor-mediated vasodilation. Dominant sites for the 움 effect are microvessels larger than 100 애m, whereas the 웁 effect is found in smaller arterioles. Both effects work in a feedforward manner, complementing the feedback control of local metabolic factors. It has been suggested that there is a beneficial effect of the combination of 움 constriction and 웁 dilation induced by the nervous system to protect the vulnerable inner layer of the heart.

2. Coronary Circulation and Hemodynamics

29

FIGURE 11 Pressure distribution as a function of diameter in epicardial vessels of the cat. Data from Chilian et al., AJP 256, H383–H390 (1989).

E. Integration of Flow Control Mechanisms It is important to be aware of the distribution of control of coronary resistance. This has been made clear by Chilian et al. (1989), who in elegant experiments measured the pressure in epicardial vessels of different diameters. Results of these experiments are provided in Fig. 11. Open circles represent data under control conditions demonstrating that most of the pressure drop in this case is within vessels smaller than 200 애m. When the vascular bed was dilated by the administration of dipyridamole, the pressure distribution is much more gradual. Pressure starts to drop at a diameter of 400 애m. The right-hand side of Fig. 11 illustrates that the larger arteries dilated as well, implying that these vessels take part in the control of coronary resistance under normal conditions. The different control mechanisms discussed earlier must work in an integrated way (Spaan, 1991; Jones et al., 1993). The concept has evolved that the small resistance vessels, the arterioles, are under the influence of the myocytes because of short diffusion distances. Hence, these will respond to the local tissue PO2 . The more proximal resistance vessels are less under the influence of metabolism but react to circumstances induced by the control action of the smallest vessels. For example, assume that oxygen consumption increases by a certain amount. This will dilate the smaller vessels but not enough to increase flow sufficiently to meet the increased demand for oxygen. However, the larger resistance vessels will feel the increase in flow by an increase in shear stress. This then will dilate these vessels by the production of NO such that the total decrease in

resistance will be sufficient to meet the increased demand. It is difficult to position the myogenic response within this integration scheme. In some vessels the pressure will decrease upon an increase in oxygen consumption and hence will contribute to the overall vasodilation. However, in some vessels the pressure will increase because of vasodilation in more proximal vessels. The reaction would be myogenic vasoconstriction, counteracting the dilatory effects at other levels. The nervous system acts to increase the speed of responses of the coronary circulation to changes in oxygen demand or to threatening developments in perfusion conditions. It may also play a role in protecting the subendocardium from steal of blood flow by the subepicardium. Many other factors affect coronary flow and the overview provided in this section is by no means complete. However, it is felt that the mechanisms discussed earlier are the main players in the short-term regulation of the perfusion of the myocardium.

VI. CARDIAC CONTRACTION AND CORONARY FLOW A. Extravascular Resistance and Waterfall Models In the history of coronary flow analysis, different mechanisms have been proposed to explain the pulsatility of coronary flow. In this century, up to the 1970s,

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I. Pumping Action and Electrical Activity of the Heart

the model of systolic extravascular resistance dominated. The idea behind this model was that cardiac contraction would compress intramural vessels or that these vessels would be deformed in a different way such that their resistance in systole was larger. Consequently, it was thought that at the end of diastole, all external dynamic effects involved in the coronary flow signal had faded away and that the ratio between coronary pressure and flow reflected the true resistance of the vascular bed, not compromised by so-called extravascular forces playing a role only in systole. This section discusses some of the important factors that have contributed to the acceptance of this concept of end-diastolic resistance. The waterfall model, introduced for the coronary circulation in a landmark paper by Downey and Kirk (1975), provided a possible explanation for the lower subendocardial perfusion compared to subepicardial perfusion. The underlying idea was that intramural venules would collapse under the influence of tissue pressure during systole. The theory of collapsible tubes then predicts that flow is determined by the pressure difference between the tube inlet and the collapse point, divided by the resistance of the tube between these two points. This theory was applied to the coronary circulation. Consequently, systolic flow will not be lower than diastolic flow because of an increased systolic resistance, but because of an increased backpressure, the collapse pressure. Based on both experimental and theoretical data, the concept evolved that radial stress in the myocardium could be equated with local tissue pressure, which decreases from left ventricular pressure at the endocardium to intrathoracic pressure at the epicardium (Arts and Reneman, 1985). This tissue pressure concept in connection with the waterfall concept formed a workable paradigm to explain the higher impediment of perfusion in the subendocardium, the region where tissue pressure is higher. However, like the extravascular resistance model, the waterfall model also considered systole and diastole as independent episodes of the heartbeat.

B. Tissue Pressure and Time-Varying Elastance in Relation to the Intramyocardial Pump Model The intramyocardial pump model was presented as an alternative explanation of the pulsatility of coronary flow. It coupled the time-varying differences between arterial and venous flow to changes in the volume of the intramural vessels by an element denoted as intramyocardial compliance (Spaan et al., 1981). Additionally, the intramyocardial compliance related the events in systole to those in diastole: the amount of blood

squeezed out of the intramural vessels in systole has to be replaced in diastole. Thus, diastolic flow must contain a component related to the preceding systole and hence is not free of the effects of cardiac contraction. In fact, the capacitive influx raises diastolic arterial flow above the level determined by the resistance of the vascular bed alone. The intramyocardial pump model provided a good basis for the interpretation of arterial and venous outof-phase waveforms. It also formed a logical concept for understanding retrograde coronary arterial flow found in some circumstances. The intramyocardial pump was first presented in its linear form and was not directed to predicting differences in subendocardial and subepicardial perfusion. For the latter, resistance changes had to be coupled to volume changes of intramural vessels, resulting in a nonlinear intramyocardial pump model (Bruinsma et al., 1988). It was thought that tissue pressure was the driving force behind the intramyocardial pump. However, the group of Westerhof emphasized that coronary arterial flow in an empty beating heart was also pulsatile. Because left ventricular pressure is zero in an empty beating heart, systolic tissue pressure is also zero at the endocardium, which does not support the tissue pressure concept. Moreover, in isolated rat heart experiments, Westerhof’s group demonstrated that the magnitude of coronary flow pulsation was rather independent of the magnitude of systolic–diastolic differences in left ventricular pressure. The varying elastance model was put forward as an alternative explanation for the generator of pulsatile flow (Krams et al., 1989a, 1989b; Westerhof, 1990). In essence, it is also an intramyocardial pump model but it is assumed that the compliance of the vascular bed varies in time throughout the cardiac cycle. This idea was based on the varying elastance model for the explanation of cardiac function (Suga et al., 1973). The varying elastance-driven intramyocardial pump model is explained in Fig. 12. The idea is that the intramural vessels can be treated as a volume compartment analogous to that of the left ventricle. For the latter, contraction of the heart increases left ventricular pressure until the aortic valve opens. The amount of blood that is then ejected depends on (1) the end-systolic pressure–volume relation, (2) the pressure at which the valve is opened, and (3) the volume at the end of diastole. Note that the maximum pressure that can be generated by the left ventricle also depends on the end-diastolic filling. In its simplest form, the elastance concept assumes that the end-systolic and end-diastolic pressure–volume curves of the left ventricle are independent of the function of the ventricle. The slope of the endsystolic pressure–volume relationship generated from

2. Coronary Circulation and Hemodynamics

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experimental condition that resulted in higher than normal values for venous pressure. However, effects of left ventricular pressure are present in arterial inflow patterns and also in epicardial lymph pressure. When systolic left ventricular pressure is increased by aortic clamping, early systolic coronary flow depicts a larger dip and is sometimes negative (see also Fig. 2) (Kouwenhoven et al., 1992). Therefore, left ventricular pressure does have an effect on intramural vessels, but only when the stiffness of the heart muscle is low, which is the case during those periods of the heart cycle when elastance is low. Similar interactions between effects of left ventricular pressure and elastance have been shown to exist in the pressure waveform of epicardial lymph pressure (Han et al., 1993; Van Teeffelen et al., 1998).

FIGURE 12 Illustration of the varying elastance concept to explain the effect of heart contraction on intramural vessels. The thin dotted line represents a ventricular pressure–volume loop, which has welldefined corners because of the heart valves. The pressure–volume loops for the intramyocardial vessels are different because these are emptied continuously during systole and filled in diastole. Used with permission from Spaan, Basic Res. Cardiol. 90, 89–103 (1995).

multiple pressure–volume curves is used as an index of the contractility of the left ventricle (see Chapter 1). The intramyocardial pump model relates the rate of change of volume of the intramural vessels to the difference in instantaneous arterial and venous flows. Because the coronary circulation has no valves, intramural vascular volume is reduced continuously in systole via the timevarying elastance by reduced or sometimes retrograde arterial inflow and by enhanced venous outflow. There are several observations in strong support of this concept. It explains why coronary arterial and venous flow are pulsatile in the absence of left ventricular filling. Although the left ventricle is empty and not generating pressure on the endocardium, the intramural vessels are filled and influenced directly by the contraction. The amplitude of coronary arterial flow pulsation increases with increasing perfusion pressure at full vasodilation. This observation fits nicely with a larger filling of the intramural vascular space (Krams et al., 1990). It has been demonstrated further that venous pressure pulsation in an obstructed vein is indeed increased when intramyocardial blood volume is increased after the end of a long diastole, even in the presence of a reduced left ventricular pressure (Vergroesen et al., 1994). Hence, the relationship between end-diastolic filling and pressure generation by contraction was demonstrated, as well as the absence of an effect of left ventricular pressure on the intramural veins, at least for the chosen

C. Distribution of Coronary Flow We have already discussed two mechanical factors that are important for understanding the waveforms of coronary arterial flow and pressure. However, in light of the vulnerability of the subendocardium, the most crucial process to understand is the distribution of flow across the myocardium. This vulnerability is caused by the fact that heart contraction impedes perfusion in this inner layer of the heart muscle more than in the outer layer, the epicardium. The classical way to measure flow distribution over the heart muscle is by the use of radioactive microspheres. Microspheres for flow measurement should not be so small that they can pass through capillaries but not so big that they block flow to large amounts of tissue. The microspheres that are used have a diameter between 10 and 15 애m. A certain amount of microspheres is injected into the coronary circulation either as a bolus or over a period of a few beats. The microspheres are carried along with the flow, but their size prevents them from passing through the capillaries. Thus, the higher the flow in a certain region the larger the concentration of microspheres will be in that region. This local concentration of microspheres can then be measured in small pieces of tissue that are cut out of the heart that is excised after the experiment. The amount of radioactivity of a certain label can then be determined per sample volume and is reported as activity per gram tissue. Up to nine different radioactive labels can be used in such experiments. Relative flow distribution can be calculated easily by taking a certain sample as a reference. The determination of absolute flow to the different tissue pieces requires a reference measurement of flow or knowledge about the activity in a reference sample withdrawn at a known rate from the circulation. In either case, the total amount of radioactivity injected

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I. Pumping Action and Electrical Activity of the Heart

per label has to be known. Recently, fluorescent microspheres have become increasingly popular because they avoid the environmental problems related to the use of radioactivity. Under normal conditions the differences between perfusion of the epicardium and the endocardium are not very great due to the fact that metabolic control will ensure a local match between demand and supply. However, in conditions where the metabolic drive cannot dilate resistance vessels any further, local perfusion will be the result of purely mechanical factors such as driving pressure and compression by cardiac contraction. Such a condition can exist, e.g., in the presence of a critical stenosis (see later), which causes compensatory vasodilation. This condition has been simulated in experimental laboratories by applying drugs such as adenosine to achieve full vasodilation. A characteristic outcome of microsphere experiments performed at full vasodilation is that flow in the subepicardium is hardly affected by cardiac contraction when arterial pressure is kept constant. In contrast, flow in the subendocardium appears to be very sensitive to heart rate. The ratio of subendocardial to subepicardial flow for a typical experiment is demonstrated in Fig. 13. This so-called endo/epi ratio is plotted versus diastolic time fraction. This indicates that time period in the heartbeat when the vessels are not compressed by contraction. The interpretation of this graph is as follows. When the heart beats at a slow pace, the contractions squeeze out some blood from the intramural vessels but not so much as to seriously affect resistance of the intramural

vessels. Hence, the time-averaged flow in the beating heart at this low heart rate will be equal to the flow in a very long diastole. In such a long diastole the endo/ epi ratio was found to be 1.5 (Wuesten et al., 1977; Domenech, 1978). When heart rate becomes higher than 60 bpm, the time spent in diastole becomes too short to completely restore the volume of blood that was expelled out of the intramural vessels during preceding systoles and diastolic resistance will be higher than in the case of a prolonged diastole. At a very high heart rate, diastole becomes so short that coronary resistance will remain quite high throughout the heartbeat. In fact, extrapolation of experimental data results in a time fraction of 0.4 at which subendocardial flow will cease completely. Hence, it is suggested that the relationship between endo/epi ratio and diastolic time fraction starts at the top right corner of the graph (Tdia ⫽ 1, Endo/ Epi ⫽ 1.5), then endo/epi remains 1.5 until TDia is about 0.75 and drops sharply to become zero at TDia ⫽ 0.4. It is important to note that this course is quite different from the relationship between Endo/Epi and TDia for the case that perfusion of the subendocardium is determined by diastole alone. Endocardial flow would then vary linearly between the value at cardiac arrest, TDia ⫽ 1, and at permanent systole, TDia ⫽ 0, as shown by the dashed line passing through the origin. The diastolic time fraction is not only dependent on heart rate. An increase of contractility at a constant heart rate will cause prolonging of the diastolic time fraction. The diastolic time fraction also increases at lower levels of perfusion pressure (Merkus et al., 1999).

FIGURE 13 Ratio of flow in subendocardium and subepicardium as a function of diastolic time fraction but at constant coronary arterial pressure. Data from R. J. Bache and F. R. Cobb, Circ. Res. 41, 648–653 (1977).

2. Coronary Circulation and Hemodynamics

This mechanism represents a natural protection against underperfusion of the subendocardium when coronary arterial pressure drops, e.g., in the presence of a severe stenosis. It is not yet understood why heart contraction hardly influences perfusion in the subepicardium but has a strong effect on perfusion in the subendocardium. The waterfall model provided a nice framework for the interpretation, at least for hearts under normal working conditions. However, it fails to explain too many other observations. Basically, the simple concept of tissue pressure being related to left ventricular pressure in the subendocardium does not hold. This is demonstrated clearly by the fact that subendocardial flow is also much lower then epicardial flow in an empty beating heart (Van Winkle et al., 1991). This fact emphasizes the importance of the findings of Westerhof’s group on the direct effect of heart contraction on coronary arterial flow. However, if the flow impediment caused by cardiac contraction is the result of the direct effect of contracting myocytes on vessels embedded between them, why is subepicardial flow not hampered? Moreover, as mentioned earlier left ventricular pressure does have an effect on intramural vessels in the early and late phases of systole. Hence, ventricular pressure can still not be ruled out as a factor.

D. Dominant Role of Heart Rate in the Balance between Demand and Supply of Flow The concept of supply–demand ratio in the context of myocardial perfusion is classic. It simply states that the oxygen needed by the heart should be supplied in order to prevent malperfusion, which can lead to ischemia or even infarction. It has been attempted to derive indirect indices for this ratio. Oxygen demand has been indexed by the product of systolic aortic pressure and systolic duration. Similarly, the product of diastolic time and mean diastolic pressure denotes an index for the supply. The advantage of these indices is that they can be derived from the aortic pressure signal alone. The idea behind the supply–demand ratio is that perfusion of the myocardium takes place in diastole and oxygen is used for the generation of pressure in systole. Obviously, there are some simplifications in this index, but conceptually, the supply and demand ratio is useful. Heart rate is a dominant factor in the supply and demand ratio because of two reasons. First, an increase in heart rate increases the oxygen consumption of the heart. Second, increasing heart rate, as is obvious from Fig. 13, reduces the maximum possible coronary flow at the subendocardium. In general, the first factor is well recognized by clinicians and motivates the adminis-

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tration of 웁 blockers to reduce heart rate. However, the second factor is often overlooked but luckily is improved by 웁 blockers as well. Oxygen consumption by the heart is determined by the sum of basic oxygen needs independent of contraction and an amount per heartbeat that depends on contractility and generated pressure (Suga et al., 1981). The maximum possible flow, hemoglobin content, and oxygen saturation of the coronary arterial blood determine the maximum supply of oxygen. The reduction of maximum flow by cardiac contraction has been discussed earlier. It can be calculated that the sensitivity of oxygen demand to heart rate is about the same or even higher than the sensitivity of maximum flow to heart rate (Spaan, 1991). The dependency of maximum flow supply and demand on heart rate is demonstrated schematically in Fig. 14 for a normal heart. Where the two relationships meet, supply falls short of demand and ischemia will occur. Obviously, a stenosis will move this intercept point to a lower heart rate, which is demonstarted in Fig. 14 as well.

E. Interaction of Mechanisms Compressing Intramural Blood Vessels Interestingly, a full understanding of myocardial perfusion requires elements from all models discussed so far. It has been demonstrated that during myocardial contraction, several types of intramural vessels are reduced in diameter such that their resistance is higher in systole than in diastole. Collapse has been demonstrated in epicardial veins, and a waterfall pressure is the backpressure of coronary flow, especially at higher diastolic left ventricular pressures. Left ventricular pressure has an influence on intramural vessels, especially in the early and late systolic phases when the elastance of the myocardial wall is low. Elastance has an effect on coronary flow, especially in systole. A factor not yet mentioned is the role of transmural arteries and veins. These vessels exhibit a considerable pressure drop when the heart is arrested and perfusion is continued at a normal level by a perfusion system. The task for the coming years will be to construct a comprehensive model that takes all these factors into account and will allow us to understand the interaction of these mechanisms.

VII. CORONARY DIASTOLIC PRESSURE–FLOW LINES A. High Zero-Flow Pressure, PZF When diastole is sufficiently prolonged, plots of coronary flow versus pressure in this period may show an

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I. Pumping Action and Electrical Activity of the Heart

FIGURE 14 Schematic representation of sensitivities of supply and demand on heart rate. The demand per unit weight of tissue is similar for the two layers in the heart, but the maximum flow at the subendocardium decreases with heart rate faster than the demand increases, especially in the presence of a stenosis. Note that the maximum flow curve for the subepicardium in the presence of a stenosis has not been drawn.

intercept with the pressure axis. This zero-flow pressure, PZF , is far above coronary venous pressure. Moreover, the curves appear to have a rather constant slope. The interpretation of diastolic pressure flow lines with a high PZF value follows the concept of the waterfall model: a constant resistance terminated with a backpressure, PZF , as the result of collapse at the level of resistance vessels. The collapse is related not only to tissue pressure as in the waterfall model, but to the combination of tissue pressure and a degree of vascular tone that may close the vessel lumen when local pressure is reduced below PZF . The classical recording of Bellamy’s (1978) paper is provided in Fig. 15 with the constructed diastolic pressure–flow relation shown in Fig. 16.

At first, the pressure–flow relations were obtained during exponential decay of coronary pressure as with the natural aortic pressure. However, it was demonstrated that with such an exponential decay of pressure the actual flow measured at the beginning of the coronary artery may overestimate the true microvascular flow by a so-called capacitive flow component needed to charge the proximal compliance in the coronary system. Furthermore, it was pointed out that when coronary arterial flow comes to a standstill there still was venous outflow and the pressure measured in the coronary artery would thus reflect the pressure in the microcirculation. Because of the large intramyocardial compliance, this pressure would decay only slowly and the decay of

FIGURE 15 In a long diastole coronary, flow decays with aortic pressure. Note that coronary flow ceases when aortic pressure has reached a value of 40 mm Hg. Also note that aortic pressure is still decreasing when flow becomes zero. From R. F. Bellamy, Circ. Res. 43, 92–101 (1978).

2. Coronary Circulation and Hemodynamics

FIGURE 16 Diastolic pressure–flow relation constructed from Fig. 15. From R. F. Bellamy, Circ. Res. 43, 92–101 (1978).

coronary arterial pressure would be more rapid and catch up with the intramyocardial vascular pressure at a level above venous pressure. Hence, PZF would equal microvascular pressure at the moment flow stops. Eng et al. (1981) introduced the so-called compliance-free method of coronary pressure–flow measurement. He advised that coronary arterial pressure should be changed stepwise to different levels and that flow should be measured after a sufficient stabilizing period. This method indeed prevents the effect of proximal compliance, but not, however, that of intramural compliance, because microvascular pressure is still changing even when pressure in the coronary artery is kept constant. The discussion about the mechanism for the high PZF value was important because if local collapse at the level of the resistance vessels would indeed be a factor, then the effects of drug administration on PZF have to be considered.

B. Incremental Linearity of Diastolic Pressure–Flow Curves A confusing feature of diastolic pressure–flow curves is that these can be so straight. This straightness suggests the assumption that the slope represents a constant resistance. However, we have to consider that interacting mechanisms may result in straight relationships and therefore may fool us into the acceptance of mechanisms other than what they truly are. For example, we have mentioned earlier that the time constants for change in coronary resistance are in the order of seconds. Hence, one may expect that vasoconstriction occurs during long diastoles and that coronary resistance is not constant. As a result, the slope of a pressure–flow relation will not reflect a constant resistance. It has been shown in chronicly instrumented dogs that diastolic pressure– flow relations measured in the restoring phase of reactive hyperemia were also straight. Therefore, one has

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to accept that even if a pressure–flow line is straight, resistance may be changing (Spaan, 1979). It was recognized that errors could be made in determining PZF by extrapolation of the diastolic pressure–flow relation to zero flow (Klocke et al., 1985). Hence experiments were performed in which pressure was changed slowly toward PZF and then stopped. Interestingly, in those few experiments where pressure was reduced below PZF , flow actually became negative, reflecting emptying of intramyocardial compliance. In conclusion, with respect to the interpretation of diastolic pressure–flow relations, one has to take into account the rate of change of resistance due to flow control, the rate of change of volume in the intramyocardial compliance, and the rate of change of volume in the epicardial arteries.

C. Rates of Equilibration of Arterial Flow and Intramural Vascular Volume in Diastole at Maximal Vasodilatation An important issue in the discussion about the role of intramyocardial compliance in diastolic pressure–flow relations is the rate by which a steady state in coronary flow is reached after a step change in arterial pressure or in transition to cardiac arrest. An example of rapid restoration of flow is provided in Fig. 17. The study was performed in an anesthetized, open chest dog with a cannulated coronary artery (Kajiya et al., 1986). The bundle of His was destroyed with the injection of formaldehyde in the AV node and the heartbeat was maintained by pacing. The perfusion line was occluded, and a few seconds into the occlusion the heart was put into a long diastole by cessation of pacing. Then, still within the long diastole, the perfusion pressure was restored. Figure 17 illustrates that coronary arterial flow quickly reaches a steady state, whereas it takes much longer for the venous flow to follow. In the dead time between restoration of arterial flow and the start of the venous outflow, the intramural vessels are being filled. The time constant for restoration of venous flow is much slower than the time constant of arterial flow restoration. The idea of a rapid equilibration of arterial flow was further supported by elegant experiments of Canty et al. (1985). Within long diastoles, they applied lowamplitude sinusoidal variations around different mean coronary arterial pressures and measured the sinusoidal response of flow to the pressure perturbation. The amplitude ratio, as well as the difference in phase between pressure and flow, was plotted as a function of frequency to obtain so-called Bode plots. These Bode plots were consistent with a resistance–capacitance–resistance

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(RCR) model in which R and C were constant at a fixed mean pressure but varied with the level of mean pressure. The sum of the two resistances was much smaller than the ratio between mean arterial presure and flow, which was explained by a large PZF value related to some point of collapse in the microcirculation. R*C products, representing a time constant for the model, were a few tenths of a second long, thus supporting the idea that coronary arterial flow stabilizes itself quickly. These small time constants obscured the influence of a large intramyocardial compliance on diastolic pressure–flow relations. That is why the magnitude of the compliance has been overlooked for a long time.

D. Intramyocardial Compliance Is Concealed in Diastole by Volume-Dependent Resistance The small time constants found from measurements at the arterial side of the coronary circulation seemed at odds with the larger time constants, in the order of 1 to 3 sec, found for the changes in intramyocardial blood volume that are induced by changing arterial pressure or heart rate. These time constants were measured either indirectly by integrating the arterial-venous flow differences or more directly by contrast subtraction angiography. It was then postulated that the continuous change of intramural vascular volume would also change resistance continuously during the response to these interventions and that these changes in resistance might result in faster equilibration times for coronary flow. The intramyocardial pump model is depicted again in Fig. 18. But without the effect of contraction, the model is simply a RCR model, similar to the one used

to interpret Bode plots of Canty and colleagues (1989). For our analysis of dynamic responses of arterial and venous flow to a stepwise change in arterial pressure, we assumed the compliance to be about 10 times larger than found in the study of Canty and co-workers. In addition, we assumed that the two resistances were equal. The model was evaluated for two conditions: (1) with resistances constant and (2) with resistances allowed to vary with vascular volume, i.e., the load on the capacitance. Responses with constant resistances, as shown in the bottom left-hand side of Fig. 18, are typical for the linear model. The rates of change of inflow and outflow are the same. The change of volume follows from the integrated difference between inflow and outflow over time. Its rate of change is therefore similar to that of inflow and outflow and agrees with experimental data on intramural volume change and venous outflow response. However, the slowness of the inflow response seems at odds with the experimental data. When the resistances were made volume dependent, a different picture develops, as is shown in the top righthand side of Fig. 18. The rate of change in volume and venous outflow remains similar to the linear case, but the arterial inflow reaches the level of steady state almost immediately after the pressure step. This is the result of variable resistance, which is reduced concomitantly with the increase in volume. One could say that inflow is trying to reach a moving target: the new steady state for flow, which is higher than the steady state for flow when resistance remains constant. Hence, a large intramyocardial compliance can be consistent with rapid changes in arterial pressure and flow signals, provided

FIGURE 17 Demonstration of rapid restoration of inflow but delayed venous outflow. The coronary artery is occluded and then the heart is arrested. Within the long diastole, pressure is restored. Tracings kindly provided by Prof. Kajiya from F. Kajiya et al., Circ. Res. 58, 476–485 (1986).

2. Coronary Circulation and Hemodynamics

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FIGURE 18 Response to pressure step obtained with a one-element intramyocardial pump model without contraction effects. (Top left) Pressure step imposed at the inlet. (Bottom left) Response of inflow and outflow when resistances are constant. (Top right) Response of inflow and outflow when resistance is allowed to change with vascular volume. (Bottom right) Resistance of the compartment for both cases. Redrawn from J. I. Hoffman and J. A. E. Spaan, Physiol. Rev. 70, 331–390 (1990).

the resistance variations during the perturbations are taken into account. This line of reasoning was confirmed by experiments using the isolated nonbeating septum preparation developed by the group of Yin (Judd et al., 1993; Resar et al., 1992). Septal thickness was continuously measured sonometrically. Using an angiographic technique, it was shown that variations in septal thickness reflected intramural vascular volume changes. Bode plots of pressure and flow were obtained similar to those of Canty and colleagues, and additionally, Bode plots of pressure and thickness were constructed. These results were interpreted on the basis of a RCR model where it was assumed that the resistances were dependent on the volume of the capacitance to which they were connected. This model fit resulted indeed in a correct description of the Bode plots. As an additional validation, direct estimates of absolute intramural vascular volume as a function of mean arterial pressure were obtained. These values were quite realistic, 5, 8, and 15 ml/100 g tissue at mean arterial pressures of 30, 50, and 70 mm Hg, respectively (Spaan et al., 1999). The septal study clearly demonstrates that resistance changes related to vascular volume variation obscure the existence of the large intramyocardial compliance from signals obtained from the arterial side alone.

VIII. PHYSIOLOGICAL EVALUATION OF CORONARY STENOSIS Atherosclerosis can cause local narrowing of coronary arteries, which presents a resistance to blood flow and thereby reduces the perfusion pressure for the tissue dependent on this artery. Based on the principles of autoregulation, one can appreciate that dilation of the resistance vessels may compensate for this fall in distal pressure. However, this dilatory capacity was intended to accommodate an increase in flow at times of an increased oxygen demand. While ischemic signs may be absent at rest, they become noticeable during exercise. Hence, the dilatory capacity, or so-called coronary flow reserve, is reduced in the presence of a stenosis. In order to understand this limitation of flow reserve, one needs to understand the hemodynamic characteristics of a stenosis.

A. Pressure Drop across a Stenosis The residual lumen of a stenosis may be circular, elliptical, or slit like, and the plaque may cover the whole wall circumference or it may be positioned eccentrically. Several pathological studies indicate that 70–75% of coronary atherosclerotic plaques are eccentric (most

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I. Pumping Action and Electrical Activity of the Heart

likely due to the curved nature of epicardial arteries) and leave an arc of the arterial wall uninvolved and flexible (Saner et al., 1985; Waller, 1989). Each geometric feature of the plaque introduces a specific element into the hemodynamic analysis. For now, we will restrict ourselves to discussing some basic characteristics of a rigid stenosis. Numerous studies have shown that the translesional pressure gradient can be adequately expressed as the sum of (1) frictional losses along the entrance and throat of the stenosis and (2) convective inertial losses at the downstream end of the throat. Due to the reduced crosssectional area, flow velocity inside the stenosis must be higher than proximal or distal to the lesion. The higher kinetic energy of blood within the stenosis is realized at the cost of pressure, which may be considered a potential energy density. Within the stenosis, the decrease in pressure through convective acceleration of blood is based on the law of Bernoulli, whereas frictional losses are related to the law of Poisseuille. At the downstream exit, the deceleration of blood should result in a recovery of kinetic energy losses, which ideally should compensate completely the pressure decrease due to acceleration. However, the sudden expansion of flow at the downstream end of the stenosis throat causes flow separation and recirculating eddies, which make this pressure recovery only partial. The total pressure drop over a stenosis has therefore a component due to frictional losses and one due to convective inertial losses, which can be described by ⌬p ⫽ Av Q ⫹ BQ

2

where Av and B are the viscous and expansion loss coefficients that depend critically on the inverse fourth power of the minimum diameter of the stenosis (Young, 1979). The mathematical expressions for these coefficients incorporate detailed elements of stenosis geometry, fluid properties, and effects of flow fields at the entrance and exit of the stenosis (Mates et al., 1978; Kirkeeide, 1991). An important implication of the pressure drop–flow equation is that the resistance (⌬p/Q) of a given stenosis is directly related to flow. Figure 19 illustrates the nonlinear dependency of the pressure gradient on blood flow through the stenosis, which becomes more prominent with increasing stenosis severity. The presence of a stenosis changes the coronary pressure flow lines as indicated schematically in Fig. 20. Because of compensatory dilation of the distal resistance vessels (autoregulation), the pressure–flow curve with tone intact will not be altered much at basal myocardial metabolism. However, comparison to autoregulation under normal conditions (Fig. 4) makes clear that there is now less reserve when the autoregulation curve

FIGURE 19 Effect of flow on pressure gradient and resistance of a single, fixed stenosis. The severity of the lesion increases from S1 to S3.

shifts upward under conditions of increased MVO2 . Essentially, the pressure–flow relation at maximum dilation is directly affected by the presence of a stenosis. Total flow resistance at maximum vasodilation is equal to the sum of stenosis resistance and the resistance of the vascular bed. Because stenosis resistance increases with flow, the shape of pressure–flow relation at maximum vasodilation changes from straight to curvilinear in the presence of a stenosis. At any given arterial pressure, the flow at full dilation is lower than without a stenosis and the reserve capacity for flow to increase above resting level is diminished.

B. In Vivo Evaluation of Functional Stenosis Severity In the past few years, the emphasis has shifted from a geometric (angiographic images) to a hemodynamic assessment of stenosis severity in the cardiac catheter-

FIGURE 20 Coronary pressure–flow relation with and without a stenosis. S1 is a milder stenosis than S2. The solid arrow illustrates the reduced coronary flow reserve related to the presence of a severe stenosis. The dashed arrow indicates the dilatory reserve lost because of the stenosis. PZF , zero-flow pressure.

2. Coronary Circulation and Hemodynamics

ization laboratory. Advances in miniaturized transducer technology have led to the development of sophisticated sensor-tipped guide wires with a diameter of only 0.014 inches (0.35 mm), which have made it possible to directly measure intracoronary pressure or Doppler flow velocity distal to a stenosis in humans. Data obtained with these transducers form the basis for the determination of the distal coronary flow velocity reserve (CFVR) and the myocardial fractional flow reserve (FFR). CFVR is defined as the ratio of mean distal flow velocity obtained during pharmacological vasodilation, e.g., after injection of adenosine into the target vessel, to that obtained at rest, CFVR ⫽ vhyp /vrest This index is based on the concepts discussed previously. It was developed in analogy to the coronary (volume) flow reserve and describes the vasodilatory capacity of a stenosed artery. A cutoff value of CFVR ⬍2 identifies lesions that can lead to inducible ischemia as determined by noninvasive stress tests (Joye et al., 1994). As can be seen from Fig. 20, CFVR for a given fixed stenosis is dependent on perfusion pressure at the time of measurement. Furthermore, being a ratio, it is influenced by conditions that modify the level of either the resting or the hyperemic flow velocity. Common factors that influence hyperemic flow include changes in heart rate, aortic pressure, and myocardial contractility. Resting flow is affected by increased workload and left ventricular hypertrophy, but often also by pathophysiological factors such as persistent partial dilation of resistance vessels. In order to circumvent these limitations of CFVR, the myocardial fractional flow reserve has been introduced (Pijls et al., 1993; De Bruyne et al., 1996). It is defined as the ratio of mean maximum hyperemic flow in the presence of a stenosis to that theoretically expected if the same vessel was normal, i.e., without a stenosis: FFR ⫽ Qs /Qn This ratio is indicated by the dashed arrow in Fig. 19. Expressing flow as the ratio of driving pressure to resistance and assuming that myocardial resistance is constant during maximal vasodilation, this equation can be written as FFR ⫽ (Pdist ⫺ Pven )/(Pao ⫺ Pven ) If we further assume that venous pressure (Pven) is constant and small compared to distal pressure (Pdist) and aortic pressure (Pao), then FFR can be determined as the ratio of mean distal-to-proximal pressure during maximum vasodilation. The major conceptual advantage of this index lies in the fact that it is not affected by changes in basal hemodynamic conditions, as it relies

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only on measurements performed during maximal hyperemia. It has a normal value equal (or close) to 1, and a cutoff value of ⬍0.75 has been shown to discriminate functionally significant lesions from those that do not cause reversible ischemia (Pijls et al., 1996). Experiments in dogs and patients suggest that FFR is not influenced significantly by changes in heart rate, myocardial contractility, or arterial blood pressure (Pijls et al., 1993; De Bruyne et al., 1996). However, such independence is not likely because of their effects on coronary resistance, as discussed earlier. It may well be that in previous experimental studies, several factors influencing FFR were not controlled independently. An increase in heart rate, for example, will decrease distal resistance and result in an increase of FFR, whereas an increase in aortic pressure will result in a higher flow at maximal vasodilatation, inducing an increased stenosis resistance (see Fig. 19) and a reduced FFR. Hence, a combined increase in heart rate and aortic pressure may result in an apparently constant FFR. Per definition, CFVR assesses coronary conductance as the combination of a stenosed conduit vessel and the vasodilatory capacity of the distal myocardial vascular bed. A reduced CFVR can thus not distinguish among epicardial disease, microvascular disease, or a combination of both. In contrast, FFR addresses the effect of an epicardial stenosis, under the assumption that maximum vasodilation is not impeded by other mechanisms. However, a normal FFR value may indicate the presence of a mild stenosis or a limited vasodilatory response due to the presence of microvascular disease in the presence of a severe coronary narrowing. Therefore, indices based only on pressure or flow signals can lead to contrasting results or erroneous conclusions regarding the physiological severity of a stenosis in the presence of additional pathological cardiac conditions (Meuwissen et al., 1999). The combined measurement of the transstenotic pressure gradient and distal flow velocity may provide a more complete and accurate description of the hemodynamics of the stenotic vessel, and the myocardial vascular bed perfused by it.

C. Effect of Stenosis on Phasic Patterns of Distal Flow and Velocity Normally, coronary arterial pressure closely follows the aortic pressure wave and coronary blood velocity is pulsatile. However, a stenosis acts as a low-pass filter on the velocity waveform, which becomes more flat. The pressure distal to a stenosis will be uncoupled from aortic pressure and becomes more dominated by the squeezing effect of myocardial contraction on the microcirculation. This effect is illustrated in Fig. 21. A sensortipped guide wire obtained the coronary pressure signal

40

I. Pumping Action and Electrical Activity of the Heart

FIGURE 21 Change in the coronary pressure profile to a left ventricular pressure pattern when passing the transducer from a proximal position to one distal to the lesion (see arrow). This tracing was obtained in a patient’s left anterior descending artery with a 75% diameter stenosis.

while being advanced through the stenosis. The pressure signal proximal to the stenosis differs somewhat from that of the aortic pressure due to a more distal location of the measurement site. In addition, the aortic pressure was measured through the guiding catheter, which has a less favorable frequency response compared to the pressure wire. In any case, there was a marked change in the coronary pressure waveform when the sensor arrived at a position distal to the stenosis. The waveform suddenly resembled more that of the left ventricle. The shape mimics that of the driving pressure generated by the intramyocardial pump. Although there is some phase shift between proximal coronary pressure and aortic pressure waveforms, it is clear that the rise of pressure in the distal signal occurs earlier in the heart cycle. From animal experiments with an artificial stenosis, we could see that the rise of distal coronary pressure coincides with the rise in left ventricular pressure. Hence, the proximal pressure rises with aortic pressure at the onset of aortic valve opening, whereas distal pressure starts to rise during the isovolumetric contraction phase of the heart. Figure 21 was obtained from a patient with a severe stenosis, as can be seen from the large pressure drop across the stenosis. Distal pressure waveforms and velocity waveforms would obviously be less affected at a lower degree of stenosis severity.

compliant stenoses is therefore not fixed, but depends on the prevailing intraluminal pressure. Passive changes in stenosis diameter are therefore possible in response to the variations in coronary pressure during the heart cycle (Siebes et al., 1988). These properties constitute an additional limitation to the angiographic evaluation of stenosis severity. Furthermore, a flow increase due to distal vasodilation can cause such a low intraluminal pressure that the stenosis partially collapses and acts as a flow-limiting resistance (Aoki and Ku, 1993; Siebes et al., 1996). The hemodynamics of a compliant, or dynamic, stenosis can no longer be described by a single curve. Instead, the pressure drop–flow relationship traverses a family of curves, each reflecting a changed stenosis diameter following a change in intravascular distending pressure. It has been suggested that blood velocity at the outlet of a stenosis in a systemic artery can be so high that pressure distal to the stenosis is reduced to the point that vascular collapse may occur at such a location (Downing and Ku, 1997). For the coronary circulation, this appears to be an unlikely possibility. We have never measured negative pressure distal to a coronary stenosis. Collapse of distal segments will certainly not occur in systole when intracoronary pressure is high due to microvascular compression. Such collapse may then potentially occur during diastole.

D. Compliant Coronary Stenosis A stenosis may be compliant if the plaque is not circumferential and a partially flexible wall segment exists at the site of the lesion. The geometry of these

E. Coronary Collateral Circulation In this chapter, we have so far considered the perfusion areas supplied by the major coronary arteries to

2. Coronary Circulation and Hemodynamics

be independent. However, at several levels within the heart muscle, vascular cross-connections exist between neighboring perfusion areas (Schaper et al., 1976; Scheel et al., 1982). These vascular connections are called collateral vessels. There is variation in the collateral vascular development between species. In pig hearts, collateral vessels are not existent, whereas these connections are prominently present in dogs. In humans, collateral blood vessels are preexisting. Ischemia stimulates collateral development by the production of vascular growth factors. Blood flow through collateral vessels can be lifesaving, if ischemia develops gradually enough to allow sufficient time for collateral development. Current research is in progress to investigate ways to initiate and promote collateral growth. The existence of collateral vessels can be determined clinically during angiography by demonstrating that contrast injected in one vessel arrives at the perfusion area of the occluded vessel. When such measurements are performed during angioplasty of atherosclerotic arteries, the pressure downstream of the stenosis can be obtained during balloon inflation when the diseased artery is occluded. In the presence of collateral vessels, this so-called wedge pressure is higher than in the absence of these vessels. When collateral vessels are present, there is also an increase in blood velocity in the nondiseased artery that feeds the collateral vessels during balloon occlusion of the troubled artery (Piek et al., 1993).

IX. SUMMARY AND FUTURE DIRECTIONS Research on the hemodynamics of the coronary circulation has been revitalized in view of recent clinical developments and new technological advances in the diagnosis and treatment of coronary disease. For the interpretation of clinically obtainable hemodynamic signals as understanding of physiological mechanisms is a prerequisite. In this chapter, the physiology and biomechanics of the coronary circulation have been outlined. The pulsatility of coronary venous and arterial flow was explained on the basis of the intramyocardial pump concept. According to this concept, the time-varying compression of the intramural microcirculation squeezes blood out of the intramyocardial compliance, thereby impeding arterial inflow and promoting venous outflow. A timevarying intramural blood volume has, however, also consequences for intramural vascular diameters. Increasing the heart rate decreases intravascular volume. At the same time, subendocardial perfusion at maximal dilation is reduced, in contrast to subepicardial perfusion which is hardly affected by heart rate. Subendocardial resistance at maximal dilation is only half of that

41

at the subepicardium at diastolic arrest, but it is 50% higher at a heart rate of 180 beats/min. In addition to the increase of oxygen demand with increasing heart rate, this mechanical compression makes the subendocardium vulnerable for ischemia. The relation between oxygen supply and demand has been explained and both the static and dynamic characteristics of flow control have been outlined. Three major mechanisms were described. The link between flow and metabolism was defined by control of oxygen tissue pressure by an as yet unknown mechanism. Since such a feedback requires a tight interaction and short diffusion distances, it is most effective at the level of the smallest arterioles. There must be a signal transfer from smaller to larger vessels upstream and an obvious candidate for this is blood flow itself causing flow-dependent dilation. Hence, a small change in flow in the smallest arterioles is magnified by flow-dependent dilation of upstream arterioles. The myogenic response of resistance vessels is an intrinsic mechanism and by its nature stabilizes flow despite arterial pressure changes. Under normal circumstances the nervous system has little effect on the steady state control of coronary blood flow, but denervation of the heart equilibrates the rates of change of coronary resistance to rapid changes in heart rate and perfusion pressure. Normally, the dilatory responses are faster than the constrictor responses. However, there is an alpha-adrenergic effect on the coronary circulation, especially during exercise and ischemia. It is assumed that this effect is directed to equalize transmural perfusion and to prevent steal from the subendocardium under these circumstances. The coronary pressure-flow lines obtained in long diastoles exhibit a zero-flow pressure that is higher than coronary venous pressure. At maximal vasodilation, this pressure is in the order of 15 mm Hg but can become as high as 40 mm Hg when autoregulation is intact. Several pitfalls in the estimation of zero-flow pressure have been discussed. Discharge of epicardial as well as intramyocardial compliance contributes to arterial zeroflow pressure with continuation of venous outflow. A second issue is the pressure dependence of coronary resistance. In order to obtain a diastolic pressure flow line, pressure has to be reduced, thereby increasing coronary resistance. This increase of resistance is especially obvious from the curvature of the pressure-flow lines at lower perfusion pressures. However, although pressureflow lines are incrementally linear at perfusion pressures higher than about 30 mm Hg, resistance is changing with pressure as well. In this range of pressures the pressure dependence of resistance conceals the existence of a large intramyocardial compliance from dynamic pressure-flow measurements. Interventional cardiologists nowadays apply the con-

42

I. Pumping Action and Electrical Activity of the Heart

cept of flow reserve to judge the severity of a coronary stenosis. This flow reserve is measured directly by a guide wire equipped with a blood velocity sensor near the tip (CFR), or indirectly as the ratio between pressures proximal and distal of the stenosis at maximal dilation by a guide wire with a pressure sensor (FFR). The complications introduced by nonlinear pressure flow-lines of a stenosis and the coronary microcirculation for the assessment of CFR and FFR have been discussed. The developments of new technological advances in the diagnosis and treatment of coronary disease have certainly not arrived at an endpoint. Many aspects pertaining to the mechanics of coronary flow, its control and distribution still need to be elucidated. An important element is the interaction and integration of all of these mechanisims. From a clinical point of view it is very important to understand not only the consequences of the disease but also the cause of it. Since the endothelium is the first line of defense against vessel wall deterioration, much insight is to be expected from research into its biochemistry and transport properties. However, since the endothelium is subject to direct mechanical interaction with flowing blood, the mechanics of blood flow will continue to play an important role. Better methods have to be developed to evaluate the physiological significance of coronary artery disease and the success of clinical inteventions. Since all intracoronary interventions carry a risk of serious negative side effects, it is important to provide the cardiologist with tools that facilitate the differentiation between needed and unneeded procedures. In the development of diagnostic tools we have to arrive at the ability to separately evaluate the characteristics of a stenosed conduit artery and that of the microcirculation. The assessment of the hemodynamic behavior of a stenosis requires at least two measurements: flow (or velocity) through the lesion and pressure drop across it. Such a combined measurement would automatically provide additional information on microvascular resistance because both distal pressure and flow are known. In addition, knowledge about the perfusion of the subendocardium, which is the most vulnerable part of the myocardium, is essential. Clinical tools are needed for the evaluation of subendocardial perfusion that are more practical then the presently available technologies, such as perfusion scintigraphy and positron emission tomography, and that can preferably be incorporated into diagnostic cardiac catheterization.

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3 Neurohumoral Control of Cardiac Function JEFFREY L. ARDELL Department of Pharmacology James H. Quillen College of Medicine Johnson City, Tennessee 37614

I. INTRODUCTION

(i.e., stellate and middle cervical ganglia) and intrinsic cardiac ganglia also contain afferent neurons whose sensory neurites lie variously within the heart, lungs, and great thoracic vessels (5, 8, 12, 30, 38). Additional sensory inputs for the control of cardiac autonomic neurons arise from baroreceptors and chemoreceptors located along the aortic arch, carotid sinus, and carotid bodies (33, 37, 63, 142), as well as from other afferent neural elements within the central nervous system (CNS), especially the hypothalamus (90). During exercise, sensory inputs arising from active skeletal muscles make additional contributions to overall reflex control of autonomic efferent outflows to the heart (105, 109). Each group of major cardiac afferent neurons will be considered in turn.

With respect to neural control of the heart, the intrathoracic ganglia and their interconnections form the final common pathway for autonomic modulation of regional cardiac function. Although recent studies have identified some of the neuroanatomical and functional characteristics of intrathoracic autonomic neurons, we are just beginning to understand how the activity generated within the network of intrathoracic ganglia ultimately affect regional cardiac function. In this review, data will be presented indicating that intrathoracic autonomic ganglia contain a heterogeneous population of cell types, including afferent, efferent, and local circuit interneurons. As discussed in this chapter, intrathoracic reflexes contained within these peripheral autonomic ganglia function in a coordinated fashion with central neurons located in the spinal cord, brain stem, and supraspinal regions to regulate cardiac output on a beatto-beat basis.

1. Nodose Ganglia Afferent Neurons Nodose ganglia are paired structures that receive cardiac afferent inputs from sensory neurites located in atrial and ventricular tissues. These sensory neurites preferentially sense chemical stimuli, with a few responding to mechanical stimuli or both modalities (22, 125). The response characteristics to induced stimuli are likewise divergent with mechanical stimuli exerting short-lived effects, and the augmentation in activity elicited by chemical stimuli far outlasting the applied stimulus (22). While inputs from these receptors contribute to overall cardiovascular regulation, they are not normally perceived by the sensorium. However, during myocardial ischemia, inputs from these sensory neurites may contribute to symptoms referred to the neck and jaw regions of the body (49).

II. AFFERENT NEURONS A. Cardiac Afferent Neurons Sensory afferent neurons provide the autonomic nervous system with information about blood pressure, blood volume, and blood gases, as well as the mechanical and chemical milieu of the heart. For sensory inputs from cardiopulmonary regions, nodose and dorsal root ganglia are classically recognized as providing sensory inputs to the brain stem and spinal cord, respectively (62). Data have indicated that intrathoracic extracardiac

Heart Physiology and Pathophysiology, Fourth Edition

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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I. Pumping Action and Electrical Activity of the Heart

2. Dorsal Root Ganglia Afferent Neurons The cell bodies of afferent neurons associated with sensory neurites localized throughout the heart are located bilaterally in C6 –T6 dorsal root ganglia (62). The sensory neurites of most of these afferent neurons transduce chemical and mechanical stimuli (66). Inputs from this subpopulation of cardiac afferent neurons subserve normal cardiovascular regulation, as well as nociception when activated excessively (49, 122). During myocardial ischemia, activation of these receptors contributes to the perception of symptoms referred to the thorax and upper limbs (49). 3. Intrathoracic Afferent Neurons Functional and anatomical data indicate that intrathoracic autonomic ganglia contain afferent soma (24, 38, 141). The sensory neurites associated with these afferent neurons are variously located in atrial, ventricular, major vascular, and pulmonary tissues. Most are responsive to mechanical and chemical stimuli (19). These afferent neurons continue to influence intrathoracic efferent postganglionic outflows to the heart after long-term decentralization of intrathoracic ganglia (5, 8, 12). It has been proposed that such intrathoracic afferent neurons provide inputs to the intrathoracic short-loop feedback control circuits that involve intrinsic cardiac and intrathoracic extracardiac neurons (15, 19). These intrathoracic neural circuits, acting in concert with CNS-mediated reflexes, dynamically control regional cardiac function throughout each cardiac cycle. 4. Aortic and Carotid Artery Baroreflexes Stretch receptors, sensitive to changes in vessel size, are found on thoracic and cervical arteries, being concentrated on the inner aspect of the aortic arch and the carotid sinus. They provide inputs to neurons within the medulla and spinal cord proportional to systemic arterial blood pressure (33, 37, 142). Inputs from these sensory neurites course centrally in the IX and X cranial nerves to synapse with neurons located in the nucleus of the medullary solitary tract (63). Via multisynaptic connections, these afferent inputs modulate the activity of cardiac parasympathetic efferent preganglionic neurons located primarily in the nucleus ambiguus, with fewer being located in the dorsal motor nucleus and regions in between (63). They also indirectly influence sympathetic efferent neuronal outflows to the heart via brain stem projections to the intermediolateral (IML) region of the spinal cord (48). The baroreflex so involved represents a negative feedback system that modulates cardiac function and peripheral vascular tone in response to everyday stressors (109). Thus, in response to changes in arterial blood pressure, sympathetic and

parasympathetic efferent inputs to the heart are activated reflexly and reciprocally to maintain normal blood pressure. 5. Muscle Pressor Reflexes During exercise the cardiovascular system must meet the increasing blood flow demands of active skeletal muscle, while maintaining adequate perfusion of all other organ systems. Activation of somatic chemosensory neurites within active muscles initiates a somatoautonomic reflex that contributes to the augmented sympathetic outflow associated with static and dynamic exercise (105, 109). Inputs from these somatic sensory neurites modify cardiovascular reflex control circuits, including those of the baroreflex, to differentially modulate autonomic inputs to the heart and the systemic vasculature (105, 109).

III. EFFERENT NEURONS A. Sympathetic Efferent Neurons Somata of sympathetic preganglionic efferent preganglionic neurons, which regulate the heart, are located within the IML cell column of the spinal cord, projecting axons via the ipsilateral rami T1–T5 to synapse with sympathetic postganglionic neurons contained within various intrathoracic extracardiac and intrinsic cardiac ganglia (13). Activation of these sympathetic efferent projections augments heart rate, changes patterns and speed of impulse conduction through the heart, and increases contractile force in atrial and ventricular tissues (7, 99, 104). While conduction velocity through atrioventricular (AV) nodal tissues is increased markedly during sympathetic excitation, it is increased only modestly in the Purkinje system and ventricular muscle (98). Thus there are qualitative similarities but quantitative differences in the functional response of the heart to stimulation of the principal cardiac sympathetic efferent neuronal projections. Activation of left-sided sympathetic efferent postganglionic neurons primarily elicits augmentation in ventricular contractile force while exerting lesser effects on heart rate (7, 98, 104). These rate changes are often associated with shifts in pacemaker activity from the sino-atrial node to inferior atrial or atrioventricular junctional sites (98, 104). In contrast, rightsided sympathetic efferent neuronal projections tend to exert greater sinus acceleration in heart rate, with less alteration in AV nodal function than left-sided nerves (7, 98, 104). Neurons located in ganglia in both sides of the thorax exert nearly equal effects on ventricular contractile force (7). Sympathetic efferent neurons that modulate cardiac automatic, conductile, and contractile tissues project ax-

3. Neurohumoral Control of Cardiac Function

ons in distinct intracardiac nerves. Right-sided sympathetic efferent neurons that influence ventricular contractile force course primarily in mediastinal nerves located at the root of the common pulmonary artery (7). However, right-sided sympathetic efferent neurons projecting to sinoatrial nodal pacemaker tissues course axons between the superior vena cava and the ascending aorta (7). Right sympathetic efferent neurons that influence conductile tissues are located primarily in nerves that converge between the common pulmonary artery and proximal right pulmonary artery (7). Left-sided sympathetic efferent neurons project axons to ventricular contractile tissues in nerves located adjacent to the common pulmonary artery as well as in the ventrolateral cardiac nerve (7, 104). Nerves carrying sympathetic efferent axons that innervate the sinoatrial and atrioventricular nodes are located adjacent to the origin of the left pulmonary artery. These nerves then course between the right pulmonary artery and the left superior pulmonary vein (7). Thus, there are parallel, yet distinct, extrapericardial and intrapericardial sympathetic efferent nerves that regulate cardiac automatic, conductile, and contractile tissues. Sympathetic efferent postganglionic somata that project axons to various cardiac effector tissues are localized in intrathoracic extracardiac and intrinsic cardiac ganglia (4, 18). Classically, the somata of sympathetic

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efferent postganglionic neurons that innervate the heart have been thought to be restricted to the stellate ganglia (75). However, cardiac sympathetic efferent postganglionic soma have been identified in thoracic middle cervical, mediastinal, and intrinsic cardiac ganglia (13, 15, 20, 21, 53). A subpopulation of intrinsic cardiac neurons express aspects of the catecholaminergic phenotype. These neurons thus contain the necessary enzymes to convert L-DOPA to dopamine and norepinephrine (27). The intrinsic cardiac nervous system also contains a separate population of small intensely fluorescent (SIF) cells that display tyrosine hydrolyase immunoreactivity (86, 87). Some of these project to adjacent principal intrinsic cardiac neurons (118). Electrical stimulation of sympathetic efferent preganglionic axons in the thoracic rami activates a subpopulation of neurons in intrathoracic extracardiac and intrinsic cardiac ganglia (9, 10, 20, 21, 53). The activation so induced can be eliminated with nicotinic blockade (20, 21, 53). Many of these intrathoracic sympathetic neurons can also be activated by 움- or 웁-adrenoreceptor agonists (11, 16, 110, 139). Furthermore, their activity can be affected by inputs from sensory neurites responsive to altered regional cardiac function (9, 10, 16). Angiotensin II, acting via AT1 receptors, likewise activates many intrathoracic augmentor neurons, thereby enhancing regional cardiac function (Fig. 1) (64). Thus,

FIGURE 1 Effects of angiotensin II (AII) injected into extracardiac and intrinsic cardiac ganglia on indices of right (RV IMP) and left (LV IMP) ventricular contractile function and evoked activity within the intrinsic cardiac nervous system (ICN). Ventricular inotropic function was assessed by induced changes in intramyocardial pressure (IMP) development. ICN activity was recorded from the dorsal right atrial ganglionated plexus (RAGP). AII injected into the extracardiac middle cervical ganglia (MCG) or intrinsic cardiac RAGP differentially activated augmentor neural responses directed to both ventricles. Losartan, a selective AT1 blocker, prevented all neural and cardiac effects elicited by subsequent AII administration. Timolol, a 웁 antagonist, blocked the cardiac responses to AII, but only slightly attenuated the evoked change in ICN activity. Adapted from Horackova and Armour (64).

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ACE inhibitors or angiotensin II AT1 receptor antagonists exert direct effects on populations of neurons in peripheral autonomic ganglia, providing a novel therapeutic approach to the treatment of cardiac dysfunction and perhaps even essential hypertension.

B. Parasympathetic Efferent Neurons Somata of cardiac parasympathetic efferent preganglionic neurons within the brain stem are located primarily within the nucleus ambiguus, with lesser numbers being located in the dorsal motor nucleus and regions in between (63, 82, 94). Axons from these preganglionic soma project via the ipsilateral X cranial nerve to synapse with parasympathetic efferent postganglionic neurons located within various intrinsic cardiac ganglia (see later). Activation of parasympathetic efferent neurons depresses heart rate, slows the speed of impulse conduction through the heart, induces major suppression of atrial muscle contractile force, and evokes negative inotropic effects on ventricular contractile force (4, 101). As with sympathetic efferent neuronal inputs, there are qualitative similarities but quantitative differences in the functional response of the heart to stimulation of different populations of principal cardiac parasympathetic efferent neurons. Activation of leftsided parasympathetic efferent postganglionic neurons slows atrial rate and atrioventricular conduction, as well as depressing atrial contractile force (4). If heart rate is allowed to vary, the tendency to evoke heart block is greater when left-sided versus right-sided parasympathetic efferent neurons are activated (4, 101). If heart rate is fixed via atrial pacing, right- and left-sided parasympathetic efferent neuronal inputs suppress atrioventricular conduction similarly (6, 101). When activated, right-sided parasympathetic efferent neuronal projections usually induce greater sinus bradycardia in conjunction with their effects to slow atrioventricular conduction and depress atrial contractile force (4, 101). If changes in preload, heart rate, and afterload are controlled, supramaximal stimulation of parasympathetic postganglionic efferent neurons suppresses ventricular contractile function by 10–15% (4, 13). Thus, autonomic neurons located in extracardiac tissues exert differential control over regional cardiac function, with the region of control depending on the location of the nerves that contain their projecting axons.

IV. LOCAL CIRCUIT NEURONS A subpopulation of neurons contained within extracardiac and intrinsic cardiac intrathoracic autonomic ganglia functions to interconnect neurons within indi-

vidual ganglia and between neurons in separate intrathoracic ganglia; these are called local circuit neurons (4, 13, 15). Preliminary data indicate that these neurons are involved in the processing of afferent information to coordinate sympathetic and parasympathetic efferent outflows to cardiac effector sites (19). Interactions within this neuron population form the substrate for generation of the basal activity within peripheral autonomic ganglia, especially when intrathoracic ganglia are disconnected from the influence of central neurons (5).

V. ORGANIZATION OF THE INTRINSIC CARDIAC NERVOUS SYSTEM Distinct and separate projections from the right and left vagi (cardiac efferent parasympathetic preganglionic neurons) or the right and left stellate and middle cervical ganglia (cardiac efferent sympathetic neurons) selectively affect the sinoatrial node, atrioventricular node, and regional contractile function (6, 7). Within the intrapericardial neural network, which controls the heart, are specific convergence points where bilateral autonomic inputs come together prior to their selective distribution to pacemaker, conductile, and contractile tissues (92, 100, 107, 108). Histological examination reveals convergence points, associated with one or more nerve trunks, containing encapsulated ganglia of varying number and size in the fatty connective tissue on the epicardium or adjacent to the root of the major arteries (14, 53, 102, 103, 136). To date, eight separate ganglia clusters have been identified within the canine intrinsic nervous system; five associated with atrial tissues and three with ventricular tissue. The five atrial ganglionated plexi include (1) the right atrial ganglionated plexus localized in fatty tissue on the ventral surface of the common right pulmonary vein complex (6, 29, 53, 103, 138); (2) the inferior vena cava–inferior atrial ganglionated plexus located on the inferior left atrium adjacent to the inferior vena cava (6, 102); (3) the dorsal atrial ganglionated plexus located on the dorsal surface of the atria between the common pulmonary veins, immediately caudal to the right pulmonary artery (140); (4) the ventral left atrial ganglionated plexus contained within fat on the caudal–ventral aspect of the left atrium adjacent to the AV groove (20); and (5) the posterior atrial ganglionated plexus (97). The three major ventricular ganglionated plexuses are (1) the right lateral ventricular ganglionated plexus located adjacent to the origin of the right marginal artery (21); (2) the left lateral ventricular ganglionated plexus located adjacent to the origin of the left marginal artery (21); and (3) the cranial medial ventricular ganglionated plexus located in fatty tissues surrounding the base of the aorta and main pulmonary

3. Neurohumoral Control of Cardiac Function

artery (21, 140). In general, intrinsic cardiac neurons located on the atria influence primarily, but not exclusively, atrial tissues, whereas intrinsic cardiac neurons located on the ventricles modify primarily, but not exclusively, ventricular tissue (140). Of these eight clusters of ganglia, functions have been ascribed to five of them: neurons in the right atrial and posterior atrial ganglionated plexus have been shown to exert control over the sinoatrial node (6, 51, 52, 97, 100, 103); those in inferior vena cava–inferior atrial ganglia exert predominant control over inferior atrial and atrioventricular conductile tissues (6, 51, 52, 100, 102). Neurons in dorsal atrial and cranial medial ventricular ganglia are principal modulators of contractile tissue (140).

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ent neurons provide mechanosensitive and chemosensitive inputs from cardiopulmonary regions directly to intrinsic cardiac and extracardiac neurons, forming the basis of the intrathoracic neural feedback system (5, 65). Superimposed on activities generated by neurons in peripheral autonomic ganglia are efferent inputs from preganglionic neurons in the brain stem and spinal cord that together exert tonic influences on regional cardiac tone (6, 7, 35). CNS preganglionic inputs are, in turn, influenced by inputs from higher centers in the central nervous system and by afferent feedback from central and peripheral sensory afferent neurons (9–11).

VII. INTERACTIONS AMONG PERIPHERAL AUTONOMIC NEURONS

VI. NEUROHUMORAL INTERACTIONS CONTRIBUTING TO CARDIAC CONTROL Figure 2 summarizes the current working hypothesis for neurohumoral interactions involved in the control of cardiac function. Data indicate that a hierarchy of peripheral autonomic neurons function interdependently via nested feedback loops to regulate cardiac function on a beat-to-beat basis. Figure 2 summarizes the emerging concept of neural control of the heart as mediated by intrathoracic extracardiac and intracardiac neurons, which are continuously influenced by descending projections from higher centers in the spinal cord, brain stem, and suprabulbar regions. Each successive synaptic relay point within this autonomic outflow, from the brain stem to the heart, is in turn influenced by afferent feedback from various cardiopulmonary and vascular afferent receptors. Accumulating evidence suggests that there may be at least four functionally distinct neuronal types within the intrinsic cardiac nerve plexus (see earlier discussion): parasympathetic postganglionic efferent neurons (1, 29, 34, 46, 51, 115, 135), local circuit neurons (20, 21, 53, 119, 136), adrenergic postganglionic efferent neurons (25, 34, 84–86, 140), and afferent neurons (5, 20, 21, 53). Local circuit and cardiac afferent neurons also lie within intrathoracic extracardiac ganglia, along with sympathetic postganglionic neurons (15, 18). With respect to intrathoracic autonomic ganglia, cholinergic and adrenergic efferent neurons in these ganglia represent the output elements that project axons to cardiac electrical and mechanical tissues. Local circuit neurons interconnect adjacent neurons within one ganglion or link neurons in separate clusters of intrathoracic ganglia (14, 136). These interneurons are presumably involved in the coordination of neuronal activity within these peripheral autonomic ganglia, likely providing the underlying inputs necessary for the maintenance of basal autonomic neuronal discharge. Intrathoracic affer-

Cardiac performance is modulated by both sympathetic and parasympathetic efferent neuronal inputs. The induced change in any regional cardiac function ultimately depends on the intrinsic characteristics of the cardiac end effector being innervated, the level of efferent activity from the CNS to the periphery, and interactions occurring within peripheral autonomic ganglia and at the respective cardiac end effectors. With respect to autonomic regulation of sinus rate, parasympathetic efferent neuronal influences predominate (77, 78), whereas sympathetic influences predominate for control of AV conduction (77, 78). Even within the same cardiac region, differential autonomic effects are evident. For example, atrioventricular conduction is preferentially sensitive to sympathoadrenergic activity (127), whereas AV junctional automaticity is more responsive to the influence of parasympathetic efferent neuronal inputs (128). Although the two efferent limbs of the autonomic nervous system are often controlled reciprocally (101), concomitant changes in efferent outflow also occur (18).

A. Interactions at the Organ Level Anatomical and functional studies indicate that sympathetic and parasympathetic efferent postganglionic nerve endings lie in close proximity to each other in the target tissues (24, 85, 86, 93, 141). Interactions among sympathetic and parasympathetic efferent projections to the heart involve pre- and postjunctional mechanisms at the end effectors in cardiac tissue. Postjunctional interactions involve differential modulation of adenylate cyclase via G-protein-coupled receptor systems (Fig. 2) (31, 32, 73, 76, 121). Catecholamines, released from sympathetic efferent projections or derived from the circulation, influence myocardial tissues by binding primarily to 웁1- and 웁2-adrenoceptors (32). Myocardial 웁-adrener-

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I. Pumping Action and Electrical Activity of the Heart

gic receptors are coupled to and stimulate adenylate cyclase via stimulatory guanine nucleotide-binding protein (Gs ) (32). Acetylcholine, released from parasympathetic efferent postganglionic neurons, binds to cardiomyocyte M2 muscarinic receptors, which, in turn, are coupled to and inhibit adenylate cyclase via inhibitory guanine nucleotide-binding protein (Gi ) (32). Interactions between these two receptor-coupled systems at the adenylate cyclase level ultimately determine the rate of formation of cAMP and thereby myocyte second messenger function (31, 32, 73, 76). The various interactions that occur at cardiac end effectors also involve the modulation of neurotransmitter release from prejunctional synaptic terminals. Neural release of principal mediators norepinephrine and acetylcholine, along with the corelease of various neuropeptides (e.g., NPY and VIP), act on specific receptors associated with sympathetic or parasympathetic efferent axon terminals (28,

78, 106, 113). These mechanisms act to modulate subsequent neurotransmitter release.

B. Interactions within the ICN Various lines of evidence indicate that peripheral sites that are separate from the end effectors contribute to mediating sympathetic–parasympathetic interactions for the control of regional cardiac function. Stimulating parasympathetic and/or sympathetic efferent projections to the heart can activate subpopulations of intrinsic cardiac neurons (20, 21, 53). These extrinsic autonomic projections converge on separate aggregates of intrinsic cardiac neurons, each of which exhibits preferential control over regional cardiac function (4, 96). As shown in Fig. 3, with respect to control of chronotropic function, surgical disruption of the right atrial ganglionated plexus eliminates direct vagal projections to the sinoatrial node

FIGURE 2 Schematic of proposed neural interactions occurring within the intrathoracic autonomic ganglia and between these peripheral networks and the central nervous system. Within the intrinsic cardiac ganglia are included sympathetic (Sympath) and parasympathetic (Parasym) efferent neurons, local circuit neurons (LCN), and afferent neurons. Contained within the extracardiac intrathoracic ganglia are sympathetic efferent neurons, local circuit neurons, and afferent neurons. These intrinsic cardiac and extracardiac networks form separate and distinct nested feedback loops that act in concert with CNS feedback loops involving the spinal cord and medulla to regulate cardiac function on a beat-to-beat basis. These nerve networks are also influenced by circulating humoral factors, including catecholamines (catechol) and angiotensin II (ANG II). Aff., afferent; DRG, dorsal root ganglia; Gs, stimulatory guanine nucleotide-binding protein; Gi , inhibitory guanine nucleotide-binding protein; AC, adenylate cyclase; 웁1 , 웁1-adrenergic receptor; M2 , muscarinic receptor.

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3. Neurohumoral Control of Cardiac Function

FIGURE 3 Atrial rate response to parasympathetic (A), sympathetic (B), and concurrent sympathetic–parasympathetic stimulation (B) before (intact) and after ablation of the ventral right atrial ganglionated plexus (RAGPx). Solid bars indicate duration of 12 Hz parasympathetic (PS) stimulation (Stim.); cross-hatched bar represents duration of 4 Hz stimulation of both stellate ganglia (sympathetic stimulation). Note that after RAGPx, vagal stimulation no longer slows atrial rate (A), but the tachycardia to sympathetic stimulation remains (B). Following RAGPx, when parasympathetic stimulation is superimposed on a background of sympathetic-induced tachycardia, the atrial rate returns to baseline rapidly. This residual sympathetic–parasympathetic interaction was eliminated by the muscarinic antagonist atropine (RAGPx ⫹ atropine). Adapted from McGuirt et al. (83).

(83). However, sympathetic efferent neuronal control of chronotropic function (83) and the vagal inhibition of the sinus tachycardia produced by cardiac sympathetic efferent neurons are maintained (83). These residual sympathetic–parasympathetic efferent neuronal interactions occur at the level of the heart and are prejunctional to the sinoatrial node (83). We have proposed that such residual interactions occur within the intrinsic cardiac nervous system (83, 97). Whether such intraganglionic autonomic interactions play correspondingly significant roles in modulation of dromotropic and inotropic function has yet to be determined.

C. Interactions within the Intrathoracic Nervous System Coordination of autonomic outflows from intrathoracic neurons to cardiomyocytes depends to a large extent on sharing of inputs from higher centers along with interactions among and between various peripheral ganglia. Interactions within and between intrathoracic ganglia involve local circuit neurons (see earlier discussion). Activities generated by neurons in intrinsic cardiac ganglia demonstrate no consistent short-term relationship to neurons in extracardiac ganglia (19). However, the sharing of cardiopulmonary afferent information acting through both intrathoracic and brain stem/spinal cord feedback loops permits an overall coordination of ef-

fector control (Fig. 4) (19). Together, these nested feedback control systems allow for a redundancy in neural control of the heart, while at the same time maintaining an intrinsic flexibility to differentially modulate regional cardiac function (4).

VIII. ELECTROPHYSIOLOGY OF INTRATHORACIC CARDIAC GANGLIA A. In Vivo Studies Cardiac neurons generate spontaneous activity in situ, frequently exhibiting activity that is temporally related to cardiac or respiratory cycles (20, 21, 53). Of the neurons that display cardiac-related activity, many are affected by mechano- or chemosensory inputs from the heart (21, 53). Trains of electrical stimuli delivered to axons in the T1–T5 ventral roots activate a substantial population of stellate and middle cervical neurons (9, 10). These data indicate a convergence of preganglionic inputs onto the extracardiac postganglionic soma, reflective of a functional amplification of such sensory input. In contrast, trains of electrical stimuli delivered to vagosympathetic trunks activate a much smaller population of intrinsic cardiac neurons (20, 21, 53). Moreover, few intrinsic cardiac neurons are activated after a fixed latency when extracardiac efferent neurons that innervate the heart are stimulated electrically, a finding

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are not absolutely required for the reflex coordination of intrathoracic neuronal activity. The medium loop (extracardiac) and short loop (intrinsic cardiac) cardiocardiac reflexes so involved are represented schematically in Fig. 2. They are distinct from the long feedback control loops associated with the various central neurons described earlier. We have demonstrated that the intrinsic cardiac feedback regulatory system continues to function in the autotransplanted heart, contributing importantly to the maintenance of cardiac function in such a state (123).

B. In Vitro Studies

FIGURE 4 Effects of activating left ventricular (LV) sensory neurites on intrathoracic intrinsic cardiac (ICN) and extracardiac (MCG, middle cervical ganglia) neural activity before (open bars) and after (filled bars) decentralizing the intrathoracic nervous system from the CNS. Neural activity is presented as percentage change from baseline. With innervation intact, activation of LV epicardial sensory neurites with mechanical stimuli (touch), chemical stimuli (veratridine and substance P), or iv isoproterenol reflexly increased activities in both intrinsic cardiac and extracardiac ganglia. Decentralization of the intrathoracic nervous system modified, but did not eliminate, reflex responses to subsequent cardiac afferent stimulations. These residual responses reflect the continued functioning of the nested intrathoracic feedback loops depicted in Fig. 2. Adapted from Armour et al. (19).

indicative of monosynaptic interconnections to such neurons. These data indicate that, in contradistinction to extracardiac ganglia, substantial spatial and temporal summation of inputs is required to modify the activity generated by neurons on the heart. Intrinsic cardiac neurons generate low-level activity in such a state consistent with a nerve network that functions as a ‘‘lowpass filter,’’ thereby minimizing the potential for imbalances within autonomic efferent neuronal inputs to the heart, a process which by itself could be arrhythmogenic (70). Intrathoracic cardiac neurons are influenced by central neuronal inputs as well as by inputs from intrathoracic autonomic efferent and cardiopulmonary afferent neurons (14). Acute transection of efferent projections to cardiac autonomic ganglia (i.e., ganglion decentralization from the central nervous system) markedly reduces the spontaneous discharge generated by neurons therein (20, 21, 53). However, within decentralized intrathoracic ganglia, many neurons exhibit basal activity, some of which can be influenced by cardiopulmonary mechanoand chemoreceptor afferent inputs (Figs. 4 and 5) (20, 21, 53). These data suggest that CNS feedback loops

As mentioned previously, intrinsic cardiac ganglia contain a heterogeneous population of neurons. Intracellular recordings from isolated whole mount aggregates of intrinsic cardiac ganglia support the concept that complex neural interactions occur within the heart. Studies on aggregates of intrinsic cardiac ganglia derived from different species indicate that the resting membrane potential of these neurons is approximately ⫺60 mV, with relatively low input resistances and thresholds for the generation of action potential being approximately 20 mV more positive than the resting membrane potential (89, 115–117, 119, 135, 138). These

FIGURE 5 Effects of chronic decentralization from the CNS on the capacity of intrinsic cardiac neurons (ICN) to generate spontaneous activity as well as to respond to alterations in the mechanical milieu of the ventricles. All afferent and efferent nerve connections between the CNS and extracardiac autonomic ganglia to intrinsic cardiac neurons were surgically interrupted 3 weeks previously. Shown are lead II electrocardiogram (EKG), left ventricular pressure (LVP), and extracellular neural activity recorded from the right atrial ganglionated plexus of the ICN in two separate animals. (Left) Basal ICN activity with cardiac-related discharge. (Right) Effects of touching the LV apex (arrows) on such activity. These data demonstrate that the ICN can generate spontaneous activity separate from the CNS and that cardiocardiac reflex loops continue to function in the decentralized ICN. Adapted from Ardell et al. (5).

3. Neurohumoral Control of Cardiac Function

properties are consistent with neurons functioning with low excitability. No evidence for ramp-like pacemaker activities has been found within mammalian intrinsic cardiac neurons in vitro (115, 117, 137). Thus spontaneous activity generated by such neurons in vivo likely reflects underlying cell–cell interactions. For orthodromic stimulation there is substantial dispersion in time of the evoked excitatory postsynaptic potentials (EPSPs) generated by a given intrinsic cardiac neuron, indicative of polysynaptic inputs to neurons within the intrinsic cardiac nervous system (115, 119, 135). After the generation of action potentials, prolonged afterhyperpolarizations are produced by these cells (115, 119, 137), an additional factor that limits the excitability of intrinsic cardiac neurons in situ. Intracellular recordings from isolated aggregates of intrinsic cardiac ganglia have identified both cholinergic and noncholinergic synaptic mechanisms coexisting within intrinsic cardiac ganglia (115, 117, 119, 135, 137, 138). In rats and pigs, only fast excitatory postsynaptic potentials are displayed by intrinsic cardiac neurons in response to orthodromic stimulation of closely adjacent intraganglionic axons (115, 117, 119). These postsynaptic potentials are substantially attenuated, but not completely eliminated, by nicotinic cholinergic blockade (117, 119). In the dog, orthodromic stimulation of presynaptic fibers in these nerves elicits fast and slow postsynaptic potentials within intrinsic cardiac neurons (135). Fast excitatory postsynaptic potentials are mediated by cholinergic nicotinic receptors (135), whereas slow excitatory and slow inhibitory potentials are mediated by cholinergic muscarinic receptors (135). In the pig, direct application of norepinephrine modifies the properties of about 25% of identified intrinsic cardiac neurons (119). These data indicate that intrinsic cardiac neurons possess muscarinic cholinergic and nicotinic cholinergic as well as adrenergic receptors. As detailed in the next section, many other putative neurotransmitters likewise modify electrical events of intrinsic cardiac neurons. These neurochemicals may play important roles in the modulation of intrinsic cardiac neuronal activity. In summary, intrathoracic autonomic ganglia do not function as obligatory synaptic stations for autonomic efferent neuronal input to the heart. Instead, they appear to be capable of complex signal integration involving afferent, local circuit as well as parasympathetic and sympathetic efferent neurons. While the physiological properties of extracardiac autonomic ganglia tend to amplify CNS and afferent feedback inputs, those of the intrinsic cardiac nervous system act to limit cardiac excitability. As such, the final common pathway of cardiac control—the intrinsic cardiac nervous system—may function as a ‘‘low-pass’’ filter to minimize transient

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neuronal imbalances arising from separate sympathetic and parasympathetic efferent neuronal inputs to the heart. In conjuction with this local afferent feedback mechanism, neurons in intrathoracic ganglia also mediate local cardiocardiac reflexes at sites separate from those on the heart and the CNS. The synaptic events underlying such intraganglionic interactions apparently involve multiple neurotransmitters that interact with various neuronal receptors to exert rapid-acting neuronal membrane conductance and/or longer-term modulation of synapses within the intrinsic cardiac nervous system. How the interplay among the various intrathoracic, spinal cord, and brain stem neurons ultimately affect regional cardiac function remains unknown. An equally important question concerns how these interactions are modified in disease states.

IX. SYNAPTIC MECHANISMS ASSOCIATED WITH NEURONS IN INTRATHORACIC AUTONOMIC GANGLIA A. Cholinergic Mechanisms Synaptic transmission in autonomic ganglia principally involves the release of acetylcholine by presynaptic terminals and subsequent binding of that neurotransmitter to nicotinic cholinergic receptors on postganglionic neurons. In mammals this synaptic junction is not obligatory, indicating that a significant convergence of inputs may be necessary to evoke postganglionic neuronal activity (115, 119, 135, 138). Thus the potential for synaptic integration exists within intrathoracic autonomic ganglia (15). Nicotinic and muscarinic cholinergic receptors have been associated with intrathoracic autonomic neurons (2, 14, 55, 68, 119, 135). Furthermore, the blockade of nicotinic receptors attenuates, but does not eliminate, activity generated by intrinsic cardiac neurons (20, 21, 53). Muscarinic blockade attenuates excitatory and inhibitory synaptic function within intrinsic cardiac ganglia as well (135). These data indicate that acetylcholine exerts both mediator and modulator effects at synapses within intrathoracic autonomic ganglia. Application of nicotine to intrathoracic autonomic neurons can alter their activity (68) and induce concomitant changes in regional cardiac function, whether the neurons are located in extracardiac or intrinsic cardiac ganglia (68, 140). Nicotinic activation of intrinsic cardiac neurons evokes a biphasic cardiac response, with initial suppression in regional cardiac function being followed by augmentation (Fig. 6). Nicotinic activation of atrial intrinsic cardiac neurons modifies primarily, but not exclusively, atrial function, whereas nicotinic activation of ventricular intrinsic cardiac neurons modifies primarily,

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FIGURE 6 Chronotropic and atrial inotropic effects elicited by injecting nicotine into a locus of the dorsal right atrial ganglionated plexus (RAGP) before (control) and following sequential selective muscarinic (atropine) and then 웁-adrenergic (propranolol ⫹ atropine) blockade. The dorsal right atrial ganglionated plexus is primarily associated with the control of right atrial function (4, 120). Before administration of blocking agents, nicotine injected into the RAGP evoked a biphasic response with an initial negative chronotropic and atrial inotropic response followed by increases in both cardiac indices. Atropine prevented the neurally evoked suppressive effects, whereas propranolol blocked the neurally evoked augmentation in atrial function. Adapted from Yuan et al. (140).

but not exclusively, ventricular function (140). Acute decentralization of intrathoracic ganglia from the CNS attenuates, but does not eliminate, such effects (140). In time, following chronic decentralization of intrathoracic ganglia, including those on the heart as with cardiac transplantation, peripheral nerve networks remodel to sustain cardiac function (123).

B. Noncholinergic Mechanisms Blockade of nicotinic cholinergic receptors attenuates, but does not eliminate, the activity generated by neurons within intrathoracic autonomic ganglia (5, 14). These data indicate that non-nicotinic putative neurotransmitters act as mediators for synaptic transmission within the intrathoracic neuronal system. Anatomical and physiological studies have identified multiple putative neurotransmitters in association with the mammalian intrinsic cardiac ganglia, which include purinergic agonists (3, 40, 47, 69), catecholamines (27, 45, 71, 110), angiotensin II (64), calcitonin gene-related peptide (79, 132, 133), neuropeptide Y (41, 54, 56–58, 91, 120, 132), substance P (26, 41, 60, 61, 79, 126, 131–134), neurokinins (124), endothelin (17), and vasoactive intestinal peptide (42, 56, 120, 131,–133). Many of these putative neurochemicals arise from neurons whose cell bodies are located in stellate, middle cervical, or mediastinal ganglia, whereas others may be synthesized by neurons intrinsic to the heart (40–42, 56–58, 72, 79, 126, 131,

133). Direct application of various neurotransmitters adjacent to neurons in intrinsic cardiac ganglia modifies the activities they generated, often resulting in concomitant changes in cardiac pacemaker and/or contractile behavior (25, 67–69). Subsequent transection of all extrinsic nerve inputs to the heart (acute decentralization) markedly attenuates, but does not eliminate, neurochemical modulation of intrinsic cardiac neuronal activity (67–69); however, it does eliminate most changes induced in regional cardiac function (25). In summary, intrinsic cardiac ganglia contain a heterogeneous population of neurons that utilize cholinergic and noncholinergic synapses to control intraganglionic, interganglionic, and nerve effector organ cell activities. Some of these neurotransmitters subserve short duration synaptic actions (e.g., acetylcholine), whereas others modulate pre- and/or postsynaptic function over longer periods of time [e.g., neuropeptide Y (95, 129, 130)]. Although studies have indicated the presence and potential effects of various putative neurotransmitters within the intrinsic cardiac nervous system, the physiological function of most of these substances in overall cardiac regulation remains to be determined.

X. INTERACTIONS BETWEEN CNS AND INTRATHORACIC NEURONAL NETWORKS: IMPLICATIONS FOR TREATMENT OF ANGINA PECTORIS Myocardial ischemia reflects an imbalance in the supply:demand balance within the heart with resultant activation of cardiac afferent neurons and, as a consequence, the perception of symptoms (i.e., angina pectoris) (49). In addition to such nociceptive responses, activating cardiac afferent neurons can elicit autonomic and somatic reflexes (18, 49). Pharmacological, surgical, and angioplasty therapies are commonly used to improve symptoms and cardiac function in patients exhibiting angina pectoris. Despite the fact that these treatments are usually successful, some patients still suffer from pain of cardiac origin following these procedures (74, 114). Epidural stimulation of the spinal cord (SCS) has been suggested as an alternative to bypass surgery in high-risk patients (81). With SCS, high-frequency, lowintensity electrical stimuli are delivered to the dorsal aspect of the T1–T2 segments of the thoracic spinal cord. This therapy decreases the frequency and intensity of anginal episodes (43, 59, 112). SCS reduces the magnitude and duration of ST segment alteration during exercise stress in patients with cardiac disease (111), improves myocardial lactate metabolism (80), and increases workload tolerance (111). The mechanisms

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whereby this mode of therapy produces such beneficial effects are poorly understood. Because intrathoracic cardiac neurons have been found to play important modulatory roles in cardiac regulation, we have begun to study SCS and its effects on the activity generated by intrinsic cardiac neurons (50). Transient cardiac ventricular ischemia increases the activities generated by intrathoracic ganglia, including those on the heart (18, 65). Excessive focal activation of intrathoracic neural circuits can induce cardiac dysrhythmias, even in normally perfused hearts (70). SCS resulted in an immediate suppression in intrinsic cardiac neuronal activity. A neurosuppressor effect imposed in the intrinsic cardiac nervous system occurs whether SCS is applied immediately before, during, or after coronary artery occlusion (50). Furthermore, the suppression of intrinsic cardiac neuronal activity persists even after cessation of SCS. That transection of the ansae subclavia eliminated these effects suggests that they primarily involve the sympathetic nervous system (50). The synaptic mechanisms and specific pathways mediating these responses have yet to be determined. They likely involve both sympathetic afferent and efferent neurons. For instance, spinal cord stimulation may activate sensory afferent fibers antidromically such that endorphins (44) or neuropeptides such as calcitonin generelated peptide or substance P (23, 25, 39) are locally released in the myocardium. It is known that opiates and neuropeptides can influence cardiac function of intrinsic cardiac neurons (see earlier discussion). Spinal cord stimulation may also suppress intrinsic cardiac adrenergic as well as local circuit neurons as the result of altered sympathetic efferent preganglionic neuronal activity. It is known that the activation of sympathetic efferent preganglionic axons can suppress many intrathoracic reflexes that are involved in cardiac regulation (4, 15). Thus these neurosuppressor effects may be due, in part, to the activation of inhibitory synapses within intrathoracic ganglia (36, 88). Clinical experience with SCS highlights the dynamic interactions that can occur between central and intrathoracic neurons, demonstrating the potential for effective clinical treatment of cardiac pathology via modulation of the intrathoracic nervous system.

XI. SUMMARY Regional control of cardiac function is dependent on the coordination of activity generated by neurons within intrathoracic autonomic ganglia and the CNS (4, 15). The hierarchy of nested feedback loops therein provides precise beat-to-beat control of regional cardiac function. Contrary to classical teaching, work from a number of

laboratories utilizing electrophysiological and neuropharmacological techniques applied from the level of whole organ to that of neurons recorded in vitro indicates that intrathoracic autonomic ganglia act as more than simple relay stations for autonomic control of the heart. Within the intrathoracic hierarchy of ganglia and nerve interconnections, complex processing takes place that involves spatial and temporal summation of sensory inputs, preganglionic inputs from central neurons, and intrathoracic ganglionic reflexes activated by local cardiopulmonary sensory inputs. The activity of neurons within intrathoracic autonomic ganglia is likewise modulated by circulating agents, chief among them being circulating catecholamines and angiotensin II. The progressive development of cardiac disease may be associated with maladaptation of these neuronal control mechanisms. Data indicate that many conventional treatments for cardiac diseases, such as heart failure, may exert some of their beneficial effects not only on cardiomyocytes, but also on peripheral autonomic nervous neurons. A comprehensive knowledge of the complex processing that occurs within the intrathoracic nervous system may provide a basis for understanding the role that these neurons play in the control of the normal heart as well as in the diseased heart. Information derived from an understanding of this complex neuronal hierarchy should provide novel therapeutic approaches for the effective treatment of cardiac dysfunction by the modulation of neurons regulating regional cardiac behavior.

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102. Randall, W. C., Ardell, J. L., Calderwood, D., Milosavljevic, M., and Goyal, S.C. (1986). Parasympathetic ganglia innervating the canine atrioventricular nodal region. J. Auton. Nerv. Syst. 16, 311–323. 103. Randall, W. C., Ardell, J. L., Wurster, R. D., and Milosavljevic, M. (1987). Vagal postganglionic innervation of the canine sinoatrial node. J. Auton. Nerv. Syst. 20, 13–23. 104. Randall, W. C., Armour, J. A., Geis, G. S., and Lippincott, D. B. (1972). Regional cardiac distribution of sympathetic nerves. Fed. Proc. 31, 1199–1208. 105. Raven, P. B., Potts, J. T., and Shi, X. (1997). Baroreflex regulation of blood pressure during dynamic exercise. Exerc. Sport Sci. Rev. 25 365–389. 106. Riley, D. A. (1988). Effects of neuropeptides on heart rate in dogs: Comparison of VIP, PHI, NPY, CGRP, and NT. Am. J.Physiol. 255, H311–H317. 107. Roberts, L. A. (1991). The sinoatrial ring bundle: A cardiac neural communication system? Am. J. Anat. 191, 250–260. 108. Roberts, L. A., Slocum, G. R., and Riley, D. A. (1989). Morphology study of the innervation pattern of the rabbit sinoatrial node. Am.J.Anat. 185, 74–88. 109. Rowell, R. B. (1993). ‘‘Human Cardiovascular Control.’’ Oxford Univ. Press, New York. 110. Saito, K., Potter, W. Z., and Saavedra, J. M. (1988). Quantitative autoradiography of 웁-adrenoceptors in the cardiac vagus ganglia of the rat. Eur. J. Pharm. 153, 289–293. 111. Sanderson, J. E., Brooksby, P., Waterhouse, D., Palmer, R. B., and Neubauer, K. (1992). Epidural spinal electrical stimulation for severe angina: A study of its effects on symptoms, exercise tolerance and degree of ischemia. Eur. Heart J. 13, 628–633. 112. Sanderson, J. E., Ibrahim, B., Waterhouse, D., and Palmer, R. B. (1994). Spinal cord stimulation for intractable angina: Long term clinical outcome and safety. Eur. Heart J. 15, 810–814. 113. Schmidt, H. H. H. W., Schurr, C., Hedler, L., and Majewski, M. (1984). Local modulation of noradrenaline release in vivo: Presynaptic 웁2-adrenoceptors and endogenous adrenaline. J. Cardiovasc. Pharmacol. 6(4), 641–649. 114. Schoebel, F. C., Frazier, O. H., Jessurun, G. A. J., DeJongste, M. J. L., Kadipasaoglu, K. A., Heintzen, M. P., Jax, T. W., Cooley, D. A., Strauer, B. E., and Leschke, M. (1997). Refractory angina pectoris in end-stage coronary artery disease: Evolving therapeutic concepts. Am. Heart J. 134, 587–602. 115. Seabrook, G. R., Fieber, L. A., and Adams, D. J. (1990). Neurotransmission in neonatal rat cardiac ganglion in situ. Am. J. Physiol. 259, H997–H1005. 116. Selyanko, A. A. (1982). Membrane properties and firing characteristics of rat cardiac neurons in vitro. J. Auton. Nerv. Syst. 39, 181–190. 117. Selyanko, A. A., and Skok, V. I. (1992). Synaptic transmission in rat cardiac neurones. J. Auton. Nerv. Syst. 39, 191–200. 118. Shvalev, V. N., and Sosunov, A. A. (1985). A light and electron microscopic study of cardiac ganglia in mammals. Z. Mikrosk. Anat. Forsch. 99, 676–694. 119. Smith, F. M., Hopkins, D. A., and Armour, J. A. (1992). Electrophysiological properties of in vitro intrinsic cardiac neurons in the pig (Sus scrofa.) Brain Res. Bull. 28, 715–725. 120. Sternini, C., and Brecha, N. (1985). Distribution and colocalization of neuropeptide Y- and tyrosine hydroxylase-like immunoreactivity in the guinea pig heart. Cell Tissue Res. 241, 93–102. 121. Summers, R. J., McMartin, L. R., Kompa, A. R., Gu, X., and Molenaar, P. (1995). Signalling pathways in cardiac failure. Clin. Exp. Pharm. Physiol. 22, 874–876. 122. Sylven, C. (1989). Angina pectoris. Clinical characteristics, neurophysiological and molecular mechanisms. Pain 36, 145–167.

3. Neurohumoral Control of Cardiac Function 123. Thompson, G., Ardell, J. L., Murphy, D. A., Sangalang, V. E., Stevenson, R., MaCraty, R., and Armour, J. A. (1999). Neural control of the autotransplanted heart. FASEB J. 13, A446. 124. Thompson, G. W., Hoover, D. B., Ardell, J. L., and Armour, J. A. (1998). Canine intrinsic cardiac neurons involved in cardiac regulation possess NK1, NK2 and NK3 receptors. Am. J. Physiol. 275, R1683–R1689. 125. Thoren, P. (1977). Characteristics of left ventricular receptors with nonmedullated vagal afferents in cats. Circ. Res. 40, 415–421. 126. Urban, L., and Papka, R. E. (1985). Origin of small primary afferent substance P-immunoreactive nerve fibers in the guinea pig heart. J. Auton. Nerv. Syst. 12, 321–331. 127. Urthaler, F., Neely, B. H., Hageman, G. R., and Smith, L. R. (1986). Differential sympathetic–parasympathetic interactions in sinus node and AV junction. Am. J. Physiol. 250, H43–H51. 128. Wallick, D. W., Felder, D., and Levy, M. N. (1978). Autonomic control of pacemaker activity in the atrioventricular junction in the dog. Am. J. Physiol. 235, H308–H313. 129. Warner, M. R., and Levy, M. N (1989). Neuropeptide Y as a putative modulator of the vagal effects on heart rate. Circ. Res. 64, 882–889. 130. Warner, M. R., Senanayake, P. D., Ferrario, C. M., and Levy, M. N. (1991). Sympathetic stimulation-evoked overflow of norepinephrine and neuropeptide Y from the heart. Circ. Res. 69, 455–465. 131. Weihe, E., Reinecke, M., and Forssman, W. G. (1984). Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart: Interrelation with neurotensin- and substance P- like immunoreactive nerves. Cell Tissue Res. 236, 527–540. 132. Wharton, J., and Gulbenkian, S. (1987). Peptides in the mammalian cardiovascular system. Experientia 43, 821–832. 133. Wharton, J., Polak, J. M., Gordon, L., Banner, N. R., Springall, D. R., Rose, M., Khagani, A., Wallwork, J., and Yacoub, M. H.

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(1990). Immunohistochemical demonstration of human cardiac innervation before and after transplantation. Circ. Res. 66, 900–912. Wharton, J., Polak, J. M., McGregor, G. P., Bishop, A. E., and Bloom, S. R. (1981). The distribution of substance P-like immunoreactive nerves in the guinea pig heart. Neuroscience 6, 2193– 2204. Xi-Moy, S. X., Randall, W. C., and Wurster, R. D. (1993). Nicotinic and muscarinic synaptic transmission in canine intracardiac ganglion cells innervating the sinoatrial node. J. Auton. Nerv. Syst. 42, 201–214. Xi, X., Randall, W. C., and Wurster, R. D. (1991). Morphology of intracellularly labeled canine intracardiac ganglion cells. J. Comp. Neurol. 314, 396–402. Xi, X., Randall, W. C., and Wurster, R. D. (1993). Intracellular recording of spontaneous activity of canine intracardiac ganglion cells. Neurosci. Lett. 128, 129–132. Xi, X., Thomas, J. X., Randall, W. C., and Wurster, R. D. Intracellular recordings from canine intracardiac ganglion cells. J. Auton. Nerv. Syst. 32, 177–182. Xu, Z. J., and Adams, D. J. (1993). 움-Adrenergic modulation of ionic currents in cultured parasympathetic neurons from rat intracardiac ganglia. J. Neurophysiol. 69, 1060–1070. Yuan, B. X., Ardell, J. L., Hopkins, D. A., and Armour, J. A. (1993). Differential cardiac responses induced by nicotine sensitive canine atrial and ventricular neurons. Cardiov. Res. 27, 760–769. Yuan, B. X., Ardell, J. L., Hopkins, D. A., Losier, A. M., and Armour, J. A. (1994). Gross and microscopic anatomy of the canine intrinsic cardiac nervous system. Anat. Rec. 239, 75–87. Zucker, I. H., Wang, W., Brandle, M., and Schultz, H. D. (1996). Baroreflex and cardiac reflex control of the circulation in pacinginduced heart failure. In ‘‘Pathophysiology of Tachycardia-Induced Heart Failure’’ (F. G. Spinale, ed.), pp. 193–226. Futura, Armonk, NY.

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4 Control of Cardiac Output and Its Alterations during Exercise and in Heart Failure JAMES M. DOWNEY

GERD HEUSCH

Department of Physiology University of South Alabama Mobile, Alabama 36688

Department of Pathophysiology University of Essen Essen, Germany

I. INTRODUCTION

ated that increases in cardiac output were generally associated with a declining right atrial pressure. This led to antegrade and retrograde hypotheses. The ‘‘retrograde’’ hypothesis reasons that if atrial pressure falls, the gradient for venous return from the periphery increases, thus accounting for the increased cardiac output. This simplistic argument, however, ignores the role played by the heart. The ‘‘antegrade’’ argument attempts to include the heart in the explanation. It is reasoned that atrial pressure falls because the heart is taking more blood out of the veins and pumping it into the arteries. An inherent circular reasoning flaws both approaches. If, as is obvious in a closed circuit, more cardiac output equates with more venous return, then how does one separate them? Although both approaches are reasonable, each fails as a useful predictor of circulatory dynamics.

An obvious function of the cardiovascular system is to maintain a continuous circulation of blood that meets the nutritional needs of the periphery. The primary index of this circulation is the cardiac output, the amount of blood pumped by one ventricle per unit time. In normal circulation the left heart is in series with the right heart. Thus, the amount of blood pumped by either the right or the left ventricle is equal. However, anatomical abnormalities such as patent ductus arteriosus or atrial/ventricular septal defects can cause shunts that disrupt the series arrangement of the two ventricles. The blood volume within the pulmonary circulation represents the running integral of the difference between left and right heart outputs. Because the capacity of the pulmonary system is limited, differences such as those occurring with respiration cannot be maintained for more than a second or two and the average outputs of the two chambers must be equal. While the heart is clearly a key determinant of the cardiac output, the beginning student is often surprised to learn that the periphery plays an equally important role in this process. Before the hemodynamic alterations of cardiovascular states, such as exercise or heart failure, can be appreciated, the fundamentals of the interplay between the heart and the periphery must first be discussed. Since the classic studies of Otto Frank and Ernest Starling it has been appreciated that cardiac filling pressure is a primary determinant of stroke volume. However, the nature of the interaction between this filling pressure and the resulting cardiac output was a long-standing and perplexing problem. It was appreci-

Heart Physiology and Pathophysiology, Fourth Edition

II. THE TWO-COMPARTMENT CAPACITANCE MODEL Arthur Guyton solved the just-discussed problem. His unique analysis modeled the peripheral circulation based on the capacitance of its compartments (Guyton et al., 1973). Capacitance refers to the relationship between the pressure and the volume of a container and is defined as volume/pressure. For example, the venous system has a very large capacitance. Transfusing a unit of blood into a patient expands the venous volume by about 400 ml with an increase in venous pressure of only 4–5 mm Hg. However, arterial capacitance is small. Adding just 60 ml of blood to the arterial system (a

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typical stroke volume) would raise arterial pressure by about 40 mm Hg. The cardiovascular system can be represented in its simplest terms by the model shown in Fig 1. The two-compartment capacitance model consists of a closed loop with a pump (the heart) directing flow through a resistor (the peripheral resistance). The arterial compartment resides between the output of the pump and the peripheral resistance. The arterial compartment is represented as a small diameter vertical tube open at the top. In this representation the height of the fluid in the tube is proportional to the arterial pressure and the arterial volume is the height times the cross-sectional area of the tube. Because the crosssectional area is small, the arterial reservoir’s capacitance will be small. Between the resistor and the pump’s inlet is the venous compartment. It is represented as a large-diameter tube reflecting the large capacitance of the venous system (about 30 times that of the arterial system). The total volume of blood in the system remains constant and the only thing that can change is the distribution between the two compartments. The first thing that becomes obvious in this model is that venous and arterial pressure will vary reciprocally. If arterial pressure increases, the arterial volume must increase as dictated by its capacitance. An increase in arterial blood volume must be met with a decrease in venous blood volume and hence venous pressure falls. Because the venous capacitance is about 30 times that of the arterial capacitance, large changes in arterial pressure cause small changes in venous pressure. The second observation is that arterial pressure will adjust itself so that a pressure head exists sufficient to force flow through the peripheral resistance that is equal to the pump’s output. If pressure is too low, then inflow into the arterial compartment will exceed outflow and blood will accumulate in the arterial system until the arterial

FIGURE 1 The simple capacitance model of circulation. Pressures are represented by the height of fluid in the compartments and capacitances by the width of the compartment.

pressure rises to a level to achieve a new steady state. Note that if the heart were stopped the system would come to rest with zero flow across the resistance. If the flow is zero then the pressure difference across the resistance connecting the two compartments must also be zero. Thus pressure would be uniform throughout the system. Guyton called this equilibrium pressure the mean systemic filling pressure (not to be confused with mean aortic, left atrial, or central venous pressure) and in dogs determined its value to be about 7 mm Hg (Guyton et al., 1973). The cardiac function curve in Fig. 2B reveals that the cardiac output is influenced greatly by the venous pressure (cardiac filling pressure). This relationship can be derived directly from the ejection loop analysis presented in Chapter 1. Note that the cardiac function curve can be shifted by two other determinants. Increasing contractility as shown in Fig. 2B shifts the curve up and to the left, whereas decreasing contractility has the opposite effect. The other determinant of stroke volume is the afterload. Increasing afterload decreases stroke volume and thus shifts the curve down and to the right. Decreasing afterload has the opposite effect. The significance of the afterload’s influence on cardiac output will become apparent when changes in peripheral resistance are explained later. If we were to replace the heart with a mechanical pump whose output could be varied, we could determine the relationship between cardiac output and central venous pressure. That effect is shown in Fig. 2A. Both arterial and venous pressures are plotted. When the pump is off, pressure is equal in both compartments (mean systemic filling pressure). Turning on the pump causes arterial pressure to rise markedly and venous pressure to fall. Because of the differences in capacitance, however, the change in venous pressure is only one-thirtieth of that in the arterial system. If venous pressure is plotted as a function of cardiac output, the venous function curve shown in Fig. 2C results. Note that the venous pressure in the model varies as an inverse function of cardiac output. Venous pressure is 7 mm Hg (mean systemic filling pressure) at zero cardiac output and falls as cardiac output increases. The fall in venous pressure is the result of translocating blood into the arterial system. As cardiac output rises so does arterial pressure and thus arterial blood volume. Note that a theoretical maximum occurs when venous pressure reaches zero. Any further increase in cardiac output will result in venous collapse, as the luminal pressure becomes negative. Thus while the heart’s output is determined by venous pressure, venous pressure is in turn determined by the heart’s output. The key is to find an analysis that considers both of these relationships at the same time.

4. Control of Cardiac Output

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FIGURE 2 (A) Pressures in the arterial and venous systems while the heart is arrested (left side). The pressure in both compartments equals mean systemic filling pressure. When the heart is started (right side), pressure in the arteries rises by a large amount whereas that in the veins falls by a lesser amount. The pressure in the veins, however, is of particular interest because that is the filling pressure of the heart. (B) The volume of blood pumped by the heart per unit time as a function of its filling pressure (venous pressure). Note that the amount of blood pumped for any level of venous pressure is increased when contractility is increased. (C) Pressure in the venous system is related inversely to cardiac output. (D) If the heart depicted in B were connected to the vascular system depicted in C, a stable equilibrium would only occur at the crossing point of the two curves.

This problem can be overcome by solving both relationships as a set of simultaneous equations. In that way the equilibrium cardiac output that satisfies both the heart and the vascular system can be determined. Because the axes for Figs. 2B and 2C are identical, the simultaneous equations can be solved graphically by simply coplotting the two curves on the same axes as shown in Fig. 2D. Note that the ordinate is now cardiac output whereas the abscissa is venous pressure. The crossing point of the two curves is the solution to the two simultaneous equations. In other words, if the heart shown in Fig. 2B were connected to the blood vessels shown in Fig. 2C, a cardiac output of 5 liters/min and a venous pressure of 3.5 mm Hg would be achieved. Note that the system is highly self-regulating. Because any momentary in-

crease in cardiac output would lower venous pressure, cardiac output would return quickly to that determined by the crossing point of the two curves. This hydraulic negative feedback ensures a highly stable cardiac output.

A. Alterations in Blood Volume and Venous Capacitance The just-described analysis is very powerful. For example, it predicts changes resulting from altered blood volume. Transfusion shifts the venous function curve up and to the right in a parallel fashion as shown in Fig. 3. The model predicts an increase in the mean systemic filling pressure as well as an equal increase in

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FIGURE 3 The effect of increasing blood volume or decreasing venous capacitance is illustrated by the dotted line. Note that both venous pressure and cardiac output are increased.

FIGURE 4 The effect of increasing cardiac contractility is illustrated by the dotted line. Note that cardiac output is increased but that venous pressure falls.

pressure for any given cardiac output. The effect is to raise venous pressure and cardiac output. Hemorrhage has the opposite effect. The analysis immediately reveals why volume replacement is the only rational treatment for hemorrhage. Vasomotion in the veins has an effect similar to that of volume alterations. Venoconstriction decreases the venous capacitance and venodilation increases it. Thus venoconstriction will raise venous pressure and therefore cardiac output. A sudden loss of venous tone has the opposite effect and is often associated with syncope.

output in which the system is at mean systemic filling pressure. Constricting the resistance would have a negligible effect on pressure in either reservoir. With the pump operating, however, arterial pressure would be higher and venous pressure lower for any level of output with the added constriction. Conversely, arterial pressure will be lower and venous pressure higher for any cardiac output at a reduced peripheral resistance. The

B. Alterations in Contractility Figure 4 shows the effect of increasing cardiac contractility. If contractility were increased, the cardiac function curve would be shifted up and to the left (greater cardiac output for any given filling pressure). The net effect of an increased contractility then becomes an increased cardiac output with a reduced filling pressure. Reducing contractility has the opposite effect. Cardiac output falls and filling pressure rises. For example, reduced contractility is typically observed in heart failure, and a cardinal sign of heart failure is elevated venous pressure.

C. Alterations in Peripheral Resistance Figure 5 shows the remaining possible maneuver: a change in total peripheral resistance. Refer back to the model in Fig. 1. Consider the condition with zero cardiac

FIGURE 5 The effect of a fall in peripheral resistance is illustrated by dotted lines. The cardiac function curve is shifted up because stroke volume will increase with a reduced afterload. Note that cardiac output is increased but the effect on venous pressure is ambiguous.

4. Control of Cardiac Output

effect of reduced peripheral resistance would be to cause the venous function curve to rotate about the x axis intercept in a clockwise fashion as shown in Fig. 5, and that of increased peripheral resistance would cause the venous function curve to rotate counterclockwise. Until now we have ignored the influence of afterload on the cardiac function curve. The decreased afterload associated with the decrease in peripheral resistance will facilitate ejection and thus increase cardiac output for any given filling pressure. The result is to shift the cardiac function curve up and to the left (Fig. 5). The net result is a clear increase in cardiac output, although the overall effect on filling pressure is ambiguous. Note that an increase in peripheral resistance primarily results from arteriolar constriction and should be differentiated from a change in venous compliance due to venoconstriction as was discussed earlier.

D. Neural Control The cardiovascular system is modulated by the autonomic nervous system. However, the control is physically constrained by the principles outlined earlier. Therefore the autonomic system can only alter the system by changing one or more of the parameters discussed earlier. This is seen as an alteration of the curves on the plots. For example, increasing sympathetic nerve activity to the heart increases contractility, which is equivalent to shifting the cardiac function curve up and to the left. Another important control point is the vascular smooth muscle in peripheral vessels. Sympathetic activation generally causes a constriction of these vessels with a twofold effect. The reduced caliber acts to elevate the resistance to blood flow through the bed, which occurs primarily at the arteriolar level. Thus widespread arteriolar constriction acts to raise the peripheral resistance. The sympathetic nervous system’s effect on the venous system is to decrease its capacitance. Because the veins offer little resistance to blood flow, the overall effect of venoconstriction on the venous bed’s resistance is small. The major effect is to force blood out of the periphery and into the heart with a resulting increase of filling pressure. While the autonomic nervous system has many complex reflexes with which it controls the cardiovascular system, in the end all of them affect the cardiac output by modulation of vascular capacitance, peripheral resistance, cardiac contractility, and/or heart rate. There are no other choices.

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rate normally has only a small influence on cardiac output. Figure 6 shows cardiac output as a function of heart rate when a dog’s heart is electrically paced (Miller et al., 1962). Note that in the physiological range of 75 to 125 beats/min there is very little change in cardiac output because cardiac output is controlled primarily by venous pressure. As heart rate increases, venous pressure falls, resulting in decreased stroke volume. At very low heart rates the cardiac output falls because a maximal filling volume has been achieved and any further reduction in the heart rate cannot be compensated by a further increase in stroke volume. At very high heart rates, stroke volume drops off disproportionately with increasing rate. At heart rates above 170 beats/ min the duration of diastole is shortened much more than that of systole. That makes for inadequate time for the ventricles to relax and to be filled between beats (decreased preload), thus decreasing stroke volume. As a result, output again falls, even though venous filling pressure is rising. These two extremes manifest themselves clinically. Tachyarrhythmias are usually associated with an inadequate cardiac output just as is the bradycardia of complete heart block. Interestingly, when the heart rate is physiologically augmented by sympathetic activation, the duration of the cardiac action potential is simultaneously shortened. This shortens the period of ejection, leaving more time for diastole. Also relaxation occurs more quickly as evidenced by the increase in negative ventricular dP/dt (the rate at which ventricular pressure falls during the isovolumetric period of relaxation), thus effectively improving ventricular filling.

E. Heart Rate Heart rate was not mentioned in the previous discussion because it plays a special role. Cardiac output equals stroke volume times heart rate. In actuality, heart

FIGURE 6 A plot of cardiac output as the heart rate of a dog is varied by electrical pacing. Note that within the physiological range, heart rate has little influence on cardiac output. Adapted from Miller et al. (1962).

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F. Respiration Moment-to-moment mismatches occur physiologically between the ventricles with respiration. Inspiration increases the stroke volume of the right ventricle as the negative intrathoracic pressure promotes enhanced filling of the right heart from the extrathoracic veins. At the same time, the negative pressure is transmitted to the blood within the pulmonary circulation, thus reducing the right ventricle’s afterload. The exact opposite occurs in the left ventricle. The left ventricle’s filling pressure is decreased by negative intrathoracic pressure. Simultaneously, the increased pressure gradient between the left ventricle and the extrathoracic aorta effectively puts the ventricle under an elevated afterload, further reducing its stroke volume. Because the average output of both ventricles must be equal, this mismatch is compensated with a reversed gradient during expiration. The profound influence of respiration on cardiac output is taken advantage of in intensive care medicine. Artificial ventilation at positive end expiratory pressure unloads the left ventricle and facilitates its ejection (Peters et al., 1989).

III. EXERCISE The circulation has an impressive ability to increase cardiac output on demand, such as in exercise. Welltrained subjects can increase their cardiac output seven to eight fold in heavy dynamic exercise. The body accomplishes this through a variety of mechanisms. Right atrial pressure is normally only 3–6 mm Hg. One of the fundamental characteristics of the venous function curve is that cardiac output can only increase up to the point where central venous pressure becomes zero. At that point the veins are at their unstressed volume. Negative venous pressure leads to venous collapse, preventing the veins from supplying any further increase in blood flow and thus putting a finite limit on the cardiac output. In simple terms, the heart is incapable of sucking blood out of the venous system. Because the normal circulation operates very close to the point of vascular collapse, the ability to increase cardiac output though an increase in cardiac contractility alone is very limited. With no adjustments to venous capacitance, cardiac output could barely double before venous pressure fell to zero. Therefore, multiple mechanisms must be activated to increase venous return during exercise.

A. Venous Capacitance Venous smooth muscle contraction will decrease venous capacitance and thus increase cardiac output (Fig. 3). Arteriolar vasoconstriction occurs in the

splanchnic circulation. The resulting decrease in its postcapillary pressure will cause translocation of blood out of the splanchnic bed and into the central veins, which again will contribute to the increased cardiac output (Fig. 3) (Barcroft and Samaan, 1935). Both decreased venous capacitance secondary to venous vasoconstriction and increased venous blood volume secondary to splanchnic arteriolar vasoconstriction are mediated by the sympathetic nervous system through the activation of vascular 움-adrenoceptors.

B. The Muscle Pump The muscle pump also plays an important part in the exercise response. The venous system in the skeletal muscles is rich in valves that direct flow back toward the heart. As the muscles contract they compress the venous segments between adjacent valves, forcibly expelling the contained blood into the central veins. It is estimated that a contracting muscle displaces about 12 ml of blood/kg of muscle (Janiki et al., 1996) Thus, vigorous voluntary muscle contraction may actually serve to translocate blood into the central circulation and maintain cardiac output during orthostatic challenge. Jet pilots and astronauts take advantage of the muscle pump’s action by tensing their muscles during intense acceleration conditions to prevent blacking out. In dynamic exercise the rhythmic contraction and relaxation of the skeletal muscles actively pump blood into the large veins. The muscle pump’s effect on the circulation would be the same as an increase in blood volume or a decrease in venous capacitance, which are indistinguishable in the venous function curve (Fig. 3). Bevega˚rd and Lodin (1962) studied patients with a congenital absence of venous valves. Although these individuals were still able to increase their cardiac output during exercise, they did so with a marked reduction in central venous pressure as compared to subjects with normal veins. Thus the decreased venous capacitance is a mixture of mechanical events within the contracting muscles as well as a systemic autonomic venoconstriction. The muscle pump offers another important benefit. In the simple model presented in Fig. 1 it was shown that reducing peripheral resistance would increase cardiac output. In the erect human the pressure of the hydrostatic columns in the leg blood vessels are of a similar order of magnitude as the blood pressure itself. An individual standing quietly has a hydrostatic column pressure of about 75 mm Hg between the heart and calf muscle. That pressure is added to the mean arterial pressure (100 mm Hg) such that the arterial pressure at the calf would be 175 mm Hg. That high pressure does not increase perfusion to the calf muscle, however, as venous pressure is also elevated by 75 mm Hg due

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to the corresponding hydrostatic column on the venous side. However, activation of the muscle pump quickly lowers venous pressure in dependent limbs to nearly zero during walking, as is shown in Fig. 7 (Noddeland et al., 1983). The resulting increase in the pressure gradient would increase muscle blood flow by 75% without any change in vascular dimensions. The system sees this effect in the dependent limbs as a reduction in vascular resistance even though it is the pressure gradient rather than the resistance that is actually altered. When this effect is added to the active hyperemia that occurs in contracting skeletal muscle, blood flow, especially to the muscles of the lower limbs, can become quite large. It should be appreciated that dynamic exercise is accompanied by a large fall in the total peripheral resistance despite the vasoconstriction that occurs in most vascular territories other than contracting muscles. The skin usually vasodilates for thermoregulatory reasons.

C. Neural Control and Central Command Exercise is accompanied by an increase in arterial blood pressure. The increased arterial blood pressure increases the irrigation of the exercising muscles by further increasing the pressure gradient across them. While the observed increase in arterial blood pressure reflects increased cardiac output and generalized vasoconstriction in all territories other than contracting muscles and skin, the neurohumoral origin of this increase in arterial blood pressure is only beginning to be understood and is attributed to elevated ‘‘central command.’’ The cardiovascular system behaves as if the set point for blood pressure regulation through the baroreflex had been increased suddenly. A number of muscle afferent nerves emanating from skeletal muscle have been identified that act to raise blood pressure. These nerves are activated by muscle contraction and may be an important component of the increased central command (Wilson and Hand, 1997). The contractility of the heart is also increased as part of the sympathetic response to exercise. The augmented venous return during exercise would result in a massive

FIGURE 8 A composite diagram illustrating the changes that contribute to an increased cardiac output during exercise.

rise in central venous pressure during heavy dynamic exercise if contractility failed to increase. As was shown in Fig. 4, increasing contractility acts to lower venous pressure. Because of the increased contractility, the actual change in central venous pressure during exercise tends to be ambiguous in healthy adults. Depending on the level of the workload and the age group studied, right atrial pressure may rise or fall slightly during exercise but never changes by much (Janiki et al., 1996). An increase in heart rate accompanies the increase in contractility and acts to keep stroke volume within a workable range. A foreshortening of the systolic period through a reduction in action potential duration and an increased rate of relaxation both aid cardiac filling during tachycardia. As mentioned previously, however, increasing heart rate alone has only a modest effect on cardiac output within the physiological range, and it is interesting to note that physical conditioning is actually accompanied by a reduction in heart rate, both at rest and at any level of exercise. In summary, dynamic exercise is met with a decrease in venous capacitance, a decrease in total peripheral resistance, an increase in cardiac contractility, and an increase in heart rate. The overall effect as shown in Fig. 8 is a marked increase in cardiac output, which is directed particularly to contracting muscles.

IV. HEART FAILURE FIGURE 7 A plot of venous pressure in a lower leg vein when a subject is standing quietly and then begins walking. The muscle pump lowers the venous pressure, thus increasing the driving pressure for blood flow through the leg muscles. Adapted from Noddeland et al. (1983).

Heart failure is a clinical condition in which cardiac output becomes inadequate to meet the nutritional needs of the periphery, almost always because of a lesion within the heart itself. The primary defect is most often

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I. Pumping Action and Electrical Activity of the Heart

a reduced contractility (Mann, 1999). As was predicted by Fig. 4, a reduction in cardiac contractility alone causes a concomitant rise in central venous pressure for any given cardiac output. Indeed elevated venous pressure is one hallmark of cardiac failure. The heart is composed of a right heart in series with a left heart and an interposed pulmonary circulation. In the healthy individual these parts function as a unit. Blood pumped by the right heart is passed through the low-resistance pulmonary circulation and is dutifully pumped by the more powerful left heart through the systemic circulation. Should the left heart’s output fall behind that of the right heart, left atrial pressure would quickly rise to bring the output back to that of the right heart. Thus, matching of left and right heart outputs is self-regulating. For this reason the right heart, lung, and left heart operate as a unit and were, therefore, considered as a single pump in the model shown in Fig. 1. With heart failure one chamber is often affected more than the other. When this occurs the two sides of the heart no longer function as a unit. The capacitance model presented earlier predicts that the maximum value venous pressure could achieve (that occurring with complete cessation of flow) would be the mean systemic filling pressure, a mere 7 mm Hg. Patients with left ventricular failure, however, often have left atrial pressures in excess of 20 mm Hg. In fact, pulmonary venous pressure is so high in these patients that pulmonary edema becomes a serious threat to life. The rise in left atrial and pulmonary venous pressures derives from several factors. The first is the continuous pumping of the healthy right ventricle, which forces blood into the pulmonary circulation in the expectation that the left ventricle will pass it on to the periphery. As the left ventricle fails, a higher left atrial pressure is required to match the left ventricle’s output to that of the healthy right ventricle. This mismatch between the ventricles is exacerbated when the overall cardiac output falls and reflex sympathetic activation and the resulting constriction of the venous beds mobilize even more venous blood to the right heart in an attempt to restore the cardiac output. In normal circulation, blood volume is adjusted for long-term regulation of cardiac output. This is accomplished both through physical filtration forces at the kidney and through humoral effects largely involving the renin–angiotensin system. If cardiac depression persists, the long-term response of the body is to retain fluids in an attempt to increase the blood volume. Normally an increased blood volume translates to increased cardiac output (see Fig. 3), but in the situation of heart failure the resulting rise in atrial pressure and the threat of pulmonary edema far outweigh any gain in cardiac output.

Pulmonary edema is a particular threat when the left ventricle is involved, as is most often the case. Because pulmonary edema interferes with normal lung function, death from asphyxia can occur in these patients. Reducing blood volume by use of diuretics and limiting salt in the diet often lowers the left atrial pressure to an acceptable level in these patients. Failure of the right ventricle has a different effect: a rise in central venous pressure. In these patients, swelling of the lower limbs, hepatic congestion, and ascites are typical problems, as it is right, rather than left, atrial pressure that is elevated. The body can compensate for a moderate decrease in contractility through elevation of preload and persistent sympathetic drive of the heart. Compensated heart failure would result in elevated venous pressure in front of the failing chamber and reduced exercise tolerance. As the cardiac lesion worsens, however, the patient may eventually be unable to mount a normal cardiac output at rest whereupon the heart failure would be considered to be decompensated. The etiology of heart failure is mixed. It can result from myocardial ischemia and infarction, persistent mechanical overload, or a myopathy of the heart. Infarction simply reduces the contractile mass of the heart through the death of ventricular muscle. Loss of more than a third of the left ventricle will result in the signs and symptoms of heart failure. The excessive workload placed on the surviving muscle can cause it to remodel and degenerate further, especially if it is poorly perfused as a result of narrowed coronary arteries. Chronic overload due to hypertension or outflow tract obstruction causes a biphasic response of first concentric hypertrophy where the ventricular mass is increased and the ventricular walls become thickened at the expense of the lumen. The thickened wall results in diastolic dysfunction so that the ventricle does not respond to a rise in filling pressure with an appropriate increase in myocardial fiber length. Remodeling of the hypertrophied ventricle is accompanied by altered gene expression, which again manifests itself in alterations of excitation–contraction coupling (Mann, 1999). For reasons that are now only beginning to be understood, the hypertrophied heart then progresses to a weakened state in which the contractility of the muscle is reduced greatly. The possible causes of this change are reviewed in Chapters 57–59. The third cause of heart failure, cardiomyopathy, is perhaps the most insidious and least understood. The etiology of primary cardiomyopathies is unknown. For no apparent reason, patients begin to undergo a slow but progressive deterioration of the heart muscle itself. There is no offending valve defect or loading condition that can be corrected to halt the progression of the disease. Because of the progressive nature of many car-

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diomyopathies, heart transplantation is often the only treatment. The underlying causes are undoubtedly varied, and identifying the cellular changes that have robbed these patients of their normal heart function is currently the object of intense research. Secondary cardiomyopathies may be of toxic origin (most notably ethanol). Infection can also cause cardiomyopathy. Acute and chronic viral infections such as with Coxsackie virus along with superimposed autoimmune responses can cause cardiomyopathy (Knowlton and Badorff, 1999). Bacteria that can cause myopathy include diphtheria and streptococcus. Even parasites can attack the heart. A good example is Chagas disease, which is widespread in Latin America.

V. SUMMARY This chapter revealed that cardiac output is as much determined by the peripheral vasculature as by the heart. Guyton’s two-compartment model is very useful for predicting the behavior of the cardiovascular system under a wide range of conditions. In exercise, the heart, peripheral vasculature, and muscle pump all act together to promote blood flow to the skeletal musculature. In heart failure, declining cardiac performance is first compensated by the periphery in an attempt to maintain a cardiac output. In later stages, however, these changes, particularly increased blood volume, actually exacerbate the condition by causing venous congestion and edema.

Bibliography Barcroft, H., and Samaan, A. (1935). Explanation of increase in systemic flow caused by occluding descending thoracic aorta. J. Physiol. (Lond.) 85, 47–61. Bevega˚rd, S., and Lodin, A. (1962). Postural circulatory changes at rest and during exercise in five patients with congenital absence of valves in the deep veins of the legs. Acta Med. Scand. 172, 21–29. Guyton, A. C., Jones, C. E., and Coleman, T. G. (1973). ‘‘Circulatory Physiology: Cardiac Output and Its Regulation,’’ 2nd Ed, Saunders, Philadelphia. Janicki, J. S., Sheriff, D. D, Robotham, J. L., and Wise, R. A. (1996). Cardiac output during exercise: contributions of the cardiac, circulatory, and respiratory systems. In ‘‘Handbook of Physiology’’ (L. B. Rowell and J. T. Shepherd, eds.), Chap. 15, pp. 649–704. Oxford Univ. Press, New York. Knowlton, K. U., and Badorff, C. (1999). The immune system in viral myocarditis. Maintaining the balance. Circ. Res. 85, 559–561. Mann, D. L. (1999). Mechanisms and models in heart failure: A combinatorial approach. Circulation 100, 999–1008. Miller, D. E., Gleason, W. L., Whalen, R. E., Morris, J. J., and Mclntosh, H. D. (1962). Effect of ventricular rate on the cardiac output in the dog with chronic heart block. Circ. Res. 10, 658– 663. Noddeland, H., Ingemansen, R., Reed, R. K., and Aukland, K. (1983). A telemetric technique for studies of venous pressure in the human leg during different positions and activities. Clin. Physiol. 3, 573–576. Peters, J., Fraser, C., Stuart, R. S., Baumgartner, W., and Robotham, J. L. (1989). Negative intrathoracic pressure decreases independently left ventricular filling and emptying. Am. J. Physiol. 257, H120–H131. Wilson, L. B., and Hand, G. A. (1997). The pressor reflex evoked by static contraction: Neurochemistry at the site of the first synapse. Brain Res. Rev. 23, 196–209.

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5 Ultrastructure of Cardiac Muscle and Blood Vessels MICHAEL S. FORBES ‘‘Merry Oaks’’ Troy, Virginia 22974

I. INTRODUCTION AND CAUTION

ogy of the heart’s circulatory elements—despite the particularly vital nature of this particular bed of blood vessels. Nor, as noted earlier, has there been any great outpouring of structurally related work on the heart in the years since the last issue of this book appeared. In part, this can be attributed to the general decline of descriptive disciplines in favor of more substructurally slanted interests. The heyday of the transmission electron microscope (TEM) as a descriptive research tool lasted about 30 years (1955–1985), but the TEM as ‘‘beall and end-all’’ lost favor over the years as investigators and funding agencies alike came to prefer the techniques allied with molecular biology, a discipline that had its beginnings in the late 1940s. The demise of anatomical– structural biology, although curtailing the careers of many, has nevertheless left an enduring legacy: detailed three-dimensional descriptions of the cellular entities that compose organs such as the heart. It is to this legacy—shopworn though it may be—that I return in this chapter to establish the structural foundation for many of the other subjects covered in this book. For example, several older reviews still provide largely valid treatments of cardiac muscle ultrastructure (Forbes and Sperelakis, 1995; McNutt and Fawcett, 1974; Simpson et al., 1973; Sommer and Johnson, 1979). Structural biologists are darkly referred to in some molecular circles as ‘‘GOMs’’ (‘‘grizzled old morphologists’’); it is nonetheless now left by default to such decrepit individuals to explain the detailed form of cells and organelles to new audiences; one can only wonder, therefore, who will prepare a chapter such as this one in the future.

This chapter seeks to summarize the fine structure of three of the essential cell types found in the heart: cardiac muscle cells (also called ‘‘myocardial cells’’ or ‘‘cardiomyocytes’’), vascular smooth muscle cells (for which a more inclusive term is ‘‘periendothelial cells’’), and endothelial cells. The first cell category constitutes most of the heart’s mass, whereas the remaining two make up the walls of the heart’s extensive system of blood vessels. My long-term mentor, colleague, and friend, Nick Sperelakis, spent a fair amount of time persuading me to prepare this chapter. All during the course of our negotiations, I argued that my own dayto-day involvement with the subject of cardiovascular ultrastructure had ceased a decade ago. My misgivings became even more profound when I found that my two chapters from the previous editions of this book would require abbreviation into one, with a consideration of endothelial cells added, no less, and the entire work sufficiently generalized so as to fit the needs of a reference-type textbook. As has historically been the case, however, Nick’s sheer perseverance overcame—more accurately, ground away—my objections. I then made a discovery that was at once relieving and disturbing: since I last contributed to the book, little new has appeared that deals with the fine structure of either myocardial cells or myocardial circulation. In addition, while the literature extant concerned with fine structure of the heart is voluminous, it is heavily weighted on the side of cardiac muscle cells, with shockingly few publications addressed to the morphol-

Heart Physiology and Pathophysiology, Fourth Edition

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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I. Pumping Action and Electrical Activity of the Heart

II. CARDIAC MUSCLE CELLS A. The Cellular Nature of Myocardium To the present-day student of biology, it would be heretical to think that a tissue or organ could be composed of anything but individual cells; however, this was not a tacit assumption a century ago. At that time, two major organs of the body, the brain and the heart, were thought by many to be syncytial in nature (a syncytium is an extensive biological system having multiple nuclei widely distributed within a single mass of cytoplasm). In the final decades of the 19th century, the brain yielded to the cellular school of thought; this realization was in large part brought about by the metal-impregnation techniques of Golgi and Cajal, which clearly demonstrated the extensive yet discrete nature of the individual nerve cells. The concept of a cardiac syncytium was more durable, however, persisting in some texts into the 1950s, despite early TEM investigations, which—in historical hindsight—clearly demonstrated the cellular borders between cardiomyocytes. Part of the reluctance to accept cellularity as a intrinsic feature of the myocardium came from lingering misinterpretations concerning the structural basis of intercalated discs (considered later in this chapter). Finally, however, the sheer weight of detailed TEM observations, together with the finding that myocardial tissue can be readily dissociated into individual muscle cells by enzymatic and mechanical dispersion techniques (Fig. 1), won the day for the cellular theory.

B. Construction of a Myocardial Cell The shape of a cardiac muscle cell is largely the product of its internal construction; the cell is a fasces-like collection of rod-like myofibrils about which an external covering, the sarcolemma, is wrapped. The enveloped myofibrils often are of different lengths, and thus form staggered cell ends that are the basis for the step-like profiles of the intercalated discs (Fig. 1). Cardiac muscle cells are not necessarily the simple cylindrical entities implied to exist by many histology texts, but in fact may be band or ribbon like and can display a significant amount of branching. The length of the ‘‘average’’ mammalian ventricular myocardial cell is commonly given as ca. 100 애m, with a diameter on the order of 20 애m. Because of their somewhat flattened overall shapes, however, major and minor diameters of ca. 27 and 8 애m have been measured for ventricular cells of ferret (Phillips et al., 1977). The binuclearity common in such cells increases their overall size and volume, furthermore, relative to cells having a single nucleus (see later). In addition to measurements made on overall dimensions of myocardial cells, considerable work has been

devoted to the determination of the relative contributions of various cellular components to the cardiomyocyte. As practiced with certain defined sampling procedures and statistical formulae, this sort of measurement is known as ‘‘stereology’’ and can be used to generate parameters such as volume fraction (VV), which is the percentage of the total cell volume occupied by a particular type of structure. It is intuitively obvious from the inspection of electron micrographs of heart that myofibrils and mitochondria are major constituents of the muscle cells, and stereological studies have repeatedly confirmed this qualitative observation. Other cell constituents are visually less obvious, and it remained for certain specialized staining techniques to be combined with stereological regimens in order to determine their extent (see Section II,G). A summary of observations made on working cells of ventricles and atria of the laboratory mouse is shown in Table I.

C. Myocardial Cell Types Myocardial cells can be categorized as either working or conductive. Working cells of the ventricles and atrium are responsible for the contractile activity of the heart muscle, whereas conductive cells are more electrical in function, with the cardiac action potential being initiated in the the pacemaker region and spreading through the rest of the atrioventricular conducting system (AVCS) to activate the working cells. Ventricular working cells are generally the largest myocardial cells, far exceeding their atrial counterparts in both length and width; in turn, atrial working cells are larger and structurally more complex than conductive cells. One notable exception to this hierarchy is seen in ungulate heart, where the Purkinje cells are huge and distended, forming grossly visible, pale fibrous networks on the inner ventricular surfaces (Hayashi, 1962). Microscopic inspection of such conductive cells reveals a watery cytoplasm containing sparse contractile material and small scattered mitochondria. In rodents and insectivores, by contrast, con-

TABLE I Percentage of Cell Volume Occupied by Cardiac Muscle Cell Components in Mousea Component

Right ventricle

Left ventricle

Right atrium

Left atrium

Myofibrils Mitochondria Sarcoplasmic reticulum Transverse tubules Nuclei

43.5 38.2 6.9 1.9 1.4

43.2 35.7 7.0 1.8 1.3

44.7 24.7 12.2 2.8 2.5

44.5 26.8 12.3 2.6 1.7

a

Adapted from Forbes et al. (1984, 1985, 1990).

5. Ultrastructure of Cardiac Muscle and Blood Vessels

FIGURE 1 Survey view of ‘‘working’’ cardiac muscle (myocardium) in right papillary of vervet monkey (Cercopithecus aethiops). Most myocardial cellular components are aligned parallel to the longitudinal axis of the cells (running vertically in this field). The myofibrils (Mf) are relaxed, showing the typical striped pattern derived from sarcomere units. Inserted among myofibrils are rows of dense mitochondria (Mi). The myocardial nucleus (Nu) is fusiform, and the myoplasm at either nuclear pole contains opaque lipofuscin bodies (Lf). At the cell tips, collections of intercellular junctions are assembled into steplike profiles, the intercalated discs (ID). (Inset) Phase-contrast light micrograph of single isolated rat heart ventricular cell. The banding pattern and longitudinal arrangement of myofibrils are evident. The myofibrils are of different lengths, thus forming the staggered cell tips seen at the intercalated disc level in intact tissue. Scale bars: 10 애m.

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I. Pumping Action and Electrical Activity of the Heart

ducting cells are only marginally distinguishable from neighboring working fibers on the basis of fine details such as their smaller diameters, the absence of atrial granules, and a lack of any transverse tubular invaginations. A mixed population of small Purkinje cells appears in some primates, with some cells being filled with contractile material whereas others are myofibril poor (Fig. 2; also see Viragh and Challice, 1973). Although ventricular working cells are usually the largest cardiomyocytes, their sizes can vary considerably in the same heart. In part this is because of the ability of cardiomyocytes to carry out nuclear duplication without any accompanying cytoplasmic partitioning. In adult rat ventricle, binucleate cells (which are twice the volume of a mononucleate cardiomyocyte) constitute 85% of the total muscle cell population. This nuclear proliferation can continue through life, to the degree that, in aged humans and swine, more than 20 nuclei may occupy a single ventricular cell.

D. Nuclei and Nuclear Core Region Nuclei of myocardial cells are often, but not always, located toward the center of the cell. Nuclei are more or less fusiform and conform to the longitudinal arrangement of major organelles such as myofibrils and mitochondria. The elongate form of myocardial nuclei is maintained in part by numerous microtubules at their periphery (see Section II,E), as well as by the surrounding myofibrils. Extending from each pole of the nucleus is a roughly conical region of myofibril-free myoplasm containing a variety of organelles, including the Golgi system, centrioles, mitochondria, endoplasmic reticulum, and lysosomal bodies. In atrial working cells (Fig. 3), the Golgi system is extensive, appearing in selectively contrasted thick sections as an array of tubules and sacs that proliferate at both nuclear poles and connect through elements that sweep longitudinally along the length of the nucleus (Rambourg et al., 1984). The nuclear pole regions of these atrial cells are characterized by prominent spheroidal, electron-opaque specific atrial granules (Fig. 3), which react strongly when exposed to antibodies prepared against the vasoactive substance atrial natriuretic protein (ANP). The region housing nuclei and their associated myoplasmic zones can be viewed as a sort of cell core, and a similar central region free of contractile elements is a characteristic of vascular smooth muscle cells (Forbes, 1982, 1995).

E. Fibrillar Components 1. Contractile Apparatus As pointed out earlier, the collections of myocardial cell contractile proteins, known as myofibrils (or some-

times ‘‘myofilamentous masses’’), form the major portion of the muscle cell. Archetypal ‘‘tonic’’ skeletal muscle, such as the frog sartorius, exhibits an extreme degree of regularity of its myofibrils, each of which is cylindrical, uniform in diameter, and closely aligned with its neighbors in terms of sarcomere register. The presence of uniform, small-diameter myofibrils is said to constitute a Fibrillenstruktur, whereas larger, amorphous masses of myofilaments are termed Felderstruktur. In the fastbeating hearts of such small mammals as shrew and mouse, the myofibrils of working cardiomyocytes tend to be of smaller diameters (i.e., more nearly a Fibrillenstruktur; e.g., see Fig. 3) than those of cells in slowerbeating hearts (e.g., dog, monkey), whose myofibrils are thicker and more variable in cross-sectional profile (Felderstruktur) (Figs. 4 and 5). An obvious consequence (or, perhaps, advantage) of having a contractile system of Fibrillenstruktur conformation is the conferral of a larger expanse of myofibrillar surface area per unit cell volume; applied to these myofibrillar surfaces is an extensive system of anastomosing tubules and saccules of sarcoplasmic reticulum, which is responsible for movements of calcium into and out of the underlying myofibrils—the very basis of the contractile cycle of the cell. The geometric interposition of various contractile proteins (notably actin, myosin, troponin, tropomyosin, and 움-actinin) gives myofibrils a characteristic repeating pattern of stripes or striations whose basic unit is known as the sarcomere (Figs. 1 and 4). Sarcomeres in mammalian ventricular cells have a characteristic resting length of approximately 2.2 애m. Their most evident components are Z bands (‘‘Z lines’’, ‘‘Z discs’’), A bands, and I bands; less noticeable segments include the Mband–L-line complex, or ‘‘pseudo-H zone’’ at the middle of the sarcomere (Figs. 4 and 5). Although a sarcomere, sensu stricto, encompasses two ‘‘half’’ I bands, one A band, and the transversely bisected halves of two Z bands (Fig. 4), popular convention considers a sarcomere to be each segment bracketed by Z bands. A (‘‘anisotropic’’) bands are zones in which actin and myosin filaments overlap, whereas I (for ‘‘isotropic’’) bands represent sarcomere zones in which the actin filaments stand alone. At the midlevel of the sarcomere, where actin filaments do not extend in the relaxed myofibrils, a pair of relatively clear L lines flanks a dark M band, whose opacity is derived from the presence of myosinto-myosin crossbridges. The presence of M bands in heart is in fact a sign of myocardial cell maturity; in rat heart, M bands appear only postnatally and are missing altogether from the embryonic heart. The intensely opaque Z bands are composed largely of 움-actinin and likely act as linchpins for the stabilization of sarcomere structure via anchoring of the actin

5. Ultrastructure of Cardiac Muscle and Blood Vessels

FIGURE 2 A so-called ‘‘type I’’ conducting cell (Purkinje fiber) in rhesus monkey ventricle. The nucleus of this cell occupies a substantial area of this transverse profile, and the myoplasm is lucent, containing scattered small mitochondria (Mi) and myofibrils composed of contractile filaments so dispersed as to be practically invisible at this magnification. Other types of conducting cells in the same heart have well-developed myofibrils and can be discerned from working cells only on the basis of their subendocardial location, small size, and lack of T tubules. Scale bar: 5 애m. FIGURE 3 Working myocardial cell in left atrium of mouse heart. Structures found in the nuclear pole myoplasm distinguish it from a ventricular myocyte, including a welldeveloped Golgi apparatus (GA) (appearing in section as collections of saccules, tubules, and vesicles), as well as the definitive atrial hallmark, specific atrial granules (SAG) that form within the Golgi and contain concentrated atrial natriuretic peptide. Different stages of SAG formation are seen, including nascent granules with small cores and distinct halos (*). Mitochondria (Mi) mass within the perinuclear region. Atrial myofibrils (Mf) are typically slender. Nu, nucleus; Ly, lysosome. Scale bar: 1 애m.

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FIGURE 4 Rhesus monkey right papillary. Relaxed myofibrils, sectioned longitudinally, display the distinct pattern of sarcomere units. Each sarcomere is delimited by opaque Z bands and also contains a dark central A band and two ‘‘half’’ I bands (entire I band denoted by I). The middlemost set of stripes in the sarcomere is known in heart as the ‘‘pseudo-H’’ zone (psH). Side-to-side register is not always exact between adjacent myofibrils (also see Fig. 1). Sarcomere patterning has its basis in organized arrays of proteinaceous filaments: actin stands alone in I bands, overlaps with myosin in all but the pseudo-H zone of the A band, and inserts into the dark substance of the Z band. Certain myocardial cell structures appear preferentially adjacent to the Z band, among them vesicles of corbular SR (C-SR), a form of ‘‘extended’’ junctional sarcoplasmic reticulum. Scale bar: 1 애m. FIGURE 5 Rhesus papillary in transverse section. Because of differences in myofibrillar register across the cell, all levels of the sarcomere are represented in this field. Z band material, at the right, the most opaque component of the myofibril, is often closely associated with tubules of sarcoplasmic reticulum (SR). The most lucent portion is the I band, where only thin actin filaments appear, whereas the A bands comprise both actin and myosin arranged in a geometric pattern. Actin does not extend into the midlevel of the sarcomere (the pseudo-H zone), and there thick myosin filaments appear either in isolation (*) or in a dinctinct pattern of hexagons formed by myosin-to-myosin crossbridges (example circled). Mitochondria (Mi) are massed in intermyofibrillar spaces, and in monkey heart are characterized by dense matrices and distinct scalloped internal membranous shelves (cristae). Scale bar: 1 애m.

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5. Ultrastructure of Cardiac Muscle and Blood Vessels

within Z band material, which in transverse view is revealed as structurally complex latticeworks (Fig. 5). Z band material may possess additional functions. The Z-line levels of cardiac myofibrils seem to have a definite organizing effect on the neighboring myoplasm, so that certain structures (Z tubules of sarcoplasmic reticulum, transverse tubules, bundles of intermediate filaments) are preferentially oriented at or across Z lines, whereas mitochondria are largely excluded from these regions of ‘‘Z-line myoplasm’’ (Forbes and Sperelakis, 1980). Overproduction of Z-line substance is a hallmark of ventricular hypertrophy, and similar rod-like, paracrystalline accumulations, known collectively as ‘‘nemaline bodies,’’ occur normally in some conducting system cells (see, e.g., Figs. 1.44 and 1.45 of Forbes and Sperelakis, 1995). 2. Structural Fibrils Filamentous structures that do not actively contribute to the contractile process, but instead support the shapes of the cell components, thence the cells themselves, constitute the so-called cytoskeleton. In heart, the cytoskeleton is made up of microtubules and intermediate filaments. As mentioned earlier in the section on nuclei, microtubules frequently appear next to nuclear edges. This is best seen in strict transverse views of cells, where cross-sectioned microtubule profiles lie adjacent to the nuclear surface. This orientation is quite constant in some species such as mouse, suggesting that a framework of longitudinally disposed microtubules surrounds each myocardial nucleus. Microtubules form helical enwrapments around individual myofibrils (Goldstein and Entman, 1979), and thin sections that graze myofibrillar surfaces often reveal microtubules coursing across I- or Z-line levels, at oblique or nearly right angles to the long axes of the myofibrils. Intermediate filaments—so named because their average diameter of ca. 10 nm is roughly intermediate between the diameters of myosin and actin—have been demonstrated in a wide variety of cell types, and wherever found consistently serve a structural role. Although similar in general appearance in a variety of different cells, biochemically different categories of intermediate filaments have been identified, including desmin, vimentin, keratin, and neurofilamin. The desmin type of intermediate filament appears to predominate in cardiac myocytes, and in them is often encountered running transversely, either as individual filaments or in the form of meshworks surrounding myofibrils at Z-line levels. These fibrillar bundles, containing as many as 50 intermediate filaments, may also attach to the inner sarcolemmal surface and the nuclear envelope, suggesting the existence of multiple nets that stabilize the cell transversely in a series of parallel, semirigid strata. Another prominent site within the cardiac muscle cell with which

intermediate filaments are specifically associated is the intercalated disc, specifically the intracellular plaques of desmosomes (see Section II,H).

F. Mitochondria Mitochondria are the second most populous constituents of heart muscle cells, constituting ca. 40% of the total volume of mouse ventricular working cells (see Table I). In ventricular muscle, this contribution is readily appreciated; while the myofibrils are evident because of their predominantly longitudinal orientation and characteristic pattern of dark and light striations, mitochondria are conspicuous on the basis of their considerable opacity and their tendency to fall into longitudinal rows between myofibrils (Figs. 1 and 5). Depending on the particular heart cell in which they are found, however, cardiac mitochondria may be isolated, small and rather simple in structure (e.g., in embryonic muscle or in adult conducting cells: Fig. 2), or may be densely packed, with each mitochondrion having substantial size, a dense matrix, and an elaborate internal substructure (typically the case in ventricular working cells: Figs. 1 and 5). In working cells, mitochondria occupy not only myofibrillar interspaces, but are sometimes massed just beneath the surface sarcolemma. A physical location near the inner sarcolemmal surface may have some effect on the properties of adjacent mitochondria, and it has in fact been proposed that two biochemically distinct populations of mitochondria exist: intermyofibrillar and subsarcolemmal (Matlib et al., 1981). One striking and frequently encountered feature of subsarcolemmal mitochondria is their close parallel apposition to gap junctions (Forbes and Sperelakis, 1982a). The most highly developed examples of cardiac mitochondria exhibit impressive internal architecture, derived from the layout of their internal membranes, or cristae, ranging from the densely packed shelf-like cristae of mouse ventricle to the elaborate scalloped cristal structures in carnivore and primate hearts (Fig. 5). Socalled giant mitochondria have been frequently reported in heart, and when found most often have been linked to pathological conditions, but in truth, given world enough and time, the electron microscopist who examines a sufficient area of heart will eventually encounter examples of unusually large mitochondria within otherwise normal myocardial cells.

G. Membrane Systems 1. Transverse-Axial Tubular System Of all the cell systems common to both skeletal and cardiac muscle cells, transverse (‘‘T’’) tubules may be

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I. Pumping Action and Electrical Activity of the Heart

the only system first discovered in cardiac muscle, having been revealed accidentally in a 19th-century staining experiment in which India ink was applied to sheep myocardium to contrast the vasculature. In this fortuitous preparation there appeared parallel arrays of tiny ink-filled incursions that invaginated from the heart cell surfaces. A century later, Forssmann and Girardier (1970) used another opaque tracer material (horseradish peroxidase) to produce similar pictures in rat heart that confirmed the existence of an extensive system of transversely oriented tubular structures that extended into the cell at Z-line levels of subsarcolemmal myofibrils. Even without special staining it is evident that T tubules are structures whose limiting membranes con-

nect to the surface sarcolemma and whose luminal contents are continuous with the extracellular fluid space. Further investigation has enlarged upon the concept of the myocardial ‘‘T system,’’ establishing that it is most highly developed in the ventricular working myocardium. Rigorous examination of a number of species has revealed arrays of longitudinal (axial) tubules that run more or less parallel to the long axis of the cell and connect at many points with the transverse tubules, thus forming a complex system of invaginations that we have termed the transverse-axial tubular system (TATS) (Fig. 6). The TATS is usually far less complex in atrial working muscle, and is virtually absent there in some species

FIGURES 6 AND 7 Mouse ventricle prepared with ferrocyanide-reduced osmium tetroxide to delineate myocardial membrane systems. To better appreciate the extent and interconnection of these system, ‘‘semithin’’ sections (1 and 0.3 애m thick, respectively) are used. Variations in the staining regimen can allow infiltration of only the membrane-limited tubes that connect to the extracellular fluid space (Fig. 6). Transverse tubules (TT) penetrate into the cell at right angles at Z-line levels, and axial tubules (AxT) are oriented obliquely or longitudinally, but all components of the ‘‘transverse-axial tubular system’’ are interconnected and communicate with the extracellular fluid space contents. Irregular ‘‘beaded’’ tubule segments are shown at arrows. In Fig. 7, the internal membrane system (sarcoplasmic reticulum, SR) has been selectively opacified. At the rightmost part of this field, a virtually continuous covering of SR tubules extends over several sarcomeres. SR is continuous over Z lines. The bulk of SR is known as network SR (N-SR), which falls into sarcomere-specific configurations, including loosely arranged elements over I bands, more closely packed parallel tubules over A bands, and perforated ‘‘fenestrated collars’’ (at arrows) at the middle of the sarcomeres. Scale bars in Figs. 6 and 7 are 2 and 1 애m, respectively.

5. Ultrastructure of Cardiac Muscle and Blood Vessels

(e.g., guinea pig). In the rat, in terms of the TATS, it has been established that two subpopulations of atrial working cells exist: one having the TATS and one lacking it (Forssmann and Girardier, 1970). In the TATs-less myocytes, the subsurface system of vesicles, or caveolae, seems to be more in evidence. Unlike the ‘‘micropinocytotic vesicles’’ of endothelial cells (q.v., later), caveolae appear to be permanently fixed in their connection to the surface membrane of the cell. However, this does not necessarily mean that they are static entities; in muscle cells of various types there have been described structures called ‘‘beaded tubules,’’ which are chains of caveolae apparently generated by a directed vectorial proliferation of these bodies from the cell surface into its interior. Beaded tubules appear to reflect the formative mechanism for definitive T tubules in skeletal muscle and, may under some circumstances—notably culture conditions or certain pathological states—generate tubulovesicular masses (‘‘labyrinths’’), which achieve considerable volume and three-dimensional complexity. Labyrinths are found routinely in certain normal mammalian hearts (mouse, rat, shrew), thus further fostering the homology between cardiac and skeletal TATS formation and structure. The ‘‘typical’’ TATS element varies widely among mammals, in many (e.g., guinea pig, hamster, dog, cat) appearing in the form of a large-bore profile (Fig. 8) lined with a distinct cell coat. Such large tubules also can be found in rat (Fig. 9), mouse, and shrew, but more often the TATS in these hearts consists largely of smalldiameter elements, many of which are anastomosed into complex transverse-axial latticeworks that permeate the cell and retain their beaded profiles (Fig. 6), thus further testifying to their mechanism of origin from caveolar proliferation. In most mammalian conducting system cells and lower vertebrate myocardial cells documented to date, there appears to be little evidence for any TATS formation. There is a lingering question regarding the necessity for the TATS in any heart muscle cell, furthermore. The long-standing reasoning concerning the function of the TATS is that it provides excitable membrane and a supply of extracellular fluid to all depths of the cell. Given that most myocardial cells are far smaller than their skeletal counterparts, it has been argued that diffusion from the outer rim of the cell should be sufficient for reasonably simultaneous contraction of myofibrils at all cell levels, once the excitation signal has arrived to elicit Ca2⫹ release from internal stores, such as the sarcoplasmic reticulum (SR). The TATS remains a prominent entity in many mammalian myocardial cells, nevertheless, and it is likely that other more subtle physiological processes are served by it. Because of the complexity and extent of the TATS, for example, there may

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not be totally free exchange of its luminal contents with the extracellular reservoir, at least on a moment-tomoment basis. Therefore, localized concentrations of ions and other metabolites could vary considerably among different regions within the TATS. It is worthwhile for the investigator to realize that, where the question as to presence or absence of TATS is raised, it is necessary to study only those cells having relaxed myofibrils. Even in cells with a definitive TATS, contraction leads to scalloping of the cell surface, forming trenches of entrained surface sarcolemma that are pulled into the cell at successive Z-line levels; these ‘‘pseudo’’ T tubules may connect with the genuine tubular invaginations only at some point considerably deeper in the contracted cell. In thin section, therefore, false T tubule profiles can appear in overly contracted cells that actually lack a definitive TATS, and this likely has been responsible for most claims of a T system in the AVCS or in lower vertebrate heart. 2. Sarcoplasmic Reticulum The sarcoplasmic reticulum is the third largest component, by volume, of the myocardial cell (see Table I) and is nothing more nor less than the muscle cell’s homologue of the endoplasmic reticulum of nonmuscle cells [‘‘sarcoplasmic,’’ derived from the Greek base word sarkos (flesh) survives in current terminology in addition to sarcolemma and sarcomeres, but over time other muscle-specific labels such as ‘‘sarcosomes’’ (mitochondria) have—probably mercifully—fallen by the wayside]. Sarcoplasmic reticulum was early recognized to be a major component of skeletal muscle, particularly in fast-twitch fibers where—because of the orderly sarcomere pattern and the small sparse mitochondria— substantial SR arrays were readily visible as ‘‘torn sleeves’’ encasing each myofibril. In cardiac muscle, however, myofibrils are uniform neither in size nor in shape, and mitochondria are large and optically dense in the transmission electron microscope; furthermore, these two components together constitute 70–80% of the cell volume. As a result, in ‘‘conventional’’ ultrathin sections of heart only those areas grazing the myofibrillar surfaces and capturing the entire thickness of SR tubules were effective in revealing the complexity of this membrane system. As a result, the statistical contribution of the SR to the cardiac muscle cell was not accurately determined in studies that utilized routine staining (usually uranium and lead, applied on section). However, certain contrasting procedures selective for the SR system became available in the 1970s, among them the ferrocyanide-reduced osmium tetroxide technique, or ‘‘osmium-ferrocyanide (OsFeCN)’’ (Forbes et al., 1977c). Furthermore, the use of ‘‘pseudo high-volt-

FIGURES 8–10 Features of the complexes known as ‘‘couplings,’’ formed by the apposition of junctional SR with the sarcolemmma or its derivative, the transverse-axial tubular system.

FIGURE 8 Guinea pig ventricle. A transverse tubule opens to the extracellular space through a small mouth (arrow) but dilates considerably along its length. In this tissue, the SR is stained dark; the T tubule surface is caught in grazing section, revealing several stained saccules of junctional SR (*) applied to its surface, seen both in side view (i.e., vertically sectioned) and en face. These complexes of SR and T tubule membranes are called interior couplings; the J-SR forms peripheral couplings with the surface sarcolemma as well (PJ-SR, peripheral J-SR, seen here in side view). Scale bar: 0.5 애m. FIGURE 9 Rat ventricle. A complex interior coupling structure has formed between two T-tubule profiles (TT) and flattened specialized saccules of junctional SR (J-SR). Identifying characteristics of J-SR include its opaque internal contents, as well as its junctional processes (arrowheads) that extend out from the J-SR surface into the gap between the apposed SR and T-tubule membranes. At the lower right, the T tubule profile is irregular where it is not part of the coupling (cf. Fig. 6). Scale bar: 0.1 애m. FIGURE 10 Mouse atrium. A very thin grazing section captures, in face view, a saccule of peripheral junctional SR (cf. Fig. 8). Although superimposed on the contents of the junctional gap, the opaque material within the J-SR does not completely obscure the junctional processes (some denoted by arrowheads), which appear as punctate opaque bodies ca. 57 nm in diameter. Such views are rare, and any consistent pattern of junctional processes in them is not obvious. Scale bar: 0.1 애m.

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age electron microscopy,’’ in which sections of OsFeCNtreated material measuring from 0.1 to 2 애m in thickness were examined at conventional accelerating voltages (up to 100 keV), reveals extensive anastomoses of ‘‘network’’ SR (N-SR) (Fig. 7). Such enhanced SR visualization was a boon to qualitative observation, but in fact selective staining could be put to an even more effective use: quantitative structure measurement (stereology) of myocardial cells. This technique allows recognition of even tiny SR profiles, which in routine sections would be invisible against the opaque background of other, larger structures. This made a profound difference in the appreciation of SR contribution. Whereas studies made on conventionally contrasted sections had judged the total percentage of the cell volume occupied by SR [SR volume fraction or VV(SR)] to be no more than 2%, it turns out in fact that total VV(SR) is ca. 6% in ventricular cells and may exceed 12% in atrial cells (where the contribution of SR is proportionately greater because of the relatively low population of myofibrils; see Table I). An alternative, more qualitative technique, which shows off myocardial SR to good advantage, combines the selective extraction of the soluble myoplasm and myofibrils—leaving the mitochondria and membrane systems intact—with high-resolution scanning electron microscopy (Ogata and Yamasaki, 1990). The interconnected meshworks of N-SR compose approximately 90% of the entire SR volume. The remainder of the SR, composing only ca. 10% of its total volume, is made up of far more readily recognizable elements, most of which are flattened saccules that resemble the distended terminal cisternae of skeletal muscle, but which are known in heart by the less restrictive term ‘‘junctional SR (J-SR),’’ as they frequently exhibit a morphology far more richly varied than that of their skeletal counterparts. This variety includes complexes formed by J-SR profiles that encircle or intertwine with the transverse-axial tubular system (Figs. 8 and 9), in addition to the simpler J-SR sacs that abut the surface sarcolemma (‘‘peripheral’’ J-SR; Figs. 8 and 10). These complexes formed by structured SR with the sarcolemma are known rather tendentiously as ‘‘couplings,’’ as they have long been presumed to play a central role in the signal transduction process known as ‘‘excitation– contraction coupling,’’ by which the arrival of an action potential at the outside of the cell is translated into calcium release within the cell. The hallmark of the coupling is its collection of rather evenly spaced, vaguely opaque bodies that extend from the J-SR membrane across a small myoplasmic gap to approach the inner surface of the apposed sarcolemma or TATS element (Fig. 9). Again, these structures were first recognized in skeletal muscle, where they were called ‘‘SR feet.’’ The term ‘‘junctional processes’’ has come to denote

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these structures in various forms of muscle, both invertebrate and vertebrate, skeletal, cardiac, and smooth. The function of junctional processes was originally thought to be that of connection between the apposed membranes, thus keeping the junctional SR connected to the sarcolemma or T tubule. Further investigation, however, has established that junctional processes are the ultramicroscopic embodiment of calcium-releasing channels of the SR (e.g., Inui et al., 1987), further defined by their sensitivity to blockage by the toxic alkaloid, ryanodine. Thus the processes have come to be known as ‘‘ryanodine receptors’’ or ‘‘ryanodine-sensitive calcium release channels.’’ Detailed microscopic examination of junctional processes often shows that they stop short of making actual contact with the sarcolemma, which would be consistent with their ability to release Ca2⫹ into the restricted junctional gap (which is sometimes referred to as the ‘‘fuzzy space’’). Thus one wonders what additional adhesive molecules or structures may be present in couplings to keep the complex intact. On occasion, in thin, high-resolution electron micrographs of muscle couplings, one can discern bridging structures, similar in appearance to unit membranes and seemingly fused with both J-SR and sarcolemma/TATS membranes (Somlyo, 1979; Forbes and Sperelakis, 1982b). SR segments that bear junctional processes, but which do not contact any surface membranes, are collectively known as ‘‘extended junctional SR.’’ These can take the form of small vesicles (corbular SR: Fig. 4), large hollow spheres, or saccules that resemble conventional junctional SR except for their lack of sarcolemmal association (Forbes et al., 1990). A distinct pattern of junctional process distribution is evident in skeletal muscle couplings, where a double row of processes runs along the length of the junctional SR cistern. Cardiac couplings are considerably flatter than skeletal triads, however, and the problem of superposition in the TEM therefore plays an obscuring role. Even in the best-oriented micrographs (Fig. 10), the most that can be concluded is that cardiac junctional processes are individually similar in appearance to skeletal SR feet, but observe no obvious pattern of twodimensional organization.

H. Surface Sarcolemma. Intermembranous Junctions, and Intercalated Discs This section is devoted mainly to the entities in heart that have long been known as intercalated discs. These are the zones where adjacent myocardial cell tips form close adhesive interdigitatations containing a patchwork of specialized zones of sarcolemma (Figs. 1 and 11). No homologue to intercalated discs exists in other types of mammalian muscle. Because of the choice of species,

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the preferential use of longitudinal sections, and the thickness of the preparations, it is not surprising that these dense, transversely oriented, seemingly disembodied linear profiles were thought by some early investigators to be discoidal collections of invaginated connective tissue or zones of supercontraction. As mentioned earlier in the section on cellularity, it took 50 years and a barrage of consistent electron microscopic evidence to dispel the concept of the cardiac syncytium and thus identify the intercalated discs as regions of cell-to-cell junctional attachment. Once this ancient controversy was laid to rest, it remained to see just what intercalated discs consisted of and what functions they performed in the heart. One difficulty in considering intercalated discs is the question of their boundaries, i.e., just where does an intercalated disc begin and end? Although cardiac muscle cells are often treated as simple cylinders, they seldom take this form. Ventricular cells in particular are more ribbon shaped, with predominating major and minor diameters. The shapes of myocardial cells are further complicated by the configurations of their tips (Fig. 1), which regions are wholly occupied by the intercalated discs. Intercalated discs, then, can be defined as those junctional complexes that incorporate all the sarcolemma composing the cell ends as well as some variable portion of the adjacent lateral cell surfaces. It is the degree and type of sarcolemmal specialization that set the different intercalated disc components apart from one another (Figs. 11 and 12). Intercalated disc sarcolemma consists of (1) relatively unspecialized or ‘‘general’’ sarcolemma that bears no obvious membrane-associated specialized structures; (2) fasciae adherentes (sing. fascia adherens), consisting of subsarcolemmal amorphous dense plaques into which the myofibrils insert; (3) maculae adherentes (‘‘desmosomes’’), noteworthy because of their structured subsarcolemmal plaques and distinct central lamella; and (4) maculae communicantes, known more commonly as ‘‘gap junctions’’ and composed of collections of subunits called ‘‘connexons.’’ All four of these sarcolemmal categories are interspersed in an organized way within the myocardial cell tips, thus constituting the structures identified long ago as intercalated discs. There is considerable interspecies and interregional variation in the profiles of intercalated discs, which in longitudinal view incorporate both transverse and longitudinal segments. In ventricles of primates and carnivores, the ‘‘discs’’ extend in a precise, geometric steplike array (Fig. 1). Recall that ventricular cells of these animals are characterized by myofibrils that are thick and pleiomorphic (‘‘Felderstruktur’’). Add to this the fact that myofibrils invariably terminate in the intercalated disc at the level of the Z line, so that the actin filaments of the I band are inserted into the fasciae

adherentes. This contributes to the formation of broader transverse expanses of intercalated disc in such cells. Where smaller-diameter myofibrils appear in a more Fibrillenstruktur form, as for example in most atrial fibers, as well as in ventricles of smaller mammals such as rat and mouse, the transverse segments of the discs are limited in extent, and therefore jagged or ‘‘wavy’’ intercalated discs are formed (Fig. 11). Because the transverse segments are mainly occupied by fasciae adherentes junctions, the longitudinally oriented segments of the discs contain the majority of the general sarcolemma, desmosomes, and gap junctions, and because longitudinal segments are considerably more prominent in the wavy sorts of discs, their desmosomes and gap junctions are usually noticeably larger. The intercalated discs formed between cells of the AVCS are even simpler in form, which is not surprising in view of the fact that force generation is not the strong point of these particular muscle cells. Desmosomes are quickly distinguished from adherens junctions by their lack of association with the myofibrils. Furthermore, each desmosome is rendered structurally distinct by its central lamella (Fig. 12), an adhesive component that lies in the extracellular space midway between apposed dense plaques and connected to the sarcolemma by filamentous strands. Opaque subsarcolemmal desmosomal plaques frequently show close association with intermediate filaments. Gap junctions are probably the most interesting constituent of intercalated discs as far as the physiologist is concerned. Note that the term ‘‘gap junction’’ is a misnomer as it is casually understood. The apparent 4-nm space between apposed cell membranes is actually a relatively lucent zone filled with hexagonally packed, transmembranous connexon subunits. The term ‘‘connexon’’ is more to the point where the structure and function(s) of this cell-to-cell attachment are concerned. The only actual extracellular spaces within a gap junction are the thin channels that wind among the connexons. Certain preparative techniques have revealed a dot in the connexon’s center, thought to be an aqueous pore through which ions can travel from cell to cell. This has contributed to the concept that myocardial cells are electrically coupled through their shared gap junctions. There is doubtless an adhesive aspect to the connection afforded to heart cells by gap junctions, however, as the structural integrity of gap junctions is unaffected by a variety of unfavorable conditions, including mechanical and enzymatic regimens designed to dissociate the myocardium into individual muscle cells. TEM examination of such separated cells reveals that the septilaminar gap junctions are not cleaved, but instead remain wholly with one cell, tearing away with them an adhering fragment of the neighboring cell’s sarcoplasm (Forbes and Sperelakis, 1985). This testifies to considerable mechani-

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FIGURE 11 Longitudinal thin section through an intercalated disc in mouse ventricle. This zone of cell-to-cell attachement has a jagged profile compared to intercalated discs of monkey ventricle (cf. Fig. 1). The transverse disc segments are composed primarily of ‘‘intermediate’’ or fasciae adherentes junctions (FA) junctions, into which actin filaments of the subjacent sarcomeres insert. Gap junctions (GJ) and desmosomes (D) are usually located in longitudinally running portions of the intercalated disc. Scale bar: 1 애m. FIGURE 12 A transversely sectioned portion of mouse ventricle intercalated disc. All four of the cell membrane components composing intercalated disc are present. These are the ‘‘general’’ sarcolemma (SL), which bears no visible specialized structures; the fascia adherens (FA), characterized by amorphous opaque intracellular material; the macula adherens or desmosome (D), identified by the central lamella in the extracellular space, layered subsarcolemmal plaques, and the associated intermediate filaments (IF); and the macula communicans or gap junction (GJ), the septilaminar junction that forms the narrowest region of the disc. Scale bar: 0.2 애m.

cal strength of the myocardial gap junction, in addition to the electrical functions it may perform. Quantitative work on gap junction contribution to various cells and species is difficult to interpret (see summary in Forbes and Sperelakis, 1985), and so despite

the greater prominence of these attachments in mouse ventricle, it cannot be said that they are more extensive overall there than in carnivore or primate ventricular cells, where individual gap junctions are usually of limited length. Perhaps the argument is in part moot, as it

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has been shown in certain cell types that a large gap junction is not necessarily an electrochemically functional one, but rather represents a region of the cell surface that is slated to be internalized and recycled.

I. Other Organelles 1. Golgi Apparatus Stacks of flattened fenestrated sacs form the major portion of the Golgi apparatus. Whereas in ventricular cells the Golgi may consist of only a few stacked profiles next to a nuclear tip, in atrial cells a veritable Golgi system is found surrounding the nucleus. In atrial cells, a major function of the Golgi is the packaging of atrial natriuretic peptide into specific atrial granules (Fig. 3) for storage and eventual release into the circulation to promote vasodilation, sodium excretion, and diuresis. Numerous vesicles, both coated and uncoated and of different sizes and appearances, are closely associated with Golgi saccules; profiles of both rough and smooth endoplasmic reticulum are found as well in the neighborhood of the Golgi. 2. Centrioles Centrioles are not prominent constituents of myocardial cells, but when encountered are most often seen in atrium. Although centrioles have classically been considered as part of the mitotic apparatus, they do not seem to be required for the completion of cytokinesis. It is curious, however, that they so seldom appear in ventricular cells, which, it will be recalled, virtually never divide in the adult mammal’s heart (whereas adult atrial cells, under certain experimental conditions, can be induced to undergo mitosis). The lack of proliferative ability in ventricle has, however, been more specifically attributed to the developmental loss in them of DNA polymerase activity, which leads in essence to a state of terminal differentiation in these cells. 3. Lysosomes and Other Myocardial Cell Inclusions Lysosomes per se are rather infrequent in heart (Fig. 3), but membrane-enclosed lipofuscin bodies (also known as aging pigment: Fig. 1) are seen in increasing numbers as the animal grows older, nearly filling the nuclear pole cytoplasm in some cells. Lipofuscin is likely a form of ‘‘residual body,’’ formed when a primary lysosome engulfs and digests some other organelle such as a mitochondrion. Large multivesicular bodies have been described in heart and are also considered to be of lysosomal origin. Microbodies (peroxisomes) are sometimes present in intermyofibrillar and subsarcolemmal spaces and can

be distinguished cytochemically from similar-sized lipid droplets on the basis of their preferential location (at the A–I levels of sarcomeres) and their specific formation of a dark reaction product when exposed to hydrogen peroxide and diaminobenzine. Glycogen particles appear with varying frequency in myocardial cells. Individual 웁 particles are common in slower-beating hearts, but are rare in fast-beating hearts such as those of rat and mouse. 웁 particles, where found, may specifically lie among the filaments of sarcomere I bands, but can also appear in myoplasmic regions, where they may be confused with individual ribosomes. Clusters of glycogen, known as 움 particles, appear most often in developing and neonatal hearts, and in some conductive cells, but are not especially common in adult working myocytes.

III. MYOCARDIAL BLOOD VESSELS A. Overall Construction As pointed out at the beginning of this chapter, the amount of dedicated ultrastructure research addressed to coronary blood vessels is pitifully small, which forces me into the immodest position of quoting mainly from my own observations, many of which themselves arose only as incidental sidebars to my main goal at the time (the investigation of cardiac muscle cells). In any event, these admittedly cursory studies have included a fairly wide range of animals, including least shrew, mouse, rat, guinea pig, cat, dog, pig, and several primates (squirrel monkey, vervet, and rhesus monkey). Certain consistent features of myocardial circulation can be found in each of these species, including the presence of an extensive vascular bed and a similar location of the major coronary vessels. Otherwise, generalizations regarding the fine structure of the vessels are hard to come by. A common diagrammatic view of the ‘‘typical’’ blood vessel would involve an endothelial cylinder covered with smooth muscle cells that wind about the cylinder in a circular configuration. In heart, such a picture is not always likely to appear with any great consistency save in some vessels on the arterial side of the circulation, and there more commonly in the smaller arteries. One rule of thumb in heart is that the larger the diameter of the vessel, the thicker and more structurally complex are its endothelial cells, so that in the smallest capillaries the endothelial layer consists of very thin cells with limited cytoplasmic contents, whereas in arterial and venular endothelium–as well as endocardium– the cells are larger and thicker, and contain a rich variety of fibrils and other inclusions (see Section III,D). Another of the limited number of generalizations to

5. Ultrastructure of Cardiac Muscle and Blood Vessels

be drawn concerning cardiac vessels is the presence of three distinct component layers in all of them (Figs. 13 and 14), even in the smallest microvessels (Fig. 20). Starting from the lumen, the first layer or ‘‘tunic’’ is the tunica intima, which in its simplest incarnation is made up of endothelial cells, and includes in larger vessels the subintimal space with its various contents (in some, for example, a discontinuous elastic layer: Fig. 13). The next layer, the tunica media, is often considered synonymous with the vessel wall and consists of cells that in their most advanced form are called vascular smooth muscle cells (VSMC). In the smaller-diameter reaches of the vasculature, less highly developed cells appear in the tunica media, where they are sometimes termed ‘‘primitive smooth muscle cells’’ or—in the smallest elements of the microvasculature—‘‘pericytes’’ (Fig. 20). Here it would be well to remind the reader that the vasculature of the heart, rather than being composed of distinct, clearly distinguishable segments, is instead a continuum of vessels that gradually make the transition from arteries to arterioles down to capillaries and enlarge just as gradually from that point to form venules and veins. Additional subdivisions of the bed are sometimes posited, including ‘‘arteriolar capillaries,’’ ‘‘postcapillary venules,’’ ‘‘muscular arteries,’’ and ‘‘small veins,’’ but this is a largely artificial distinction, more indicative of the desire of the anatomist to affix different labels to as many structures as possible. Not only do the vessels themselves almost imperceptibly change in overall appearance, but their endothelial and medial components also undergo gradual transformation, such that (for example) the circular configuration of the arterial VSMC gives way at the capillary level to pericytes whose long dimensions run parallel to the vessel axis. The outermost tunic of the blood vessel is the tunica adventitia. This layer is the most heterogeneous of the three and contains a hodgepodge of elements, which may include adipose cells, collagen, elastin, fibroblasts, Schwann cells, nerve axons (some of them myelinated), and terminal boutons filled with neurotransmitter vesicles. Some form of adventitial layer is found associated with all myocardial blood vessels, even though in thin sections of capillaries it may consist of no more than a collagen fibril, a wisp of fibroblast cytoplasm, or— surprisingly often—an axon terminal (a more detailed discussion of this is made in Section III,C,6).

B. Vessel Classification 1. Arteries These are the major sources of pressure regulation and resistance within the cardiac circulation. The com-

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position of their walls serves to demonstrate a venerable tenet of anatomy, namely that an elephant is not bigger than a mouse because its individual cells are proportionately larger, but because there are more of them. Applied to coronary arteries, this principle is demonstrated in a segment of a coronary artery in a squirrel monkey (Fig. 15) compared with an equivalent vessel in mouse (Fig. 16). Diameters of the VSMC profiles are similar in the two animals; the greater thickness of the monkey artery is primarily attributable to its greater number of VSMC, routinely arranged in several layers as opposed to the mouse vessel, which in some places consists of only a single VSMC layer. Profiles of the monkey VSMC are noticeably more complex, incorporating various notches and irregular indentations (Fig. 15). The monkey vessel contains a considerable adventitial layer as well, which contributes substantially to the overall wall thickness of this vessel. Among the animals examined, there is considerable variation in VSMC arrangement within the medial layer. In shrew and mouse the VSMC observe a largely circular disposition along the lengths of the coronary arteries. In dog right coronary artery, however, while a prominent inner circular VSMC component exists, bundles of longitudinally oriented VSMC appear at the periphery of the tunica media (Forbes, 1982). An even more unusual architecture was encountered in vervet RCA, where there are three distinct medial layers: an innermost VSMC layer running parallel to the vessel axis, a thick middle stratum consisting of circularly arranged VSMC, and an outer detached layer of smooth muscle cells that wind in a lazy spiral at the periphery of the tunica media (Forbes, 1995). Arterial VSMC can be envisioned overall as flattened, tapered, strap-like cells. While in smaller-bore arteries in general, regardless of species, the VSMC are tightly packed like bricks in a wall (Fig. 16), muscle cells of larger arteries are farther apart (Fig. 15) and embedded in a matrix composed of cell coat material, connective tissue elements such as elastin and— particularly in older individuals—accumulations of opaque granular material. It has been proposed that this material is derived from the exocytotic liberation into the extracellular spaces of VSMC breakdown products derived from lysosomal activity, and that the entrapment and accumulation of such cellular waste may contribute to the literal hardening of artery walls during the course of aging (Joris and Majno, 1974). The VSMC of larger arteries also display rugose profiles, and their tips, rather than being flattened or rounded, instead form complex fingers and indentations within which cell coat material forms whorls and filigrees (Fig. 15).

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FIGURES 13 AND 14 Anterior descending coronary artery (Fig. 13) and its accompanying vein (Fig. 14) in heart of a squirrel monkey (Saimiri sciureus). Both vessels are cut in cross section, oriented with vessel lumina to the right, and printed at the same magnification for direct comparison of their structure and dimensions. FIGURE 13 In the artery, four layers of vascular smooth muscle cells (VSMC) constitute the medial layer and are arranged circularly within the cylindrical vessel wall. Where the tips of the VSMC overlap (*), they form finger-like extensions enmeshed in structured cell coat material. The outer layer of the artery (adventitia) contains collagen and fibroblasts (FB). The innermost lining of the vessel is called the tunica intima and is composed here of a discontinuous internal elastic layer (EI) and a monolayer of endothelial cells (EN, endothelial cell nucleus). Scale bar: 2 애m. FIGURE 14 Same labeling as in Fig. 13. The coronary vein is comparatively thin walled, in some places containing a single VSMC layer. Adventitial connective tissue makes a proportionately greater contribution to the vessel wall, and endothelial cells are thinner than those of the artery. Scale bar: 2 애m.

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FIGURES 15 AND 16 Longitudinally-cut coronary arteries (therefore VSMC appear in cross section) to compare wall structure of comparable vessels in two different species. FIGURE 15 Squirrel monkey right coronary artery. VSMC are strikingly pleiomorphic and arranged in several layers. Their profiles display involved sarcolemmal indentations and cavities (*). The muscle cells bear thick structured surface coats and are separated by wide intercellular gaps. EN, nucleus of endothelial cell. Scale bar: 5 애m. FIGURE 16 Mouse anterior descending coronary artery. Medial layer consists primarily of a single layer of VSMC, which cells are comparatively smooth surfaced and closely applied to one another. Scale bar: 5 애m.

2. Veins Given that the ultrastructure of myocardial vessels is poorly studied, then that of the venous side of the cardiac circulation is the worst documented of all. Veins may simply be dismissed as being thin walled and of larger diameter than the accompanying arteries, but this ignores the astonishing variety seen in the arrangement of venous VSMC. In the anterior vein of rhesus monkey, for example (Forbes, 1995), segments of vessel wall contain mixtures of circumferentially and longitudinally oriented VSMC, whereas contiguous regions may be composed of fibroblasts and collagen lying adjacent to the endothelium, with the VSMC displaced abluminally.

Some stretches may lack VSMC entirely, with bands of collagen taking the place of the tunica media. Some venous VSMC resemble the primitive smooth muscle cells and pericytes of the microcirculation (see later), but there is to date very little information available that is addressed to the comparative structure of mammalian cardiac veins.

3. Microvessels As implied earlier, the terminological separation of any circulatory bed into arteries, veins, and ‘‘microvessels’’ is more a convenience than the reflection of strict

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underlying anatomical segmentation. However, the term ‘‘microvasculature’’ always includes the smallest blood vessels, namely the capillaries. The medial components of these vessels, the pericytes, form a discontinuous coating about the endothelial cylinder. Pericytes have also been shown by serial section reconstruction techniques, freeze-fracture replication (Forbes et al. 1977a; Forbes, 1995), and scanning electron microscopy (Sims, 1986) to possess both extensive longitudinal stems that run parallel to the vessel axis and shorter transverse processes that partly enwrap the capillary. They can be envisioned therefore as so many elongate starfish lined up and clasping the endothelial tube. In this respect they resemble ‘‘Rouget cells,’’ medial elements of amphibian capillaries, which were described in the 1800s and found to be capable of contractile activity similar to that of VSMC. In the mammalian heart, direct evidence for pericyte contractility has not been obtained; it does appear, however, that pericytes meet many of the structural criteria necessary to exert some constrictive influence on their accompanying endothelial cells.

C. Organelles and Cell Systems of Perivascular Cells VSMC, primitive smooth muscle cells, and pericytes alike can be lumped together under the category of perivascular cells. The majority of their various cell structures can be found in one form or another in all of these cells and at all levels of the myocardial circulation. It is for this reason that perivascular cells of any type are likely to carry out functions that are least qualitatively similar in terms of the vessel segment in which they are located, including stabilization and maintenance of the vessel’s integrity, as well as some variable control of the shape and diameter of the vessel lumen. In general, perivascular cells of large vessels are themselves larger and more richly endowed with fine structural elements than medial cells found on microvessels. A similar scheme applies to endothelial cells, which will be considered toward the end of this chapter. 1. Nucleus and Nuclear Core Region Arterial VSMC display the highest degree of organization among all the perivascular cells, and the following sections concentrate on them for that reason. Early TEM descriptions of smooth muscle, whether vascular or visceral, tended to leave the reader with the impression that these cells were primitive, vague, and nondescript in appearance, resembling ‘‘bags of mostly water,’’ to paraphrase from a popular television show. In large part the preparative techniques were to blame; although

primary fixation with osmium solution is marginally adequate to paint—albeit with broad strokes—tissues such as striated muscle that are mostly composed of tough protein fibrils, this procedure destroys the delicate substructure of smooth muscle and many other cell types. Marked improvements in overall structural preservation came about with the advent of aldehyde fixation and more beam-stable embedding resins. With these improvements came the realization that smooth muscle cells in particular were complex, well-organized entities. Although they lack a strict segmentation of their contractile filaments into obvious myofibrils and sarcomeres, definitive VSMC nevertheless display a marked longitudinal orientation overall (Figs. 13, 17, and 18), along with a distinct compartmentation of their cytoplasm. The contractile system is segregated into an outer cortical cytoplasmic shell, whereas the inner, central core region is the zone to which the nucleus, mitochondria, cytoskeleton, centrioles, and lysosomes are confined (Fig. 18). Cardiac VSMC usually contain a single nucleus (Figs. 13 and 14), which in some cases can attain a length over 30 애m. The nucleus is strictly aligned along the cell axis, and the associated central core of myoplasm surrounds it and extends out toward the cell ends. At the farther reaches of the cell, particularly in larger VSMC, the core ramifies into thin extensions that penetrate among the collections of contractile fibrils, in transverse view appearing as small myoplasmic zones containing mitochondria, intermediate filaments, and microtubules. 2. Fibrils (Contractile System and Cytoskeleton) In arterial and venous VSMC, the outer bulk of the cell is filled with contractile filaments (Figs. 13, 14, 17, and 18), all of which are aligned with the longitudinal axis of these fusiform or strap-like cells. In less highly developed perivascular cells, bundles of these filaments divert away from the main axis to follow the smaller, transversely oriented circumferential processes (Fig. 19). The filaments become more regionalized as well, so that in pericytes they fill the tips of the thinnest cell processes, but in the thicker portions of the cell are present only at the adluminal surface of the cell that abuts the endothelial layer (Fig. 20). Although actin and myosin filaments are routinely preserved in striated muscle almost regardless of the preservation technique employed there, this has not been the case with smooth muscle. More often than not, particularly in VSMC, thin filaments are abundant, but the larger myosin profiles cannot be found or may appear only in some cells (Fig. 17) and not in others within the same vessel wall. Various techniques, including the incorporation of physiological salt solutions into the

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FIGURE 17 High-magnification view of transversely-cut VSMC in squirrel monkey right coronary artery showing all the categories of structural and contractile filaments. Structural filaments include microtubules (MT), which appear in the vicinity of the cell nucleus (Nu) as well as in more peripheral cytoplasm. Intermediate filaments (IF) are concentrated in the perinuclear region (cf. Fig. 18). The cortical region of the cell is occupied in the main by large numbers of actin filaments (A) among which scattered myosin filaments (M) appear. At the cell surface, caveolae alternate with subsarcolemmal dense bodies (DB; also see Fig. 18). Small interior dense bodies also exist (*), and actin filament profiles can be seen within these opaque regions. In this transverse section, mitochondria (Mi) appear small, but are revealed as having considerable length when viewed in longitudinal section (e.g., Fig. 18). Scale bar: 0.1 애m.

fixative carrier, have been tried with limited success in producing myosin profiles. In all probability, this lability of smooth muscle myosin is related to the fact that the contractile system in these cells is not locked into a geometric latticework such as that of skeletal or cardiac myofibrils. It is now generally presumed that myosin is present in VSMC in filamentous form, but it is not likely

to be consistently preserved even in the best-fixed tissues. There is immunological evidence for the existence of both smooth muscle type actin and tropomyosin in pericytes, thus further supporting their homology with smooth muscle cells of larger blood vessels. The actin filaments of VSMC anchor within accumulations of dense material, the majority of which in coro-

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FIGURE 18 Vervet monkey right coronary artery. This longitudinal section passes through the center of the left-hand VSMC, revealing its compartmentation into a cortex packed with bundles of contractile filaments and a central cell core here occupied by elongate mitochondria (Mi), skeins of intermediate filaments (IF), and a lysosome (Ly). The section grazes the sarcolemmal surface of the right-hand VSMC, showing stripes of subsarcolemmal dense bodies (DB) that alternate with rows of surface-connected caveolae (C); compare this arrangement with the transverse view shown in Fig. 17. Scale bar: 1 애m. FIGURE 19 Squirrel monkey anterior descending artery. En face section along the VSMC surface, similar to that shown in Fig. 18. Tubules of sarcoplasmic reticulum (SR) wind among the numerous caveolar profiles (C). Scale bar: 0.2 애m.

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FIGURE 20 Transverse section through the wall of a capillary in rhesus monkey papillary muscle. In this microvessel, the medial layer is composed of a VSMC homologue known as a pericyte (PC). Surface caveolae (C) of pericytes are restricted to the upper (abluminal) surfaces. Actin-like microfilaments (MF) are concentrated in the ventral cytoplasm where it abuts the endothelial layer, and microfilaments extend out to fill the attenuated pericyte processes. The underlying endothelial cell profile is dominated by its large nucleus (EN). The detail of the pericyte shows numerous microtubules (MT) running longitudinally in the main body of the cell. Rough endoplasmic reticulum (RER) is present, along with a subsarcolemmal sac (J-SR) that resembles junctional SR of VSMC. Scale bars: 1 and 0.2 애m, respectively.

nary VSMC lie just underneath the surface sarcolemma (Figs. 17–19), with a few densities existing deeper in the cell (Fig. 17). An analogy can be drawn between subsarcolemmal dense bodies and the fasciae adherentes of cardiac muscle intercalated discs, as actin filaments are closely associated with both. Aside from scattered intercellular junctions between VSMC, however, there is no further evidence to suggest any sort of intercalated disc-like formation within the tunica media of blood vessels. VSMC subsarcolemmal dense bodies are subject to the general rule of longitudinal alignment of cell components and segment much of the sarcolemma lengthwise into a series of dense bands that alternate with longitudinal arrays of caveola-bearing surface membrane (Fig. 18; also see Section III,C,3).

Cytoskeletal elements are generally restricted to the central core and its ramifications; intermediate filaments and microtubules all run within the longitudinal axis of the cell, parallel to the surrounding collections of myofilaments (Figs. 17 and 18). VSMC containing either the desmin or the vimentin category of intermediate filament can coexist in larger vessels such as carotid artery, but no clear evidence is available as to the type(s) occurring in cardiac perivascular cells. 3. Membrane Systems The definitive VSMC is a cell characterized by longitudinal orientation of many of its components, and this alignment extends out to the cell surface, where stripes

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of subsarcolemmal densities, into which actin filaments insert, alternate with bands of sarcolemma decorated profusely with the mouths of caveolae. This longitudinal segmentation is best appreciated in sections that graze the VSMC surface (Fig. 18) or in freeze-fracture replicas of the tunica media. The caveolae can be thought of as the equivalent to T tubules of cardiac muscle, and in fact frequently form beaded arrays—sometimes branching—which contain from 2 to as many as 17 fused vesicular subunits (Forbes et al., 1979). Caveolae are more numerous on the dorsal (i.e., abluminal) surfaces of VSMC than on the adluminal, ventral side. The internal membrane system of perivascular cells is now universally referred to as ‘‘sarcoplasmic reticulum,’’ just as in any other type of muscle. This SR, like that of cardiac and skeletal muscle, appears in both peripheral (subsarcolemmal) and deeper regions. At the cell periphery, the SR forms peripheral couplings with the sarcolemma, which wind about the caveolae (Fig. 19), sometimes appearing to form attachments with them through dense spanning structures (Forbes, 1982, 1995; Forbes and Sperelakis, 1982b; Forbes et al., 1979). These bodies are presumably equivalent to the ryanodine-sensitive Ca2⫹release channels (RYR) that have now been isolated from both skeletal and cardiac muscle (as well as from neurons, where they appear in couplinglike complexes known as ‘‘subsurface cisterns’’ and are biochemically quite similar to cardiac RYR). 4. Other Organelles Such internal structures as mitochondria, Golgi, centrioles, and lysosomes are generally restricted to the central core region of VSMC (Fig. 18) and, in less welldeveloped perivascular cells, are likewise excluded from those cell regions containing contractile filaments (Fig. 20). In transversely sectioned VSMC, mitochondria appear small and not particularly remarkable, but like many other organelles they are longitudinally oriented within the central core and actually may achieve 4–5 애m in length (Fig. 18). VSMC mitochondria have been implicated in the metabolism of Ca2⫹ in VSMC (Devine et al., 1973). The Golgi system is usually reduced to small stacks of parallel cisterns, with no hint of formation of dense-cored granules. Centrioles are encountered, sometimes in groups of four, which may correspond to the rare binucleate VSMC. Lysosomes of myocardial VSMC can assume a number of forms, including opaque membrane-bounded spheroids, variegated bodies (probably secondary lysosomes), and multivesicular structures. The occurrence of collections of vesicles in the extracellular spaces between rat coronary artery VSMC has suggested a mechanism by which waste products are liberated to the out-

side of cells via exocytosis of lysosomes, a process that may contribute to a loss of resiliency within the medial layer of such vessels (Joris and Majno, 1974). 5. Junctions Despite the failure of the system of myocardial VSMC to form elaborate organized attachments, they are connected to one another nevertheless, both through their conjoined embedment within complexes of thick cell coat material and their formation of small but distinct intercellular junctions. In larger vessels, such as the coronary arteries of primates, VSMC cell coats are particularly well developed and thrown into elaborate whorls and filigrees of material (Figs. 13 and 15); in smaller-diameter arteries, the smooth muscle cells are fitted more closely together in the manner of masonry blocks, with cell coats constituting the mortar (Fig. 16). In both cases, one can envision this extracellular material to constitute a sort of gel in which the formed elements of the tunica media are suspended and held in a rather consistent spatial relationship to one another. Actual membrane-to-membrane attachments have been described in the medial layers of a variety of blood vessels. In the myocardial vessel bed, in fact, three categories and several forms of junctions have been found. That is, attachments can form between individual perivascular cells (homocellular/intercellular), can be formed by the same cell folding over on itself (homocellular/intracellular), or can connect a perivascular cell with the underlying endothelium (heterocellular). Such junctions can take the form of simple appositions, where the cell membranes come into close approximation but are devoid of any dense intra- or extracellular specializations. Where fuzzy opaque material is clustered on the cytoplasmic side of the apposed membranes, and sometimes forms vague structures in the extracellular space, the junctions are known as intermediate contacts, really for lack of any better term. It is not known whether this plaque material is in any way biochemically related to the dense bodies in VSMC into which actin filaments project, but the overall appearance of such junctions suggests at least an analogous relation between them and the fasciae adherentes of cardiac intercalated discs. In rare instances, a desmosome— complete with discrete subsarcolemmal plaques and extracellular lamella—appears between adjacent VSMC (Forbes, 1982). More commonly, short yet distinct gap junctions are represented in all three categories of cardiac VSMC appositions. This includes heterocellular gap junctions, which have to date been documented only in mouse myocardial circulation, and are found in both coronary arteries and microvessels (Forbes, 1982, 1995).

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6. Effector Systems (Innervation) The concept of homology among all levels of the myocardial circulation may appear a bit strained when one considers the microvasculature. In particular, definitive resistance vessels such as coronary arteries consist of the three distinct layers: intimal, medial, and adventitial. While capillaries consist of a complete, albeit thin, cylinder of endothelium, and a case can be readily made for a medial component in the person of the partial covering afforded by pericytes (Fig. 20), the presence and composition of a microvascular adventitial tunic offers more grist for debate. For one thing, the perivascular space, i.e., that volume system which separates the blood vessel from the major consituents of its associated organ, in this case the myocardial cells, becomes progressively narrower and more confined as one progresses from the arteries down to the capillaries. It is largely through the perivascular space that the growth and penetration of nerve processes can occur. While it is accepted that axon terminals, filled with vesicles of neurosecretory substance (usually norepinephrine or acetylcholine), are specifically involved in the control of coronary arteries, TEM examination reveals that the terminals may be separated from their VSMC targets by a considerable distance, in part because of the large perivascular space typically available at that particular vascular level. In contrast, very narrow perivascular spaces abut the capillaries, and detailed examination of the microvasculature of a variety of hearts has revealed a rather consistent presence of efferent axon terminals. Many of these ‘‘terminal boutons’’ in fact are apposed to pericytes rather than to the endothelial cell (Forbes et al., 1977b). Two questions arise: ‘‘Is there a definitive tunica adventitia in microvessels?’’ and ‘‘Do efferent terminals exert an effect on microvessels? First, recalling that the division of the vascular tree into segments known as ‘‘arteries,’’ ‘‘arterioles,’’ ‘‘postcapillary venules,’’ and so on is partly an artifice, the compartmentation of blood vessels into distinct layers is as well a distinction of convenience, as all the components are to varying degrees interactive and thus functionally if not physically interdigitated. However, by and large, the answer to the first question is ‘‘yes,’’ as all the components of the adventitia can appear in the microvessel’s perivascular space, including fibroblasts, collagen fibrils, Schwann cells, and of course nerve elements such as terminal axons. This is not to say that one will find all of these components in every thin section, nor along certain microvessel stretches, but they can and do exist in the microvascular milieu and can be tallied if one is willing to spend time looking. The second question falls—or at least deserves to fall—more into the laps of physiologists and molecular

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biologists. To argue from the stance of the null hypothesis, it is not surprising to find axon terminals in association with microvessel walls because nerves by their nature use the system of perivascular channels as conduits through which they reach all organ levels; because the perivascular space is most narrow at this vascular level, the terminals by default lie close to the vessels. Upon release, furthermore, the neurotransmitter could just as well have an effect on the nearby myocardial cells—and it is well established that myocardial cells are responsive to these substances—as to serve some role in terms of microvessel function. At this point, I will belabor the subject only a little more, by stating once again how many parallels have been found in structure and biochemistry among perivascular cells at all levels of the heart circulation. These include the presence of organized collections of potentially contractile filaments, which in pericytes are collected against the adluminal cell surface, adjacent to the endothelial cylinder (Fig. 20). The filaments are arrayed in the long axes of the numerous circumferential processes that wind about the endothelium, and traction between the two vessel layers is achievable through heterocellular junctions demonstrated to exist there (Forbes et al., 1977b; Forbes, 1995). To be sure, an active and complete closure of the myocardial capillary by pericyte contracture is an unlikely and perhaps unwanted possibility. Given the rather passive appearance of capillary endothelial cell cytoplasm, however (see Section III,D), even an amoeboid-like movement of the pericyte, or of several pericytes, could participate in a sort of fine-tuning of vascular dynamics at the most delicate levels of the heart’s circulatory system.

D. Endothelial Cells 1. Shape and Orientation The patterns and shapes of endothelial cells forming the lining of blood vessels have been studied rather thoroughly in major vessels such as aorta and carotid artery, whose sheer size make them easier to process and view with such techniques as scanning electron microscopy. Extrapolating from these larger vessels, then, one can generalize to say that, en face, endothelial cells appear either as rounded polygonal bodies arranged in a cobblestone pattern or as more elongated profiles whose long dimensions are aligned with the direction of blood current flow. Both shapes may be found along stretches of the same vessel and their distributions appear to be related to hemodynamics, such that characteristic patterns exist around the regions of vessel branch points, for example. Within the extensive vasculature of the heart, endothelial cells generally appear to be of the more elongate

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variety; whereas the majority of VSMC components are oriented circumferentially around the vessel cylinder, both endothelial cells and their contained structures are oriented longitudinally along the vessel (Fig. 21). Endothelial cells are large and thick in bigger vessels such as arteries and are smaller and quite thin at microvascular levels, where presumably form is following function, as the bulk of metabolite exchange occurs across the capillary endothelial layer. As might be expected, in microvessels, each endothelial cell consists in large part of a single nucleus (Fig. 20), and its attenuated cytoplasm contains fewer and less complex arrays of intracellular organelles when compared to the arterial or venous endothelium. The following sections, which describe the contents of myocardial endothelial cells, are therefore weighted on the side of arterial endothelium, but will point out significant differences where other vessel types are concerned. 2. Fibrils The overall look of arterial and venous endothelial cells is of structures designed to maintain a barrier between the lumen and the underlying vessel wall. Intermediate filaments are abundant and, along with varying numbers of microtubules, are usually strictly aligned with the long axis of the vessel. Particularly in arteries, microfilaments (i.e., thin filaments of 5–7 nm diameter) are tightly bundled together to form prominent rod-like structures (known in some cells as ‘‘stress fibers’’) that may exceed a micrometer in diameter (Fig. 21). These endothelial microfilament bundles sometimes display vague longitudinal densities and irregularly spaced cross-striations. 3. Junctions and Vesicles These two components are presented together because a major function of endothelium—in contrast to the other blood vessel tunics—is transport and exchange. The myocardial vessel bed does not possess the vanishingly thin pores that are the hallmarks of fenestrated endothelium such as seen in kidney glomerulus. Therefore the exchange of materials between blood and myocardial tissue must take place largely across or between endothelial cells. A distinct difference in junctional distribution in the endothelial layer is evident at different levels of the cardiac vascular tree. The borders of adjacent endothelial cells in arteries and veins often exhibit considerable overlap; again, this would be expected in vessels that are handling a great deal of blood flow. Where the two categories differ is in the type of specialized junctions that appear in these appositions, veins usually being characterized by regions of ‘‘tight

junctions,’’ where in essence the outer membrane layers of the two apposed cells have fused. Although tight junctions are also found in arterial endothelium (Fig. 22) definitive gap junctions, sometimes quite extensive in length, are more characteristic here (Fig. 22). In arterial endothelium, then, the adhesive function of gap junctions may well come into play. In microvessels, particularly the smallest capillaries, there is minimal overlap of the endothelial edges, and only intermediate appositions and tight junctions are formed. Tight junctions are so named partly because of their presumed function: they form a barrier to penetration of most substances through intercellular spaces and, in fact, are generally conceded to be the basis of the celebrated ‘‘blood–brain barrier.’’ In published electron micrographs, blood vessels for one reason or another are most often presented in transverse section. This can convey the mistaken impression that endothelial cells form junctions that are only punctate, like spot welds. This disregards the three-dimensional construction of the endothelial tube, which is composed of cells laid down to resemble an unbroken mosaic of ellipsoidal tiles. Grazing sections that pass longitudinally through the endothelium reveal long, virtually unbroken stretches of dense material that correspond to extensive junctional complexes—containing gap junctions, intermediate-type contacts, tight junctions, or some combination, depending on the level of the vasculature—that hold the cells tightly together in the manner of a continuous bead of welding, to further that analogy. Such tenacious attachments make the endothelial cylinder a rather stout structure. At the very least, these extensive junctions obstruct the intercellular spaces to pose a considerable impediment to bulk passage of materials; it seems better therefore to look elsewhere for the anatomical construct that subserves transendothelial transport. Such a mechanism appears to reside in the system of cytoplasmic vesicles. As implied earlier, the microvascular endothelium is invested with only a thin, rather watery-appearing cytoplasm as compared with the major vessels (Figs. 20 and 24). However, a prominent constituent of this endothelial layer is its population of small membranous vesicles, sometimes called ‘‘micropinocytotic’’ vesicles. These should not be equated with the caveolae of the overlying perivascular cells (nor for that matter those of the myocardial cells). In fact, endothelial vesicles appear to be freely mobile within the cell, presumably able to shuttle across from one side to the other, as well as perhaps to form—on a transitory basis—transendothelial channels through which a more directed flow of substances could occur. The overall thinness of the endothelial layer of capillaries would seem therefore to facilitate transport, with

5. Ultrastructure of Cardiac Muscle and Blood Vessels

FIGURES 21 AND 22 Details of endothelium in a transversely-sectioned papillary artery of rhesus monkey heart. Vessel lumen at right in both figures.

FIGURE 21 Arterial endothelial cells are typically thicker than their venous or microvascular counterparts and are filled with a variety of structures, many of them running longitudinally within the cell, and therefore in this preparation appearing in transverse section. Rod-like bundles of microfilaments are commonly present in such cells (MF); microtubules (MT) and intermediate filaments (IF) contribute to the endothelial cytoskeleton. The transport system of endothelial vesicles (V) is seen in the form of circular profiles that are both connected to the cell surface as well as free in the cytoplasm. Tubules and cisternae of endoplasmic reticulum (ER) are also plentiful in such cells. Scale bar: 1 애m. FIGURE 22 Region of overlap between margins of adjacent endothelial cells. This extensive apposition is dominated by gap junctions (GJ), but regions of cell-to-cell membrane fusion (tight junctions, TJ) are also prominent. Scale bar: 0.2 애m.

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FIGURE 23 Mouse heart. Weibel–Palade granules (WP) are commonly found in thicker endothelial cells of the heart such as those of arteries, veins, and endocardium (shown), but are absent from the endothelium of smaller vessels. Characteristically the contents of these bodies are filamentous, tubular, or quasicrystalline in appearance. V, endothelial vesicles. Scale bar: 0.5 애m. FIGURE 24 Capillary in shrew heart. This tissue was prepared by ferrocyanide–osmium contrasting, which resulted in the appearance of an irregular, amorphous, opaque layer (arrows) on luminal surfaces of the endothelium. This phenomenon can be elicited by a variety of dense stains, and although there is physiological evidence for the existence of an endothelial luminal coat, it is unclear in such fixed preparations whether this opacity represents luminal coat material or results from an accidental deposition of stain particles. At the least, this micrograph depicts how such an endothelial luminal coating would be expected to appear. Scale bar: 1 애m.

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tightly bound interendothelial junctional complexes serving mainly to hold the vessel lining together. 4. Miscellaneous Organelles A structure seemingly unique to endothelial cells, the Weibel–Palade granule, was named in honor of its founders. ‘‘W-P’’ granules are membrane-bounded, rodshaped bodies whose contents display a more or less structured appearance, usually taking the form of small tubules (Fig. 22). It is still unclear what function these serve, although amphibian organelles similar in appearance have been linked with histamine content. Weibel– Palade granules appear most prominently in the thickest endothelial cells, namely the endocardium, arteries, and veins, but seem utterly absent from microvascular endothelium. Both free and membrane-bound ribosomes are plentiful in endothelial cells. The Golgi apparatus can be surprisingly robust, and rough endoplasmic reticulum is evident even in microvessels (see, e.g., Fig. 10, Forbes et al., 1977a). Overall the viewer gets the impression that artery and vein endothelium has as its primary purpose the maintenance of stability of vessel architecture in the face of demanding blood-flow dynamics. In contrast, capillaries tread a fine line between maintaining the integrity of the barrier between blood and tissue, while offering as little impediment as possible to metabolite and gas exchange. 5. Surface Coating The abluminal surfaces of endothelial cells (i.e., those not bordering on the vessel lumen) are covered with a thick cell coat (‘‘glycocalyx’’), which fuses with a similar surface coating on the adjacent perivascular cells; in fact, the presence of such a covering is diagnostic for distinguishing pericytes from perivascular fibroblasts, the latter of which do not display an opaque cell coat in conventional TEM preparations. This brings us to the endothelial surfaces that border on the luminal space of blood vessels. In the majority of preparations, these ‘‘adluminal’’ endothelial surfaces appear absolutely bare (e.g., Fig. 23). In fact, however, some sort of endothelial luminal surface coat has long been supposed to exist, but its structure seems chronically to be obliterated by the preparative steps (notably dehydration) used for conventional TEM processing. Luft’s (1966) use of ruthenium red to delineate cell coat material showed a reactive layer within capillaries, and some form of luminal coating has been demonstrated by various workers using lanthanum, uranium, and other electron-opaque substances (Simionescu and Simionescu, 1986; Fig. 24). Multiple components (proteins,

proteoglycans, enzymes, etc.) are likely present in this coat, which is thought to achieve half a micrometer in thickness, thus physically diminishing the effective flow volume of the vessel, and its functions are likewise supposed to be manifold (filtration, selective adsorption, differential transport, and so forth). It remains for correlated physiological and morphological studies to more fully reveal the true scope of this coating’s extent and its functional contributions (Vink and Duling, 1996).

IV. SUMMARY Despite my taking pains to point out how little there is that is new in the field of heart ultrastructure, and how little is being done at present, this should not be taken to mean that there is nothing left to discover. From my own observations, both dedicated and cursory, I can suggest a few avenues of research that would depend at least in part on ultramicroscopic documentation. The first concerns quantitative morphology of heart. Although rather comprehensive surveys are available for certain hearts (mouse, guinea pig), stereological data available for such animals as rat, cat, dog, and various primates, including humans, are meager or nonexistent, and what data exist are intrinsically suspect on the basis of a substantial underestimation of certain cell systems, notably the sarcoplasmic reticulum. I once had a colleague remark to me that he had discontinued any further work where heart morphometry was concerned, simply because it took too much time to do, and thereby severely decreased his rate of publication. Just as it has been suggested that science would be well served by having funding specifically directed toward research that consisted exclusively of the preparation of review articles, I would think there would be benefit as well in supporting scientists whose efforts are devoted to the generation of dependable quantitative descriptions of tissue. Other structurally related questions to be addressed pertain to the study of excitation–contraction coupling. One of these concerns further elucidation of junctional process distribution and density on the junctional SR saccule’s face, as well as a determination as to whether these processes serve solely as Ca2⫹ release channels, or if they might also have an adhesive function, or whether some other structures in the ‘‘fuzzy space’’—perhaps based on unit membranes—are responsible for the integrity of the coupling, which persists even after tissue homogenization. I have repeatedly bemoaned the paucity of structural literature concerning cardiac circulatory elements. Chief among these elements, in terms of a lack of understanding, are the vessels on the venous side of the myocardial

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circulation. Chance observations in just a few different species have revealed a wide structural variety among positionally equivalent cardiac veins. There is also the continuing question of the influence that efferent innervation may exert at the different levels of the heart’s circulatory bed, perhaps down to its smallest reaches, and there likely through the medium of the pericyte, which may be a very primitive form of contractile cell. There is also the question of communication among the vessel’s components, notably the endothelial cylinder and the overlying perivascular cells, whether VSMC, ‘‘primitive smooth muscle cell,’’ or pericyte. It seems most unlikely that heterocellular gap junctions, which could serve both electrocommunicative and adhesive functions between the two layers, should be present solely in mouse heart. A more dedicated survey of other species’ circulatory beds should be undertaken in search of such potentially important junctional complexes.

Bibliography Devine, C. E., Somlyo, A. V., and Somlyo, A. P. (1973). Sarcoplasmic reticulum and mitochondria as cation accumulation sites in smooth muscle. Philos. Trans. R. Soc. Lond. (B) 265, 17–23. Forbes, M. S. (1982). Ultrastructure of vascular smooth-muscle cells in mammalian heart. In ‘‘The Coronary Artery’’ (S. Kalsner, ed.), pp. 3–58. Croom-Helm, London. Forbes, M. S. (1995). Vascular smooth muscle cells and other periendothelial cells of mammalian heart. In ‘‘Physiology and Pathophysiology of the Heart’’ (N. Sperelakis, ed.), 3rd Ed., pp. 803–826. Kluwer, Boston. Forbes, M. S., and Sperelakis, N. (1980). Structures located at the level of the Z bands in mouse ventricular myocardial cells. Tissue Cell 12, 467–489. Forbes, M. S., and Sperelakis, N. (1982a). Association between mitochondria and gap junctions in mammalian myocardial cells. Tissue Cell 14, 25–37. Forbes, M. S., and Sperelakis, N. (1982b). Bridging junctional processes in couplings of skeletal, cardiac, and smooth muscle cells. Muscle Nerve 5, 674–681. Forbes, M. S., and Sperelakis, N. (1985). Intercalated discs of mammalian heart: A review of structure and function. Tissue Cell 17, 605–648. Forbes, M. S., and Sperelakis, N. (1995). Ultrastructure of mammalian cardiac muscle. In ‘‘Physiology and Pathophysiology of the Heart’’ (N. Sperelakis, ed.), 3rd Ed., pp. 1–36. Kluwer, Boston. Forbes, M. S., Rennels, M. L., and Nelson, E. (1977a). Ultrastructure of pericytes in mouse heart. Am. J. Anat. 149, 47–70. Forbes, M. S., Rennels, M. L., and Nelson, E. (1977b). Innervation of myocardial microcirculation: Terminal autonomic axons associated with capillaries and postcapillary venules in mouse heart. Am. J. Anat. 149, 71–92. Forbes, M. S., Plantholt, B. A., and Sperelakis, N. (1977c). Cytochemical staining procedures selective for sarcotubular systems of muscle: Applications and modifications. J. Ultrastruct. Res. 60, 306–327. Forbes, M. S., Rennels, M. L., and Nelson, E. (1979). Caveolar systems and sarcoplasmic reticulum in coronary smooth muscle cells of the mouse. J. Ultrastruct. Res. 67, 325–339.

Forbes, M. S., Hawkey, L. A., and Sperelakis, N. (1984). The transverse-axial tubular system (TATS) of mouse myocardium: Its morphology in the developing and adult animal. Am. J. Anat. 170, 143–162. Forbes, M. S., Hawkey, L. A., Jirge, S. K., and Sperelakis, N. (1985). The sarcoplasmic reticulum of mouse heart: Its divisions, configurations, and distribution. J. Ultrastruct. Res. 93, 1–16. Forbes, M. S., Van Niel, E. E., and Purdy-Ramos, S. I. (1990). The atrial myocardial cells of mouse heart: A structural and stereological study. J. Struct. Biol. 103, 266–279. Forssmann, W. G., and Girardier, L. (1970). A study of the T system in rat heart. J. Cell Biol. 44, 1–19. Goldstein, M. A., and Entman, M. L. (1979). Microtubules in mammalian heart muscle. J. Cell Biol. 80, 183–195. Hayashi, K. (1962). An electron microscope study on the conduction system of the cow heart. Jpn. Circ. J. 26, 765–842. Inui, M., Saito, A., and Fleischer, S. (1987). Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures. J. Biol. Chem. 262, 15637–15642. Joris, I., and Majno, G. (1974). Cellular breakdown within the arterial wall: An ultrastructural study of the coronary artery in young and aging rats. Virch. Arch. (Pathol. Anat.) 364, 111–127. Luft, J. H. (1966). Fine structures of capillary and endocapillary layer as revealed by ruthenium red. Fed. Proc. 25, 1773–1783. Matlib, M. A., Rebman, D., Ashraf, M., Rouslin, W., and Schwartz, A. (1981). Differential activities of putative subsarcolemmal and interfibrillar mitochondria from cardiac muscle. J. Mol. Cell. Cardiol. 13, 163–170. McNutt, N. S., and Fawcett, D. W. (1974). Myocardial ultrastructure. In ‘‘The Mammalian Myocardium’’ (G. A. Langer and A. J. Brady, eds.), pp. 1–49. Wiley, New York. Ogata, T., and Yamasaki, Y. (1990). High-resolution scanning electron microscopic studies on the three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum and intercalated disc of the rat myocardium. Anat. Rec. 228, 277– 287. Phillips, S. J., Dacey, D. M., Bove, A., and Conger, A. D. (1977). Quantitative data on the shape of the mammalian ventricular heart cell. Fed. Proc. 36, 601. Rambourg, A., Segretain, D., and Clermont, Y. (1984). Tridimensional architecture of the Golgi apparatus in the atrial muscle cell of the rat. Am. J. Anat. 170, 163–179. Simionescu, M., and Simionescu, N. (1986). Functions of the endothelial cell surface. Annu. Rev. Physiol. 48, 279–293. Simpson, F. O., Rayns, D. G., and Ledingham, J. M. (1973). The ultrastructure of ventricular and atrial myocardium. In ‘‘Ultrastructure of the Mammalian Heart’’ (C. E. Challice and S. Viragh, eds.), pp. 1–41. Academic Press, New York. Sims, D. E. (1986). The pericyte: A review. Tissue Cell 18, 153–174. Somlyo, A. V. (1979). Bridging structures spanning the junctional gap at the triad of skeletal muscle. J. Cell Biol. 80, 743–750. Sommer, J. R., and Johnson, E. A. (1979). Ultrastructure of cardiac muscle. In ‘‘Handbook of Physiology’’ (R. M. Berne, N. Sperelakis, and S. R. Geiger, eds.), Vol. 1, pp. 113–186. American Physiological Society, Bethesda, MD. Vink, H., and Duling, B. R. (1996). Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries. Circ. Res. 79, 581–589. Viragh, S., and Challice, C. E. (1973). The impulse generation and conduction system of the heart. In ‘‘Ultrastructure of the Mammalian Heart’’ (C. E. Challice and S. Viragh, eds.), pp. 43–90. Academic Press, New York.

C

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6 Excitability and Impulse Propagation MORTON F. ARNSDORF

JONATHAN C. MAKIELSKI

Department of Medicine, Section of Cardiology University of Chicago Chicago, Illinois 60637

Departments of Medicine and Physiology University of Wisconsin Madison, Wisconsin 53792

I. INTRODUCTION

details and equations, in large part, have been moved to an Appendix.

Cardiac excitability has an intuitive meaning, suggesting the ease with which cardiac cells undergo individual and sequential regenerative depolarization and repolarization, the manner in which cells communicate with each, and the eventual propagation of the impulse. Electrophysiologically, the heartbeat arises from a highly organized control of ionic flow through channels in the cardiac membrane, the myoplasm, and the extracellular space. These bioelectrical events are regulated within very tight limits to allow the coordinated propagation of excitation and contraction of the heart that is necessary for an efficient cardiac output. Abnormalities in the regulatory mechanisms often accompany cardiac disease. Structurally, impulse propagation is much affected by the organization of cell membrane structure; the functionality, density, and distribution of gap junctions; and by characteristics of the extracellular space. A complete biophysical consideration of cardiac function would include neurohumoral control, excitation–contraction coupling, and analysis of the stress and strain relations of myocardial tissue during contraction, as well as the effects of contraction and mechanical loading on cellular electrophysiological properties. These feedback phenomena must control cardiac excitability closely under dynamic beat-to-beat conditions. Together, structure and function may result in abnormalities in impulse propagation underlying clinical conduction disorders and reentrant arrhythmias. In contrast to previous editions of this book, technical

Heart Physiology and Pathophysiology, Fourth Edition

II. MATRICAL CONCEPT OF CARDIAC EXCITABILITY A. Linear and Nonlinear, Continuous and Discontinuous Relationships between Stimulus and Response Electrophysiologists often distinguish cellular properties as being either active or passive. Passive properties are those that are more or less constant and are characterized by a response proportional to the stimulus. Intra- and extracellular ionic activities and membrane capacitance are usually considered passive properties. Gap junctions are also often considered passive. Although they may open and close in response to stimuli, an effect usually considered active, they do so slowly enough to render their effects constant. The effects of passive properties may be linear for small stimuli and, therefore, follow Ohm’s law (V ⫽ IR, where V is voltage, I is current, and R is resistance). Even more complex functions, such as decremental conduction through cable-like tissues, can be described using linear differential equations. Most biological systems, however, are nonlinear. While the relationship between current and resistance may be linear for small changes in potential, it becomes nonlinear for larger stimuli, e.g., in the relationship be-

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tween current and voltage in the subthreshold range of potentials. Active properties are nonlinear and are characterized by a response out of proportion to the stimulus. Properties considered active are primarily the transmembrane voltage- and time-dependent ionic conductances responsible for the depolarization and repolarization of the action potential. Subthreshold responses in transmembrane voltage to steps in intracellular current injection may be fairly continuous, although nonlinear, until the threshold is attained when there is a sudden explosive, discontinuous response to a stimulus of the same magnitude. Conceptually, active properties can be considered the ‘‘source’’ and passive properties the ‘‘sink.’’ The nonlinear nature of a system is frequently evident. Nonlinear and discontinuous events are quite common in response to stimuli and may demonstrate, for example, bifurcations, bistability, and hysteresis. In bifurcations, the character of the response evolves in time or, as a parameter is changed, in a specific sequence. One of us has suggested that bifurcations, particularly those assisted by conditions, occur commonly in clinical electrophysiology, and that this is an important principle in arrhythmogenesis and in the actions of antiarrhythmic drugs (see Ginsburg and Arnsdorf, 1995a,b; Arnsdorf and Dudley, 1998; original articles cited in these reviews). Bistable systems are characterized by a stimulus causing two or more kinds of response, e.g., a propagated response and the initiation of triggered activity. In hysteresis, the response after a stimulus has reached some fixed amplitude differs, depending on how fast and/or in what direction the stimulus had been changed previously in the course of reaching that amplitude. The response of a nonlinear system to a periodic input the response may include harmonic and/or subharmonic frequencies or can fall in arbitrary ratios (N : M) of integers, with respect to the input period.

B. Electrophysiologic Matrix The concept of an electrophysiologic matrix proposed previously takes into account the essential nonlinear character of cardiac excitability and propagation in an intuitive way without requiring explicit mathematical equations. A matrical diagram (see Fig. 1) is drawn to contain the system parameters, and lines are drawn to show interrelationships. Figure 1 depicts a bifurcation diagram depicting multiple stabilities and the rapid transition from one matrix to another from matrices deduced from data in our cellular electrophysiologic study on the interactions between changes in extracellular potassium concentration,

[K⫹]o , and flecainide (Arnsdorf and Sawicki, 1996). The ‘‘normal’’ matrix is at a physiologic [K⫹]o and has a regular hexagonal shape indicative of a normal state. Abnormal states, presumably the arrhythmogenic matrical configurations, are represented by matrices of irregular polygonal shape. Bonds between matrix elements show interactions and mutual dependencies. This matrix includes the resting potential (Vr), threshold voltage (Vth), Na⫹ conductance (gNa), membrane resistance (Rm), the length constant (␭), and, as a measure of overall excitability, the liminal length (LL), all of which will be defined later. The matrix has many more dimensions than are shown here, as there are many more ionic channels, including gap junctions, and each element, in turn, is determined by underlying properties, such as ion channel conductances. The active (source) and passive (sink) properties, which form the elements of the matrix, are not usually independent of each other. For example, gap junctional conductance determines in large part all these depicted parameters. The interaction between the antiarrhythmic drug flecainide and the normal matrix at point B results in a new matrix at B*, which in this experiment was largely unchanged except for a slight decrease in sodium conductance indicated by gNa moving toward the center of the hexagon. Hyperkalemia alone, in contrast, caused multiple electrophysiologic changes in both active and passive properties and drove the equilibrium from C to a new equilibrium at point C* characterized by decreases in Rm , ␭, Vth , Vr , and gNa . Hyperkalemia consistently produced this type of matrical configuration and was responsible for what is termed an assisted bifurcation that consistently moved the system from point A to point C*. If the tissue with the electrophysiologic matrix created by hyperkalemia at C* was exposed to flecainide, a second bifurcation occurred, leading to the equilibrium at D*. Further bifurcations occur that depend on the rate of stimulation resulting in a situation at point D** in which the liminal length requirements were either not met, resulting in inexcitability, or were met intermittently, resulting in a 2:1 or some other response. Note how similar the matrical configurations are after the ‘‘arrhythmogenic’’ intervention of increasing [K⫹]o and after the application of flecainide, suggesting a narrow toxic to therapeutic ratio. The dashed lines represent paths that might be taken were another drug used (B⬘), were [K⫹]o lowered below 5.4 mM (C⬘), or were [K⫹]o returned from 10 to 5.4 mM in the presence of flecainide (C* to B*). The point of this exercise is to underscore the complexity of the interrelationships between active and passive properties that determine the source.

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FIGURE 1 A simplified normal matrix of active and passive cellular electrophysiologic properties, which determine cardiac excitability. The elements include resting potential (Vr), threshold voltage (Vth), sodium conductance (gNa), membrane resistance (Rm), length constant (␭), and liminal length (LL), all of which are defined in the text. Each element in turn depends on a set of more basic processes. Bonds connecting the elements indicate interactions and mutual dependencies. A normal state is indicated by a regular hexagonal shape. When quantities represented in the matrix change, the matrix changes shape: when a quantity decreases, the corresponding element shifts toward the center of the hexagon, whereas when the quantity increases, the corresponding element shifts away from the center. Note that conditions cause a change from one steady state to another, resulting in bifurcations. From Arnsdorf and Sawicki (1996), with permission.

C. Individual Myocardial Cells 1. Components of the Cell Membrane The plasma membrane is mainly a thin, phospholipid bilayer that separates aqueous phases inside and outside the cells. The phospholipids are amphipathic, with their hydrophobic portions stably oriented toward the membrane interior and their charged hydrophilic portions oriented toward the internal and external aqueous phases. Phospholipids and other membrane constituents are subject to dielectric polarization, with the result that the membrane can store charge as an electrical capacitor (denoted cm in Fig. 2). This lipid membrane bilayer by itself is an insulator and is a barrier to the flow of ions. Transmembrane proteins, however, including ion channels and pumps or exchangers, mediate and impart

specific ion and water permeation properties to the membrane measurable as electrical conductance or resistance (denoted Rm in Fig. 2). Figure 2B shows an electrical equivalent membrane model. When a potential difference exists across the membrane, current flow through any one unit length of cable (e.g., im in element A-B) consists of two components: a capacitative current (ic) through the capacitor (cm) and an ionic current (ii) through a resistor. In studies on isolated cells, Rm , Cm , Im , expressed respectively as ohms (⍀), farads (F), and amperes (A), normally represent totals for the entire area of the cell membrane. In studies on isolated cable-like fibers or chains of cells end to end, with fixed geometry, the corresponding quantities, denoted in lowercase as rm , cm , and im , can be expressed per unit length and have the dimensions

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FIGURE 2 (A) Experimental arrangement for cable analysis. Stimulus is sudden application of a constant current, injected intracellularly through a microelectrode near the ligated end of a cardiac Purkinje fiber (S). Response is a change in transmembrane voltage (Vm), recorded by microelectrodes at several points along the preparation (V1 , V2 , V3 , etc). Stimulating current Im is monitored via the bath ground. (B, top) An electrical analog for a cable-like preparation, showing membrane resistance (rm), membrane capacitance (cm), internal longitudinal resistance (ri) due to myoplasm and gap junctions, and external resistance (ro) due to the extracellular space. (Bottom) Transmembrane voltage as a function of distance in steady state after intracellular current application. Arrow marks length constant (␭). (C) An analog that represents membrane behavior more accurately by inclusion of series elements rs and cs in addition to rm and cm .

6. Excitability and Impulse Propagation

ohm-meter (resistivity; typical unit ⍀-cm), farad/meter (typically 애F/cm), and ampere/meter (typically 애A/ cm). Equations (1) through (4) in the Appendix are based on this structure and on characteristics of Ohm’s law, whereas Eqs. (5) through (7) show the relationship between conductance (the reciprocal of resistance) and the driving voltage gradient that contribute, to the total current flow. In reality, the electrical behavior of a cell membrane is more complex. The membrane behaves as a capacitor, part of which is in series with a significant amount of resistance. This property of Cm originates partly in details of the composition of the membrane, including the presence of proteins, which can polarize without a conductance change. However, it is due mainly to the T-tubule system, transverse tubules that conduct action potentials into the interior of many types of cardiac cells and link the membrane of the sarcoplasmic reticulum (SR) with the extracellular membrane. Portions of the cell membrane (and thus of Cm) are accessible to the cell exterior only through the T-tubule system, which, although filled with extracellular medium, are narrow and resistive to ionic flow. As a consequence, the effective value of Cm is not truly constant, but rather depends on the frequency or rate of change of Vm . Ultrastructurally, the boundary of a cardiac muscle fiber (a cell) also includes extracellular matrix components such as glycocalyx, in addition to the cell. These features establish and maintain cell apposition and adhesion, but they also restrict the extracellular space and can thereby affect excitation and propagation by restricting the flow and redistribution of ions. Three-dimensional geometry adds further complexity to the electrical analogue of membrane geometry. Nevertheless, the equivalence of the transmembrane current to the sum of the capacitative and resistive current serves as a useful approximation, particularly when a preparation is used as its own control and not much anatomical change is anticipated. 2. Cellular Excitability and Electrotonic Potentials Cardiac excitability, as mentioned, is the ability of cardiac cells regeneratively to depolarize and repolarize during the action potential, as well as the ease with which electrical activity propagates from cell to cell. These processes result from ionic flow through paths involving the cardiac cell membrane, the myoplasm, gap junctions between cells, and the extracellular space. By convention, the outside of the cell is considered to be at ground or zero potential, and positive ions flowing into the cell are called inward currents and tend to depolarize the cell, positive ions flowing out of the cell are outward currents and tend to repolarize the cell

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(see Chapters 7 and 10). If the net amount of positive charge flowing into the cell through a population of ion channels should increase transiently by a small amount, Vm will be reduced (made less negative). For a small change in Vm , there is little or no change in Rm , and Vm will return to its original value when the transient disturbance is over, according to Ohm’s law. Such a disturbance in Vm is called an electrotonic or decremental potential. If a larger current disturbance occurs where Vm decreases (becomes less negative) beyond a point called threshold (determined by the properties of the ion channels in the membrane), then Rm will decrease, allowing transmembrane current Im to increase, resulting in a regenerative action potential. The changes in Vm , which occur during the generation of electrotonic and action potentials, result in voltage differences among various points in a cell and between any one cell and its neighbors. These potential differences will cause currents to flow through regions encompassing many cells. As will be described these currents, when properly regulated, lead to the coordinated propagation of excitation, which underlies normal contraction of the heart and an efficient cardiac output. 3. Action Potentials of Fast and Slow Response Tissues A few additional comments regarding the membrane action potential, which provides the current source for propagation, are in order. The determinants of depolarization and repolarization of the action potential in heart cells are covered in detail elsewhere (Chapters 7 and 10), and the individual ion channels are also covered in individual chapters, but the overall process is briefly summarized here. The depolarization phase (phase 0) is determined mainly by the properties of the inward Na⫹ current (Chapter 11) and to a lesser extent by Ca2⫹ currents (Chapter 12) in the so-called ‘‘fast response’’ tissues, which include the atria, cells of Purkinje fibers, ventricles, and accessory bypass tracts. Fast response tissues show action potentials with a faster rate of rise ˙ max) and have shorter of phase 0 of the action potential (V refractory periods, faster conduction velocities, and other properties listed in Table I. In the so-called slow response tissues of SA and AV nodes, the kinetically fast INa is not evident. This is so either because Vm rests at around ⫺60 mV, at which potential INa is completely inactivated and therefore eliminated functionally, or because fast Na⫹ channels are simply not expressed. Instead, phase 0 depolarization is dominantly due to a current called ICa , carried by a Ca2⫹ channel (L type), which can be blocked by phenylalkylamines (e.g., verapamil) and dihydropyridines (e.g., nifedipine). Fast response tissues can behave like slow response tissues if

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TABLE I Comparison of Electrophysiologic Characteristics of So-Called Fast and Slow Response Tissues Properties

‘‘Fast’’ response tissues

‘‘Slow’’ response tissues

Geographic location

Atria; Purkinje fibers of the infranodal specialized conduction system; ventricles; AV bypass tracts (accessory pathways)

SA and AV nodes; perhaps valves and coronary sinus Depolarized ‘‘fast’’ response tissues in which sodium channels are inactivated and phase 0 depends on calcium current.

Normal resting potential (Vr)

⫺80 to ⫺95 mV

⫺40 to ⫺65 mV

Subthreshold conductance

Primarily components of potassium conductance, particularly gK1

Probably a component of gK

Current responsible for phase 0

INa

Largely ICa (can be a mixed current with Na⫹, often called ‘‘slow inward current’’

Phase 0 channel kinetics of activation and inactivation

Rapid

Slow. Activation multistep

Maximal rate of ris of phase 0 (dV/dtmax) ˙ max) or (V

300–1000 V/sec

1–50 V/sec

Peak overshoot (Vov)

20 to 40 mV

⫺5 to 20 mV

Action potential amplitude

90 to 135 mV

30 to 70 mV

⫺60 to ⫺75 mV Rapid: 0.5 to 5 m/sec High source over sink Partial reactivation during phase 3 with complete reactivation in normal tissue 10 to 50 msec after return to normal Vr

⫺40 to ⫺60 mV Slow: 0.01 to 0.1 m/sec Low source over sink Partial and complete reactivation returns after attainment of Vr (⬎100 msec)

Marked change Steep curve Independent Independent Only with inactivation of sodium system with marked slowing of conduction velocity

Slight change ‘‘Flat’’ curve Varies directly with frequency Decays with frequency Present even in normally ICa tissues (SA and AV nodes)

Automaticity

Yes. Depends on changing balance between inward currents and an increasing outward IK

Yes

Automaticity depressed by physiologic increases in [K⫹]o

Yes

No to slightly

Properties importantly dependent on the interaction between active and passive properties ‘‘Threshold’’ voltage (Vth) Conduction Safety factor Refractoriness and reactivation

Relationship of rate to Action potential duration Refractory period duration Threshold Conduction velocity Characteristics conducive to reentry

they are depolarized by injury or treated with drugs modifying the kinetics and states of the fast Na⫹ channel. In these cases, standing Na⫹ channel inactivation without recovery occurs. Action potentials developed by fast tissues in these states depend on ICa . In contrast to depolarization, the plateau phase and repolarization of the action potential is a more complex process. An initial rapid repolarization, which may inscribe a notch (phase 1), is caused by the rapid early decay of the Na⫹ current combined with the activation of transient outward currents carried by a voltage-activated transient outward K⫹ current (Chapter 13), a calcium-activated transient outward Cl current (Chapter 19), and electrogenic Na–Ca exchange (Chapter 23).

During the action potential plateau (phase 2), membrane resistance is relatively high. Slowly decaying late residual inward currents through Na⫹ and Ca2⫹ channels are balanced against voltage and time-dependent activating outward K⫹ currents carried by the slow and rapidly activating delayed rectifier K⫹ channels (Chapter 13). Eventually the balance tips toward rapid repolarization (phase 3), and inward rectifier K⫹ channels (Chapter 14) provide a final assist to repolarize the membrane. After initiation of an action potential, tissues are refractory to further stimulation. In fast response tissues, INa normally inactivates rapidly, once activated by depolarization, but recovers partially as Vm repolarizes

6. Excitability and Impulse Propagation

during the later part of the action potential. Partial recovery of INa-dependent excitability is associated with properties such as accommodation. As soon as Vm returns to its maximal diastolic value (⫺80 to ⫺95 mV) at the end of the action potential, fast Na⫹ channels recover fully and maximal excitability returns. In contrast, in slow response tissues, ICa recovers much more slowly, and reactivation cannot occur until well into electrical diastole, limiting the rate at which action potentials can be reinitiated. In the AV node, this property would limit the maximal impulse rate thereby protecting the ventricles in case of atrial fibrillation or flutter.

III. INTERCELLULAR COMMUNICATION A. Gap Junctional Structure Gap junctions will be considered in detail in Chapter 21. A few points, however, deserve emphasis to tie together the following discussion. In the heart, myocardial cells are electrically connected. The awareness of communication among cardiac cells dates back before the start of this century. The work of Heidenheim (1901) and others led to the idea that the heart was an anatomical and electrical syncytium. Although Engelmann (1875a) had noted a sealing off of cells after injury in the frog heart, cells at a short distance from a cut surface remained excitable. In small myocardial segments connected by bridges of intact tissue, stimulation at any point caused contraction through the entire preparation, indicating that healthy cells were electrically connected with each other. In 1952, Weidmann noted that the influence of stimulation extended over many cell lengths. Some of this history has been considered in the review by DeMello (1998). The electron microscopic studies of Sjo¨strand and Anderson in 1954 described the intercalated disk and showed that cardiac cells were bound by membranes without any direct cytoplasmic connection between cells. The structure responsible for intercellular communication is the gap junction. The gap junction is a specialized location where the membranes of neighboring cells meet within some 3 nm and are linked by hydrophilic channels that connect the interiors of the two cells and allow intercellular transport of small molecules and ions. The main (43-kDa) protein moiety of cardiac gap junction channels, which differs from those of other tissues, is called a connexin and has four membrane-spanning regions and so terminates at both ends in the cytoplasm (Beyer et al., 1998). Connexins group to form a hexameric structure containing a central pore. This structure is termed a connexon or hemichannel. A working gap junction channel is formed when a connexon in one

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cell becomes localized in a cell membrane and matches sterically with a connexon from a neighboring cell. How this coupling of hemichannels is driven to occur is unknown. At least an initial period assembly into working channels can occur without de novo protein synthesis, as embryonic chick heart cells, reaggregating in culture, synchronize their rhythms within minutes after making physical contact. A review on gap junctions and the development of the heart has appeared (Gourdie et al., 1998). Electrically, a gap junction channel connecting the interiors of two cells behaves as though it were shaped as a right cylinder. Gap junction channels are minimally ion selective; in fact the pore is large enough to pass low molecular weight dyes or ATP. Because the long cylindrical path of a gap junction channel slows permeation, the conductance of the channel is comparable to those of nonjunctional channels. The efficacy of intercellular communication is determined in large part by the conductance of gap junctions. Under some conditions, gap junction conductance depends on transjunctional voltage, which can change rapidly (within msec) during the normal generation and propagation of action potentials. Exogenous agents, such as alkanols, strophanthidin, anesthetics, changes in ionic milieu, including low [Na⫹]i , addition of Sr2⫹, La3⫹, or Mn2⫹, hypertonicity, or Ca2⫹-free EGTA solution as well as factors related to ischemia and injury, can also rapidly modify gap junction conductance. The latter include transients in internal Ca2⫹ and/or pH presence of lipophiles, arachidonic acid pathway intermediates, and metabolic poisons (e.g., see DeMello, 1998). Rapid modulation could also occur via phosphaterelated phenomena, and there are much data to support this view, but the phosphorylation site is not an absolute requirement for gap junction channel activity (Moreno et al., 1992). These rapid modulations indicate that gap junction conductance is at some times an active physiological property. At the same time, gap junction conductance can be considered a passive property, as indicated by the following considerations. Gap junction channels cluster together in plaques, driven to do so in part by interprotein forces and in part by forces originating in the tortuosity and other higher-order properties of the cell membrane, the glycocalyx, and/or other cell surface components. Clustering profoundly affects electrical properties of gap junction channels, as will be seen later. The localization and distribution of gap junction plaques in adult mammals are similar (Gourdie et al., 1998). The specific distribution depends on the stage of development (Gourdie et al., 1992) and becomes less organized after injury (Luke and Safitz, 1991, Smith et

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al., 1991). In adult ventricular tissue, junctional plaques appear to form exclusively and very densely within intercalated discs but at least in atria they also participate in lateral communication. In SA node tissue, gap junctions are more sparse, consistent with the total conductance between cell pairs, which is much smaller than in ventricles. Taken together, these features suggest that the number of functioning junctional channels in a tissue is a point of physiological regulation. The connexin phenotype may vary among regions of the heart (see Beyer et al., 1998). Cx40, Cx43, and Cx45 have been identified with certainty in cardiac myocytes. Cx45 has been found in all cardiac tissues. Cx43 is quite abundant in the atrium, ventricles, and most Purkinje fibers, but has not been detected in the canine sinus node, the atrioventricular node, or the proximal Purkinjes. Cx40 is present in abundance in the atria, bundle branches, and Purkinje fibers and is scant in the sinus and AV nodes as well as ventricles. Multiple connexins have been expressed within a single tissue and cell, suggesting that they may intermingle with each other. The functions and implications of such mixed connexins are poorly understood. Models suggested for the structure of gap junctional channels are illustrated in Fig. 3.

B. Electrical Characteristics of Gap Junctional Channels In contrast with Vm-dependent ion channels responsible for excitability, gap junction channels are less accessible and have been more difficult to characterize experimentally. This section summarizes some properties of

the gap junction channel relevant to its crucial role in propagation. Voltage clamp analysis of gap junctional channels has been a powerful tool in revealing much new information on the conductance of gap junctions. Several approaches have been used, but usually each cell is clamped with separate microelectrodes, which allows manipulation of the transjunctional voltage and the measurement of current flow. Relevant equations are found in the Appendix [Eqs. (8) and (9)]. Voltage clamp studies agree that cardiac gap junction conductance is symmetric with respect to the direction of current flow. As discussed later, a localized unidirectional disturbance of conduction is generally thought to be a substrate for reentrant cardiac arrhythmias. Unidrectional block could be explained by inhomogeneities in refractoriness and other tissue properties. It is not known whether asymmetric gap junction transmission, as occurs in invertebrate nervous systems, could also be a contributing factor. Dependence on transjunctional electric field (voltage gradient), a key issue in understanding how gap junction conductance is regulated dynamically, has proven difficult to assess. In a number of adult ventricular preparations, gj was high and constant over the full range of variation of Vj expected during action potential generation and propagation. In embryonic and neonatal cardiac preparations, gj showed transiency and voltage dependence. Voltage-dependent conductance has also been found in gap junctions of adult rabbit SA node. Biophysics, as mentioned previously, have been reviewed (Veenstra and Wang, 1998). A feature that unifies many results on Vj dependence

FIGURE 3 Models for structure of gap junctional channels. (A) Hexameric arrangement of connexins; end view of a hemichannel from the cytoplasmic side. The four membrane-spanning regions of each connexin form a left-handed helix; the pore region is formed when membranespanning regions 3 of all six connexins associate as a right-handed helix. (B and C) Open and closed states, respectively, of the pore. When connexins undergo torsion, possibly in response to a transjunctional voltage difference, phenylalanine residues, represented by dark triangles, move into the channel lumen and block permeation. (D) Topological model showing situation of connexins in cell membrane. (A–C) From Bennett et al. (1991), with permission, and (D) from Beyer et al. (1990), with permission.

6. Excitability and Impulse Propagation

is that, regardless of age or type of preparation, it only appears when the total gj is relatively small. Many investigators have assumed junctional resistance to be much higher than cytoplasmic resistances due to the very limited cross-sectional area represented by gap junctions. However, physical considerations predict that when many active gap junction channels are clustered together in a plaque, enough current can flow through their large total conductance to drop significant voltage through the cytoplasmic resistance. Most of this loss will occur within submillimeter distance of the gap junction channels. The lack of voltage dependence in situations of large junctional conductance is consistent with this prediction. Junctional channels can also demonstrate multiple conductive states. Sizes and distributions of the conductive states depend on the relative expression of isoforms of the junctional channel proteins and, for a given from, on the phosphorylation state. Further, unlike nonjunctional transmembrane ion channels, which show only abrupt transitions between conducting and nonconducting states, gap junction channels appear able to make graded and/or slow conductance changes. This behavior may represent constriction or relaxation of the gap junction channel pore, which has been proposed to be formed as an array of helical screws (e.g., Bennett et al., 1991).

C. Control of Gap Junctions As mentioned previously, the work of Heidenheim (1901) around the turn of the century suggested that the heart was an anatomical and electrical syncytium. Engelmann’s earlier physiologic studies, however, conflicted with this view (Engelmann, 1875b, 1877). He observed a decrease in injury current and a sealing off of cells after injury to the frog heart. When Engelmann cut the heart with scissors, cells at the cut surface became inexcitable whereas those at a short distance remained excitable and the current of injury decreased with time. Small myocardial segments, connected by bridges of intact tissue, could be stimulated at any point, and stimulation at any point caused contraction throughout the entire preparation. Microscopically, Engelmann could not detect nerves connecting the cells and concluded that the impulse was conducted from one cell to the next. Cells in healthy tissue were connected electrically with each other, but cell injury and death resulted in a sealing off of the normal from injured or dead cells. This led to Engelmann’s famous dictum that ‘‘Cells live together but die singly.’’ Aspects of gap junctional control have been summarized (Ginsburg and Arnsdorf, 1995a,b; Arnsdorf and Dudley, 1998; various chapters from a book edited by

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DeMello, 1998). To briefly summarize, gap junctional conductance may be nearly ohmic or, under some conditions, voltage dependent. We have suggested that voltage dependency may be important in poorly coupled cells and that perhaps this voltage dependency ‘‘fine tunes’’ cell–cell communication in injury (Lal and Arnsdorf, 1992). Gap junctional conductance can be modified by transients in internal calcium concentration and/ or pH, the presence of lipophiles, arachidonic acid pathway intermediates, hypoxia, strophanthidin, hypertonicity and other changes that may be part of the ischemic process or therapeutic intervention. The specific distribution of gap junctions becomes less organized in injury. There may also be tissue-specific differences in the density and distribution of gap junctions. The distribution in SA nodal tissue, atria, the AV node, Purkinje fibers, and ventricular muscle differs and may well play an important role in physiologic regulation.

IV. CABLE THEORY AND ELECTROTONIC POTENTIALS A. Spatial Factors and Anisotropy Among the cells encountered along the pathway normally followed by cardiac excitation and propagation (SA node, atrial muscle, AV node, His–Purkinje fiber system, ventricular muscle) is marked heterogeneity, which generally serves a useful function in the orderly conduction of the cardiac impulse. Differences exist not only in electrical properties of individual cells within each tissue type (Spach et al., 1989b), which depend on their complements of ion channels, but also in the spatial arrangement and types of gap junctions and connective tissues. Major phenomena that depend on these differences include not only AP waveform and automaticity, as described previously, but also speed, safety, directional properties of propagation, and, finally, susceptibility to pathological disturbances. Excitation normally spreads broadly in three dimensions through the masses of atrial and ventricular muscle. The pattern is not uniform; rather, it has specific anisotropy, i.e., there is a measured difference of a physical property related to the direction in which the measurement is made. In contrast, the AV node and Purkinje fibers both form narrow pathways for the spread of excitation. The Purkinje fiber system provides very rapid (0.5–5 m/sec) and safe conduction of action potentials from the AV node to ventricular muscle via the bundle of His, bundle branches and fascicles, and the terminal branching system. In Purkinje fibers, cells are organized in many cases into essentially cylindrical columns two to three cells in

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diameter, surrounded by connective tissue. End-to-end (as well as side-to-side) connections of cells allow the system to extend and transmit excitation over many cell lengths. Because of this structure, as well as their electrophysiology, long Purkinje fiber preparations resemble cables. The AV node is not cable like and supports much slower conduction than the His–Purkinje system. AV nodal conduction is relatively susceptible to failure, and many disturbances of heart rhythm originate there. In 1952, Weidmann studied the electrophysiological properties of cardiac Purkinje fibers. He applied subthreshold depolarizing and hyperpolarizing currents (im) intracellularly through a microelectrode and recorded Vm of the fiber at various points along its length. Vm decreased in graded fashion with increasing distance from the point of stimulation, but could be detected even several millimeters from the point of current application. The several cells that lay between the stimulating and recording microelectrodes must have been connected by a pathway with a low resistance to ionic flow. As the magnitude of the change in Vm was related linearly to the applied currents, electrical, rather than chemical, coupling appeared to be the mechanism of communication. Weidmann (1966) later also demonstrated long-range communication in ventricular muscle. He showed that 42 K diffused freely between cells as would be predicted by cable theory. The upper limit for resistance between cells, 3 ⍀-cm, was almost 700-fold less than for the outer cell membrane. The observations of Weidmann were well described by uniform cable theory. This theory was used by Lord Kelvin in the mid-19th century to model the decrement in the signal carried by the transatlantic telegraph cable. The model included terms corresponding with ri , ro , and rm in Fig. 2. The basic cable equation [Eq. (10) in Appendix] is im ⫽

1 ⭸2Vm Vm ⭸V ⫽ ⫹ cm ri ⭸x rm ⭸t

(10 in Appendix)

where im is the current flow through any unit length of membrane (amperes/cm), ri is the longitudinal resistance of a unit length of the inside conductor or core of the cable (⍀/cm), Vm is the transmembrane voltage, and rm and Cm as defined previously, are membrane resistance and capacitance for a unit length of cable (⍀ cm and F/cm, respectively). Derivation of the cable question is considered in the Appendix [Eqs. (11)– (17)]. The passive membrane properties whose analogs appear in cable theory are of central importance for coordinated excitation and conduction under both normal and abnormal conditions in that electrotonic

interactions between cells cause local regenerative depolarization, which in turn initiates further electrotonic interactions further down the Purkinje fiber or other myocardial tissues. Theory and cable equations are considered in the Appendix.

B. Nondecremental Propagation of Action Potentials In his classic study of Purkinje fibers, Weidmann (1952) used small current steps that did not perturb Vm enough to induce action potentials regeneratively. Electrotonic transmission, which occurs in this range of Vm , where Vm and Im are related linearly, is important, at least in regions where propagation has failed or blocked, and may influence propagation in normal tissues as well. Nonlinear Vm dependence of rm is the basis for normal nondecremental propagation, just as it is the basis for normal cellular excitability. To account for the fact that rm (or gm) is voltage dependent, not constant, cable properties are studied with inputs that model action potentials. The relationship is linear for very small perturbations in Im , when Vm is considerably negative of the threshold for regenerative depolarization (Vth), but not otherwise. The dependence of Vm on current density im may more accurately represent how a uniformly depolarized cable would behave than dependence on total current Im . Study of cable properties becomes much more complex in the range of Vm where rm is voltage dependent. For example, the space or length constant, a measure of the distance or space over which an electronic interaction occurs in a tissue (see Appendix for derivation), can be expressed by the relationship ␭ ⫽ 兹rm /ri, which considers the membrane resistance and the longitudinal resistance between cells cannot be considered constant. It can fall by more than an order of magnitude when dVm /dt is at maximum, as compared to during repolarization. Voltage-dependent changes in rm appeared similarly to reduce Rin and ␶m in sheep Purkinje fibers on repetitive pacing. Longitudinal resistance ri may also change. Pacing of Purkinje fibers does not change ri or cm (Pressler, 1984), but many modifiers of ri , such as Ca2⫹ accumulation, hypoxia, or a lowered pH, are themselves activity dependent.

C. Threshold and Liminal Length An excitable cell has to be depolarized beyond a certain voltage to permit regenerative production of an action potential, but this threshold voltage, Vth , cannot be defined as a scalar quantity because the ability of a stimulus to cause a regenerative response depends on many factors.

6. Excitability and Impulse Propagation

Fozzard and Schoenberg (1972) applied the concept of liminal length to excitability data in short and long Purkinje fiber preparations. In a unidimensional cable, a regenerative response will occur when the depolarizing current flowing into a certain length of tissue (the liminal length) exceeds the outward repolarizing current being sunk into adjacent segments of tissue. Near the tied end of a semi-infinite cable, the liminal length is directly proportional to the charge threshold and inversely proportional to radius, capacitance, length constant, and the threshold potential (see Appendix for equation).

D. Local Circuit Currents and Speed of Propagation When threshold is attained in some region within a cable-like fiber or other spatially extensive tissue, inward currents INa and ICa grow regeneratively, rendering the region positive as compared to neighboring regions. This locally positive Vm drives current, primarily of K⫹, longitudinally through the myoplasm toward more negative regions, through longitudinal resistance ri , which, as we have stated, is composed of both gap junctional resistance rj and cytoplasmic resistance rcyt . As is true under small signal (linear, subthreshold, electrotonic) conditions, a fraction of the longitudinal current charges cm and flows out through conductances gm . A complete circuit is formed as current flows back to the initially depolarized region via the extracellular space. As the initially depolarized region depolarizes further, beyond Vth , more longitudinal current will flow toward neighboring regions and may depolarize them enough to meet the liminal length requirement. Regenerative depolarization will ensue in these new regions, and as this occurs, enough longitudinal current will soon flow to bring yet another region to threshold, and so on. In this way, action potentials can be generated successively over extensive distances at a finite rate. Normally, propagation can only occur in directions away from the region where suprathreshold depolarization first occurred. Once a given region has sustained an action potential it will become refractory to stimulation by currents that may flow back longitudinally from newly excited regions along the exact same path. As will be shown, backflow by alternative paths can be significant. We now consider how active and passive cellular properties affect speed of propagation. Considering first ˙ max during the active properties, in an isolated cell, V phase 0 of the action potential is expected to be proportional to the maximal ionic current of Na⫹ or Ca2⫹ because most of the initial current flow recharges Cm . When action potentials propagate through a cable or extensive array of cells, ionic current flows longitudi-

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nally to neighboring regions, not just inward. Nonethe˙ max still mealess, in a one-dimensional linear cable, V sures the intensity of the maximal phase 0 ionic current. When excitation propagates along the cable at a constant conduction velocity ␪ (in m/sec), the dependence of Vm (measured at a fixed instant of time) on distance along the cable should have the same form as its dependence on time (measured at a fixed point). Modulation of gap junctional resistance rj can exert a large and possibly dominating effect on ri , and so can partly control ␪. This has been predicted by modeling, as well as in experiments in which exogenous uncoupling treatments, such as alkanols and ischemia, were shown to reduce ␪. Particularly to be noted is that increased ri can lead to outright failure of impulse transmission. Larger fibers should conduct faster. ␪ has actually been found either to increase with diameter (Draper and Mya-Tu, 1959) or to be almost constant, independent of diameter (Schoenberg et al., 1975). In the latter case, internal resistivity Ri was larger in larger fibers, possibly opposing the expected effect of diameter. In large Purkinje fibers the extracellular clefts are more prominent, contributing possibly significant ro , which would also reduce ␪. Another possibility is that large Purkinje fibers may consist of many parallel strands of small-diameter cells rather than a few strands of large diameter cells (Pressler, 1984). In situ, all strands would be largely independent and so would retain ri , rm , and therefore ␪ characteristic of the cell diameter, not the fiber diameter.

V. PROPAGATION IN SPATIALLY EXTENSIVE ARRAYS OF CELLS Propagation through massive three-dimensional tissues such as ventricular myocardium is far more complex than one-dimensional propagation along cablelike fibers.

A. Anisotropic and Discontinuous Propagation Patterns Referring now to Fig. 4, we first consider whether ri , ␶m , and other factors determining the velocity ␪ and safety of propagation are uniform in all dimensions (Spach et al., 1990). In a two-dimensional sheet of rat atrial cells, Woodbury and Crill (1961) observed that electrotonic potentials did not decay exponentially with distance from a point source of current as in a onedimensional cable, but rather decayed as a Bessel function. Potential decay was much sharper with increasing distance transverse to the fiber axis than along the axis. It has also long been known that the velocity of nondec-

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FIGURE 4 Characteristics of tissues viewed as conductive media supporting propagation. (A1) Medium whose resistivity is constant and the same in all directions. (A2) Medium whose resistivity is constant in a given direction but differs in different directions. (B1) Medium with discrete impedances, such as high-resistance junctions, in the transverse direction. The resistivity is not constant, but the distribution of these impedances is similar throughout the medium. (B2) Medium with irregularly arranged discrete transverse barriers. Transverse propagation can occur simultaneously via multiple paths. ␾c symbolizes extracellularly recorded potentials. From Spach et al. (1990), with permission.

remental propagation is anisotropic, i.e., ␪ differs when measured at points that lie in different directions relative to the orientation of cells from a stimulation site (Sano et al., 1959). Figure 4 depicts several possible characteristics of tissue as conductive media, and the situations are discussed in the legend. Given that propagation velocity is different in two directions, say x and y, we can then ask if it is constant when measured in a given direction, say y, regardless of the distance along the x direction, say x1 or x2 . Propagation meeting this condition can be called uniformly anisotropic. Figure 5 shows anisotropy of the action potential waveform and the propagation velocity in the crista terminalis and is an example of uniform anisotropy. It also shows that propagation velocity and other features of propagation are anisotropic in different ways in that despite faster propagation in ˙ max was faster and ␶foot was the longitudinal direction, V shorter transversely.

To describe anisotropic propagation empirically on a macroscopic scale, Spach et al. (1981, 1982) proposed measurement of the effective axial resistivity, Ra. Ra is the value of internal resistivity that accounts for the observed speed of propagation along any direction, not just along the long axis of muscle fibers. Unlike Ri in linear continuous cable theory, which incorporates only axial cytoplasmic resistivity and end-to-end gap junction conductivity, Ra also includes implicitly the influences of cellular geometry and packing, extracellular resistivities, side-to-side couplings, and other features. The equation, included in the Appendix, has successfully predicted propagation on a macroscopic scale (several millimeters or more), even along pathways of complex or heterogeneous structure (Spach et al., 1981, 1982). On a microscopic scale (between a cell length and a few millimeters), uniform anisotropy may not hold. As Fig. 6 (Spach and Dolber, 1986) shows, extracellular potentials and their derivatives were recorded at multiple sites with high spatial resolution (separations of the order of 100 애m). In atrial tissue from a 2-year-old male, excitation spread rapidly and along smooth contours in the axial direction (Fig. 6A) and also spread along smooth contours in directions off the long axis, indicating uniform anisotropy (Fig. 4B1). In older preparations, in this case atria from a 42-year-old man, the fast longitudinal path was narrow and had abrupt borders. Off the long axis, excitation spread very slowly and in irregular or zigzag fashion (Fig. 6B). Excitation often reached a transverse site multiphasically (see also Fig. 4B2). This dissociation or fractionation indicates propagation by multiple paths, which could not occur in uniformly anisotropic tissue. Propagation with these features has been called discontinuous (Spach and Dolber, 1986), dissociated microscopic (Spach et al., 1988), or fractionated. Microscopically anisotropic propagation has also been confirmed using fast optical recording (Mu¨ller et al., 1989). Anisotropic propagation must originate with structural features of cardiac muscle. Sperelakis and MacDonald (1974) found that the bulk resistivity of ventricular muscle was much lower longitudinally than transversely. The fine structure of normal cardiac muscle is such that myocytes form ‘‘unit’’ bundles of 2–15 cells that have connections every 0.1 to 0.2 mm. These unit bundles are arranged into separate fascicles, connected with each other at longer distances, possibly related to diameter (Sommer and Dolber, 1981). The fascicles, in turn, group into macroscopic bundles that have complex and varying interconnections. The anisotropic distribution of gap junctions, i.e., the localization (especially in adult tissue) of gap junctions dominantly or exclusively in end-to-end connections, leaves transverse electrical coupling with a smaller magnitude and less uniformity

6. Excitability and Impulse Propagation

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FIGURE 5 Anisotropy of AP waveform and propagation velocity (␪) in the crista terminalis. (Left) Points of stimulation are indicated by square pulses; dots indicate sites of extracellular recording. (Right) Extracellular (␾c) and intracellular (␾i) potentials were recorded at points (circled and arrowed) longitudinal (dotted traces) and transverse (solid traces) to a point of stimulation. The index of sodium conductance, dV/dtmax , was higher and ␶foot was longer in the transverse direction. Isochrone maps (left), constructed from extracellular recordings, indicate uniform anisotropy; conduction velocity, calculated as the distance traveled normal to an isochrone per unit time, was lower transversely, despite higher dVm /dtmax . From Spach et al. (1981), with permission.

than longitudinal coupling, consistent with slower and more indirect propagation off the long axis. Nonuniform anisotropic propagation cannot be described using the cable equation that used limiting arguments for infinitesimal distances. Another situation in which an infinitesimal limit cannot apply is propagation through regions where conduction is decremental. If action potentials fail to propagate through a region, current transfer must have become electrotonic and have decremented to the point where the liminal length requirement at a downstream site is not met. The converse of this is not true. As shown in Fig. 7, electrotonic conduction through an inexcitable gap can support action potential propagation (Antzelevitch and Moe, 1981). Purkinje fibers were put into a three-compart-

ment bath in which the central (1.5 mm long) segment of tissue was rendered inexcitable by superfusing it with a solution that had [K⫹]o of 15–20 mM. Enough current could be made to flow from the proximal compartment through the gap junctions in the inexcitable segment and into the distal compartment to evoke a regenerative action potential. Nonuniformly anisotropic propagation, involving indirect pathways and possibly regions of decremental conduction, is a feature of normal cardiac tissue. Transverse propagation via relatively sparse lateral connections, possibly electrotonic, may help stabilize the overall pattern of propagation through the myocardium. Anisotropies are also relevant to pathology. As considered later, anisotropic structure changes in the

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FIGURE 6 Nonuniform anisotropic propagation in atrial tissue. (A) Preparation from a 2-year-old male. Excitation spread smoothly from the site of initiation (*), as shown by continuous isochrones (top). Smooth extracellular voltage waveforms (␾c) and their time derivatives, recorded from circled points numbered 1–3, indicate monophasic excitation (bottom). Thin dashed lines show orientation of the fibers. Arrows at the bottom mark the times of dVm /dtmax of the underlying action potentials, which were used to construct the isochrones. Isochrones are separated by 1 msec. (B) Preparation from a 42-year-old male. The prominent open arrow on the preparation (top) indicates that in a narrow longitudinal region, propagation was fast and uniform as shown in trace 1 (bottom). The sawtooth indicates that, in directions not collinear with the fiber axis, excitation spread along an irregular zigzag course. The corresponding extracellular waveforms and derivatives, seen in traces 2 and 3 (bottom), are multiply peaked, indicating that excitation spread nonuniformly by multiple paths. From Spach and Dolber, (1986), with permission.

FIGURE 7 An ischemic gap preparation demonstrating that electronic interaction can bridge an area of inexcitable cable and activate nondecremental conduction in a distal segment. (A) Impulses were initiated by stimulating proximal segment P. They propagated as far as the border of the block and stopped there. Not enough electrotonic axial and local circuit current flowed in the inexcitable gap to bring the distal segment (D) to threshold. (B) Same as A, but sufficient current did flow to trigger action potentials in the distal segment. (C) Experimental arrangement. The middle compartment was rendered inexcitable by depolarization with a high [K⫹]o solution, which inactivated both INa and ICa . From Antzelevitch and Moe (1981), with permission.

6. Excitability and Impulse Propagation

chronic phase after injury. Damaged longitudinal pathways can be supplanted by intact transverse– longitudinal–transverse alternates, which may be very long and have less than the normal strength of coupling. Propagation through such restructured tissue could become very substantially slower and more variable than normal, and electrotonic coupling may become more prominent (Luke and Safitz, 1991; Smith et al., 1991). These features can support stable arrhythmic patterns of propagation.

B. Safety Factor and Propagation Failure Clinicians are well aware that propagation can fail more readily in certain tissues such as the AV node, whereas propagation rarely fails in the His–Purkinje system or in atrial and ventricular muscle. The safety factor is the excess of activating current or charge over that just required to produce a regenerative propagated response, the excess of source over sink. Propagation should not fail in a continuous linear cable whose diameter and other properties are constant. Nor should it fail in uniformly anisotropic multidimen˙ max , higher Ri , longer ␶m , or lower sional tissues. Lower V Rm will slow propagation, but if Ri is finite and if the liminal length criterion is met anywhere in the cable, traveling waves should exist throughout. Empirically, however, these expectations are not met. Both active and passive properties influence the safety of propagation. Regarding active properties, the tissues supporting safer propagation are those dependent on INa for phase 0 depolarization, whereas the safety factor is poorer in tissues depending on ICa . Fast tissues depolarized during injury have a lower safety factor because of partial inactivation of INa . Passive properties also critically affect the success of propagation. Failure can occur at points where passive properties change discontinuously, e.g., at points of branching. If the cross-sectional area and length of a segment are constant, membrane surface area will increase and thus Rm will decrease at a branch point. ␪ will change when fibers change their diameter or branch (Goldstein and Rall, 1974). In general, increased diameter or more branching led to smaller ␪ as the sinking of current downstream was greater. Critically slowed conduction at times resulted in the failure of propagation and, at other times, in an echo beat, reflections, and other signs of mismatch. Extensive branching, as found in the AV node, is associated with lower safety. Longitudinal propagation is faster, but may or may not be safer than transverse propagation. Heptanol uncoupling of gap junctions and events generally associated with injury blocked slow transverse propagation earlier than fast longitudinal propagation (e.g., Kleber

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and Janse, 1990). In contrast, forcing normal tissue with increasingly premature extra stimuli blocked longitudinal propagation earlier than transverse (Spach et al., 1981, 1982, 1990). Fast longitudinal propagation became first decremental and then ceased, whereas transverse propagation, although fractionated, continued. When longitudinal propagation is less safe, one causal factor may be the loading effect of coupling on the action potential waveform. Referring to Fig. 5, in the transverse direction, higher coupling resistance leaves ˙ max higher, conpropagation speed slower, but leaves V trary to expectation for a uniform linear continuous cable. The underlying reason for disparate observations with respect to safety or failure of propagation is most likely differences in the microscopic nonuniform anisotropy of tissue structure. A bit more advanced in concept are the spatially discrete cable theories, which are considered briefly in the Appendix. A key difference between continuous and discrete cable theories is in the relations governing propagation speed and safety. In continuous cable theory, traveling waves will propagate when nonlinearly voltage-dependent excitation of an appropriate waveform is applied. If ri should increase, propagation will slow, but traveling waves can be sustained as long as ri is finite. However, in the discrete theory, there is a maximum permissible value of ri , dependent on the waveform of excitation, above which propagation will fail. Continuous cable theory with boundary conditions takes into account that cytoplasmic resistance may be of greater or lesser significance to intercellular coupling. The theory predicts that larger cytoplasmic resistance reduces the ratio of longitudinal to transverse ri . Thus, a higher cytoplasmic resistance would make longitudinal propagation relatively more safe, whereas a lower rcyt would make transverse propagation safer. This prediction may partly explain the disparate observations noted earlier with respect to directional differences in safety. Apparent safety may also depend on the preparation used and the manipulation applied. The safety of transfer may also be looked at in terms of numbers of open gap junctional channels. The magnitudes of these resistances differ markedly among tissues. When input resistance is high, as in the SA node, junctional resistance can be low enough to allow partial synchronization when there are only a few channels. When input resistance is low, as in ventricular muscle, the rapid conduction needed for ventricular contraction requires several tens or hundreds of active channels. As mentioned, the spatial distribution and overall density of gap junctions are changed in injured tissue. It may be predicted that injury, by reducing coupling, renders coupling-dependent properties, such as conduc-

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tion velocity, more susceptible to modification by ratedependent factors such as pH, [Ca2⫹]i , or transjunctional voltage than normal tissue. These influences may operate on a beat-to-beat time scale.

VI. CLINICAL IMPLICATIONS A. Further Comments on the Hypothesis of Altered Excitability and the Electrophysiologic Matrix The interaction of active and passive cellular properties as depicted in the electrophysiological matrix determines normal cardiac excitability. In the normal state, the heart is resistant to the development of cardiac arrhythmias even in the presence of potential triggering beats. The normal matrical configuration must be altered by arrhythmogenic influences that affect one or several determinants of excitability. The result is a change in the matrical configuration that results in abnormal excitability and a proarrhythmic precursor state, sometimes also called the arrhythmia substrate. In this state, premature ventricular beats and other triggers may give rise to reentrant or automatic arrhythmias. Figure 1 shows matrices encountered in a study on the electrophysiologic effects of flecainide (Arnsdorf and Sawicki, 1996). As compared to the control (A), flecainide at [K⫹]o of 5.4 mM depressed gNa slightly without much affecting other properties or overall excitability (B). Hyperkalemia itself would decrease Rm , shorten ␭, make Vr less negativexs, inactivate the sodium channel, decrease gNa , and increase the liminal length (C). Flecainide at the elevated [K⫹]o would depress gNa (D) further and, at a critical rate, would render the tissue inexcitable by expanding the liminal length beyond the capability of activating currents to overcome the depolarizing currents of neighboring tissues.

B. Bifurcations, Symmetry Breaking, and Assisted Bifurcations We have commented on the seeming paradox that multiplicities, discontinuities, and dynamic interactions that exist among the active and passive cellular properties determining excitability should result in unpredictably complex behavior, yet there seems to be order in this apparent chaos, and the resolution to the paradox is the realization that the electrophysiologic matrix responds as a system to an arrhythmogenic influence or an antiarrhythmic drug (Arnsdorf, 1990). This can be conceptualized by using bifurcation diagrams in which experimental results from this study are used to show the

transition from one equilibrium to another. Referring to Fig. 1, point A is the normal dynamic equilibrium shown as a normal matrix. The interaction between flecainide and the normal matrix at point B results in a new matrix at B*, which in this experiment is largely unchanged except for a slight decrease in sodium conductance. Hyperkalemia alone, causing the multiple electrophysiologic changes noted earlier in the experimental results, drives the equilibrium primarily upward from C to a new equilibrium at point C*. Hyperkalemia, then, is responsible for what is termed an assisted bifurcation that will consistently drive the system from point A to point C*. If the tissue with the electrophysiologic matrix created by hyperkalemia at C* is exposed to flecainide, a second bifurcation occurs, leading to the equilibrium at D*. Further bifurcations occur that depend on the rate of stimulation, resulting in a situation at point D** in which the liminal length requirements are not met, resulting in inexcitability, or are met intermittently, resulting in a 2:1 or some other response. Note how similar the matrical configurations are after the ‘‘arrhythmogenic’’ intervention of increasing [K⫹]o and after the application of flecainide, suggesting a narrow toxic-totherapeutic ratio. The dashed lines represent paths that might be taken were another drug used (B⬘), were [K⫹]o lowered below 5.4 mM (C⬘), or were [K⫹]o returned from 10 to 5.4 mM in the presence of flecainide (C* to B*).

C. Assisted Bifurcations and Predominant Drug Effects The predominant drug effect, then, depends on the matrix encountered. Given the predictability of the matrix at C* in ischemia, flecainide will consistently create the type of matrix noted at D* that presumably is antiarrhythmic or at D** that presumably is proarrhythmic. In the presence of a normal local [K⫹]o , the matrical configuration at B* will be obtained, which may suppress premature ventricular beats but which has less proarrhythmic potential. Ischemia is more complicated than a simple increase in [K⫹]o , but a low pH, acidosis, and the accumulation of active metabolites also tend to produce changes similar to the matrix at C*, so presumably the more complicated experiments that would include these other parameters on the X axis (or more properly, multidimensional axes) would give similar results. In terms of the Coronary Arrhythmia Suppression Trial (CAST) study, the simple suppression of premature ventricular beats may have occurred with a matrical configuration such as that at B*, but the sudden deaths that were linearly distributed in time may have occurred due to the coincidence of ischemia with the underlying

6. Excitability and Impulse Propagation

drug effect, resulting in a proarrhythmic configuration such as at D**. These results underscore the importance of considering electrophysiologic changes as alterations in a complex interacting system.

D. The Action Potential as the Local Event; Fulfillment of Liminal Length as the Propagated Event The study on flecainide led us to a somewhat different premise about impulse propagation, i.e., the action potential is a local membrane event and fulfillment of the liminal length requirement by the local circuit currents is the propagated event. The reasoning summarizes much of what has already been discussed. As mentioned, liminal length is the amount of tissue that must be raised above threshold so that the inward depolarizing current from that patch of tissue exceeds the repolarizing influences of adjacent tissues and results in an action potential, and liminal length depends both on active and passive cellular properties. Because the local transmembrane voltage is now more positive than in neighboring patches of membrane, a driving force is established for the longitudinal flow of current in the myoplasm, which, in turn, displaces the negative charge from the interior of the membrane and depolarizes these adjacent patches toward threshold. The local circuit current is completed by capacitative current flow across the membrane and by the flow of current in the extracellular space. If the local electrotonic currents are sufficient to fulfill the liminal length requirements of the neighboring patch or patches of the membrane, these patches will also produce action potentials. These events repeat and sequential depolarization of patches of membrane results. If the liminal length requirement of a patch is not fulfilled, this patch and its neighboring tissue will not be activated. The experiment depicted in Fig. 1 supports this concept. At a [K⫹]o of 10 mM, flecainide caused impulse propagation to fail when excitability failed due to a frequency-dependent decrease in sodium conductance and some small changes in other determinants of the charge threshold. If, however, the liminal length requirements were fulfilled, propagation occurred, albeit with a somewhat slower conduction velocity than was observed for the control tissue at the same [K⫹]o .

E. Arrhythmogenesis and Changes in Impulse Propagation The situation most generally thought to lead to arrhythmias is reentry, which in a number of models has a basis in slow conduction and unidirectional block of

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propagation (see Chapter 67). Unidirectional block may originate with spatially inhomogeneous refractoriness. In fast tissues, any localized influence reducing maximal Na⫹ conductance could also slow conduction and/or cause partial block, as could any inhomogeneity of conducting pathway geometry, or of ion distribution. Excitation can propagate around an area of block by a long indirect path or it can propagate slowly through it. In either case, it can ultimately reach back to and reenter an area of previously excited tissue, which will have recovered. This reentrant mechanism is illustrated in Fig. 8. Normally (Fig. 8A), excitation propagates along Purkinje fibers (labeled 2 and 3) to excite ventricular tissue (labeled 4). Should injury produce unidirectional block of conduction (Fig. 8B) in one fiber (labeled 3), excitation would propagate down the fiber (labeled 2) and possibly back along the fiber branch labeled 3. Due to the length of the path and/or the condition of fiber 3, excitation may propagate quite slowly back to site 1. Should site 1 have recovered excitability, paths (1 and 2) will be reactivated, possibly in a sustained manner. The duration of absolute refractoriness and the conduction velocity interact to determine a minimum path length over which reentrant propagation (sometimes called circus movement) could occur, as described approximately by the relationship: Lc ⫽ ␪ RP, where Lc is the length of the reentrant path, ␪ is the conduction velocity, and RP is the refractory period. In a normal Purkinje fiber, ␪ is about 3 m/sec and RP is about 300 msec, so Lc would be 1 m, which is anatomically unlikely. However, if ␪ is reduced to 0.01 m/sec, then Lc is only 3 mm, a distance comparable to those over which discontinuities of propagation have been measured in normal tissue. Arrhythmias could also occur when a previously excited region of tissue is reexcited electrotonically by way of a delayed reflected wave or when propagating wavefronts collide. These events may cause waves of excitation to propagate off in a new direction, which is not necessarily along, say, a defined reentrant loop. Experimentally, reflection can also terminate arrhythmias. When drug interventions (heptanol, propafenone, and others) are effective in terminating reentrant arrhythmias, they may do so by changing the character of propagation in the involved circuit so that reflections occur. Reflected waves then collide with and annihilate those propagating orthodromically through the circuit (e.g., LaBarre et al., 1979). Reentrant excitation, as we have described it so far, is implied to follow circumscribed paths defined by anatomical changes such as infarcts; it is ordered. However, this need not be the case. Inhomogeneous properties of conduction velocity or refractoriness in themselves are

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FIGURE 8 Scheme of unidirectional block and reentrant propagation. (A) Normally, impulses propagate from a central Purkinje fiber (1) down branches 2 and 3 and activate the ventricle (4), producing a QRS complex. (B) Injury to branch 3 produces a depressed segment that unidirectionally blocks the impulse in that branch. The impulse travels normally down branch 2 and activates the ventricle, producing a normal QRS complex, but also conducts very slowly in the retrograde direction through the depressed segment in branch 3. If the tissue at the bifurcation has recovered its excitability by the time the impulse emerges from the depressed segment, this tissue will be reexcited. Excitation may travel down branch 2 to the ventricle, producing a premature depolarization (dashed arrow). Such reentrant excitation can occur repeatedly, leading to a sustained tachycardia. Excitation can also travel back up the central Purkinje fiber 1 (dashed arrow). (C) A reentrant loop can be abolished by conversion of unidirectional to bidirectional block. If unidirectional block was induced by depression of INa and lowering of dVm /dtmax , further depression of INa will lead to bidirectional block. (D) The conditions for reentry are eliminated by abolishing the area of unidirectional block.

enough to support untoward propagation phenomena (e.g., Allessie et al., 1990). A stable anatomical substrate, a fixed proarrhythmic matrix, may be a more likely concomitant of a chronic arrhythmia. Transient or random modes of reentry, although usually associated with acute ischemia, may have no evident histological basis and can be induced in normal tissue. Transient arrhythmias reflect nonstationarity in time and/or space of the supporting properties (e.g., refractoriness, propagation velocity). A representative random reentry phenomenon is fibrillation, in which excitation progresses simultaneously along multiple wave fronts. The reentrant circuit may be very short and functional or, as Allessie puts it, the head of the circulating waving front may be ‘‘biting on its own tail’’ of relative refractoriness (Allessie et al., 1977). Allessie called this smallest possible circuit where the wavelength equals the pathway length the ‘‘leading circle’’. This type of

reentrant movement does not require gross anatomical obstacles, but creates its own refractory center.

F. Integrated and Fragmented Wave Fronts in Impulse Propagation Historically, altered active properties have been thought more important than passive properties as the cause of arrhythmogenic conduction disturbances. Inactivated sodium channel conductance, as reflected in an ˙ max , as noted, not only affects index such as a lowered V individual cells, but can abet formation of reentrant pathways, as it is a sufficient cause of slowed conduction. Conduction can be slowed or fragmented in the presence of normal excitation (e.g., Gardner et al., 1985; Ursell et al., 1985; Spach and Dolber, 1990). An important function of gap junctions is that they are the structures through which electrotonic interactions occur be-

6. Excitability and Impulse Propagation

tween cells, thereby importantly integrating the wave front. For example, given two neighboring columns of cells, such as occurs in Purkinje fibers and elsewhere in which one column conducts more rapidly than the other, the faster fiber through electrotonic interaction will speed up the slower fiber and the slower fiber will slow down the faster fiber. The result is an electrotonically integrated unit of two columns, traveling at a uniform, intermediate speed. In the heart, many such interactions produce a smooth, integrated wave front of activation that is responsible for the highly controlled and efficient contraction of the heart needed to maximize cardiac output. An integrated wave front, moreover, is unlikely to produce reentry. Experimentally, fractionated electrograms have been be recorded from the surface of a myocardial infarction in a dog 8 weeks following the acute event. These fractionated electrograms are the fingerprint of delayed conduction of the type that underlies reentry. Interestingly, action potentials recorded from myocytes within these zones are normal (Gardner et al., 1985; Ursell et al., 1985). When looked at under the microscope, the muscle fibers were widely separated and disoriented by connective tissues. The slow, fragmented activation that gave rise to the fractionated electrograms, therefore, occurred in the presence of normal action potentials, indicating that the fragmentation and fractionation were not due to changes in the active generator properties of the tissue, but rather to disruption in the integrative electrotonic interaction between cells caused by fibrosis that physically disconnected the cells. This type of fragmented conduction is thought to be an important substrate for reentrant arrhythmias. Because these signals are recorded by the signal-averaged electrocardiogram and are sought during ablation studies, they are very important markers diagnostically and clinically.

G. Anisotropy in Arrhythmogenesis The concept of anisotropy has been the subject of a number of reviews (e.g., Lesh et al., 1990; Ginsburg and Arnsdorf, 1995a; Keener and Panfilov, 1995; Spach, 1995; Wikswo, 1995; Wit et al., 1995; Arnsdorf and Dudley, 1998). Anisotropy is a measured difference of a physical property related to the direction in which the measurement is made. The inherent organization of cells within the heart results in what has been called structural anisotropy, which, therefore, is intrinsic to whatever myocardial structure exists in health and disease. Functional anisotropy can result from the establishment of lines of functional block. Because gap junctions probably play little role in functional anisotropy, with the

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possible exception of common atrial flutter, this will not be discussed in any detail. Much of what follows is speculation as it is difficult to study cellular coupling in humans. The velocity of impulse propagation most commonly is faster parallel to fiber orientation then perpendicular to orientation, and these velocities are termed longitudinal and transverse, respectively. Passive resistivity within and between cells depends on gap junctional connections and on geometry, including tapering shape and bifurcations, and several of these influences have been elegantly modeled. As mentioned previously, some studies have found a rough relationship between diameter and conduction velocity (Draper and Mya-Tu, 1959), but others have found conduction velocity to be quite constant regardless of diameter, as in Purkinje fibers (Schoenberg et al., 1975). Pressler (1984) has suggested that anisotropy within a fiber may explain in part the failure of conduction velocity to correlate with diameter in the Purkinje fiber. The reasoning is that the local circuit currents at the edge of the propagating wave front preferentially flow longitudinally within a column of cells. Conduction velocity, then, would be independent of diameter if the ‘‘fiber size’’ was determined not by the size of the individual cells, but rather by the number of cells. The extracellular space also influences conduction. Perhaps another reason that conduction velocity often fails to correlate with diameter in Purkinje fibers is that the extracellular clefts are more important in larger cells so that the effect of ro ⫹ ri may counter the influence of diameter. Goldstein and Rall (1974) modeled the change in conduction velocity in situations of changing fiber and geometry and found that with a step reduction in diameter, conduction velocity increases, whereas with a step increase in diameter, conduction velocity decreases. This follows from the amount of current lost downstream in the sink. When branching was considered, the impulse approaching the branching site first decreased in velocity as the branches provided a larger sink, and once beyond the junction and into a smaller branch, the conduction velocity increased. Interestingly, extracellular space anisotropy may be discordant directionally with intracellular anisotropy (Sepulveda et al., 1983). Propagation through three-dimensional tissues is far more complex than one-dimensional propagation along cable-like fibers such as Purkinje fibers. As already discussed in some detail, the concept of axial resistivity is the value of internal resistivity, which would account for the observed speed of propagation along any direction, not just along the long axis of muscle fibers (Spach et al., 1981, 1982; Spach, 1995). Ri in linear continuous

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cable theory incorporates only axial cytoplasmic resistivity and end-to-end gap junctional conductivity. Axial resistivity also includes implicitly the influences of cellular geometry and packing, extracellular resistivities, side-to-side couplings, and other features. Referring back to Fig. 6, excitation in young hearts spreads along smooth contours in directions off the long axis, indicating uniform anisotropy, but in older preparations, the fast longitudinal path can be narrow with abrupt borders (Spach and Dolber, 1986; Spach and Dolber, 1990; Spach et al., 1990). Off the long axis, excitation spreads very slowly and in an irregular or zigzag fashion, often reaching a transverse site multiphasically. This dissociation or fractionation indicates propagation by multiple paths, which could not occur in uniformly anisotropic tissue. Propagation with these features has been called discontinuous, dissociated microscopic, or fractionated. The structural features of cardiac muscle in large part are responsible for anisotropic activation and propagation. Although published in 1979, the elegant review by Sommer and Johnson remains an important source of information. The fine structure of normal cardiac muscle suggest that myocytes form ‘‘unit’’ bundles of 2–15 cells that have connections every 0.1 to 0.2 mm. These unit bundles are arranged into separate fascicles that connect with each other at longer distances, possibly related to diameter. The fascicles, in turn, form macroscopic bundles that have complex and varying interconnections. The localization and distribution of gap junctions have already been discussed. As mentioned, adult tissues have dominantly end-to-end connections, leaving transverse electrical coupling with a smaller magnitude and less uniformity than longitudinal coupling. This is consistent with slower and more indirect propagation off the long axis (Pressler et al., 1995). The density of immunostained Cx43 per cell is less in the AV node than in either atrial or ventricular myocardium with a punctate distribution within and along the borders of the nodal cells as well as variation in intensity of Cx43 staining in different portions of the AV node (Pressler et al., 1995). The predominantly end-to-end distribution in atrial, Purkinje, and ventricular tissues favors anisotropic conduction, whereas the punctate distribution in the AV node would favor current distribution in all directions. Pathology may change or even create anisotropies. Some of these issues were discussed in Section VI,F. As a further example, damaged longitudinal pathways can be supplanted by intact transverse–longitudinaltrans–verse alternates, which may be very long and have less than the normal strength of coupling (e.g., Wit et al., 1995). Propagation through such restructured tissue becomes slower and more variable than normal, and electrotonic coupling may become more prominent

(Luke and Safitz, 1991; Smith et al., 1991). These electrophysiologic features can support stable arrhythmogenic patterns of propagation (Spach, 1991; Wit et al., 1995). Figure 9 illustrates the hypothesis of anisotropic reentry in infarct border zones as proposed by Wit (1989). In Fig. 9A, the heavy arrows and isochronal lines indicate that excitation is progressing along a long line where conduction appeared to be blocked. Figure 9B enlarges the hatched area in Fig. 9A showing closely spaced isochrones. Slow transverse propagation seems to have occurred across the line of apparent block. Wit, Allessie, and colleagues proposed that anisotropic reentry is the basis for sustained ventricular tachycardias, which can often be induced by a single stimulus in healed tissue (Dillon et al., 1988). During tachycardia, excitation can circulate through a narrow long strip of surviving ventricular tissue whose longer dimension was coaxial with the long axis of the cells (Wit, 1989). Activation traveled longitudinally at about the normal rate and transversely at a slow rate, possibly not much different from that in normal anisotropic tissues. Spear and colleagues (1983) observed that wave fronts traveling mainly longitudinally propagated faster but followed longer paths, compared with those traveling mainly transversely, which moved slowly, but over short distances, and possibly electrotonically. There was some dispersion or fractionation in the rearrival of these wave

FIGURE 9 Hypothesis of anisotropic reentry in infarct border zones. (A) Heavy arrows and isochronal lines indicate excitation progressing around a long line where conduction appeared to be blocked (horizontal thick black lines). (B) Expansion of area within gridwork from (A) showing closely spaced isochrones. Slow transverse propagation is proposed to have occurred across the line of apparent block. From Wit (1989), with permission.

6. Excitability and Impulse Propagation

fronts at the start point, but regardless of the path, the transit time was long enough to allow the starting subarea to recover, so sustained reentry was supportable. It is reasonable to conclude then that anisotropic conduction can cause slow conduction, the area of unidirectional conduction block, or both involved in reentry. Anatomical pathways may be involved, but anisotropy can also occur without an anatomical pathway. Wit et al. (1995) have attempted to distinguish between functional and anisotropic reentry. They propose that the functional characteristic that causes the leading circle type of reentry is a difference in refractory periods in adjacent areas caused by inhomogeneous conduction. In reentry caused by anisotropy, the essential feature is a difference in effective axial resistance to impulse propagation dependent on fiber direction. Most likely, the two overlap, resulting in a more complex relationship. To return to events in ischemia and infarction, disturbances of conduction in acute ischemia have been thought mainly to be associated with action potential generation. For instance, metabolic inhibition via DNP or lowered [ATP]i led to radical decreases of Rin , producing a ‘‘leaky’’ cell membrane and a loss of action potential transfer in pairs of ventricular cells (Morley et al., 1992). The decreased Rin in injured cells creates large sinks into which injury currents would flow, possibly causing arrhythmogenic heterogeneities in tissue properties, without anatomical barriers. Junctional resistance rj increased minimally, but not enough to prevent electrotonic current transfer. After the initial phases of acute ischemia are past, longitudinal resistances increase in specific sequence. External longitudinal resistivity Ro begins to increase gradually within a few minutes, whereas internal resistivity Ri increases only after 10 min or more, but does so quite suddenly, completing its increase within 1 min or so, following which conduction is blocked (Kleber et al., 1987; Riegger et al., 1989). In chronically infarcted myocardium, increased Ri enhances discontinuity of propagation above that normally predicted formally and found experimentally by a number of authors (see Ginsburg and Arnsdorf, 1995a,b; Arnsdorf and Dudley, 1998), as well as decreasing conduction velocity and space constant. Increased Ri is a consequence of derangement of gap junction organization after injury. Associated with reduced gap junction conductance is a greater variance of conduction velocity as well as predicted inhomogeneities in voltage distribution in response to injected current, which should be evident at a macroscopic scale (Ben-Haim and Palti, 1992). These alterations may be destabilizing and lead to late potential and arrhythmias or may help improve the stability of propagation in surviving tissue

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(Spach et al., 1989a). Tissues with structural heterogeneities, such as Purkinje-to-ventricular paths (e.g., Veenstra et al., 1984) or AV node (Ikeda et al., 1980), may be affected more potently when Ri increases. We assume that the influences that reduce gap junction conductance during injury ultimately lead, in the healed infarct period, to all-or-none loss of and redistribution in space of gap junction channels, which otherwise function normally. This assumption is based on the observed all-or-none modification of gap junction conductance by injury-related influences, as described in reviewing intercellular communication. Particularly to be noted is the synergism between increased [Ca2⫹]i and cytoplasmic acidification (Burt, 1987). Increasing [Ca2⫹]i affects cell–cell communication, but does not decrease Gj substantially until [Ca2⫹]i ⫽ 12 애M is reached, a concentration high enough to induce contractures (Dahl and Isenberg, 1980; Spray et al., 1985; Weingart and Maurer, 1988). Normal transient increases in [Ca2⫹]i due to ICa and Ca2⫹ release from the SR seem insufficient to affect Gj, which supports the earlier view of Weidmann (1970). Only changes in the phosphorylation state (Moreno et al., 1992) and the possible expression of the gap junction channel isoforms in altered proportions (Kanter et al., 1991; Veenstra et al., 1992) have been identified as factors likely to change the behavior of individual channels. The view that a fraction of gap junction channels remains intact is also supported by the fact that normal action potentials can be recorded within tissue areas infarcted after chronic ischemia, where propagation was delayed and fractionated (Spear et al., 1983; Gardner et al., 1985; Ursell et al., 1985a). One factor associated with closure of gap junction channels in the face of intact or minimally disturbed excitability is the accumulation of nonesterified fatty acids in membranes. At concentrations comparable to those found in ischemia, normal action potentials can be recorded in one member of a pair of cells, but there is no transmission between cells. (Burt et al., 1991). The mechanism of this disruption of gap junctions was proposed to be membrane disordering localized to channels, rather than gross membrane disruption or specific channel binding. Some cells must survive in an infarcted area if it participates in reentrant circuits; otherwise the region would not support conduction at all (Wit, 1989). In these cells, as mentioned earlier, essentially normal action potentials can be recorded, although there is evidence of electrotonic influences as well. In thin strands of surviving tissue, propagation over very short distances may be almost normal. However, the borders of infarcted regions are not gradual but sharp and microscopically irregular (Janse and Wit, 1989). Details of the border zone may correspond at least in scale with details of

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discontinuous propagation. Where arrhythmogenic circuits are sustained, they may be constrained to follow paths that are microscopically much longer than the apparent lengths, which would be determined by interelectrode distances. Teleologically, the shutdown of gap junctions by increased [Ca2⫹]i , and/or lowered pH may serve mainly to isolate tissue elements that have undergone chronic ischemic/hypoxic injury from normal ones. This isolation means that normal cells will not lose their cytoplasmic contents or be depolarized by injured ones as a result of continued flow of injury currents. In summary of observations on injured tissues, many observations support the idea that a decrease of cell membrane resistance Rm is the major initial acute effect of injury, whereas decrease of Rj is the major late or chronic effect (Janse and Wit, 1989; Kleber and Janse, 1990).

VII. CONTROL OF ARRHYTHMIAS A. Phenomenology of Drug Action We have already touched on this topic. Traditionally, antiarrhythmic drugs have been classified according to some perceived predominant action (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991). The major classes are (I) those that modify sodium channels in some combination during the resting, active, and/or inactive states and that slow conduction (Na⫹ conductance modifiers), (II) 웁adrenergic blockers, (III) those that extend action potential durations (K⫹ channel blockers) and that often have reverse rate dependence, and (IV) those that shorten action potential duration (Ca2⫹ channel conductance blockers). Within each class, drugs have been placed in a hierarchy. This scheme does not recognize drugs that modify ionic activities or metabolic processes, e.g., those that lower the activities or Mg2⫹, K⫹, or Cl⫺. Passive properties are not considered. Antiarrhythmic drugs of Vaughan Williams class I (local anesthetic or membrane stabilizing), including quinidine, procainamide, encainide, tocainide, and lidocaine, all seem similar if one simply looks at the ‘‘source’’ effect, the reduction of gNa . Indeed, all inhibit gNa ; class I drugs bind specifically to and modify Na⫹ channels directly, rather than acting indirectly, for instance by modifying the cell membrane (Sheldon et al., 1991). To some extent, the traditional classification has recognized differential effects. All class I drugs reduce conduction velocity ␪, but those of class IA (e.g., quinidine, procainamide) prolong refractoriness, those of class IB (e.g., lidocaine, mexilitine) shorten refractoriness, and those of class IC (e.g., flecainide) leave refractoriness unaf-

fected (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991). However, these drugs differ fundamentally in structure and in physical properties such as lipophilicity and so must act at the Na⫹ channel in measurably different ways. Conversely, drugs that evoke phenomenologically similar responses can act by very different mechanisms. Experimentally, in ischemic isolated rat hearts, the L and D isomers of verapamil are equally protective against induced ventricular fibrillation, but D-verapamil blocks fast Na⫹ channels, whereas L-verapamil blocks Ca2⫹ channels (Levy, 1991). These complexities make the classification of antiarrhythmic drugs difficult. Any classification system must be considered a set of opinions, but the strength and usefulness of the system will improve as new knowledge validates or invalidates the underlying assumptions. Particularly important is new insight into the biophysical mechanisms of drug action at the cell membrane (pump, receptor, or ion channel) level (Grant, 1992); a classification framework that extends the Vaughan Williams scheme in this direction and is adaptable to new knowledge and newly developed drugs has been offered (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991).

B. Sites and Mechanisms of Drug Action Stated most generally, drugs interact with different affinities to a channel depending on the state (resting, activated, or inactivated) of the channel. When a drug molecule interacts with a channel in a certain state, it can change the likelihoods and rates with which the channel can enter other states. Finally, a drug molecule can modify or block ion permeation through a channel. These interactions can occur simultaneously or separately. Models of particular importance to antiarrhythmic drug actions are the modulated and the guarded receptor hypotheses to which the interested reader is referred (Hondeghem and Katzung, 1984; Starmer et al., 1984). It is not possible to determine exhaustively how drugs and channels interact because it is not possible to detect unambiguously the state of a channel. We cite here some common interactions that have been verified to occur at the unitary channel level. Many drugs show use or rate-dependent effects. Some drugs active on Na⫹ channels modify phase 0 of the action potential strongly only after one or more action potentials have occurred. This is consistent with the idea that these drugs affect Na⫹ channels in the open state and/or inactivated states. Certain K⫹ channel modifiers lengthen action potential duration more when the heart rate is low, at which time the action potential duration is normally longer in any case. This reverse use dependence is the

6. Excitability and Impulse Propagation

opposite of what would be desired to better regulate the action potential duration (Grant, 1992). Certain drugs slow the rate at which channels inactivate after opening or cause channels to open at a more negative Vm than normal, or prevent inactivation altogether. Classification of drugs by the traditional scheme has been useful empirically, but has failed to recognize variations in mechanisms of action. It may seem paradoxical, for instance, that class I antiarrhythmics, acting at Na⫹ channels, should be efficacious when Ca2⫹ fluxes would appear to be a more sensitive point for regulation (Levy, 1991), presumably because of their roles in triggered activity and in isolation of injured regions. Figure 10 shows bistability in which a Purkinje fiber has a normal resting potential and upstroke, but then fails to repolarize normally (Arnsdorf, 1977). In this example, triggered activity depending on Ca2⫹ for phase 0 in the presence of likely intracellular Ca2⫹ overloading occurs. In other examples, a second steady state without triggered activity occurs at the plateau, and the membrane potential

FIGURE 10 Termination by lidocaine of triggered sustained rhythmic activity in a cardiac Purkinje fiber having an abnormality of repolarization. (A and B) An electrically induced action potential triggered oscillatory activity, which increased in amplitude and persisted indefinitely until terminated 45 min later by a hyperpolarizing intracellular current (arrow in B). (C) After lidocaine treatment, this fiber repolarized and produced normal action potentials. (D–F) During washout of lidocaine, the action potential duration was progressively prolonged until a triggering stimulus was once again able to induce sustained rhythmic activity. From Arnsdorf (1977), with permission.

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can be switched from one steady state to another by injecting small intracellular currents. In Fig. 10, the steady state with oscillatory activity can be terminated using a small hyperpolarizing intracellular current (B) or by exposing the tissue to lidocaine (C), with the latter likely causing a decrease in depolarizing sodium current during the plateau. Research from one of our laboratories (MFA) provides a plausible basis for explaining such unexpected behaviors. It has been shown that the effects of antiarrhythmics differ, depending on the specific prior state of the tissue. This is illustrated by the bifurcation diagrams in Fig. 1 and described at that point in the text. We have also shown that the ischemic metabolite LPC has effects on active and passive properties; the balance varying with concentration and the effect of lidocaine depending on the balance encountered (Arnsdorf, 1977; Sawicki and Arnsdorf, 1985). We have expressed this in terms of bifurcation diagrams in which the path chosen depends on the initial conditions (Arnsdorf, 1992). We now cite several examples to support the statedependent duality of drug effects. In normal sheep Purkinje fibers, we found that procainamide, encainide, and quinidine increase membrane resistance rm , input resistance rin , and length constant ␭, whereas lidocaine and the 웁-adrenoreceptor blocker tolamolol decrease the same parameters [see Ginsburg and Arnsdorf (1995a,b) for original citations]. Increased rm and ␭ mean that excitation in given region has a greater than normal effect on tissue regions at a distance, whether electrotonically or by nondecremental propagation. Such an increase in excitability may be arrhythmogenic, as seems to be the case after quinidine in normal tissue. However, it may be antiarrhythmic; drug-increased excitability may allow electrotonic conduction through an injured region that had been a site of unidirectional block that participated critically in a reentrant circuit. Our laboratory has studied the effect of procainamide and lidocaine in concentrations equivalent to clinically effective plasma levels, on excitability in normal Purkinje fibers. Both drugs shifted the strength– duration curve upward, indicating less excitability; more current was required to attain threshold for any duration. However, procainamide decreased excitability by making Vth less negative whereas lidocaine affected Vth little but decreased rm in the subthreshold range. Procainamide increased and lidocaine decreased ␭, indicating different effects on passive properties. Subsequently, studies have indicated that the effect of lidocaine on the Na⫹ system depends greatly on the activation voltage, with the effect being much more intense in depolarized tissue. Other drugs for which we have found significant effects on passive properties include encainide and quinidine, both of which increase rm .

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State-dependent duality of drug action has demonstrated itself dramatically in the clinical situation. That antiarrhythmics could exacerbate rather than control arrhythmias has long been known. Proarrhythmia was initially thought to be relatively uncommon, but awareness increased. More recently, the class IC drugs flecainide and encainide proved to have an unacceptably high association with fatal ventricular arrhythmias in the CAST [CAST Investigators, 1989; Cardiac Arrhythmia Suppression Trial II (CAST-II) Investigators, 1992]. Patients in CAST had coronary artery disease and existing ventricular arrhythmias that had been well suppressed by these drugs. These patients were randomized either to placebo or to treatment with the drug that had suppressed the ventricular arrhythmia. An interesting observation was that the deaths were distributed equally throughout the period of drug treatment. As the task force of the working group on Arrhythmias of the European Society of Cardiology wrote about this aspect of the CAST trial (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1990), this result suggested that ‘‘mechanisms other than early proarrhythmic effects must have been operative.’’ The several possibilities discussed, such as transient ischemia, would be the type of perturbations that would transiently alter the electrophysiologic matrix, changing a presumably antiarrhythmic matrix to a proarrhythmic matrix. Most likely, many patients with coronary and perhaps other types of heart disease have a fixed substrate and transient ischemia or autonomic surges that deform the matrix further and lead to potentially lethal arrhythmias. CAST suggested that antiarrhythmic drugs may also contribute. Subsequently, the moricizine arm of CAST was also discontinued. Proarrhythmia can be considered to represent either bifurcating chaotic or bistable system behavior. As discussed in the introductory portion of this section, the similarity of the arrhythmogenic and the allegedly antiarrhythmic matrices in Fig. 1 is apparent, and it may be difficult or impossible to distinguish the inability of an antiarrhythmic drug to produce an antiarrhythmic matrical configuration from a drug-induced proarrhythmic matrix. Effects of class I and other drugs whose primary action is on ‘‘source’’ or active membrane properties on ‘‘sink’’ or passive properties also need to be considered. In fact, it is reasonable to ask how these primarily membrane-active agents work as antiarrhythmics when a reentrant pathway extends over a finite area. It has been suggested that the depression of sodium conductance by class I agents may block reentry by turning a unidirectionally blocked or partly refractive area within a circuit into an area of total block. The effectiveness of these drugs would then depend on both the degree

of reduction of sodium conductance and the geometry of the abnormal area (Wit, 1989). To date, no clinical drugs have been proposed that would be effective on gap junctional properties (Wit, 1989). In view of the complex anisotropic structures of circuits in healed tissues supporting reentry, the possibility of mechanisms relating to either modifying the electropharmacological properties of the existing gap junctions in surviving tissue elements or changing the number and/or types of channels expressed merit consideration. The fact that classifications such as that by Vaughan– Williams work well clinically is because disease, such as ischemia, causes the electrophysiologic matrix to change as a system. In ischemia, a rather predictable electrophysiologic matrix occurs. Because the predominant drug effect depends on the preexisting conditions encountered, a group I drug, for example, will have a reliable group I action under these conditions. If the initial conditions result from some other causes, say hypokalemia, the drug actions of a Group I may be quite different.

C. Electrical Control of Arrhythmias An understanding of electronic interactions between cells is important in planning the strategies for antiarrhythmic devices that pace, defibrillate, and ablate. Historically, the development of electrical control, such as pacing and defibrillation techniques, has been driven by clinical and engineering considerations. Empirical success has not depended on rigorous biophysical modeling. In electrical control, as in surgical ablation approaches, success has not required microscopically accurate electrocardiographic mapping of arrhythmic conduction pathways. Mechanisms underlying current approaches to electrical control should not, however, be considered simple. Responses to extrinsic stimuli such as pacing or defibrillation pulses depend on the interactions between current flows forced by potential gradients over fairly extensive distances and the refractory state of the tissue. The resulting three-dimensional patterns of excitation can be exceedingly complex. It can be predicted that strategies based more directly on system analysis will soon come into use. These strategies might take advantage of the extreme sensitivity of nonlinear systems to changes in initial state and/or parameters, particularly as manifest in bistability. The bistable, triggerable behavior described for Purkinje fibers has long been observed in patients with sustained ventricular tachycardias, i.e., such tachycardias can frequently be started or stopped by a single extra beat or stimulus.

6. Excitability and Impulse Propagation

In the years after its initiation using fixed rate stimulators, clinical pacing has grown steadily in capabilities. We mention as highlights the development of demand pacing and similar adaptations related to heart rate changes, the ability to omit or delay a stimulating pulse, or to change automatically to an alternate pacing program, in response to a sensed event, and sequential pacing of atrium then ventricle (with appropriate refractoriness). Devices can now sense two separate phenomena, such as activity and minute volume, and respond to both either according to fixed weights or selectively according to a criterion on the sensed events (Hayes, 1992). Despite all these advances, most pacing techniques applied to date clinically have relied on what can be called open-loop control. This means that all decisions as to what is the best corrective pacing algorithm to perform when any of various contingencies occur are predetermined (Ward and Garratt, 1990). One or more experimental devices use closed-loop or feedback control logic. In a closed-loop device, pacing is adjusted to minimize the difference between the value of a sensed variable and a criterion. Ideally the sensed variable would be an index of overall metabolic demand (Ward and Garratt, 1990). Closed-loop devices have the advantages of fast response to changes in demand (possibly within times as short as beat to beat) and robustness against unwanted sources of noise and variability.

123

Closed-loop control can be useful in preventing ventricular tachycardias (VT). Most attempts at interceding against VTs have relied on periodic overdrive pacing; however, Garfinkel et al. (1992) have shown that control based on chaotic system behavior might also be feasible. As shown in Fig. 11, ouabain was used to induce arrhythmia in a preparation of rabbit interventricular septum, during which interbeat intervals varied chaotically (B, left segment). Analysis of the intervals showed specific patterns of sequential dependence. These are shown in Fig. 11, in which the lengths of intervals are plotted as dependent on the lengths of previous intervals. At some times, the lengths of the intervals tended toward stability, i.e., a given interval tended to be similar in length to the immediately previous interval, and closer to the average length (intervals 163–165). At other times, the sequence tended to diverge, i.e., a long interval tended to be followed by a short one, which was in turn followed by an even longer interval, and so on (intervals 165– 167). An approximate linear dynamic model for a stable rhythm was used to predict the expected length of each future interval. If a beat did not occur within this interval, it was concluded that a divergent sequence was beginning and that correction was needed. Then an electrical stimulus was delivered. This stimulus was made strong enough to induce a beat, rather than merely producing electrotonically an advance or delay (phase resetting; see Anumonwo et al., 1991). Interrupting chaotically lengthened intervals in this way regularized the

FIGURE 11 Experimental closed-loop control of arrhythmia. Arrhythmia was induced by ouabain in a slice of rabbit interventricular septum. (A) Chaotic arrhythmic sequence represented by a phase plot of the length of each interval as dependent on the length of the previous interval. In the sequence 163–167, intervals 163–165 appear to converge toward stability, whereas intervals 165–167 appear to diverge away from stability. (B) In the left segment, interval length depended chaotically on time. In the middle segment, closed-loop control triggered a beat whenever the beat-to-beat interval was predicted to be divergently long. The rhythm regularized. Corrective stimuli were delivered occasionally. A periodic or overdrive correction was not effective. From Garfinkel et al. (1992), with permission.

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I. Pumping Action and Electrical Activity of the Heart

rhythm (Fig. 11.B, middle). The stimulus pattern that successfully controlled the rhythm was not overdrive or any similar form of periodic entrainment; in fact, during maintained control, only about one-third of intervals were found to diverge and needed to be terminated prematurely. Ablation techniques could be considered what we have called ‘‘anisotropy by intention’’ (Arnsdorf and Dudley, 1998) in that lines of resistive change are inserted using radiofrequency ‘‘burns’’ in the atrial isthmus for atrial flutter, areas of slow or fast pathways in AV nodal reentrant arrhythmias, accessory pathways in arrhythmias that utilize the pathway as a conduit from the atrium to the ventricle or as part of a reentrant loop, ventricular loops in the myocardium following injury, and the like. Surgical strategies for channeling or interrupting activation have been used with some success in the treatment of atrial flutter (e.g., Cox et al., 1993), and attempts have been made to duplicate such approaches using intracardiac catheter ablation techniques (e.g., Swartz et al., 1994).

genic to antiarrhythmic or proarrhythmic. The concept of assisted bifurcation is closely related to a predominant drug effect, as the drug effect depends on the matrix encountered. A somewhat different view is proposed in that the action potential is a local event whereas impulse propagation requires successful cell–cell communication. The cell–cell communication through gap junctions, moreover, is important in normally integrating wave front activation. Fragmented wave fronts are arrhythmogenic. The clinical correlates of the discussion based on the electrophysiologic matrix include the phenomenology as well as the sites and mechanism of drug action. The electrical control of arrhythmias is considered in terms of open- and closed-loop control, bistabilities, and triggerable electrophysiologic behavior. The concept that ablation techniques are ‘‘anisotropy by intention,’’ which we introduced in 1998, is revisited.

VIII. SUMMARY

In contrast to previous editions of this book, the equations relating to the discussion in the text are presented in the Appendix. The references are not comprehensive, but should serve as a useful starting point for the more mathematically inclined.

The intention was to provide an intellectual framework for both the clinician and the investigator. The complexity ranges from the intuitive to the equations in the Appendix. Cardiac excitability is a complex process that results from complex, highly controlled, electrophysiologic events through channels in the cardiac membrane, the myoplasm, and the extracellular space. Impulse propagation depends on cardiac excitability and the manner in which fibers are connected by gap junctions and separated by insulators. The matrical concept of cardiac excitability is discussed in detail in terms of an electrophysiologic matrix with many active and passive electrophysiologic properties that form an electrophysiologic universe. This concept takes into account the essential nonlinear character of cardiac excitability and impulse propagation. Emphasis is given to the hypothesis that the electrophysiologic matrix responds as a system to arrhythmogenic influences or antiarrhythmic drugs. Otherwise the multiplicities, discontinuities, and dynamic actions and interactions that exist among the active and passive cellular properties that determine excitability should result in unpredictably complex behavior. However, there is order in chaos, and nonlinear analyses, particularly bifurcations, symmetry breaking, and assisted bifurcations, were used to show how the electrophysiologic matrix may move from one equilibrium to another, e.g., from normal to arrhythmogenic and from arrhythmo-

IX. APPENDIX

A. Individual Myocardial Cells As Fig. 2 shows, the arrangement consisting of the lipid membrane, ion channels, and other membrane proteins together behaves electrically as a parallel resistive and capacitative network with components Rm and Cm . Whenever a potential difference Vm exist across the membrane, a current Im will flow, which consists of two components: Im ⫽ Ic ⫹ Iion

(1)

At any membrane potential Vm , the charge distribution held by membrane capacitance Cm represents stored energy. Whenever Vm changes, the physical work required to add or remove charge can only be done at a finite rate. This is described by Ic ⫽ C m

dVm dt

(2)

The relationship among Iion , Vm , and Rm is given by Ohm’s law: Iion ⫽

Vm Rm

(3)

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6. Excitability and Impulse Propagation

Combining Eqs. (1–3): Im ⫽ C m

dVm Vm ⫹ dt Rm

(4)

B. Ionic Currents and Channel States The contribution to Im from each ionic species is determined by both conductance for that species and the driving force behind that species. Conductance g is the reciprocal of resistance. For a given ionic species, say y, the driving force is the difference between the instantaneous membrane voltage Vm and the equilibrium potential (Ey) so that Iy ⫽ gy(Vm ⫺ Ey)

(5)

Ey is the value of Vm , which would have to exist at any instant to establish and maintain the existing transmembrane concentration gradient of ion species y. For any ion, when Vm ⫽ Ey , Iy ⫽ 0, regardless of gy . A hydraulic analogy is that water flows when there is a pressure gradient and the tap is opened. Recognizing that each ion species, say x, y, z, and so on, exists at a particular concentration inside and outside a cell and so has its own equilibrium potential Ex , Ey , or Ez and that each ion conductance gx , gy , or gz may depend on voltage, time, or both, in its own nonlinear way, we can write: Im 앒 Iion ⫽

Vm ⫽ VmGm ⫽ gx(Vm ⫺ Ex) Rm

(6)

⫹ gy(Vm ⫺ Ey) ⫹ gz(Vm ⫺ Ez) ⫹ . . . We have neglected capacitative current Ic and currents due to pump/exchange mechanisms, which determine intracellular ionic activities in a Vm-dependent manner (e.g., see Makielski et al., 1987), to show that Im is mainly determined by ionic conductances. The Hodgkin–Huxley model of sodium conductance can be viewed as a modification of Eq. (6): INa ⫽ gNa m3 h(Vm ⫺ ENa)

using the double whole cell patch clamp technique. Current injected into either cell is conducted by both the sarcolemmal membrane (rm) and the GJ (rj) channels, and the clamp currents can be described by Cobb et al. (1968): I1 ⫽

V1 (V1 ⫺ V2) ⫹ ⫽ Im1 ⫹ I rm 1 rj

(8)

I2 ⫽

V2 (V2 ⫺ V1) ⫹ ⫽ Im2 ⫹ I rm 2 rj

(9)

If experiments are designed so that V1 is changed stepwise to various constant values while V2 is held at zero, application of Eqs. (8) and (9) is particularly simple. Substituting V2 ⫽ 0, we have I1 ⫽ Im1 ⫹ Ij , V1 ⫽ Vj , and I2 ⫽ Ij , so that measurement of I2 yields gj ⫽ I2 /V1 ⫽ 1/rj directly.

D. One-Dimensional Cable Theory We will attribute the following properties to any preparation for which we claim a resemblance to the transmission line or one-dimensional cable of Fig. 2: the cells form a column in which the pathway through the cytoplasm and the connections between cells at their ends (Ri) has a low resistance. The outer membranes of the cells have a high resistance (Rm). Both Ri and Rm are assumed ohmic and linear, i.e., they do not depend on Vm , at least for small changes in Vm . The column of cells is assumed to be cylindrical. Surfaces of the cells membranes are uncomplicated by invaginations or other specializations so that ions will not accumulate or become depleted in the intercellular space. The lipid component of the membrane will behave as an ideal capacitor. The volume of the solution outside the cells is assumed large so that the resistance of the outside (ro in Fig. 2) is negligible. Longitudinal current flow through the cable is considered uniform, and radial currents are considered negligible. The basic cable equation is

(7)

⫹

In this equation, INa is the Na current per unit area, ENa is the equilibrium potential for Na⫹, gNa is the maximal value of gNa , and m and h are dimensionless variables, each of which weights gNa in a voltage-dependent fashion.

C. Voltage Clamp Analysis of Gap Junctional Channels Conductance of GJ channels has been studied by simultaneous voltage clamp of a pair of cells, mainly

im ⫽

⭸Vm 1 ⭸2Vm Vm ⫽ ⫹ cm ri ⭸x rm ⭸t

(10)

where im is the current flow through any unit length of membrane (amperes/cm), ri is the longitudinal resistance of a unit length of the inside conductor or core of the cable (⍀/cm), Vm is the transmembrane voltage, and rm and cm , as defined previously, are the membrane resistance and capacitance for a unit length of cable (⍀ cm and F/cm, respectively). The derivation of the cable question is considered in Eqs. (11)–(17). To understand this partial differential equation, refer to Fig. 2B. Any current ii flowing to the right past a

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I. Pumping Action and Electrical Activity of the Heart

given point through the inside conductor must return to the same point by flowing leftward through the parallel segment of the outer conductor. The resistance for a given length (termed ⌬x) of the inside and outside conductors would be ri ⌬x and ro⌬x, respectively. According to Ohm’s law, the potential difference across any resistor spanning length ⌬x, say resistor ri between elements B and D in Fig. 2, is ⌬Vi ⫽ Iiri ⌬x

(11)

The length can be made smaller and smaller so that ⌬x approaches zero. Mathematically, this is written as ⭸Vi ⌬Vi ⫽ lim ⫽ ⫺iiri ⭸x ⌬x

(12)

The negative sign indicates that the potential drops as the current passes through the amount of resistance corresponding with incremental length ⌬x. Although ro could be treated in the same manner as ri , we consider ro negligible because the outside solution is large. For this reason, the extracellular voltage will be constant. Conventionally, the extracellular voltage is taken as zero so that Vi ⫽ Vm at any point. Equation (9) can therefore be rewritten as ⭸Vm ⫽ ⫺iiri ⭸x

(13)

The membrane of the cell is leaky, i.e., a certain amount of current (⫺im) is lost through the membrane to the outside (via elements rm and cm) per unit length ⌬x of cable. This loss through the membrane (which can be described by dimensionless variables, each of which weights gNa in a voltage-dependent fashion; Eq. (4) with Im substituted by im , Rm by rm , and so on) reduces the current flowing longitudinally through the core. The loss in longitudinal current (⌬ii) in length ⌬x is ⌬ii ⫽ ⫺im⌬x

(14)

Once again, we make the length of membrane smaller and smaller, letting ⌬x approach zero: ⭸ii ⌬ii ⫽ lim ⫽ ii ⭸x ⌬x

(15)

Differentiating Eq. (13) and dividing through by ri gives another expression for ⭸ii /⭸x :

冉冊

⭸ 2V m ⭸ii 2 ⫽ ri ⭸x ⭸x

(16)

and, substituting from Eq. (13), the relationship becomes ⭸2 V m ⫽ r ii m ⭸x 2

(17)

We obtain the cable equation, Eq. (10), by substitution from Eq. (4) (again with Im substituted by im , Rm by rm , and so on) and rearranging: im ⫽

1 ⭸2V m V m ⭸V ⫽ ⫹ cm ri ⭸x rm ⭸t

(10 again)

These experimentally observable electrical terms will now be defined using the quantities in Fig. 2B. Although the cable equation defines a relationship between im and Vm , this relationship cannot be known explicitly and used to compare with experimental results until the equation is solved for a particular pattern of im , such as a sudden step to a constant value. Solution of this equation requires transform methods and a consideration of error functions. Any solution also depends on boundary conditions, which describe whether a fiber is long, or terminates by branching, with a short circuit, or with a cut end, the latter cases being relevant to injured tissues (Weidmann, 1952; Fozzard and Schoenberg, 1972). Here we illustrate instructive special cases. After a sudden step change in im is forced (by current injection), Vm changes gradually, a reflection of the fact that work is required to add or remove charge from membrane capacitance cm . After a time equal to the time constant ␶m , the change in Vm will have reached a fraction of its final value, which is characteristic of the structure. In a short fiber or cell, where ␶m ⫽ RmCm , Vm(t) ⫽ Vm(0)(1 ⫺ e t/␶m)

(18a)

so that the fraction is 63%, whereas in a long cable-like structure, in which ␶m ⫽ rmCm : Vm(t) ⫽ Vm(t ⫽ 0) erf(兹t/␶m)

(18b)

so that, because erf(1) ⫽ 0.84, the fraction is 84%. ␶m also describes the rate at which Im changes after a step change in Vm is forced. When a constant current Io has been injected into a long cable for a long duration, Vm at the point of stimulation Vm(x ⫽ 0,t ⫽ 앝) is Vo ⫽

r i Io ␭ 2

(19)

The space constant, ␭, is defined next; the denominator of 2 appears because half the current flows in one direction down the cable and the other half flows in the opposite direction. If current is introduced near a highresistance barrier such as the cut and ligated end of a Purkinje fiber, division by 2 is not required. The distribution of Vm along the cable at a point x other than 0 along the cable (again, in steady state some time after starting the injection of a constant current) is approximated by Vm(x) ⫽ Vm(x ⫽ 0) e⫺x/␭

(20)

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6. Excitability and Impulse Propagation

In Eqs. (19) and (20), the length or space constant ␭ is the distance away from the point injection over which Vm falls to e⫺1 (about 37%) of its value at the point of injection. This is a function of the leakiness of the membrane, the resistance within the cell and across the gap junctions, and the extracellular resistance, i.e.,

␭ ⫽ 兹rm /(ri ⫹ ro)

(21a)

(the T system). Not only does Cm behave as if part of it is in series with a resistance above (Fig. 2), the narrow intercellular clefts also enlarge the surface area radically and hence presumably the apparent magnitude of Cm . Mobley and Page (1972) found that 80% of the total membrane surface area was in the extracellular clefts, meaning that the total area was 10–12 times greater than assumed by Weidmann.

or, when ro can be neglected,

␭ ⫽ 兹rm /ri

(21b)

Cable Eq. (10) can be expressed using ␶m and ␭ by multiplying through by rm , substituting, and rearranging: ⫺ ␭2

冉 冊 冉

冊

⭸ 2 Vm ⭸Vm ⫹ ␶m ⫹ Vm ⫽ 0 2 ⭸x ⭸t

(22)

Equation (22) provides a relationship among Vm , x, and t that is amenable to experimental description. Input resistance (Rin , measured in ohms) is the ratio of Vm to Io at x ⫽ 0, the point of stimulation. It is normally measured with constant current in the steady state, i.e., Rin ⫽ Vo /Io

(23)

Realizing from Eq. (21b) that ␭ ⫽ 兹rm /ri and combining Eqs. (17) and (20), the input resistance can be expressed as Rin ⫽

兹rmri

(24)

2

When the diameter of a cable-like preparation, assumed to have a circular cross section, is known, we can define the specific resistance [the resistance of a unit area of membrane (in ⍀-cm2)], Rm ⫽ 2앟a2ri

(25)

as well as the internal or longitudinal resistivity (in ⍀cm), Ri ⫽ 앟a2ri

(26) 2

and the specific capacitance (in F/cm ) of the membrane Cm ⫽

cm 2앟a

(27)

The assumptions underlying one-dimensional cable analysis as described in the Appendix are not strictly true and limit the method. Deviations originate in nonideal properties of each cm , rm , and ri , as well as ro , with the latter being of special concern in tissues in situ and in injury (Kleber et al., 1987). One source of discrepancy is the nonideal capacitative properties that result from the complexly infolded structure of the cell membrane

E. Liminal Length The dependence of cardiac excitability on passive and geometrical properties is illustrated by the liminal length equation proposed by Fozzard and Schoenberg (1972): LL ⫽

兹2e⫺1 Qth

앟3aCm␭Vth

앒

0.855 Qth 2앟3aCm␭Vth

(28)

F. Speed of Propagation When APs propagate through a cable or extensive array of cells, ionic current flows longitudinally to neighboring regions, not just inward. Nonetheless, in a one˙ max still dimensional linear cable governed by Eq. (10), V measures the intensity of the maximal phase 0 ionic current, as we show here. When excitation propagates along the cable at a constant conduction velocity ␪ (in m/sec), the dependence of Vm (measured at a fixed instant of time) on distance along the cable should have the same form as its dependence on time (measured at a fixed point):

冉冊

⭸ 2 Vm 1 ⭸2Vm 2 ⫽ ⭸x ␪ 2 ⭸t 2

(29)

Combining the basic cable Eq. (10) with Eq. (29), and remembering that the ionic current is Vm /rm , we have dVm 1 d 2 Vm ⫽ cm ⫹ iion ri␪ 2 dt 2 dt

(30)

At the moment when dVm /dt is maximal, the second derivative, d 2Vm /dt becomes zero. Equation (30) then becomes ˙ max ⫽ V

iion cm

(31)

˙ max to the transmemIn practice, the relationship of V brane current im is nonlinear in cable-like fibers (e.g., see Cohen et al., 1984). Loading by neighboring cells, input impedances changes the shape of the action poten˙ max during phase 0 is pretial waveform. Distortion of V sumably somewhat mitigated by the fact that Rin is at its lowest then.

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I. Pumping Action and Electrical Activity of the Heart

We can assume that maximal phase 0 INa or ICa is ˙ max . Intuitively, an increase of acceptably measured as V ˙ max should be expected. In fact, in ␪ with increase of V ˙ max slow-response tissues where ICa generates phase 0, V and ␪ are much slower than in fast tissues where INa generates phase 0. Cable theory predicts that ␪ will ˙ max . In depend approximately on the square root of V addition to active membrane generator properties, passive properties, some created by anisotropy, are also of importance in impulse propagation. One measure of the relationship of passive properties and ␪ is the duration of the subthreshold rising phase of the action potential. During propagation, along a given direction, the earliest effect of current spreading to a previously unexcited region is electrotonic depolarization. As Cm charges, Vm decreases exponentially until Vth is reached and rapid depolarization ensues. This early rise of Vm has been called the foot of the action potential. It can be predicted for an action potential approximately by solving Eq. (30) for a step change in im (e.g., Tasaki and Hagiwara, 1957) and the solution is the sum of two exponentials, of which the dominant term is Vm[foot](t) ⫽ Aericmt ⫽ Ae t/␶foot

(32)

where A is an arbitrary constant. Recalling that ␭ ⫽

兹rm /ri ,

␶foot ⫽

冉冊

␭m 1 ⫽ ricm␪ 2 ␪

2

1 a ⫽ ␶m 2RiCm␪

(33)

Analytical predictions of how passive cable properties affect ␪ during the active propagation of action potentials suggest that ␪ decreases if cm or ri increases. This may be seen intuitively, as a larger cm requires more charging current and a larger ri leads to a steeper loss of Vm per unit fiber length at any instant. In either case, the length of fiber newly brought to Vth at each successive instant is correspondingly smaller. Active propagation has a specific speed, which is given by

␪⫽

k Cm 兹RmRi

(34)

in which k is a constant that depends on the diameter of the cable and on the specific function chosen to represent how im depends on Vm .

H. Axial Resistivity Substituting Ra, the axial resistivity, for Ri in the cable equation (Eq. 10), we have 앟a2⭸2Vm ⭸Vm ⫽ cm ⫹ iion Ra⭸x 2 ⭸t

(36)

As mentioned in the text, this equation has been used successfully to predict propagation on a macroscopic scale and even along pathways of complex or heterogeneous structure. See text for application. See Spach et al. (1981, 1982) and text for further discussion.

I. Spatially Discrete Cable Theories Spatially discrete cable theories have been discussed by Keener (1991). To provide a basis for a discrete cable theory, we return to the current balance equation, Eq. (4), which applies to a single isolated cell, considered to be electrically homogeneous. We now modify this equation to describe a cell in contact with two neighbors (a segment of a one-dimensional cable). To the total capacitative current and the total ionic current of the cell, we add the total current flowing into or out of the cell from its neighbors: Cm A

dVm ⫽ AIion(Vm, t) ⫹ Inbr dt

(37a)

in which A is the total membrane surface area. We have written Iion as dependent on Vm and t to remind the reader that a discrete cable, like a continuous one, must serve mainly to support nondecremental action potential propagation. The currents (which total Inbr) flowing in or out of a cell from its neighbors are driven by the potential differences between the cells and are controlled by the respective gap junctional resistances. Thus, for cell n having neighbors n ⫺ 1 and n ⫹ 1: Inbr ⫽

Vn⫹1 ⫺ Vn Vn ⫺ Vn⫺1 Vn⫹1 ⫺ 2Vn ⫹ Vn⫺1 ⫺ ⫽ rj rj rj (37b)

Combining and dividing through by A, we arrive at the discrete cable equation:

G. Uniformly Anistropic Propagation In uniformly anisotropic propagation, how ␪ depends on cable properties can be expressed mathematically as separate from how it depends on properties in the y direction (Kootsey, 1991):

␪ ⫽ fx,y(x, y) ⫽ fx(x)fy( y)

In this equation, fxy(x, y), fx(x), and fy( y) are all functions that describe the dependence of ␪ on cable properties.

(35)

Cm

dVm Vn⫹1 ⫺ 2Vn ⫹ Vn⫺1 ⫽ Iion(Vm, t) ⫹ dt Arj

(37c)

We assume that rj is the same between all pairs of cells. More critically, we assume junctional resistance (rj) to be large compared with cytoplasmic resistance. When

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6. Excitability and Impulse Propagation

rj between cells is low, there is more intercellular current flow. Voltage loss in rcyt becomes significant, and cells then cannot be considered homogeneous units. In highly coupled tissues such as ventricular muscle, macroscopic measures of resistivity [Eq. (36)] or voltage distribution may show smooth variation. This smoothness does not preclude the existence of microscopic discontinuities of ri , but may rather depend on the summed conductance of large numbers of parallel intercellular pathways in both transverse and longitudinal directions. The discrete Eq. (37c) is the same as continuous Eq. (10) with spatial derivative ⭸Vm /⭸x substituted by a finite difference and internal longitudinal resistance ri substituted by junctional resistance rj . Because of this formal identity, Eq. (37c) could be used to simulate numerically a continuous cable. However, Eq. (37c) does not in general behave like Eq. (10); the approximation is usefully close only when ⭸ri /⭸x is constant; moreover, the spatial step size has no physical meaning and must be kept small. In contrast, in the discrete cable equation, the step size represents a cell length. When using a discrete theory, it must be decided if quantities are to be measured relative to a unit of one cell (the step size) or along a continuous length scale. For instance, when measured on a continuous scale, longitudinal propagation is normally faster than transverse. However, if measured relative to numbers of cells, transverse propagation is normally faster (Keener, 1991).

J. Functional Properties of Gap Junctions Metzger and Weingart (1985) studied communication between paired adult rat ventricular cells by injecting current into one cell or the other. Current flowed through the nonjunctional membrane of the injected cell and also through rj into the second or follower cell, where it perturbed the recorded Vm . As with voltage clamp studies of intercell communication (see earlier discussion), the responses were analyzed using the idealized lumped model. From Kirchoff’s laws, the input resistance (dVi /dIi) for either cell i (i ⫽ 1, 2) is given by rm1(rm2 ⫹ rj) dV1 ⫽ dI1 rm1 ⫹ rm2 ⫹ rj

(38)

which is the slope of the voltage–current relation for the injected cell, whereas the coupling coefficient (dVm2 /dVm1 or dVm1 /dVm2) is given by dVm2 dVm1

⫽

rm 2 rm 2 ⫹ r j

(39)

which is the ratio of the voltage change in the follower cell over the voltage change in the injected cell. Measuring these quantities in both cells in turn generated four equations, which determined nonjunctional membrane resistances (rm1 and rm2) as well as junctional resistance rj . As is true for voltage clamp measurements of rj [Eq. (8) and Eq. (9)], the accuracy of rj measurements under current clamp is also limited. Both rm1 and rm2 are of course voltage dependent and are often much larger than rj .

Bibliography Allessie, M. A., et al. (1977). Circus movement in rabbit atrial muscle as a mechanism of tachycardia. III. The ‘‘leading circle’’ concept: A new model of circus movement in cardiac tissue without the involvement of an anatomic obstacle. Circ. Res. 41, 9–18. Allessie, M. A., et al. (1990). Atrial reentry. In ‘‘Cardiac Electrophysiology: A Textbook’’ (M. R. Rosen et al., ed.), pp. 555–571. Futura, New York. Antzelevitch, C., and Moe, G. K. (1981). Electrotonically mediated delayed conduction and reentry in relation to ventricular conducting tissue. Circ. Res. 49, 1129–1139. Anumonwo, J. M. B., et al. (1991). Phase resetting and entrainment of pacemaker activity in single sinus nodal cells. Circ. Res. 68, 1138– 1153. Arnsdorf, M. F. (1977). The effect of antiarrhythmic drugs on triggered sustained rhythmic activity in cardiac Purkinje fibers. J. Pharmacol. Exp. Ther. 201, 689–700. Arnsdorf, M. F. (1990). Arnsdorf’s paradox, J. Cardiovas. Electrophys. 1, 42–52. Arnsdorf, M. F. (1992). Cardiac excitability, the electrophysiologic matrix, and electrically induced ventricular arrhythmias: Order and reproducibility in seeming electrophysiologic chaos. J. Am. Coll. Cardiol. 17, 139–142. Arnsdorf, M. F., and Dudley, S. (1998). Gap junctions, cardiac excitability and clinical arrhythmias. In ‘‘Heart Cell Communication in Health and Disease’’ (W. DeMello, ed.), 1st Ed., pp. 217–288. Kluwer, Philadelphia. Arnsdorf, M. F., and Sawicki, G. J. (1996). Flecainide and the electrophysiologic matrix: The effects of flecainide acetate on the determinants of cardiac excitability in sheep Purkinje fibers. J. Cardiovasc. Electrophysiol. 7, 1172–1182. Ben-Haim, S. A., and Palti, Y. (1992). Intercellular conduction velocity variability as the basis for reentrant arrhythmias in the ischemic myocardium. J. Theor. Biol. 154, 317–330. Bennett, M. V. L., et al. (1991). Gap junctions: New tools, new answers, new questions. Neuron 6, 305–320. Beyer, E. C., et al. (1990). Connexin family of gap junction proteins. J. Membr. Biol. 116, 187–194. Beyer, E., et al. (1998). Cardiovascular gap junction proteins: Molecular characterization and biochemical regulation. In ‘‘Heart Cell Communication in Health and Disease’’ (W. C. DeMello, and M. J. Janse, eds.), pp. 45–72. Kluwer, Boston. Burt, J. M. (1987). Block of intercellular communication: Interaction of intracellular H⫹ and Ca⫹⫹. Am. J. Physiol. 253, C607–C612. Burt, J. M., et al. (1991). Uncoupling of cardiac cells by fatty acids: Structure-activity relationships. Am. J. Physiol. 260(3 Pt 1), C439– C448. Cardiac Arrhythmia Suppression Trial (CAST) Investigators (1989). Increased mortality due to encainide or flecainide in a randomized

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¨ ber die Structur des menschlichen HerzHeidenheim, M. (1901). U muskels. Anat. Anz. 20, 3–79. Hondeghem, L. M., and Katzung, B. G. (1984). Antiarrhythmic agents: the modulated receptor mechanism of action of sodium and calcium channel-blocking drugs. Ann. Rev. Pharmacol. Toxicol. 24, 387–423. Ikeda, N., et al. (1980). The role of electrical uncoupling in the genesis of atrioventricular conduction disturbance. J. Mol. Cell. Cardiol. 12, 809–826. Janse, M. J., and Wit, A. L. (1989). Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol. Rev. 69, 1049–1169. Kanter, H. L., et al. (1991). Cardiac myocytes express multiple gap junction proteins. Circ. Res. 70, 438–444. Keener, J. P. (1991). Wave propagation in myocardium. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (L. Glass et al., eds.), pp. 405–436. Springer, New York. Keener, J. P., and Panfilov, A. V. (1995). Three-dimensional propagation in the heart. The effects of geometry and fiber orientation on propagation in myocardium. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (D. P. Zipes and J. Jalife, eds.), 2nd Ed., pp. 335–347. Saunders, Philadelphia. Kleber, A. G., and Janse, M. J. (1990). Impulse propagation in myocardial ischemia. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (D. P. Zipes and J. Jalife, eds.), 1st Ed., pp. 156–161. Saunders, Philadelphia. Kleber, A. G., et al. (1987). Electrical uncoupling and increase of extracellular resistance after induction of ischemia in isolated, arterially perfused rabbit papillary muscle. Circ. Res. 61, 271–279. Kootsey, J. M. (1991). Electrical propagation in distributed cardiac tissue. In ‘‘Theory of Heart: Biomechanics, Biophysics, and Nonlinear Dynamics of Cardiac Function’’ (L. Glass et al., eds.), pp. 391–403. Springer, New York. LaBarre, A., et al. (1979). Electrophysiologic effects of disopyramide phosphate on sinus node function in patients with sinus node dysfunction. Circulation 59, 226–235. Lal, R., and Arnsdorf, M. F. (1992). Voltage-dependent gating and single channel conductance of adult mammalian atrial gap junctions. Circ. Res. 71, 737–743. Lesh, M. D., et al. (1990). Myocardial anisotropy: Basic electrophysiology and role in cardiac arrhythmias. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (D. P. Zipes and J. Jalife, eds.), pp. 364–376. Saunders, Philadelphia. Levy, M. N., and Wiseman, M. N. (1991). Electrophysiologic mechanisms for ventricular arrhythmias in left ventricular dysfunction: Electrolytes, catecholamines, and drugs. J. Clin. Pharmacol. 31, 1053–1060. Luke, R. A., and Safitz, J. E. (1991). Remodeling of ventricular conduction pathways in healed canine infarct border zones. J. Clin. Invest. 87, 1594–1602. Makielski, J. C., et al. (1987). Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells. Biophys. J. 52, 1–11. Metzger, P., and Weingart, R. (1985). Electric current flow in cell pairs isolated from adult rat hearts. J. Physiol. (Lond). 366, 177–195. Mobley, B. A., and Page, E. (1972). The surface area of sheep cardiac Purkinje fibers. J. Physiol. 220, 547–563. Moreno, A. P., et al. (1992). Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin43 gap junction channels. Biophys. J. 62, 51–53. Morley, G. E., et al. (1992). Effects of 2,4-dinitrophenol or low [ATPi] on cell excitability and action potential propagation in guinea pig ventricular myocytes. Circ. Res. 71, 821–830.

6. Excitability and Impulse Propagation Mu¨ller, W., et al. (1989). Fast optical monitoring of microscopic excitation patterns in cardiac muscle. Biophys. J. 56, 623–629. Pressler, M. L. (1984). Cable analysis in quiescent and active sheep Purkinje fibres. J. Physiol. (Lond). 352, 739–757. Pressler, M. L., et al. (1995). Gap junction distribution in the heart: Functional relevance. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (D. P. Zipes and J. Jalife, eds.), 2nd Ed., pp. 144–181. Saunders, Philadelphia. Riegger, C. B., et al. (1989). Effect of oxygen withdrawal on active and passive electrical properties of arterially perfused rabbit ventricular muscle. Circ. Res. 64, 532–541. Sano, T. N., et al. (1959). Directional difference of conduction velocity in cardiac ventricular syncytium studied by microelectrodes. Circ. Res. 7, 262–267. Sawicki, G. J., and Arnsdorf, M. F. (1985). Electrophysiologic actions and interactions between lysophosphatidylcholine and lidocaine in the non-steady state: The match between multiphasic arrhythmogenic mechanisms and multiple drug effects in cardiac Purkinje fibers. J. Pharmacol. Exp. Ther. 235, 829–838. Schoenberg, M., et al. (1975). Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers. J. Gen. Physiol. 65, 441–458. Sepulveda, N. G., et al. (1983). Finite element analysis of current pathways with implanted electrodes. J. Biomed. Eng. 5, 41–48. Sheldon, R. S., et al. (1991). Class I anti-arrhytmic drugs: Structure and function at the cardiac sodium channel. Clin. Invest. Med. 14, 458–465. Sjostrand, F. S., and Andersson, E. (1954). Electron microscopy of the intercalated discs of cardiac muscle tissue. Experientia 10, 369–372. Smith, J. H., et al. (1991). Altered patterns of gap junction distribution in ischemic heart disease. Am. J. Pathol. 139, 801–821. Sommer, J. R., and Dolber, P. C. (1981). Cardiac muscle: The ultrastructure of its cells and bundles. In ‘‘Normal and Abnormal Conduction of the Heart Beat’’ (A. Paes de Carvalho et al., eds.). Futura, Mt. Kisco, NY. Sommer, J. R., and Johnson, E. A. (1979). Ultrastructure of cardiac muscle. In ‘‘The Handbook of Physiology’’ (R. M. Berne, ed.), Vol. I, pp. 113–186. The American Physiological Society, Williams and Wilkins, Baltimore. Spach, M. S. (1991). Anisotropic structural complexities in the genesis of reentrant arrhythmias. Circulation 84, 1447–1450. Spach, M. S. (1995). Microscopic basis of anisotropic propagation in the heart: The nature of current flow at a cellular level. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (D. P. Zipes and J. Jalife, eds.), 2nd Ed., pp. 204–215. Saunders, Philadelphia. Spach, M. S., and Dolber, P. C. (1990). Discontinuous anisotropic propagation. In ‘‘Cardiac Electrophysiology: A Textbook’’ (M. Rosen et al., eds.), pp. 517–534. Futura, Mt Kisco, NY. Spach, M. S., and Dolber, P. C. (1986). Relating extracellular potentials: Evidence for electrical uncoupling of side-to-side fiber connections with increasing age. Circ. Res. 58, 356–371. Spach, M. S., et al. (1981). The discontinuous nature of propagation in normal canine cardiac muscle: Evidence for recurrent discontinuities of intracellular resistance that affect membrane currents. Circ. Res. 48, 39–54. Spach, M. S., et al. (1982). The functional role of structural complexities in the propagation of depolarization in the atrium of the dog: Cardiac conduction disturbances due to discontinuities of effective axial resistivity. Circ. Res. 50, 175–191. Spach, M. S., et al. (1988). Influence of the passive anisotropic properties on directional differences in propagation following modification of the sodium conductance in human atrial muscle: A model of reentry based on anisotropic discontinuous propagation. Circ. Res. 62, 811–832.

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Spach, M. S., et al. (1989a). Interaction of inhomogeneities of repolarization with anisotropic propagation in dog atria: A mechanism for both preventing and initiating reentry. Circ. Res. 65, 1612– 1631. Spach, M. S., et al. (1989b). Multiple regional differences in cellular properties that regulate repolarization and contraction in the right atrium of adult and newborn dogs. Circ. Res. 65, 1594–1611. Spach, M. S., et al. (1990). Properties of discontinuous anisotropic propagation at a microscopic level. Ann. N.Y. Acad. Sci. 591, 62–74. Spear, J. F., et al. (1983). Cellular electrophysiologic characteristics of chronically infarcted myocardium in dogs susceptible to sustained ventricular tachyarrhythmias. J. Am. Coll. Cardiol. 1, 1099– 1110. Sperelakis, N., and MacDonald, R. L. (1974). Ratio of transverse to longitudinal resistivities of isolated cardiac muscle fiber bundles. J Electrocardio. 7, 301–314. Spray, D. C., et al. (1985). Regulation of gap junctional conductance. Am. J. Physiol. 248 (Heart Circ. Physiol. 17), H753–H764. Starmer, C. F., Grant, A. O., and Strauss, H. (1984). Mechanisms of use-dependent block of sodium channels in excitable membranes by local anesthetics. Biophys. J. 46, 15–27. Swartz, J. F., et al. (1994). A catheter-based curative approach to atrial fibrillation in humans. Circulation 90, I–335. Tasaki, I., and Hagiwara, S. (1957). Capacity of muscle fiber membrane. Am. J. Physiol. 188, 423–429. Task Force of the Working Group on Arrhythmias of the European Society of Cardiology (1990). CAST and beyond: Implications of the Cardiac Arrhythmia Suppression Trial. Circulation 81, 1123– 1127. Task Force of the Working Group on Arrhythmias of the European Society of Cardiology (1991). The Sicilian gambit: A new approach to the classification of antiarrhythmic drugs based on their actions on arrhythmogenic mechanisms. Circulation 84, 1831–1851. Ursell, P. C., et al. (1985a). Structural and electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing. Circ. Res. 56, 436–451. Veenstra, R. D., and Wang, H.-Z. (1998). Biophysics of gap junction channels. In ‘‘Heart Cell Communication in Health and Disease’’ (W. C. DeMello and M. J. Janse, eds.), pp. 73–104. Kluwer, Boston. Veenstra, R. D., et al. (1984). Purkinje and ventricular activation sequences of canine papillary muscle. Circ. Res. 54, 500–515. Veenstra, R. D., et al. (1992). Multiple connexins confer distinct regulatory and conductance properties of gap junctions in developing heart. Circ. Res. 71, 1277–1283. Ward, D., and Garratt, C. (1990). Rate-responsive pacing and sensors. In ‘‘Electrical Therapy for Cardiac Arrhythmias’’ (S. Saksena and N. Goldschlager, eds.), pp. 343–353. Sauders, Philadelphia. Weidmann, S. (1952). The electrical constants of Purkinje fibres. J. Physiol. (Lond). 118, 348–360. Weidmann, S. (1966). The diffusion of radiopotassium across intercalated disks of mammalian cardiac muscle. J. Physiol. (Lond). 187, 323–342. Weidmann, S. (1970). Electrical constants of trabecular muscle from mammalian heart. J. Physiol. 210, 1041–1054. Weingart, R., and Maurer, P. (1988). Action potential transfer in cell pairs isolated from adult rat and guinea pig ventricles. Circ. Res. 63, 72–80. Wikswo, J. P. (1995). Tissue anisotropy, the cardiac biodomain, and the virtual cathode effect. In ‘‘Cardiac Electrophysiology: From Cell to Bedside’’ (D. P. Zipes and J. Jalife, eds.), 2nd Ed., pp. 348–362. Saunders, Philadelphia. Wit, A. L. (1989). Anisotropic reentry: A model of arrhythmias that may necessitate a new approach to antiarrhythmic drug development. In ‘‘Lethal Arrhythmias Resulting from Myocardial Isch-

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7 Electrocardiogram and Cardiac Excitation YORAM RUDY Cardiac Bioelectricity Research and Training Center Case Western Reserve University Cleveland, Ohio 44106

I. INTRODUCTION

the cellular and subcellular level has provided important information on cellular processes that generate cardiac excitation and has helped us to characterize better the electrocardiographic sources on a microscopic (cellular) scale. Advances in electronics and computers have made possible simultaneous recordings of electrical activity from many sites. These mapping techniques have been used to study the pattern of excitation and recovery in the myocardium, providing the basis for a macroscopic description of the cardiac sources as a function of time and space. Mapping techniques have also been used to record the potential distributions over the entire torso (the total surface ECG), extending the standard ECG approach that samples this potential distribution at only a small number of points. Advances in computers and digital computing have also led to the development of detailed computer models and simulations of the electrocardiographic process, which have provided invaluable insights into the relationships between cardiac activity and electrocardiographic fields. Considerable progress has also been made in attempts to solve the electrocardiographic inverse problem in which cardiac activity can be computed noninvasively from body surface electrocardiographic data. This chapter is an attempt to synthesize many of these concepts and to describe the genesis of the electrocardiographic potentials in terms of basic physiological processes and concepts from electromagnetic field theory. The discussion focuses on macroscopic phenomena (cellular and membrane processes are discussed extensively in other chapters of this volume) and relies on both quantitative and descriptive approaches. The emphasis is on basic principles rather than on specific questions,

The electrical activity of cardiac muscle cells is projected to the surface of the torso by means of the intervening conducting medium. The surface potentials that are recorded as electrocardiograms reflect, therefore, the heart generators as well as the surrounding volume conductor. The cardiac current sources are distributed throughout the myocardium and can be specified by giving the space–time distribution of dipole elements in the entire heart. A crude approximation is to represent the heart electrically by a single dipole, which is the vector sum of all these dipole elements. This is the basis of the dipole hypothesis introduced by Einthoven and associates (1) as early as 1913. Interpretation of most clinical electrocardiograms (ECG) and vectorcardiograms (VCG) recorded today is still based on this simplified representation of the cardiac sources as a single heart vector. Moreover, almost all of the clinical ECG and VCG practiced today neglect the effects of the medium, i.e., the torso volume-conductor inhomogeneities, on the electrocardiogram. Despite these simplifications, electrocardiography has been an extremely useful and reliable diagnostic tool for almost a century. The last 40 years have seen major progress in several areas related to basic understanding of the electrocardiogram. In the 1950s the microelectrode was introduced, permitting direct recording of the membrane current–voltage relationships of cardiac cells. The recently developed patch clamp technique has allowed scientists to record the electrical activity of individual membrane ionic channels. Molecular biologists have started to determine the molecular structure of the channel proteins and to relate it to function. This activity at

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such as different designs of electrocardiographic lead systems. The two major determinants of the electrocardiographic potentials, namely cardiac sources and the torso volume conductor, are discussed first. Processes underlying electrocardiographic body surface potential maps (BSPMs) are described. Finally, the principles introduced are used to describe an emerging electrocardiographic imaging modality (ECGI) for noninvasive reconstruction of the electrical function of the heart.

determine the action potential. A simulated ventricular action potential, together with the major ionic currents, is shown in Fig. 1 (2–4). The action potential is characterized by a fast upstroke, which is generated by voltagedependent activation of a depolarizing sodium current INa ; a prolonged plateau, which results from a balance between an inward current, ICa(L) , carried mainly by calcium ions and a time-dependent outward potassium

II. ELECTRICAL SOURCES IN THE HEART During the cardiac excitation process current sources arise in cell membranes throughout the heart. These sources can be characterized by a vector function ជJ, an impressed current density that depends on time and space. ជJ is nonzero only in cellular membranes, where it can be identified with ionic currents that are carried mainly by sodium (Na), calcium (Ca), and potassium (K) ions. While this constitutes a rigorous formal description of the cardiac sources, it is desirable to formulate alternative descriptions that lend themselves to quantitative evaluation, provide a basis for quantitative simulations of electric fields generated by the heart, and help relate the electrocardiographic potentials to the underlying cardiac activity. The goal of this section is to develop such representations of the electrocardiographic sources.

A. Single Cardiac Cell Histologically the heart (cardiac tissue) is composed of many individual cells, each with a typical length of about 100 애m and a diameter of roughly 15 애m. Under normal conditions, these cells are electrically coupled through low-resistance pathways (gap junctions) that permit current flow (carried by ions) between cells. This current provides the mechanism for conduction of the action potential and for the formation of an activation wave front that propagates through the heart and constitutes the locus of electrical sources during cardiac excitation. Because the single cardiac cell is the elementary unit of excitation, it is logical to begin characterization of the cardiac electrical sources by first formulating the elementary source associated with a single cell. The cellular action potential and the currents and membrane processes that are involved in its generation are discussed extensively in other chapters of this volume. Because the focus here is on the cell as a source of extracellular potential rather than on membrane mechanisms of excitation, our discussion is limited to a brief summary of only the major ionic currents that

FIGURE 1 A cardiac ventricular action potential (A) and major membrane ionic currents that determine its shape (C–G). The calcium transient during the action potential is also shown (B). The fast kinetics and large amplitude of the sodium current INa (C) result in the upstroke of the action potential. The inward calcium current ICa(L) (D) supports the action potential plateau against the repolarizing outward potassium current IK [E; IK is known as the delayed rectifier current and is composed of IKr (‘‘rapid’’) and IKs (‘‘slow’’)]. During the late repolarization phase, a large increase in IK together with a sharp late peak of IK1 (the time-independent potassium current) repolarize the membrane back to the rest potential (the late IK1 peak generates the late Iv peak seen in F; Iv is the sum of all time-independent currents lumped together for the purpose of presentation). The sodium– calcium exchanger INaCa (G) contributes a depolarizing inward current during this phase, acting to slow repolarization and prolong the action potential duration. Data from Luo and Rudy (2) and Zeng et al. (3), with permission.

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current, IK , composed of two components Ikr (rapid) and Iks (slow); and a fast repolarization phase, which involves both time-dependent (IK) and time-independent (IK1) outward potassium currents and an inward depolarizing current carried by the Na⫹ –Ca2⫹ exchanger, INaCa . The action potential is the transmembrane potential during excitation, defined as Vm ⫽ ⌽i ⫺ ⌽o where ⌽i is the intracellular potential and ⌽o is the extracellular potential immediately adjacent to the cell membrane. Consider a single cell immersed in a uniform conducting medium (Fig. 2). Because the plasma membrane is ex˚ ), it can be treated as a mathematitremely thin (앑75 A cal surface of zero thickness that separates the intracellular and extracellular domains. Two conditions must hold across this surface: ⌽ i ⫺ ⌽o ⫽ Vm ⬆ 0

(1)

(the potential difference is the transmembrane potential Vm , which is clearly not identically zero, and, in fact, varies with time during excitation, as shown in Fig. 1A) and

␴i

⭸⌽i ⭸⌽o ⫽ ␴o ⭸n ⭸n

(2)

(the current density normal to the surface is continuous, no current is lost or gained in crossing the surface). We now define the scalar function ⌿ (5) ⌿ ⫽ ␴⌽

(3)

where ⌽ is the desired potential and ␴ is the piecewise constant conductivity function, ␴ ⫽ ␴i intracellularly and ␴ ⫽ ␴o extracellularly. Because ⌽ satisfies Laplace’s equation in source-free intracellular and extracellular domains, so does ⌿. Expressing Eqs. (1) and (2) in terms of ⌿ as defined in Eq. (3) gives ⌿i ⫺ ⌿o ⫽ ␴i⌽i ⫺ ␴o⌽o ⬆ 0

(4)

FIGURE 2 A schematic representation of the single cell in a volume conductor. ␴i and ␴o are the intracellular and extracellular conductivities, respectively. ⌽i is the intracellular potential, and ⌽o is the extracellular potential immediately adjacent to the cell membrane. The transmembrane potential Vm is given by Vm ⫽ ⌽i ⫺ ⌽o .

and ⭸⌿i ⭸⌿o ⫽ ⭸n ⭸n

(5)

In other words, ⌿ is discontinuous across the cell membrane (surface S) while its normal derivative is continuous. This behavior identifies the surface S as the location of a double layer of strength equal to the discontinuity in ⌿ [i.e., the strength of the double layer is given by ជ␶ ⫽ (⌿i ⫺ ⌿o)nˆ] (5) [Comment: A double layer is a surface distribution of dipole sources. A dipole is a source-sink pair separated by a short distance. The reader is referred to Jackson (6) for a review of these concepts.] The potential field, ⌿, generated by this dipole source distribution is (6) ⌿⫽⫺

1 4앟

冕 (⌿ ⫺ ⌿ )ⵜ 冉1r冊 ⭈ dsជ i

S

o

(6)

where r is the distance from source to field point, ⵜ is the gradient operator, and integration is performed over the entire cell surface, S. Replacing ⌿ by ␴ ⌽ according to Eq. (3) and restricting the field points to the extracellular domain (i.e., ␴ ⫽ ␴o at the field points), we obtain the desired expression for the extracellular potential field, ⌽o , generated by an active cell: ⌽o ⫽ ⫺

1 4앟␴o

冕 (␴ ⌽ ⫺ ␴ ⌽ )ⵜ 冉1r冊 ⭈ dsជ i

S

i

o

o

(7)

In this expression (␴i⌽i ⫺ ␴o⌽o)dsជ constitutes a dipole element in a small membrane area ds. Integration over the entire cell membrane, S, provides the potential generated by the entire cell. Equation (7) identifies the source of the extracellular field arising from a single active cell to be a double layer located in the cell membrane. The strength of the double layer, ␴i⌽i ⫺ ␴o⌽o , is determined by intracellular and extracellular conductivities and the transmembrane potential. This source is no longer identified with ionic currents within the membrane. It serves as an equivalent source that can be used to obtain [through Eq. (7)] the potential field in the extracellular domain. An alternative expression to Eq. (7) can be derived by noting that the quantity ⫺ⵜ(1/r) ⭈ dsជ is an element of solid angle d⍀ so that Eq. (7) can be written: ⌽o ⫽

1 4앟␴o

冕 (␴ ⌽ ⫺ ␴ ⌽ ) d⍀ S

i

i

o

o

(8)

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[Comment: Definition of a Solid Angle: The solid angle, ⍀, is proportional to the extent of opening of the cone that is formed by connecting all points on the boundary of the double layer, S, with the field point, P. Precisely, the solid angle is defined as the area intercepted on a unit sphere, centered at the field point, by this cone (Fig. 3).] For a cell at rest, the transmembrane potential Vm ⫽ ⌽i ⫺ ⌽o is constant over the entire cell membrane, which constitutes a closed surface. Because ␴i and ␴o are constants, the double-layer strength, ␴i⌽i ⫺ ␴o⌽o , is also constant (uniform) over the cell surface. Therefore, the double-layer strength term can be removed from under the integral sign to give ⌽o ⫽ ⫽

1 (␴i⌽i ⫺ ␴o⌽o) 4앟␴o

冕 d⍀ S

1 (␴i⌽i ⫺ ␴o⌽o)⍀ ⫽ 0 4앟␴o

since, for exterior points, the total solid angle subtended by a closed surface (the entire cell membrane in this case) is zero. Hence, a cell at rest does not contribute to the extracellular potential field, as expected. Similarly, when the cell is fully depolarized (at the plateau phase of Vm , see Fig. 1), Vm is constant (to a good approximation) on the entire cell membrane and so is ␴i⌽i ⫺ ␴o⌽o . Therefore, a fully depolarized cell does not contribute significantly to the extracellular potential as well. It follows that a substantial contribution to the external potential field from a given cell occurs only during the rapid upstroke of the action potential and during the repolarization phase. In the electrocardiogram, QRS reflects the contribution of depolarization, whereas repolarization is manifest in the T wave.

FIGURE 3 A solid angle, ⍀, associated with a surface, S, and an observation point, P. The area intercepted on a unit sphere is shown by the shaded area.

B. Sources Associated with Ventricular Activation During normal (sinus rhythm) activation of the heart, ventricular excitation is initiated at many subendocardial sites more or less simultaneously as a consequence of the anatomy (geometrical arrangement) and fast conduction velocity of the His–Purkinje system. Once excitation in the working myocardium is initiated, it spreads from cell to cell by means of current flow through gap junctions, which provide low-resistance pathways between neighboring cells (7, 8). The distribution and properties of gap junctions (9) and the geometrical arrangement of the cardiac fibers in the myocardium (10) are such that the resistance to intracellular current flow is higher in the direction perpendicular to the fibers than along the fibers; a typical resistivity ratio is 9:1 (11, 12) (cardiac fibers are elongated structures so that a longitudinal axis and a transverse axis can be defined). As a result of this antisotropic property, excitation propagates at a higher velocity in the direction of the fibers than across fibers [a 3:1 velocity ratio is typical (12–14)]. The fibrous architecture of the heart is such that the cardiac fibers are parallel to the endocardial and epicardial surfaces (15, 16). The two factors discussed earlier (i.e., the Purkinje system and the anisotropic conduction velocity) lead to the formation of an activation front that is generally uniform and parallel to the endocardial surface. This activation front propagates (in both left and right ventricles) from endocardium to epicardium (17). An interesting (and somewhat paradoxical) observation is that propagation of the activation front proceeds across fibers, i.e., in the direction of slow conduction. Other observations regarding the sequence of normal excitation relate to the termination of activation; the latest parts to be activated are (1) the posterobasal area or the posterolateral area of the left ventricle and (2) the pulmonary conus and the posterobasal area in the right ventricle [the reader is referred to Durrer et al. (17) for a detailed description]. As discussed later, the activation fronts are loci of the electrocardiographic sources during the excitation process. Electrically these sources can be characterized as double layers. For a typical propagation velocity of 50 cm/sec and a rise time of 1 msec for the rising phase of the action potential, the spatial extent of the rising phase is (1 msec) ⭈ (50 cm/sec) ⫽ 0.5 mm (a schematic description is provided in Fig. 4). For each cell within this active region, the strength of the double-layer sources in the membrane is not uniform (as Vm is not constant) and the integral expression (8) yields a finite (nonzero) contribution to the external potential. By superposition, the individual cellular double layers in this region of

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FIGURE 4 Schematic representation of equivalent electrical sources associated with an action potential. (A) Idealized spatial action potential, propagating in the positive x direction. (B) Magnitude (absolute value) of the gradient of Vm , which is proportional to the volume dipole source density. Note that the source density is zero during the (idealized) plateau phase and that the source density is more confined in space and of much higher magnitude during depolarization (QRS) than during repolarization (T wave).

activation can be combined and replaced by an average dipole moment per unit volume. Because the extent of the rising phase is small compared with wall thickness, it can be regarded as a two-dimensional surface of activation (isochrone) that constitutes a double layer. A formal description of this averaging procedure can be obtained (18) by redefining the membrane discontinuity function, ⌬⌿ ⫽ ⌿i ⫺ ⌿o , to be an average of all values over the dimensions of a cell. With this definition, ⌬⌿ becomes a volume function that exists at all points and is not restricted to cell surfaces. As a result of this property, the divergence theorem (16) can be used to convert the surface integral [Eq. (6)] to a volume integral: ⌿⫽⫺

1 4앟

冕 ⵜ · 冋(⌿ ⫺ ⌿ )ⵜ 冉1r冊册 dv i

v

o

(9)

Using the vector identity

ជ) ⫽ A ជ · ⵜ⌽ ⫹ ⌽ⵜ · A ជ ⵜ · (⌽A and noting that r ⬆ 0 for extracellular field points so that ⵜ 2(1/r) ⫽ 0, we obtain ⌿⫽⫺

1 4앟

冕 ⵜ(⌿ ⫺ ⌿ ) · ⵜ 冉1r冊 dv i

v

o

(10)

Using the definition of ⌿ [Eq. (3)], ⌽o ⫽ ⫺

1 4앟␴o

冕 ⵜ(␴ ⌽ ⫺ ␴ ⌽ ) · ⵜ 冉1r冊 dv v

i

i

o

o

(11)

where integration is over the volume of cardiac tissue. We can now define

ជJ ⫽ ⵜ(␴i⌽i ⫺ ␴o⌽o)

(12)

which is identified as the (space-averaged) volume dipole moment source density. As stated previously, during activation this volume density can be approximated by a surface density (double layer) associated with the activation fronts in the heart. Equation (12) relates the distribution and strength of the cardiac sources to the spatial behavior of intracellular (⌽i) and extracellular (⌽o) potentials. With certain simplifications it could be related directly to the transmembrane action potential, Vm . If ␴i ⫽ ␴o or if the extracellular space is extensive (i.e., ⌽o Ⰶ ⌽i and Vm ⫽ ⌽i ⫺ ⌽o 앒 ⌽i), then

ជJ ⫽ ␴iⵜVm Note that ⵜVm ⫽ 0 and the source density is zero in regions where Vm does not vary spatially (i.e., where cells are at rest or in an idealized plateau state). These regions do not contribute, therefore, to the extracellular potential field. In contrast, Vm is steep and ⵜVm is large, implying a large source density, in regions that are being activated (rising phase of the action potential) and that constitute the activation front (Fig. 4). During the repolarization phase, Vm does vary spatially; however, ⵜVm during this phase is much smaller (앑100 times smaller) than during the activation phase, as the change in Vm is much more gradual. Therefore, recovery is associated with a source density that is much smaller than the source density associated with the activation process. Note, however, that the activation sources are concentrated in a shell that is about 0.5 mm thick, whereas the recovery sources are widely distributed. As a result, the total source strength is roughly of the same order of magnitude for both processes. A diagramatic illustration of double-layer activation fronts during normal ventricular excitation is provided in Fig. 5. The double-layer strength is assumed uniform on the entire front, and its dipole components are perpendicular to the activation front, pointing toward the resting tissue. As will be discussed later, this uniform double-layer model is an adequate representation of the sources during normal activation. Solid angles associated with these activation fronts, as viewed from three selected field points (‘‘electrodes’’) on the body surface, are also shown. Note that the potential magnitude at each ‘‘electrode’’ is proportional to the solid angle, which reflects the extent of the activation front, its orientation relative to the field point, and its proximity to the field point. Polarity of the body surface potentials is also indicated in Fig. 5; the potential ahead of the advancing wave front is positive (⫹), whereas behind it the potential is negative (⫺). While extremely helpful in the interpretation of ECG potentials measured on the body surface, this principle should be used cautiously. Realistic cardiac activation patterns may involve several

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I. Pumping Action and Electrical Activity of the Heart

FIGURE 5 Schematic description of broad double-layer fronts during normal activation of the right ventricle (RV, A) and of the left ventricle (LV, B). Polarity of poential and solid angles are shown for three torso sites (‘‘ECG electrodes’’). Note that polarity (plus or minus) reflects orientation of the wave front relative to the electrode (plus reflects an approaching front whereas minus reflects a receding front). The potential magnitude is proportional to the solid angle.

simultaneous wave fronts. The potential is a superposition of contributions from all wave fronts and reflects their complex three-dimensional geometry. In addition, for certain activation patterns (e.g., point stimulation), the double layer cannot be considered uniform (see later), and the potential ahead of some sections of the wave front may in fact be negative. Another illustration of the usefulness of the uniform double-layer model for interpreting body surface potentials during normal activation is provided in Fig. 6. Figure 6 depicts the well-characterized event of right ventricular (RV) breakthrough (arrival of the wave front at the epicardial surface of the right ventricle). Prior to breakthrough (Fig. 6A), the potential on the body surface region that overlies the RV free wall (sternal area) is positive, due mostly to the proximity of the wave front advancing toward the breakthrough point. Once breakthrough has occurred, a ‘‘window’’ is formed in the front (Fig. 6B) and the sternal region above the

FIGURE 6 Schematic description of double-layer activation fronts in the right ventricle. (A) Prior to breakthrough. (B) Immediately after breakthrough. A plus and minus indicate polarity of potential on the torso surface. The breakthrough ‘‘window’’ that is formed in the activation front is reflected as a local negative minimum (indicated by a minus) in the surface potential.

breakthrough point is influenced mostly by the receding wave fronts in the free wall and in the septum. The result is the appearance of a local minimum in this region (Fig. 6B). The sudden appearance of an intense and highly localized minimum in the upper sternal area, reflecting the occurrence of RV epicardial breakthrough on the body surface, has been observed consistently in body surface potential maps recorded from normal human subjects (19). Equation (12) provides an expression for the cardiac sources in terms of intracellular and extracellular potentials and conductivities. This expression is general and does not require that the myocardium behaves isotropically on a maroscopic scale. During normal ventricular excitation, broad activation fronts are formed early in the process and propagate from the endocardium to epicardium in a direction that is perpendicular to the orientation of the myocardial fibers. On a macroscopic scale, this implies that all parts of the activation fronts propagate in a direction of low conductivity and slow velocity, i.e., in Eq. (12) ␴i and ␴o in the direction of propagation are constant (or close to constant) everywhere on the wave front. For normal excitation, therefore, the source density ជJ [Eq. (12)] is approximately constant on a given wave front and the equivalent macroscopic cardiac sources can be represented to a good approximation as uniform double layers (20). The situation is entirely different when one considers the activation front produced by point stimulation (e.g., activation from an ectopic focus). For this condition the wave front is nearly ellipsoidal, with its major axis along the fiber direction (the direction of high conductivity and fast velocity) and its minor axes perpendicular to the fiber direction (the direction of low conductivity and slow velocity; Fig. 7A). It is clear that different sections

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of the wave front ‘‘see’’ different conductivities in the direction of propagation, with the extremes being along (highest conductivity) and perpendicular (lowest conductivity) to the fiber direction. The conductivities in Eq. (12) vary with the position on the wave front and the double-layer source density is no longer uniform. An analysis of the effects of myocardial anisotropy on the strength of the electrical sources can be found in Plonsey and Rudy (21). An equivalent source representation that incorporates myocardial anisotropy is the oblique dipole-layer model (22, 23). The model consists of a double layer that is situated on the wave front and that can be viewed as the superposition of axial (along fiber direction) and transverse (perpendicular to fiber direction) component dipole densities, denoted as ml and mt , respectively. In general, the combination of axial and transverse components results in a dipole layer whose dipoles are oblique to the activation front (unlike the uniform double layer where the dipoles are normal to the front). The potential is the superposition of contributions from axial and transverse components: ⌽o ⫽

1 4앟␴o

冕 [m (nˆ · aˆ )aˆ ⫹ m (nˆ · aˆ )aˆ ] · ⵜ 冉1r冊 ds S

l

axial

l

l

t t t transverse

(13)

In this expression, nˆ is a unit vector normal to the wave front and aˆl and aˆt are unit vectors parallel and perpendicular to the local fiber direction, respectively (all unit vectors point toward the tissue at rest). Note that when the front is parallel to the fibers (propagation is across fibers) nˆ ⭈ aˆl ⫽ 0, aˆt ⫽ nˆ, and the potential is due to a uniform double layer of density mt that is normal to the front and to the fiber direction (this is the case during normal ventricular excitation). When the front is perpendicular to the fibers (propagation is along the fiber direction), nˆ ⭈ aˆt ⫽ 0, aˆl ⫽ nˆ, and the potential is due to a uniform double layer of density ml that is normal to the front and along the fiber direction. Colli-Franzone et al. (22) showed that the ratio ml /mt equals the ratio of the intracellular axial and transverse conductivity coefficients, ␴ li / ␴ ti , which is typically 9:1 [in fact, ml /mt ⱖ15 was required to reproduce the measured potential fields in Colli-Franzone et al. (22)]. This implies that for point stimulation the maximum axial contribution (at the section of the ellipsoidal wave front that propagates along the fibers) is an order of magnitude greater than the maximum transverse contribution (at the sections of the wave front that propagate across fibers), suggesting that the source can be approximated (for the purpose of potentials sufficiently far from the activation front) by two equal and opposite axial dipoles, located on the long axis of the ellipsoidal wave front (see Fig. 7A). An alternative (and equivalent) form of Eq. (13) is derived in Colli-Franzone et al. (22) by defining ma ⫽

ml ⫺ mt and mu ⫽ mt (the subscripts a and u denote axial and uniform, respectively): ⌽o ⫽

1 4앟␴o

冕 [m nˆ ⫹ m (nˆ · aˆ )aˆ ] · ⵜ 冉1r冊 ds u S uniform normal

a

l

l

(14)

axial

This form represents the source as a superposition of a uniform double layer that is normal to the activation front and a nonuniform, axial double layer. From this perspective the oblique dipole layer model generalizes the classical, uniform double-layer model by adding a nonuniform, axial component to it. As discussed earlier, the uniform double-layer model provides the basis for the solid angle theory of electrocardiography. Note that the influence of the axial component decreases as the front surface becomes more parallel to the fiber direction as nˆ ⭈ aˆl decreases. The situation is realized by extensive wave fronts, such as those during normal ventricular activation (Fig. 5), and use of the uniform double-layer model and of the solid angle theory is justified under these conditions. In contrast, during early ectopic activation the axial component dominates (22), again suggesting that a simplified representation of the source by two axial dipoles may be used to provide a qualitative description of the potential field. Note that for a closed wave front the contribution of the uniform component is zero as the solid angle subtended by a closed surface at any exterior point is equal to zero. For this situation, the entire contribution to the potential is from the axial component in Eq. (14). An example of the potential field generated by point stimulation is shown in Fig. 7B. The pattern is characterized by a central negative area and two maxima that are positioned on a straight line that correlates well with the direction of the fibers near the pacing site. Such a pattern reflects the presence of a dominant axial source component in the fiber direction; in fact, similar potential patterns were generated experimentally by two dipoles oriented at 180⬚ along a straight line (24), validating the usefulness of the simple model in Fig. 7A for a qualitative interpretation of the potentials associated with ectopic activation. A fascinating consequence of this source distribution is the counterclockwise rotation of the epicardial potential pattern (i.e., of the straight line connecting the potential maxima) with increasing intramural depth of stimulation relative to the epicardial surface (25). This rotation reflects the intramural rotation of the fiber direction (16), which implies a similar rotation of the axial component of the dipole layer source. We conclude the discussion of electrocardiographic cardiac sources by reemphasizing that all dipole layer formulations assume the activation front to be a twodimensional surface (i.e., the thickness of the front is small compared to its distance to the field point). This

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I. Pumping Action and Electrical Activity of the Heart

implies that such models are not suitable for studying the potential in the immediate vicinity of the front. An intracellular current model (13) that does not assume the sources to be restricted to the wave front surface may be more suitable for this purpose. Obviously, an accurate, quantitative description of the field in the vicinity of the front requires a realistic model of the membrane currents and of the anisotropic intracellular– extracellular (‘‘bidomain’’) structure of the myocardium. However, for the purpose of relating cardiac activity to electrocardiographic potentials in regions that are sufficiently remote from the front, particularly outside the myocardium and on the body surface, the dipole layer representations of myocardial sources are extremely useful.

III. THE TORSO VOLUME CONDUCTOR The electrical activity of cardiac muscle cells is projected to the surface of the torso by means of the intervening conducting medium. The surface potentials that are recorded as electrocardiograms reflect, therefore, the properties of both the heart electrical generators (discussed in the previous section) and the surrounding passive volume conductor. Because the goal of electrocardiography is to reconstruct cardiac electrical events from body surface potential data, understanding the role played by the torso volume conductor in determining the surface potential distribution is essential. This section provides a short summary of the electrical properties of the torso volume conductor and their effects on electrocardiographic body surface potentials. A more detailed presentation of this material can be found in Rudy (26).

A. Quasi Static Approximation Although the bioelectric sources within the myocardium are time varying, most of the models that describe the potential fields generated by these sources in the surrounding volume conductor are static. In fact, they consider the spatial distribution of the sources at a certain instant of time and solve for the potential distribution assuming steady-state conditions. The justification for this simplified representation of the electrophysiological system has been considered by Plonsey and Heppner (27). Their analysis demonstrates that for the physiological range of frequencies and the typical dimensions of the human body, capacitive effects, propagation effects, and inductive effects can be neglected to a good approximation. Therefore, the medium can be considered as purely resistive and the electrocardiographic problem can be treated at any instant of time

as if steady-state conditions were in effect, i.e., at each instant of time the potential field in the torso volume satisfied Poisson’s equation ⵜ 2⌽ ⫽ ⵜ ⭈ ជJ / ␴, where ⌽ is the potential, ␴ is conductivity of the medium, and ជJ represents cardiac electrical sources. Outside the heart ជJ ⫽ 0 and ⌽ satisfies Laplace’s equation ⵜ 2 ⌽ ⫽ 0.

B. Torso Inhomogeneities The torso volume conductor is inhomogeneous and contains regions with different conductivities. The heart consists of blood cavities (high conductivity) bounded by the myocardium whose conductivity is about onethird that of blood. The heart structure is enveloped by the very extensive lung region, which is a poor conductor. Surrounding the lungs is the high conductivity skeletal muscle layer, which in turn is surrounded by low conductivity subcutaneous fat. The presence of regions with different conductivities modifies the electrocardiographic field through secondary sources that arise at the interfaces between these regions (28, 29). Based on experimental and modeling studies summarized in Rudy (26) and Gulrajani (30) several important observations can be made regarding the effects of torso inhomogeneities on ECG potentials: (1) The inhomogeneities do not add complexity to the general pattern of the body surface potential distribution; multipeaked surface potentials reflect nondipolarity in the cardiac electrical source itself rather than influences of the inhomogeneous torso volume conductor. (2) The torso volume conductor acts to smooth the body surface potential distribution and to reduce its spatial resolution; this smoothing effect is illustrated in Fig. 8. (3) An integrated effect of all inhomogeneities is an augmentation of body surface potential magnitudes; augmentation is caused by the intracavitary blood and (for anterior points on the torso) by the lungs. (4) The skeletal muscle layer acts to attenuate body surface potentials and is an important contributor to the smoothing effect of the volume conductor.

IV. EMERGING NEW APPROACHES TO ELECTROCARDIOGRAPHY: ECGI The goal of electrocardiography is to noninvasively characterize the electrical function of the heart from potentials measured on the body surface. The limitations of standard electrocardiographic techniques (i.e., ECG and VCG) were mentioned in Section I. These techniques sample body surface potentials at a small subset of points. As discussed earlier, cardiac electrical activity is a process that is distributed spatially throughout the myocardium. However, the standard techniques

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7. ECG and Cardiac

are very limited in their ability to provide information on regional electrical activity and to locate bioelectric events (e.g., foci of arrhythomogenic activity) in the heart. In fact, VCG lumps all cardiac sources in a single dipole, located at the ‘‘center’’ of the heart (the socalled heart vector). With recent advances in electronics and computers, simultaneous potential recording from many (100 to 250) torso sites has become practical and inexpensive (31). The resulting body surface potential maps (BSPMs) over the entire torso surface constitute the complete body surface ECG and contain information that is not recorded by conventional limited-lead ECG (32–36). However, even the BSPM provides only a very low resolution projection of the cardiac electrical activity as reflected on the body surface (37, 38). In contrast, potential distributions over the epicardial surface of the heart accurately mirror details of electrophysiological processes within the myocardium with high resolution (37–42). As a result, mapping of potentials directly from the epicardium has become an important experimental tool in studying cardiac arrhythmias (43). It has also become an important clinical tool for the localization of arrhythmogenic foci or of key components of a reentry circuit (e.g., the common pathway in a figure eight reentry) prior to surgical ablation (43, 44). With the increasingly widespread use of nonpharmacological antiarrhythmic interventions (e.g., ablation), there is a growing need for fast and precise localization of such electrocardiac events. It is highly desirable, therefore, to develop a true electrocardiographic imaging modality (ECGI) for the noninvasive reconstruction of epicardial potentials from BSPM data. Another important potential use of ECGI is in the area of identifying patients at risk of cardiac arrhythmias and sudden death so that preventive measures (antiarrhythmic drugs or implantable defibrillators) can be administered. Invasive methods that are currently used (i.e., EP catheterization studies) for evaluation of risk and of the efficacy of therapy are too expensive and risky for widespread application. Empirical body surface ECG indices [T-wave alternans (45, 46), dispersion of QT intervals (47), late potentials (48)] have been used with mixed results. This is not surprising, as the ECG provides a remote, integrated measure of complex cardiac events. For example, QT dispersion is determined by taking the differences between individual body surface ECG leads. Because each body surface lead reflects activity in the entire heart, this measure cannot be related to actual spatial heterogeneity of repolarization in the heart itself, which is a recognized arrhythmogenic property. ECGI could open the possibility of ‘‘recording’’ noninvasively from the heart, thereby increasing detection sensitivity and providing information

about the actual spatial heterogeneity in the heart itself, including location of regions that contribute to increased dispersion (and, therefore, to arrhythmogenesis). Advances in molecular biology and cellular electrophysiologic techniques have made it possible to relate a clinical arrhythmogenic condition to its genetic basis and to structure–function alterations in a specific ion channel (the long QT syndrome provides such an example) (49–53). This opens the exciting possibility of targeting therapy specifically toward the abnormal channel (54–56). An intervention with such a high degree of specificity will require diagnostic tools that are both highly sensitive and very specific. These developments and the exciting future of molecular medicine provide yet another motivation for the development of ECGI as a functional imaging modality for noninvasive determination of the electrophysiologic state of the heart. This section provides a brief description of ECGI methodology and shows selected examples of ECGI reconstructions performed in our laboratory.

A. Methodology The following is a brief description of the mathematical procedure used to compute epicardial potentials from body surface potentials. Details can be found elsewhere (57, 58, 59–64). The electric field generated by the beating heart within the torso volume is governed by Laplace’s equation (6, 30, 65). ECGI noninvasively computes the electrical potentials on the surface of the heart by solving Laplace’s equation within the torso volume using noninvasively recorded torso surface potentials and the geometric relationship between epicardial and torso surfaces as inputs. Discretization of Laplace’s equation is performed through the use of the boundary element method (BEM) (66), resulting in the following linear matrix relationship: VT ⫽ AVE

(15)

where VT and VE are vectors of torso surface and epicardial potentials, respectively. The matrix A provides the geometric relationship between epicardial and torso surfaces. A forward solution of Eq. (15) to obtain torso surface potentials (VT) based on knowledge of the epicardial potentials (VE) and the geometric relationship (A) is stable and accurate. This means that given the geometric relationship between the heart and torso and a set of epicardial potentials, the body surface potentials could be computed with very high accuracy (67). The goal of ECGI, however, is to compute a solution to the inverse problem associated with Eq. (15), i.e., to compute epicardial potentials (VE) from measured torso potentials (VT). This procedure requires inversion of the large matrix A, a process that is ill-posed (68) and

I. Pumping Action and Electrical Activity of the Heart

A

B

n

+

-

S

ctio

To illustrate the usefulness of ECGI, we provide an example of reconstructed epicardial potentials, electrograms, and activation sequences during pacing from single or dual epicardial sites (73). The experiments and data collection were performed at the University of Utah in Dr. Taccardi’s laboratory using a torso-tank experimental setup. This setup consists of a tank in the shape of a 10-year-old boy’s torso, with a perfused dog heart (connected to the circulation of a support dog) suspended in the correct anatomical position within the torso. Torso surface potentials are measured with 384 electrodes and are used to compute epicardial potentials noninvasively. Epicardial potentials are measured with 144 electrodes and provide a ‘‘gold standard’’ for evaluation of noninvasively reconstructed epicardial potentials through direct comparison. The early epicardial potential pattern associated with epicardial pacing is characterized by a central negative region (approximately elliptical) surrounding the pacing site, flanked by one or two positive maxima (25). A line drawn through the maxima and the center of the

ire

B. Examples of Noninvasively Reconstructed Epicardial Potentials, Electrograms, and Isochrones

negative region reflects the local epicardial fiber orientation (Fig. 7). The pacing site is located at the center of the quasi-elliptical negative region. Figure 8 provides examples of ECGI reconstructions for single-site pacing (plate 1) and for dual-site pacing with progressively smaller intersite distances (plates 2 and 3). Torso potentials are shown on the left of each plate and provide the input for ECGI. Epicardial potentials, computed noninvasively from the torso potentials, are shown on the bottom right, and measured epicardial potentials are provided as ‘‘gold standard’’ for comparison on the right top (measured and reconstructed pacing sites are marked by asterisks). For single-site pacing (plate 1), the minimum surrounding the pacing site is reconstructed with good accuracy. In addition, maxima flanking the minimum are present in both measured and computed epicardial maps. The reconstructed pacing site is approximately 7 mm from its measured position. The two maxima are reconstructed 20 and 28 mm from their measured locations. Plate 2 shows potential distributions for two simultaneous pacing sites (anterior and posterolateral left ventricle, 52 mm apart). The measured epicardial potential maps contain two negative areas that reflect the two sites of early activation. The torso potential contains only a single minimum, does not reflect the existence of two initiation sites, and obviously does not indicate their positions on the heart. However, using torso data to reconstruct the epicardial potential (right, bottom row), both epicardial minima are reconstructed as approximate negative ellipses, and the position errors of their center points (pacing sites) relative to the measured ones are 7 and 4 mm. Plate 3 shows potentials associated with two pacing sites that are only 35 mm apart. The torso potentials look almost identical

rD

very sensitive to small errors in the recorded potentials and geometry. This difficulty is overcome by imposing physiological constraints on the epicardial potential solutions, accepting only solutions that are consistent with known electrophysiologic principles and properties of cardiac electric fields. Such properties include bounds on the amplitudes or derivatives of the epicardial potentials in space (57–62, 64, 69, 70), in time (58, 63), or in both space and time (71, 72). We have introduced such constraints through the use of Tikhonov regularization (68) to obtain stable and close estimates of the epicardial potentials. ECGI implemented using these methods requires knowledge of potentials over the torso surface and of the geometric relationship between the torso and the heart. This information can be obtained noninvasively: torso potentials using a multielectrode mapping system and geometry using noninvasive imaging modalities such as CT, MRI, or biplane X-ray. Once epicardial potential maps are reconstructed for all times during the cardiac cycle, epicardial electrograms (potential over time) can be generated at various locations on the epicardial surface. Local activation time is determined at each location as the time of maximum negative dV/dt (‘‘intrinsic deflection’’) in the electrogram. Epicardial activation sequences are constructed from these activation times and are displayed as isochrone maps.

Fib e

142

+ FIGURE 7 (A) Hypothetical isochrone early after epicardial pacing from site S, with the approximate source configuration of two opposite axial dipoles oriented along fibers. (B) The corresponding epicardial potential distribution, showing a quasi-elliptical center region of negative potential flanked by two potential maxima (marked by plus signs). This pattern is aligned with the fiber direction. Negative potential contours are drawn with dashed lines; positive with solid lines.

7. ECG and Cardiac

FIGURE 8 Each plate shows measured torso potentials (left, anterior and posterior views), measured epicardial potentials (right, top), and noninvasively computed epicardial potentials (right, bottom). Positive potential contours are drawn with solid lines; negative with dashed lines. The gray scale of potential magnitudes (in microvolts) is provided. Maxima are identified with pluses, minima with minuses, and pacing sites with asterisks. Potentials correspond to 20 msec (plates 1 and 2) or 13 msec (plate 3) from the pacing stimulus. (Plate 1) Single anterior pacing site. (Plate 2) Dual pacing sites, 52 mm apart. (Plate 3) Dual pacing sites, 35 mm apart. From Oster et al. (73), with permission.

143

144

I. Pumping Action and Electrical Activity of the Heart

FIGURE 9 Epicardial electrograms (measured on left, noninvasively computed on right) from various locations on the epicardium for single-site pacing. (A) An asterisk identifies the pacing site; numbers identify the locations of the nine electrograms displayed in B–D. (B) Monophasic (Q-wave) electrograms from sites 1,2, and 3. (C) Biphasic electrograms from sites 4,5, and 6. (D) Monophasic (R wave) electrograms from sites 7,8, and 9. CC is the cross-correlation between computed and measured electrograms. From Oster et al. (73), with permission.

7. ECG and Cardiac

to those of the single pacing site in Plate 1. The reconstructed epicardial potentials, however, resolve both minima distinctly in their exact locations. An even more challenging test of the method [not shown in Fig. 8; see Fig. 7 in Oster et al. (73)] is two posterolateral pacing sites (located in a region of the heart that is remote from the torso surface) that are very close together (intersite distance of only 17 mm). ECGI reconstructs both pacing sites distinctly with position errors of approximately 5 and 4 mm. Again, body surface potentials show only one minimum, failing to reflect these two distinct pacing sites.

C. Reconstruction of Epicardial Electrograms Epicardial potential maps display the potential distribution over the epicardial surface at one instant of time. Electrograms (potential over time at a given site) provide information on the temporal progression of activity in localized areas and are used extensively in clinical electrophysiology (EP) studies. Figure 9 demonstrates the ability to reconstruct epicardial electrograms noninvasively. Sample electrograms are displayed for sites close to (1, 2, and 3; Fig. 9B), partially away from (4, 5, and 6; Fig. 9C), and far away from (7, 8, and 9; Fig. 9D) the pacing site (asterisk in Fig. 9A). Three types of waveforms—monophasic negative (B), biphasic (C), and monophasic positive (D)—are reconstructed. In

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each panel, both measured (left) and reconstructed (right) electrograms are displayed. Notice the close resemblance of noninvasively reconstructed electrograms as compared with measured epicardial electrograms. Cross-correlations (CC) for the plotted electrograms are printed in Fig. 9 and range from 0.810 to 0.998. Inspecting electrograms from the entire epicardium (not shown), CC is greater than 0.9 for 72% of all epicardial sites (54% with CC ⬎ 0.95).

D. Reconstruction of Epicardial Activation Sequences (Isochrones) An isochrone (equi-time line) connects all sites that are activated at the same time. An isochrone map provides the entire sequence of activation in a single map and is, therefore, of great practical use, especially in the clinical setting. Figure 10 shows noninvasively computed isochrones (bottom) and measured isochrones (top) for single site pacing (same anterior site as in Fig. 8, plate 1). The regions of earliest activation are reproduced in the computed isochrones, matching the measured ones. There is also very good reconstruction of the entire activation sequence. Note that spatial nonuniformities of isochrone density, indicating nonuniformities of activation spread, are reconstructed by the noninvasive approach. For example, in the anterior view there is a region of relatively slow spread (crowded isochrones)

FIGURE 10 Isochrones for the pacing site of Fig. 8, plate 1 (labeled in milliseconds, measured from the stimulus). (Top) Measured isochrones. (Bottom) Noninvasively computed isochrones. Note that the region of earliest activation corresponds to the location of the pacing site (asterisk In Fig 8, plate 1) and that nonuniformities in isochronal density are reconstructed. From Oster et al. (73), with permission.

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between 47 and 69 msec, which is reproduced in the noninvasively computed isochrone map. The examples of Figs. 8 to 10 demonstrate the ability of ECGI to noninvasively reconstruct epicardial potentials, electrograms, and isochrones during epicardial activation initiated by epicardial pacing. However, important electrical events occur within the three-dimensional volume of the ventricular wall. The ECGI approach is not limited to epicardial activation. It was shown to provide information on the depth of initiation sites within the myocardium and on the spatiotemporal propagation of intramural activation as the activation wave front traverses the ventricular wall. These studies have been reported previously, and the interested reader is referred elsewhere (74–76) for a detailed description.

V. SUMMARY It is interesting (and surprising) to note that the electrocardiogram has been used extensively and successfully as a clinical diagnostic tool before the principles of its genesis have been understood. Many of the principles that relate the electrocardiographic potentials to cardiac electrical events have been elucidated in recent years. However, the complete relationship between the ECG and the cardiac electrical excitation process is not yet fully established. Active research is being conducted in many laboratories toward this goal. Very significant progress is being made in elucidating cellular mechanisms at the level of channel function and molecular structure, which will undoubtedly enhance our understanding of the basic biophysical processes underlying the ECG. Mapping studies of activation patterns and potentials are being conducted with increasing resolution and better signal quality. Combined with sophisticated visualization and graphics tools that are now available on computers, this activity will provide a more accurate description of the macroscopic distribution of sources in the heart in relation to the myocardial structure (e.g., anisotropy). A parallel effort involves theoretical computer models of cellular processes and of macroscopic phenomena. As our understanding of the basic processes that generate the electrocardiographic potentials improves, our ability to interpret these potentials and to relate them to cardiac function will be enhanced significantly. If put into practice, this knowledge is likely to completely change the way in which electrocardiography is practiced as a research and clinical tool.

Acknowledgment Thanks to Brenda Hudson for her help in preparing the manuscript.

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8 Gap Junction Channels and Healing-Over of Injury DAVID C. SPRAY,*,† SYLVIA O. SUADICANI,*,‡ MONIQUE J. VINK,* and MIDUTURU SRINIVAS* Departments of *Neuroscience and †Medicine (Molecular Cardiology), Albert Einstein College of Medicine, Bronx, New York 10461 ‡ Universidade Sao Judas Tadeu, Sao Paulo, Brazil

I. INTRODUCTION

studies into current concepts regarding conduction in both normal and pathological heart (see Spooner et al., 1997).

Gap junctions are aggregates of channels extending from one cell to another across extracellular space, thereby allowing the intercellular diffusion of currentcarrying ions and signaling molecules. Like other membrane channels, those forming gap junctions possess mechanisms that regulate their opening and closing (a phenomenon termed ‘‘gating’’) and rapidly change their expression patterns in response to physiological and pathological stimuli. Such flexibility provides the opportunity for global or regional alterations in conduction within the heart and thus provides a substrate for disturbances of cardiac rhythm. Because gap junctions are abundant between the cells of the heart, cardiac tissue has long been regarded as being syncytial. For example, the space constants measured for injected current may correspond to more than 10 cell lengths in ventricular myocardium and may even be twice as long in Purkinje fibers. At a microscopic level, however, the higher resistance of gap junction channels than cytoplasm produces conduction discontinuities in the heart so that action potentials regenerated in each cell are propagated from one cell to the next with tiny, but measurable, delays. Because gap junctions are not distributed uniformly on the myocyte surface and because myocytes are longer than they are wide, the discontinuities at gap junctional contacts give rise to anisotropic conduction. Although most knowledge regarding the physiology and cell biology of cardiac gap junction channels has been determined using cell pairs from dissociated cardiovascular tissues and transfected cell lines, it is an important next step to integrate such

Heart Physiology and Pathophysiology, Fourth Edition

II. CARDIAC GAP JUNCTION PROTEINS A. Ultrastructural Features In the adult mammalian heart, gap junctions are mainly concentrated at the ends of the myocytes, where they are located primarily in intercalated disks (Page and Manjunath, 1986; Luke and Saffitz, 1991). Revel and Karnovsky (1967) initially distinguished two different types of junctions in the intercalated disks of mouse heart. One type (the tight junction) brought the membranes closely into contact, thereby occluding extracellular space; the other (the gap junction) was characterized by a 1.8-nm gap between apposing membranes (Fig. 1A), and in en face views showed the hexagonal subunits. The further application of freeze-fracture techniques to cardiac tissue (McNutt and Weinstein, 1970; Steere and Sommer, 1972) provided additional structural details of gap junction subunits and overall organization. These studies and those of Ernest Page and his group revealed that in freeze fracture the gap junction is composed of closely packed hexagonal arrays of pits on the E face (the internal aspect of the lipid monolayer that is on the outside of the cell) and as particles on the P (protoplasmic) face (see Fig. 1B; Page and Manjunath, 1986). A prominent feature of gap junctions in freeze-fractured or negatively stained material is the high degree

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FIGURE 1 Ultrastructural features of cardiac gap junctions: From thin section (A) and freeze-fracture (B) electron microscopy to projection density maps (C) of gap junctions. (A) High-power electron micrograph of gap junction between rat ventricular myocytes showing the characteristic seven-layered structure and the conspicuous fuzzy coating at both cytoplasmic surfaces. Fixed by vascular perfusion with osmium tetroxide and stained with uranyl acetate and lead citrate (magnification 156 ⫻ 103). From Manjunath et al. (1984), with permission. (B) Electron micrograph of replica of a freeze-fractured gap junction between rat ventricular myocytes. Fixed by vascular perfusion with glutaraldehyde and unidirectionally shadowed with platinum– carbon. The particulate P face and the pitted E face of the junction are seen on the top and bottom sides of the picture, respectively (magnification 134 ⫻ 103). From Page and Manjunath (1986). (C) Projection density maps of rat cardiac gap junction obtained at 0. 7 nm resolution showing symmetrical circular densities that are interpreted as helices lining the channels, 움 helices that are most exposed to lipids (arrow), and two continuous dense bands that presumably represent the other two transmembrane 움 helices. Spacing between grid bars is 4 nm. From Yeager and Nicholson (1996), with permission from Elsevier Science.

of order in the structure (see Fig. 1B). The application of digital image processing techniques to electron microscope images initially resulted in two- and then threedimensional projection maps of structure in which negatively stained and frozen hydrated liver gap junctions were shown to consist of units with six rod-like subunits, each believed to be one connexin molecule. These studies further revealed that hemichannels or connexons project about 2 nm into the extracellular space, accounting for the ‘‘gap’’ of 4 nm between the cells. The highest resolution projection images currently available have been obtained by Mark Yaeger using recombinant Cx43 protein truncated so as to remove most of the carboxyl terminus. At 0.7 nm resolution, the projection density maps of negatively stained material reveal that each connexin has a diameter of 6.5 nm, with the connexin molecules forming each connexon arranged in a hexameric cluster around the channel pore (Fig. 1C; see Yaeger and Nicholson, 1996). The 1.7nm central pore is immediately surrounded by circular densities interpreted as transmembrane domains roughly perpendicular to the membrane, a 3.3-nm ring that is farthest from the pore and closest to the lipid (interpreted as representing one of the other 움-helical transmembrane domains), and a continuous band at 2.5 nm radius that presumably represents the other two transmembrane regions that are not individually resolved. Inner and outer rings of densities are displaced from one another by 30⬚, which is interpreted as providing rotational staggering of connexins so as to increase

interconnexin adhesion (Perkins et al., 1998). In this latter model, connexons interlock tightly across a surface area that is maximized through corresponding convexities and concavities in each connexon subunit.

B. Connexin Multigene Family The first transcript encoding a gap junction protein was identified using antibodies generated against an isolated liver gap junction protein, connexin32 (Cx32), which were used to screen a complementary liver DNA library (Paul, 1986; Kumar and Gilula, 1986; Heynkes et al., 1986). Subsequently, clones encoding Cx43 were identified by screening a rat heart cDNA library with the liver isoform at reduced stringency (Beyer et al., 1987). These initial studies have led to an explosion in sequences determined in mammals: 15 distinct connexins have now been detected in rodents, and for most of these, human isologues are also recognized. The connexin genes have maintained highly conserved nucleic acid and amino acid sequence homology, and in most cases the genomic organization (Fig. 2D) is a hallmark feature of the family. This conservation implies critical constraints on the coding region of the gene to ensure functional protein as well as transcriptional control mechanisms; the virtual uniformity in gene structure implies that a single precursor gene encoding an intercellular channel protein subunit underwent repeated duplications and subsequent mutations during verte-

8. Gap Junction Channels

brate evolution, resulting in the formation of this large multigene family. Each gap junction channel is made of two mirrorsymmetric components contributed by each cell, called connexons or hemichannels (Figs. 2A and 2B; see Bennett et al., 1991; Kumar and Gilula, 1996). Each connexon or hemichannel in turn is made up of six homologous subunits, the connexin molecules (Figs. 2B and 2C). Connexin proteins share common membrane topology; as illustrated in Fig. 2C, each connexin crosses the membrane four times (segments M1, M2, M3, and M4) and has both its amino terminus (NT domain) and its carboxyl terminus (CT domain) on the cytoplasmic

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aspect of the channel (Bennett et al., 1991). Extracellular loops (C1 and C2) are structurally conserved, with three cysteines in each loop positioned identically in all of the known connexins. Presumably this feature accounts for the high-affinity interactions between many (but not all) different connexin molecules such that pairing cells expressing individual connexins may form heterotypic channels from the end-to-end alignment of homomeric connexons. The part of the connexin molecule between M2 and M3 forms a loop or hinge region in the cytoplasm (CL). The third transmembrane domain of connexin molecules is the most amphipathic with charged amino acid residues occurring at every third or fourth

FIGURE 2 Gap junction channels (A) are formed of hemichannels or connexons (B) composed of connexin proteins (C) encoded by connexin genes (D). (A) Schematic drawing of gap junction structure deduced from the classical study applying X-ray diffraction to gap junctions isolated from mouse liver (Makowski et al., 1977, with permission of The Rockefeller University Press). (B) Two connexons dock across extracellular space to form the complete gap junction channel. (C) The connexin protein and its membrane topology: two extracellular loops (C1 and C2), four transmembrane domains (M1, M2, M3, and M4), the intracellular loop (CL, short in group I or 움 subfamily and long in group II or 웁 connexin subfamily), and cytoplasmic amino- and carboxyl-terminal domains (NT and CT). (D, top) Connexin gene (exons E1 and E2, intron IVS) and (bottom) connexin transcript. Transmembrane domains of encoded protein are indicated by dark bars.

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position in an otherwise predominantly hydrophobic sequence; six of these M3 regions are believed to provide the hydrophilic face lining the lumen of the channel [Bennett et al. (1991), Yaeger and Gilula (1992), but see Pfahnl et al. (1996) for evidence that other sequences may also participate in providing the pore-lining part of the molecular assembly]. The regions of greatest divergence among connexins are the cytoplasmic loop (CL) region (connecting M2 and M3) and the carboxyl-terminal tail (CT) extending beyond M4. These latter unique peptide sequences have proven quite useful in generating connexin-specific antibody probes; because isoform sequences are nearly identical for most connexins in mammals, antibodies generated against peptides corresponding to rodent sequences have universally recognized the corresponding human connexin type.

C. Regional Connexin Expression in the Cardiovascular System Three connexins are expressed in the heart (Cx40, Cx43, and Cx45), with overlapping temporal and spatial profiles. Although there is considerable interspecies variation in the types and abundance of different connexins in different regions of the cardiovascular system, general patterns of expression are outlined later and summarized in Table I. Connexin43 is the most abundant connexin expressed in the mammalian heart; it is widely expressed in atrial and ventricular myocytes, in most but not all components of the conduction system, and in smooth muscle and endothelial cells of vessel walls. Although several reports have noted the absence of Cx43 in nodal tissues, immunocytochemistry on human AV node and dog sinus node has detected Cx43 expression in some individual cells (Davis et al., 1995; Kwong et al., 1998). In rodent hearts, Cx43 has been identified as early as 10 days postcoitum in trabeculae and subendocardial regions of developing ventricles, the outflow tract, the interventricular septum, and the endocardial free wall (Gourdie et

TABLE I Distribution of Connexins in Cardiovascular Tissue

SA node Atrium AV node Conduction system Ventricle Vessel wall Smooth muscle Endothelium

Cx37

Cx40

Cx43

Cx45

⫺ ⫺ ⫺ ⫺ ⫺

⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫺/⫹

⫺/⫹ ⫹⫹⫹ ⫺/⫹ ⫹? ⫹⫹⫹

⫹ ⫹⫹ ⫹ ⫹⫹ ⫹

⫺ ⫹⫹

⫹ ⫹⫹

⫹⫹ ⫹⫹

⫺ ⫺

al., 1992; Fromaget et al., 1992). Transcript levels increase substantially between midgestation and early neonatal stages (Fromaget et al., 1992; Fishman et al., 1991) and decline modestly in older animals. Connexin40 appears to be the second most abundant gap junction protein in the heart and cardiovascular system. In the heart, Cx40 is expressed in nodal tissue, in bundles within the conduction system, and in the atrium, whereas in ventricular muscle it is almost completely absent except in the coronary vasculature. In both muscular and elastic arteries, Cx40 expression is prominent in endothelial cells and may also be present in smooth muscle cells. In embryonic mouse heart, Cx40 is widely expressed at atrial and ventricular primordia at 11 days postcoitum; as embryonic development progresses, Cx40 expression is maintained in atrium, whereas its initial ventricular expression becomes confined to the differentiating conduction system (Delorme et al., 1995; Van Kempen et al., 1996). Connexin45 was originally reported to be detected throughout the heart, in most cases colocalizing with Cx43. However, recent use of more specific antibodies has revealed that Cx45 is in fact highly expressed in the conduction system, where it surrounds a core of tissue in which Cx40 is expressed (Coppen et al., 1999).

III. REGULATION OF GAP JUNCTION EXPRESSION, FORMATION, AND DEGRADATION A. Life and Death of Gap Junctions Cardiac gap junction proteins have remarkably short lifetimes. For example, in Langendorff perfused rat hearts, the measured monoexponential decay of radioactivity in immunoprecipitated Cx43 was best fit by a half-life of only 1.3 hr (Beardslee et al., 1998). Thus, in cardiac tissue, Cx43 protein is completely turned over multiple times every day. The rapid turnover of gap junction proteins reemphasizes the possibility that remodeling of communicating compartments might occur over a short period of time and also stresses the importance of understanding the intracellular trafficking events that occur during this time frame (see Fig. 3). Most connexins, including Cx43, appear to be inserted cotranslationally into the endoplasmic reticulum (ER) membrane. Newly synthesized connexins apparently remain as independent monomers as they begin their voyage along the secretory transport route from ER to Golgi apparatus, only becoming hexameric near the ER–Golgi transition (Diez et al., 1998; Falk et al., 1994). It is also unclear how connexons move from the distal Golgi to the junctional membrane; delivery might be random, to anywhere on the cell surface,

8. Gap Junction Channels

153

FIGURE 3 Regulation of gap junction expression and degradation. Steps are indicated by encircled numbers and letters. (1) MRNA is transcribed in the nucleus and translocated to the cytoplasm. (2) Protein is translated and inserted into the endoplasmic reticulum. Mistranslated or misfolded protein is proteolyzed by the proteasome (A). Along the way from ER (2) to Golgi (3), connexons are formed by connexin oligomerization. The connexons are then transported (4) to the cell surface where they meet and dock (5 and 6) with connexons of adjacent cells to form gap junction channels. Removal of gap junctions is through both proteasomal (B) and lysosomal (C) pathways. Modified from Laing et al. (1998).

followed by diffusion until trapped by high-affinity binding to an apposing hemichannel, or connexons might somehow be targeted to junctional plaques. It is possible that interaction with other proteins, such as occurs between Cx43 and the tight junction-associated protein ZO-1, plays such a role, but studies of interactions between connexins and other molecules are only just beginning. Connexins undergo other types of posttranslational modification, including phosphorylation and ubiquitination (Laing and Beyer, 1995). Where these modifications occur is not entirely clear, although Cx43 can be phosphorylated while in junctional plaques and phosphorylation has been suggested to facilitate channel formation (Musil and Goodenough, 1991). Whether such modifications as phosphorylation/dephosphorylation and ubiquitin incorporation into Cx43 provide binding sites for

retrieval of connexin proteins from the membrane and ultimate degradation is unknown. Beardslee et al. (1998) reported that selective pharmacological blockade of either lysosomal and proteosomal routes of degradation results in accumulation of junctional Cx43, indicating that both pathways normally contribute. An intriguing aspect of that study is that proteasomal inhibition caused accumulation of dephosphorylated Cx43, whereas treatment with lysosomal inhibitors increased the amount of phosphorylated Cx43 remaining in junctional membranes, suggesting that the phosphorylation state might provide one signal to direct the degradation pathway. Internalized annular gap junctions have been detected in dissociated cells (Mazet et al., 1985; Larsen et al., 1979; Severs et al., 1989) and more rarely in cells maintained in culture (e.g., Spray et al., 1991), and it

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I. Pumping Action and Electrical Activity of the Heart

has been suggested that one cell may degrade both the connexins in its own connexons and those phagocytosed from the neighboring cell (Laird, 1996). A sequential contribution of proteasomal and lysosomal degradation pathways might allow such internalization and gap junction breakdown. Consistent with such a possibility, Laing et al. (1997) reported using cell lines that Cx43 remained at the surface when the proteasome was inhibited but appeared in intracellular vesicles when the lysosomal pathway was blocked.

B. Long-Term Changes in Gap Junction Expression The rapid turnover dynamics of gap junction channels implies that gap junction-mediated circuitry in tissues might be a dynamic process that responds to local demands. The first example of a tissue in which gap junction expression was shown to undergo rapid changes was the pregnant myometrium (Garfield et al., 1978), in which Cx43 expression is now known to be upregulated transcriptionally at the time of labor (Chen et al., 1995), presumably acting to coordinate and thereby intensify the contractions of the uterus. The rapid dynamics of gap junction turnover and the plasticity of their expression in response to various stimuli offer the possibility for remodeling of the intercellular circuits both within and between communication compartments in the cardiovascular system. In the heart, such remodeling could exaggerate conduction discontinuities due to tissue anisotropy and thus be arrhythmogenic (Spach and Heidlage, 1995). Studies thus far investigating transcriptional regulation of cardiac connexins are quite limited, despite the importance of such studies as indicated by the increasing number of reports indicating altered connexin expression in pathological situations (e.g., Haefliger et al., 1997; Bastide et al., 1993). The first such studies (De Leon et al., 1994) examined transcriptional control of Cx43 in the heart by using genomic clones encompassing putative 5⬘ flanking regulation sequences of the human gene. Fusion of these sequences to that encoding firefly luciferase enzyme permitted assay of transcriptional activity both in cultured neonatal myocytes and after delivery into hearts in vivo using intracardiac DNA injection. In both culture and in vivo systems, substantial expression of the exogenous reporter gene was obtained by inclusion of as little as 175 bp of the Cx43 5⬘ flanking sequence. Inclusion of a longer genomic sequence resulted in high levels of expression in vivo, but not in the culture system, suggesting that upstream regions between ⫺175 and ⫺2400 might be uniquely responsive to hemodynamic or neurohormonal effects. The critical 100-bp sequence upstream of transcrip-

tion start sites is quite similar in Cx43 genes of human, rat, and mouse (De Leon et al., 1994; Sullivan et al., 1993; Yu et al., 1994). The use of CAT constructs with deletions within this region has demonstrated both positive and negative cis-acting regulatory sequences in the mouse gene when expressed in myometrial cells (Chen et al., 1995). In addition, an AP-1 site in this region has been demonstrated to be responsive to phorbol esters in the human promoter (Geimonen et al., 1996), and an estrogen response element has been found in the human sequence (Yu et al., 1994). Analysis of Cx40 and Cx45 promoter regions might be expected to reveal regulatory elements that would explain their differential expression patterns compared to Cx43 in the cardiovascular system and could also lead to the generation of constructs that would be selectively targeted to endothelium or conduction system. Although such studies are only just beginning, promoter analysis of the mouse Cx40 gene has identified both positive and negative regulatory elements near the transcription start site (Seul et al., 1997).

IV. FUNCTIONAL PROPERTIES OF CARDIOVASCULAR GAP JUNCTIONS In excitable tissues, including the heart, the preeminent role of gap junctions is in the electrotonic spread of current, resulting in rapid and synchronized signal relay from one cell to the next. In some regions of the heart, such as Purkinje fibers, actual rapidity appears to be most important, and in other tissue areas, such as nodal structures, a paucity of junctional channels results in a delay that provides the timing necessary for rhythmic muscular contracture. Current spread from one cardiac myocyte to the next is mediated by the flow of ions. Because K⫹ is the most abundant and most mobile intracellular ion, it is responsible for carrying most of the current between cells. In their role as electrotonic synapses, gap junction channels functionally operate as K⫹ channels. In addition to electrical coupling, gap junction channel permeability to larger ions and molecules (⬍1 kDa) allows them to couple cells from the metabolic point of view, providing an intercellular pathway for the communication of second messenger molecules such as cAMP, cGMP, IP3 , ATP, and ADP. Despite abundant older literature implicating Ca2⫹ ions in uncoupling cardiac cells, it is now generally recognized that the Ca2⫹ sensitivity of gap junctions is low. Thus, instead of closing junctional channels, Ca2⫹ can actually diffuse through gap junction channels between cells, thereby acting as an intercellular messenger or signaling molecule.

8. Gap Junction Channels

A. Slow Intercellular Ca2⫹ Waves The initial demonstration of intercellular signaling by Ca2⫹ was made by Dunlap and colleagues (1987) on the hydrozoan Obelia, in which light-sensing cells are coupled to cells containing a Ca2⫹-sensitive, lightemitting protein related to aequorin. Subsequent studies that involved the direct injection of Ca2⫹ and IP3 into hepatocytes or mechanical stimulation of airway epithelial cells and detecting local Ca2⫹ elevations with EGTAbased ratiometric indicators developed by Roger Tsien clearly showed that the Ca2⫹ rise evoked in one cell led to an elevation in adjacent cells and that this spread was blocked by substances that closed gap junction channels (Saez et al., 1989; Sanderson, 1996). Spread of slow Ca2⫹ waves was soon demonstrated in a variety of other cell types with a characteristic velocity, generally ranging from about 5 to 25 애m/sec. In many cell types, brief intracellular Ca2⫹ transients or oscillations can occur that are not transmitted to adjacent cells, indicating that a threshold concentration of messenger must diffuse to the second cell in order to elicit a propagated response. The current model for propagation of Ca2⫹ waves throughout a tissue (see Fig. 4) involves the initial generation of IP3 within the stimulated cell, which diffuses to adjacent cells through gap junction channels, liberating Ca2⫹ from intracellular stores through activation of IP3 receptors (IP3R). The spatial localization of IP3R quite close to intercalated disk regions demonstrated in adult ventricular myocytes

155

(Kijima et al., 1993) provides an anatomical organization that might be ideal for such intercellular signaling. In some cell types, the spread is depressed by the removal of extracellular Ca2⫹, implying the participation of Ca2⫹ entry through surface membrane cation channels. Whether ryanodine receptors (RYR) can sustain such Ca2⫹ waves is unknown. Intercellular Ca2⫹ waves can also be propagated through the release of ATP from a stimulated cell and activation of purinergic receptors on its neighbors. Such an extracellular route of intercellular Ca2⫹ wave spread was first shown in rat mast cells by Opsichuk and Cahallan (1992). This pathway apparently accounts for the observation that Ca2⫹ waves in cultured glial cells can leap small cell-free boundaries (Hassinger et al., 1996). In the perfused whole rat heart, spontaneous intercellular Ca2⫹ waves can be seen propagating from one ventricular myocyte to others until they penetrate deep inside the myocardium (Minamikawa et al., 1997). In neonatal mouse cardiac myocyte cultures, slow Ca2⫹ waves initiated by mechanical stimulation of a single cell propagate with a velocity of 5 to 50 애m/sec (Fig. 5A). Spread is inhibited by the gap junction channel blocker heptanol and is partially attenuated by suramin, a P2-receptor blocker. Considering that ATP can be released by cardiac cells during ischemic injury, it is possible that such release in addition to direct Ca2⫹ entry into damaged cells could initiate or sustain slow Ca2⫹

FIGURE 4 Model for slow intercellular calcium wave propagation proposed by Sanderson and colleagues (1998). Mechanical stimulation of a single cell in culture can induce the activation of phospholipase C (PLC) and synthesis of inositol trisphosphate (IP3), thereby activating IP3 receptors (IP3R) to release calcium from intracellular stores. The diffusion of IP3 to the neighboring cells, passing through gap junction channels, triggers the propagation of calcium waves from cell to cell. An extracellular pathway can also operate in parallel with the intercellular pathway. In this case, an extracellular messenger released from the stimulated cell diffuses from cell to cell and communicates the signal through activation of membrane receptors. In the majority of cells that exhibit this mechanism, ATP has proven to be the extracellular messenger mediating the propagation of the calcium waves through activation of purinoceptors.

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I. Pumping Action and Electrical Activity of the Heart

FIGURE 5 Propagation of slow intercellular calcium waves between wild-type (A) and Cx43null (B) neonatal mouse cardiac myocytes loaded with an intracellular calcium indicator (Indo1 AM) and imaged with real-time confocal microscopy (Nikon RCM 8000). The mechanical stimulation of a single myocyte in culture (cell A) initiates the propagation of the intercellular calcium wave. This figure is a graphic representation of the phenomenon as a function of time; arrows indicate moment of stimulation. Note that the absence of Cx43 expression does not prevent communication of the calcium signal, but reduces the efficacy of calcium wave spread, which extends to fewer cells per field than in wild-type myocytes.

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8. Gap Junction Channels

wave propagation in this tissue. In Cx43-null mouse cardiac myocytes the velocity of Ca2⫹ wave propagation is not greatly different than in wild-type littermates (Fig. 5B), although the efficacy of signal spread is attenuated significantly (Spray et al., 1998). In cardiac myocytes the intercellular communication of Ca2⫹ waves seems to have a high safety factor, such that signal transmission in the absence of Cx43 can still be supported by the remaining connexins present as well as by the ATP-mediated activation of purinoceptors. Application of ratiometric imaging techniques should allow a more complete evaluation of this type of Ca2⫹ signaling in cardiac tissue, in much the same way that voltage-sensitive dyes are providing detailed maps of propagation of the much more rapid action potential wave front. The nature of recruitment of elemental Ca2⫹ release events (‘‘Ca2⫹ sparks’’) into propagated responses and the relative contribution of RYR and IP3R in the spread between cardiac myocytes are major issues that remain to be determined. Slow Ca2⫹ wave propagation mediated through both gap junction-dependent and gap junction-independent pathways may underlie ‘‘triggered propagated contractions’’ (TPC) in cardiac tissue. Studies on both rat ventricular and human atrial trabeculae by Henk ter Keurs and colleagues (see Mulder et al., 1989; Daniels and ter Keurs, 1990; Daniels et al., 1993; Wier et al., 1997) have demonstrated that following damage induced by local stretch, aftercontractions occur due to Ca2⫹ overload and can lead to triggered arrhythmias as the aftercontractions spread to neighboring myocardial regions (Fig. 6). The velocity of these propagated contractions along the undamaged parts of the trabeculae is constant with distance, at about 0.1 to 15 mm/sec, which is much slower than the propagation of electrical excitability in this tissue (nearly 1 m/sec), although the velocity appears to be somewhat faster than that of Ca2⫹ waves elicited in cultured cardiac myocytes by focal mechanical stimulation. Involvement of gap junctions in propagation is suggested by the reduction of propagation velocity, triggering rate, and force of TPC in the presence of gap junction channel blockers heptanol and octanol, without affecting the twitch force. The model proposed to account for the phenomenon of TPC includes local Ca2⫹ release from the sarcoplasmic reticulum in the damaged cell, leading to slow Ca2⫹ waves that are communicated to the neighboring cells by Ca2⫹ diffusion through gap junction channels (Daniels and ter Keurs, 1990; Miura et al., 1998), with Ca2⫹-induced Ca2⫹ release in the adjoining cell leading to the spreading contraction. Such Ca2⫹ entry and mobilization are also thought to result in depolarization (delayed with respect to the more rapidly conducted electrical activation), which may reach threshold and thereby trigger arrhythmias.

FIGURE 6 Triggered propagated contraction (TPC) in rat trabeculae. The constant interval between the peak of sarcomere shortening (vertical dashed lines) recorded simultaneously at five different points (300 애m apart) along the trabeculae (length 2.9 mm) during a TPC indicates that the contraction propagates with a constant velocity (1.4 mm/sec) along the preparation. SL, sarcomere length; F, force of contraction. From ter Keurs and Zhang (1997), with permission.

B. Biophysical Properties of Junctional Channels The function of gap junction channels is to provide a pathway through which ions and small molecules (Mr ⬍ 1000 Da) can diffuse from one cell to another. The ability of molecules to pass through gap junctions depends on the electrical conductance of the junctional membrane and its permeability to ions and molecules of different size and charge. The electrical conductance of the junctional membrane (gj) depends on the number of junctional channels, the single channel conductance, and the open probability of individual channels, whereas the junctional permeability to specific ions and molecules additionally depends on the extent to which passage is permitted by the connexins forming the gap junction channels. Both macroscopic and single channel conductances can be evaluated using electrophysiological techniques on pairs of cells. The dual voltage clamp technique uses two microelectrodes in each cell of a pair to voltage clamp the cell and to measure the junctional current (Spray et al., 1979). This technique still remains the method of choice for measuring macroscopic junctional conductance accurately in oocytes and other large cells.

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The patch clamp version of the technique uses one lowresistance patch electrode per cell and made it possible to record from mammalian cells (Neyton and Trautman, 1985; White et al., 1985). Moreover, this technique has allowed the detection of single channel currents under conditions when the input resistance of cells is high (ⱖ1 G⍀) and when the number of channels between cells is few. The open probability of gap junction channels is regulated by a wide range of stimuli, including transjunctional voltage (i.e., the voltage difference across the junction), intracellular calcium and pH, and the phosphorylation state of connexin molecules and can be altered experimentally through the application of certain lipophilic molecules and other agents. The gating of junctional conductance by transjunctional voltage (Vj) has been the best studied of these manipulations, although a physiological role for the voltage dependence exhibited by gap junction channels has not yet been demonstrated.

C. Gating of Gap Junctional Channels by Transjunctional Voltage The dependence of junctional conductance on transjunctional voltage was first demonstrated in amphibian embryonic cell pairs using the dual voltage clamp technique (Spray et al., 1979). In these cells, application of large Vj steps of either polarity caused a strong decline of the junctional current to a nonzero steady-state level, whereas small Vj steps produced no appreciable change in junctional conductance. The sensitivity of junctional conductance to Vj of either polarity was found to be well described by the Boltzmann equation: gj ⫽ gss ⫽ (gmax ⫺ gmin)/兵1 ⫹ exp[A(V ⫺ V0)]其 ⫹ gmin where gmax and gmin are maximal and minimal conductanes obtained at lowest and highest Vj , V0 is the voltage at which the voltage-sensitive component of gj (gmax ⫺ gmin) is reduced by 50%, and A is a slope factor from which the equivalent number of gating changes, n, can be calculated (Spray et al., 1981a). Evaluation of voltage sensitivity after the exogenous expression of connexins in mammalian cells or in weakly endogenously coupled cell pairs has indicated that for most connexins, the steady-state conductance is symmetric around 0 mV and is well fit to Boltzmann relationships (Fig. 7). Moreover, Boltzmann parameters are now known to be distinct for gap junction channels formed of each connexin subtype, as is described in more detail in Section D. For most of the gap junction channels that have been studied, the relaxation of junctional current from its initial to steady-state levels is well fit by a single

exponential decay function for each voltage. This implies that a first-order process underlies channel voltagedependent transitions. Measurement of single gap junction channels from poorly coupled cells provided additional insight into the gating of gap junction channels (Fig. 8). At the microscopic level, junctional currents of most connexins exhibit direct, interconverting transitions between the fully open state and the voltage-insensitive or residual conductance substate (Bukauskas et al., 1995; Moreno et al., 1994a). The ratio of unitary conductances of the main open state and the subconductance state has in all cases been found to be similar to the gmin /gmax ratio, thus indicating that the residual conductance gmin seen at high Vj arises from channel transitions occurring from the main state to the residual subconductance state (Moreno et al., 1994a). Single channel open probability measurements further indicate that the voltage sensitivity of the macroscopic conductance is due to the ensemble activity of identical and independent channels (Srinivas et al., 1999; Bukauskas et al., 1995). Domains involved in voltage-dependent gating have not been explicitly identified. Unlike other voltagegated channels, connexins do not contain a highly charged helical motif upon which the voltage gradient is likely to act. Thus, it is conceivable that voltage dependence may arise from interactions between several regions of the channel macromolecule. For example, mutation of Pro87 in M2 of Cx26 reverses the sign of voltage sensitivity when the mutant is paired with wild-type Cx26 in oocyte expression experiments (Suchyna et al., 1993). More detailed mutagenesis experiments on Cx32 and Cx26 have further implicated charged residues in the amino terminus and at the M1–E1 margin of these connexins (Verselis et al., 1994). Nevertheless, conceptual understanding of just which residues are the voltage sensors and how sensing of the voltage field is transduced in vivo into conformational change resulting in channel closure is almost completely lacking.

D. Properties of Specific Connexins Expressed in Exogenous Systems 1. Connexin43 Expressed in Exogenous Systems Gap junction channels formed by Cx43 have been the best studied of all connexins known to date. Evaluation of Cx43 gap junction channel properties after expression in oocytes initially indicated little or no sensitivity of junctional conductance to transjunctional voltage (which was consistent with the earliest reports on heart cells using moderate transjunctional voltages; see later). Most studies in which space clamp conditions were im-

8. Gap Junction Channels

159

FIGURE 7 Dependence of junctional conductance of Cx40, Cx43, and Cx45 gap junction channels on transjunctional voltage (Vj). (Left) Recordings of junctional currents measured in one cell of a pair expressing each connexin individually in response to 8- to 15-sec-long pulses from 0 to ⫾ 80 mV (in 20-mV increments) that were applied to the other cell. Junctional currents are maximal at the beginning of pulses and decline to steadystate values in a time- and voltage-dependent manner. (Right) Relationship between Vj and steady-state junctional conductance of Cx40, Cx43, and Cx45 gap junction channels (solid lines) and of Cx37 and Cx50 (dotted lines). The lines are fits of the normalized steady-state junctional conductance (Gj , the ratio of the steady-state gj to maximal gj –Vj relationship to a two-state Boltzmann equation. Boltzmann parameters for channels formed by each connexin type are listed in Table II.

proved by limiting analysis to lower conductance cell pairs however, indicated that these channels are weakly sensitive to Vj (White et al., 1994). Macroscopic conductance was shown to be reduced by Vj pulses of either polarity, although even at very high Vj values a large voltage-insensitive component (gmin) remains (Fig. 7; also see Moreno et al., 1994a; Valiunas et al., 1997). Parameters describing voltage sensitivity of Cx43 channels are given in Table II. Note that the proportion of junctional conductance that is voltage insensitive is quite high (40% of total gj), as is the voltage at which the voltagesensitive component is reduced by 50% (V0 ⫽ 60 mV). Human connexin43 channels expressed in SKHep1 cells exhibit at least three discrete, nonzero conductance states—30, 60–70, and 90–110 pS—when recordings are

done using internal solutions with highly mobile anions and cations (Fig. 8). The 60- to 70- and 90- to 110-pS sizes represent phosphorylated and dephosphorylated unitary conductances of Cx43 channels, respectively (Moreno et al., 1994b; Kwak et al., 1995), and 30-pS events represent the residual subconductance state induced at high Vj values (Moreno et al., 1994b). Cx43 channels do not discriminate to any great degree between anions and cations. They are highly permeant to the anionic dye Lucifer yellow and appear to be similarly permeant to both anions and cations. This conclusion is based on ion substitution experiments in which single channel conductances were determined and compared to the aqueous diffusion coefficients of various ionic species (Wang and Veenstra, 1997).

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FIGURE 8 Unitary conductances of gap junction channels formed by Cx40 (A), Cx43 (B), and Cx45 (C) measured with pipettes containing 130 mM CsCl. (A, top) Junctional currents measured in one cell of a Cx40transfected N2A cell pair in response to a 16-sec voltage step to 30 mV from a holding potential of 0 mV that was applied to the other cell. Single Cx40 channels were maximally open (O) from the baseline level (C) immediately upon application of a voltage pulse. The amplitude of the unitary current measured from the allpoints amplitude histogram was 5.8 pA, corresponding to a single channel conductance of 193 pS. (A, bottom) Single channel current–voltage (Ij-Vj) relationships of the junctional current constructed from responses of Cx40-transfected N2A cells to a series of ramps from ⫺100 to 100 mV. The unitary conductance of the main state (웂j, main) and the substate (웂j, sub), measured as slopes of current–voltage relationships (dotted lines), were 170 and 40 pS, respectively. The ratio of 웂j, sub and 웂j, main for Cx40 channels is similar to the gmin /gmax value obtained from macroscopic recordings. (B and C) Single channel current from a Cx43- and Cx45-transfected cell pair at voltages close to the respective V0 values obtained from macroscopic recordings. Single channel current through Cx43 gap junction channels predominantly exhibits transitions between a subconductance state (S) and the open state (O) at a Vj of 75 mV (B). Transitions to the baseline level (C) were rare; only one such brief transition was observed during this 40-sec pulse. Unitary conductances of the fully open state and the subconductance state were 80 and 26 pS, respectively. In contrast, transitions of single channel current from a Cx45-transfected cell pair at a Vj of 30 mV to the subconductance state are not clearly detected due to the small unitary conductance of these channels (웂j, main ⫽ 35 pS) and to the low gmin /gmax ratio for these channels (C). In addition, note that at these low voltages, open probability of Cx45 channels was low and comparable to that of Cx43 at a Vj of 75 mV.

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8. Gap Junction Channels

TABLE II Properties of Cardiac Gap Junction Channels after Exogenous Expression in Mammalian Cells and Oocytes Voltage dependence

Unitary conductance

Connexin

V0(mV)

n

gmin /gmax

Main state

Substate

References

mCx40 rCx40 hCx43 rCx43 hCx45 hCx37 rCx37 mCx50

⫾46 ⫾48 ⫾60 ⫾54 ⫾13 ⫾28 ⫾29 ⫾38

4.5 3 2.5 2.5 2.7 3 2.5 4

0.25 0.28 0.37 0.35 0.06 0.27 0.10 0.20

190 180 60–100 60–90 32 220 260 230

37 30–40 30 30 ND 63 80 42

Bukauskus and Weingart (1994), Traub et al. (1994) Beblo et al. (1995), Hellman et al. (1996) Moreno et al. (1994) White et al. (1994), Veenstra et al. (1995) Moreno et al. (1995) Reed et al. (1993) Waltzman and Spray (1994) White et al. (1994), Srinivas et al. (1999)

2. Connexin40 Expressed in Exogenous Systems

3. Cx45 Expressed in SKHep1 Cells and Elsewhere

The expression of rodent Cx40 in exogenous systems results in gap junction channels that are moderately sensitive to transjunctional voltage, with a half-inactivation voltage (V0) of ⫾45 mV, a gmin /gmax ratio of about 0.25 (Fig. 7; also see Hellmann et al., 1996; Traub et al., 1994; Hennemann et al., 1992; Beyer et al., 1992). These values are distinct from Boltzmann parameters for Cx43 gap junction channels. Mouse and rat Cx40 channels expressed in communcation-deficient cells have a main state single channel conductance of 200 pS that is favored at low Vj values (Fig. 8; also see Beblo et al., 1995; Bukasuskas et al., 1995; Hellmann et al., 1996). In addition, transitions to a subconductance state of 40 pS were detected at high transjunctional voltages. The ratio 웂j,sub /웂j,main (⫽0.2 for Cx40 channels) corresponds well with the ratio of gmin /gmax obtained from macroscopic gj measurements, thus providing further support for the proposal that the residual conductance gmin seen at high Vj arises from channel transitions occurring from the main-state to the residual subconductance state (Moreno et al., 1994a). Cx40 is known to contain consensus sequence motifs for phosphorylation by several protein kinases. Traub et al., (1994) have demonstrated that Cx40 can be phosphorylated by cAMP in transfected cells. Thus, it is conceivable that the channel properties of Cx40 can be modulated by phosphorylation, although such an effect remains to be demonstrated. Gap junction channels formed by Cx40 and Cx45 are modestly more selective for cations than anions (Beblo et al., 1995, 1997). However, the functional implications of this observation are unclear: because of the much higher unitary conductance of Cx40 channels, the total amount of current carried by anions through channels formed by both of these connexins may be similar.

Cx45 forms gap junction channels that are more senstive to transjunctional voltage than those of any other mammalian connexin that has been characterized to date (Fig. 7). Junctional currents through Cx45 channels in SKHepl cells, where they are expressed endogenously, are reduced even at low transjunctional voltages (Moreno et al., 1995). Boltzmann parameters of hCx45 channels are listed in Table II. Cx45 forms gap junction channels that have the lowest unitary conductance of all known connexins. The single channel conductance of Cx45 ranges between 20 and 30 pS, depending on the species (hCx45: Moreno et al., 1995; chCx45: Veenstra et al., 1994). Subconductance states of 10–15 pS were also reported. Cx45 channels do not allow the passage of Lucifer yellow. Consistent with this finding, Cx45 channels have been shown to be only weakly permeable to anions (Veenstra et al., 1994). The lower anion permeability of Cx40 and Cx45 channels may have some functional relevance. Although there has been no direct demonstration of significance of the different selectivity profiles of cardiac gap junction channels, it may be speculated that such a low permeability would restrict the diffusion of second messenger molecules that are predominantly negatively charged moieties under physiological conditions. 4. Heterologous Pairings of Cells Exogenously Expressing Different Junctional Proteins The distribution of connexins in cardiac tissue is such that pairing of hemichannels formed of different connexins would be expected to occur throughout the heart and to be more prominent at compartmental boundaries. Other channel-forming proteins either require multiple subunits for function or permit functional

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participation of multiple subunits. For gap junction channels, multiple connexins have been detected at junctional interfaces, and immunoprecipitation with connexin-specific antibodies has revealed that distinct connexins in certain tissues (lens, liver) are close enough to be cross-linked. Cells exogenously expressing individual connexins (as homomeric hemichannels) can be paired, and properties of these heterotypic homomers have been evaluated, primarily in Xenopus oocytes. Such studies indicate that certain heterotypic pairings are functional, whereas others are not. From the standpoint of cardiac function, the most important of these nonfunctional heterotypic channels is probably Cx43– Cx40. Because Cx40 is a major component of the atrium and conduction system, whereas Cx43 is the predominant connexin in ventricular muscle, nonfunctional Cx43–Cx40 channels could enhance the boundary between these compartments, limiting dispersion along the conduction pathway. Nevertheless, three recent, practically simultaneous reports provide strong evidence that connexins 40 and 43 may mix together to form functional heteromeric channels (Li and Sinard, 1999; Elenes et al., 1999; He et al., 1999). Whether such mixing is the rule or the exception awaits further study. Lack of coupling between Cx43 and Cx50 hemichannels has also been found in Xenopus expression studies. The detection of Cx50 in heart valves raises the possibility that this functional compartment boundary might arise in part due to heterologous connexin expression. The only heterologous pairings of cardiac connexins thus far performed on mammalian transfectants indicate that Cx43–Cx45 form functional high-affinity interactions. Both voltage sensitivity and single channel properties are as expected for the sum of properties contributed by hemichannels made of each connexin. Because of the great difference between voltage sensitivities of Cx43 and Cx45, the sum of the properties results in a strongly rectifying Gj –Vj relationship, such that steadystate Gj in response to depolarization of the Cx43 side is much lower than for depolarization of the Cx45 side (A.P. Moreno and D.C. Spray, unpublished observation). This biophysical asymmetry of Cx45–Cx43 heterotypic channels may be functionally important in reducing reentry from Purkinje cells to ventricular muscle or in impedance matching in nodal tissue.

E. Properties of Gap Junctions Evaluated in Cardiovascular Cells Dual whole cell voltage clamping of pairs of freshly dissociated adult cardiac myocytes has revealed that junctional conductance values range from 30 to 1000 nS. Contrary to studies of connexins in expression systems, junctional conductance between cardiac myocytes ex-

hibits only a slight sensitivity to the transjunctional voltage. Voltage sensitivity was at first regarded as absent in gap junctions from cardiac tissue. In part, this was because the range of Vj values tested in most studies was below ⫾50 mV. Also, however, the underestimation of voltage sensitivity and inadequacy of space clamp on adult myocytes due to series resistance was not appreciated (Moreno et al., 1991; Wilders et al., 1992). In most weakly coupled cell pairs or in performed cell pairs where there is a sequential insertion of the gap junction protein, junctional conductance was shown to be sensitive to the transjunctional voltage (Valinaus et al., 1997). Junctional conductance between atrial myocytes in culture (for 18 hr) ranges from 0.3 to 2 nS and is moderately sensitive to transjunctional voltage with a V0 of 43 mV, A ⫽ 0.2, and Gmin ⫽ 0.22 (Lal and Ansdorf, 1992). Similarly, Boltzmann parameters in preformed cell pairs of neonatal rat myocytes have been estimated (V0 ⫽ ⫾51, A ⫽ 0.11, and Gmin ⫽ 0.28; Valiunas et al., 1997). Single channel properties of cardiac gap junctions have now been evaluated from rat, guinea pig, and rabbit. Single channel conductance of gap junctions between neonatal rat ventricular myocytes and SA nodal cells of various species range from 50 to 100 pS, a range of values that is in agreement with the expression of Cx43 in these cells (Burt and Spray, 1988; Rook et al., 1988; Anumonwo et al., 1992; Verheule et al., 1997). In contrast, two different conductances corresponding to 185 and 100 pS were observed in atrial myocytes due to the expression of Cx40 in addition to Cx43 in this cell type (Verheule et al., 1997).

F. Gating of Gap Junctions by Other Stimuli 1. Sensitivity of Gap Junction Channels to Protons and Ca2⫹ Gap junction channels are closed by intracellular acidification and by high levels of cytoplasmic Ca2⫹. More than a century ago, Engelmann (1877) discovered that when a small region of myocardium was cut in normal physiological salt solution, adjacent tissue was initially rendered quiescent but then recovered its responsiveness and contractility. Subsequent studies found that the recovery occurred less rapidly when Ca2⫹ was removed from the medium (De Mello et al., 1969; Deleze, 1970), although it was accelerated in low Ca2⫹ solution if pH was lowered (De Mello et al. 1983). These findings have been interpreted as indicating that both Ca2⫹ and H⫹ ions can close gap junction channels, thereby allowing uninjured cells to repolarize when uncoupled from damaged cells. There has been quite a bit of controversy regarding the issue of whether gap junctions are more sensitive

8. Gap Junction Channels

to Ca2⫹ or H⫹ ions, and a number of studies have suggested that effects are synergistic, perhaps involving intermediary molecules (see Spray and Scemes, 1998). Nevertheless, gap junction channels composed of different connexins seem to have different pH sensitivities; whether Ca2⫹ sensitivity varies with connexin type is unexplored. Thus, Cx45 channels expressed in SKHep1 cells are mostly closed at an intracellular pH value of 6.7 (Hermans et al., 1995), implying a pK value near pH 7 and a high Hill coefficient, whereas the apparent pK measured for Cx43 in SKHep1 cells, in cardiac myocytes, or in paired oocytes is about 6.5, with a low Hill coefficient (Hermans et al., 1995; Morley et al., 1996; Spray and Bennett, 1985). The pH sensitivity of channels formed by Cx40 is slightly less than that of Cx43 (Stergiopoulos et al., 1999). In early quantitation studies of gap junction sensitivity to pH, it was suggested that the Hill coefficient might reflect the cooperative titration of sites on connexin molecules within the connexon (Spray et al., 1981b). Based on the nearly neutral pK of the pH sensitivity, histidine residues initially seemed likely candidates for the pH sensor, and it was proposed that perhaps those residues located within the cytoplasmic loop of the connexin molecules might participate (Spray and Burt, 1990). Mutagenesis and expression of altered Cx43 and Cx32 sequences in Xenopus oocytes have revealed that this region does play a role in Cx43 but may not in Cx32. For Cx43, Mario Delmar, Steve Taffet, and colleagues have shown that the amino-terminal histidine residues present within the cytoplasmic loop are key players in the acidification-induced channel closure. Instead of being titrated, these residues appear to act as a binding site for a region within the carboxyl terminus (see Ek et al., 1994; Ek-Vitorin et al., 1996). Remarkably, pH sensitivity is low in Cx43 mutants lacking the carboxyl terminus or specific domains located therein, but is restored by coexpression of a peptide corresponding to this region. In the view of these authors, pH gating involves a particle–receptor interaction of the carboxyl terminus with the cytoplasmic loop comparable to mechanisms proposed for N-type inactivation of the potassium channel (see Fig. 9). The mechanism of pH-induced gap junction channel closure differs from that caused by transjunctional voltage in several notable respects. First, there is no detectable substate associated with acidification-induced gating; closure of the channel by weak acids is thus complete. Second, the gating by pH and by Vj appears to be independently manipulatable. Certain pharmacological compounds (glutaraldehyde, EEDQ, retinoic acid) have been reported to effectively immobilize the pH gate without appreciably affecting voltage sensitivity (Spray et al., 1986). Moreover, although intracellular

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FIGURE 9 pH sensitivity of Cx43 is mediated through a docking interaction between different domains of the connexin molecules. (A) Under control conditions, the carboxyl terminus (CT) hangs freely in the cytoplasm; acidification causes a conformational change in which a domain in CT interacts with a binding site located in the cytoplasmic hinge region to close the channel. (B) In experiments in which truncated Cx43, lacking CT, is coexpressed with a peptide corresponding to CT, acidification causes similar channel closure, which is envisioned to result from the same interaction between the blocking particle and the binding site. Modified from Calero et al. (1998).

injection of strong acid can change the shape of the voltage sensitivity in amphibian blastomeres (Bennett et al., 1988), substantial voltage sensitivity is retained at pH values sufficiently acidic to reduce open probability to very low levels (Spray et al., 1986). Further evidence that pH and voltage-sensitive gating involve distinct mechanisms is provided by single channel studies on Cx43-transfected HeLa cells, in which acidification produced slow transitions between open and closed states, in contrast to the rapid transitions between O and Os caused by Vj (Bukauskas and Peracchia, 1997). 2. Sensitivity to Lipophilic Molecules Alcohols, specifically heptanol and octanol, were discovered by Fidel Ramon and colleagues to uncouple crayfish axons (Johnston et al., 1980). These compounds have subsequently been shown to be effective in every mammalian tissue examined, and in cardiac myocytes the reduction in junctional conductance is rapid, complete, and reversible at concentrations in the range of 0.1 to 3 mM (e.g., Burt and Spray, 1988). Halothane, a volatile anesthetic with arrhythmogenic properties, also totally and reversibly uncouples cardiac cell pairs from adult and neonatal rodents (Burt and Spray, 1988; Niggli et al., 1989). This action can occur at concentrations lower than those affecting excitability, implying relative specificity of their action on junctional channels. It has

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been proposed that these agents may act through membrane fluidity changes, due to either increased bulk fluidity (Takens-Kwak et al., 1992) or decreased fluidity of cholesterol-rich domains (Bastiaanse et al., 1993), although an alternative mode of action involving domains at specific depths within the lipid bilayer is suggested by experiments performed on cardiac myocytes with doxyl stearic acid probes (Burt, 1989) and on astrocytes exposed to a series of oleamide derivatives with different lengths and side groups (Guan et al., 1997). Like acidification-induced uncoupling, lipophile uncoupling does not induce substates in gap junction channels, but rather completely closes the channels to zero conductance. It seems likely that the phenomenon of lipophileinduced uncoupling could play an important role in the ischemic heart. Arachiodonic acid (4–20 애M) has been shown to uncouple neonatal rat cardiac myocytes (Massey et al., 1992; Fluri et al., 1990), and its mechanism of action is presumably similar to that of other lipophiles. Furthermore, Yamada et al. (1994) showed that longchain acyl carnitines increased rapidly after the onset of ischemia and uncoupled pairs of myocytes. Whether certain lipophiles may be more potent in reducing open times of some gap junction channels than others, as suggested by studies of oleic acid (Hirschi et al., 1993), is an important question with potential to provide the opportunity to selectively diminish electrical coupling within or between specific cardiac compartments. 3. Effects of Phosphorylation on Channel Function Cx43 is a phosphoprotein, and each of the other cardiac connexins contains consensus domains for phosphorylation by protein kinases. In heart and in most other Cx43-expressing types as well, two phosphorylated forms of Cx43 can be detected by autoradiographic analysis of 32P-labeled Cx43 separated by SDS–PAGE. These phosphorylated bands are recognizable in immunoblots due to their mobility shifts: unphosphorylated Cx43 runs at about 41 kDa, whereas phosphorylated forms are retarded by about 2 and about 4 kDa in SDS–PAGE gels. Why the incorporation of only one or two phosphate molecules induces such a profound retardation in mobility is unanswered. The nonphosphorylated forms of Cx43 may predominantly reside in the intracellular pools of this protein, and at least some phosphorylation occurs prior to plasma membrane insertion as indicated by the accumulation of intermediate phosphorylation states of Cx43 after treatment with agents that inhibit trafficking along the intracellular route (Musil and Goodenough, 1991,1993). Numerous studies have shown that second messenger activation affects junctional communication in adult and

neonatal cardiac myocytes, including PKA, PKC, and PKG (Munster and Weingart, 1993; Kwak et al., 1995; Burt and Spray, 1988). In addition, coupling in other cell types expressing endogenous or exogenous Cx43 is sensitive to MAP kinase and tyrosine kinases (Swenson et al., 1990; Lau et al., 1996). The turnover rate of phosphate in Cx43 in cardiac myocytes has been reported to be similar to the half-life of the protein, suggesting that the protein stays phosphorylated for most of its lifetime (Laird, 1991). However, the results of other studies have shown rapid exchange of phosphate in at least a small portion of the Cx43 pool (Saez et al., 1997). In cardiac myocytes, treatment with the protein kinase inhibitor staurosporine was shown to reduce electrical and dye coupling between neonatal rat myocytes and to reduce steady-state incorporatin of 32P into Cx43 (Saez et al., 1997), whereas protein kinase C (PKC) has been found to increase coupling (Spray and Burt, 1990). Further, acute PKC activation reversed the uncoupling effect of staurosporine, presumably by overcoming the staurosporine inhibition. PKC uncouples other cell types and has been shown in several instances to increase phosphate incorporation into Cx43 (see Saez et al., 1997). Identification of residues in Cx43 that are phosphorylated by protein kinase C has not been completely straightforward. Although PKC consensus sites Ser368 and Ser372 are phosphorylated by PKC in cardiac myocytes and in a recombinant Cx43 polypeptide (AA360–375), two-dimensional tryptic digests of a longer recombinant Cx43(AA243–382) showed additional phosphorylated residues, possibly due to activation of another kinase. Phosphorylation of serine residues 255, 279, and 282 has been associated with cell uncoupling (Warn-Cramer et al., 1998), and candidate protein kinases mediating this effect include MAP kinase, which can be activated by PKC. In another cell type, Cx43 has been shown to be phosphorylated by a protein kinase A pathway (Granot and Dekel, 1994), and some studies on cardiac cells have shown increased coupling after treatment with cAMP (Burt and Spray, 1988; De Mello, 1984), whereas others have not found a change (Takens-Kwak and Jongsma, 1992). Interestingly, rodent Cx43 contains a consensus phosphorylation site for protein kinase G, and coupling is reduced when rCx43 is phosphorylated by membranepermeant cGMP derivatives (Takens-Kwak and Jongsma, 1992; Kwak et al., 1995), whereas the human sequence lacks this residue, and coupling and phosphorylation in SKHep1 cells transfected with hCx43 are not affected by elevated cGMP (Kwak et al., 1995). These studies imply that adrenoreceptor activation should exert changes in the heart through elevation of cAMP (웁 adrenoceptors) or activation of PKC (움

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adrenoreceptors, through release of diacylglycerol). The extent to which these biochemical changes in Cx43 modulate coupling and contribute to the inotropic effects induced by the activation of adrenoreceptors on cardiac tissue remains to be completely understood. Moreover, the relevance of these phosphorylation sites for normal function of Cx43 during development remains controversial. Whereas mutations in the consensus Cx43 phosphorylation sites were reported to be associated with a subset of patients with the severe axis deformity heterovisceral atriotaxia (Britz-Cunningham et al., 1995), studies of more extensive patient populations have not confirmed this finding (Penman-Splitt et al., 1997). Functional consequences of phosphorylation of consensus sites in other cardiovascular connexins are virtually unexplored, despite the potential importance of such studies in revealing changes that might occur in selected cardiac compartments in response to neurotransmitters and hormones with their associated second messenger cascades. 4. Other Agents That Affect Gap Junction Function and Expression The search for a ‘‘silver bullet’’ that would block (or enhance) gap junctional communication without altering other ion channels or other cellular functions continues. Both environmental toxins such as dieldrin (Matesic et al., 1994) and active ingredients in herbal remedies (such as glycyrrhetinic acid derivatives found in licorice; Davidson et al., 1988) appear to be potent inhibitors of at least some gap junction channel types in some systems. Although the mechanisms of action are presently unknown, the selective action reported for 움 and 웁 glycyrrhetinic acids on gap junction channels has led to their use in studies evaluating the effects of cell uncoupling in various noncardiac tissues (e.g., Munari-Silem et al., 1995). Certainly, the nonvolatility of these agents simplifies their application in long-term studies. However, their effects in most preparations appear to be limited to moderate reductions in junctional conductance rather than producing total blockade. A totally different approach followed by several laboratories has been to apply connexin antibodies (Hofer and Dermietzel, 1998; Boitano et al., 1998) or polypeptides (e.g., Chaytor et al., 1997) corresponding to extracellular domains in the hope of occupying ‘‘binding sites’’ on the extracellular aspect of connexons, thereby inhibiting channel formation. The extent of blockade of functional coupling produced by these agents has been remarkable, as has also been achieved with oligonucleotides corresponding to antisense sequences of Cx43 and Cx40 (Goldberg et al., 1994; Singh et al., 1997). The use of dominant-negative strategies in which a connexin

construct is expressed that inhibits functional channel formation by other connexins has also been used successfully in studies of Xenopus and mouse development (Paul et al., 1995; Sullivan and Lo, 1995). The refinement of these strategies is a current goal of several laboratories and should prove useful in future studies of transgenic animals and of cultured cells. If coupling between cells of the heart is important for cardiac function, agents that increase coupling might be expected to improve cardiac performance and could be therapeutically useful. Stefan Dhein and colleagues (see Dhein and Tudyka, 1995; Dhein et al., 1995) have pursued the mode of action and structure–activity relations of antiarrhythmic peptides (AAPs). Synchronized beating of chick heart cell cultures promoted by treatment with hexapeptides isolated from bovine atria was originally reported in 1980 by Kohana and colleagues (Aonuma et al., 1980, 1983). These AAPs have subsequently been reported by Kohana and Dhein’s groups to be antiarrhythmic in certain mammalian cardiac preparations, including aconitine-induced arrhythmias in mice, ventricular fibrillation in dogs and rats, and ischemia–reperfusion-associated arrhythmias (Aonuma et al., 1983; Dhein and Tudyka, 1995). Although the mechanism of action of AAPs has not been unambiguously identified, voltage clamp studies of pairs of guinea pig ventricular myocytes using 10 nM concentrations of the synthetic AAP10 have revealed that junctional conductance increases by as much as 25–30%, and it was proposed that the increased coupling may be responsible for decreased susceptibility to arrhythmias (Muller et al., 1995).

V. SOMATIC AND GENETIC DISEASE STATES IN WHICH GAP JUNCTION EXPRESSION OR FUNCTION IS ALTERED A. Somatic Cardiac Abnormalities A number of naturally occurring human diseases or animal models have been examined to understand the potential relationship of altered gap junction expression or function and clinically relevant sequelae (see Severs, 1994; Spooner et al., 1997). Chagas’ disease, a multisystem disorder caused by the parasite Trypanosoma cruzi, often manifests as a cardiomyopathy with prominent rhythm disturbances (Elizari and Chiale, 1993). Interestingly, despite evidence for preserved connexin43 gap junctional abundance in chagasic hearts, marked disturbances in subcellular localization are observed, with a prominent loss of appositional plaque formation (Campos de Carvalho et al., 1992, 1994). These data suggest that perturbations of connexin43 biotrafficking

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may contribute to the high incidence of arrhythmias in the chagasic heart. Substantial changes in gap junction expression are also reported following experimental myocardial infarction in rodents and larger mammalian species, such as the dog. Most reports describe a relative loss and/or reorganization of Cx43 within the epicardial border zones, a disturbance that may facilitate the development of reentrant ventricular tachycardia (Luke et al., 1991; Matsushita and Takamatsu, 1997; Peters et al., 1997). Downregulation of Cx43 has also been observed at sites distant from the infarct, although in contrast to epicardial border zones, the subcellular localization appears unchanged from normal myocardium. Similar quantitative and qualitative alterations in gap junction expression have also been observed in tissue samples taken from humans with either ischemic or hypertrophic heart disease (Peters, 1996; Severs, 1994; Kaprielian et al., 1998), supporting the utility of these animal models to understand the pathogenetic relationships between gap junction dysregulation and arrhythmogenesis.

B. Reversed Physiology: Inferring Gene Function from Its Absence in Knockout Mice Mice in which Cx43 was deleted by homologous recombination (Cx43 KO mice) were generated by a collaboration between Janet Roussant and Gerry Kidder (Reaume et al., 1995) and are now available commercially from the Jackson Laboratory (Bar Harbor, ME). These animals die at birth due to hyperplasia of the right ventricle and stenosis of the pulmonary artery, leading to reduced or total loss of circulation to the lung. However, cells derived from these animals can be maintained in culture, allowing comparison of their properties with those obtained from wild-type littermates. In cardiac myocytes obtained from Cx43 KO mice and wild-type littermates, we have compared junctional conductance, single junctional channel properties, and dye coupling, as well as synchrony of spontaneous contractions (Spray et al., 1998). Junctional conductance in pairs of myocytes isolated primarily from ventricular tissue was lower in Cx43 KO than in wild-type mice (10.7 ⫾ 1.6 vs 4.6 ⫾ 0.9 nS; p ⬍ 0.05). However, transfer of intracellularly injected Lucifer yellow was reduced to barely detectable in Cx43 KO myocytes, as contrasted with strong dye coupling to contiguous and second-order myocytes from wild-type littermates. Consistent with the reduced junctional conductance and Lucifer yellow transfer, propagation of Ca2⫹ waves between mechanically stimulated myocytes was markedly less extensive in Cx43 KO than in wild-type primary cultures. When contraction synchrony was compared, interbeat

intervals of Cx43 KO myocytes were twice as long as for wild-type cells and showed much greater dispersion, reflected in a fivefold greater variability in interbeat intervals. We conclude that cardiac myocytes of Cx43 KO mice are deficient in intercellular coupling. The more profound reduction in dye coupling than electrical coupling presumably reflects the decreased anion permeability of the other cardiac gap junction proteins (Cx40 and Cx45) compared to Cx43. Conceivably, the reduced anion permeability of gap junctions in Cx43 KO ventricular myocytes may contribute to the developmental abnormality by limiting the diffusion of anionic morphogens. Mice heterozygous for expression of Cx43 (Cx43 ⫹/⫺ animals) exhibit about half as much Cx43 in the heart as compared to their wild-type littermates (Guerrero et al., 1997; Thomas et al., 1998); perhaps surprisingly, our studies indicate that junctional conductance, dye spread, and calcium wave velocity were not significantly different between wild types and heterozygotes. Nevertheless, electrical mapping of ventricular conduction on these heterozygotes revealed that such conduction was significantly slower than wild types, although action potential properties were similar and there were no detectable anatomical abnormalities. This difference results in significant prolongation of the QRS complex in ECGs of the heterozygotes (see Figs. 10C and 10D). More recent studies have now reported that atrial conduction in Cx43 (⫹/⫺) mice is normal, with no differences in P wave parameters or heart beat rates and no increased propensity for arrhythmogenesis (Thomas et al., 1998). This difference in behavior of conduction in atria and ventricles of Cx43 (⫹/⫺) mice is attributed to the abundant expression of Cx40 in the atrial muscle, which serves to compensate functionally for the reduction in Cx43 expression. Studies on mice lacking Cx40 have revealed abnormal ECGs (Figs. 10A and 10B), with evidence of conduction slowing; in 6 of 31 Cx40 (⫺/⫺) mice, atrial rhythm disturbances were noted (Kirchoff et al., 1998). Heart rates in Cx40 (⫹/⫹) and Cx40 (⫺/⫺) mice were similar and sinus rhythms were normal but atrioventricular conduction was slower (PR interval 21% longer) as was intra-atrial conduction (QRS complexes 34% larger, presumably due to slowed conduction in the Purkinje system). Ninety percent of Cx40 (⫺/⫺) mice showed differences in ventricular activation as evidenced by split QRS complexes and atrial P waves 9% longer than wild types. All findings are similar to first-degree AV block and associated bundle branch block, and these findings have been attributed primarily to effects on Purkinje fibers (Simon et al., 1998).

8. Gap Junction Channels

FIGURE 10 Representative ECG recordings from wild-type, connexin40-null homozygous (Cx40⫺/⫺) and connexin43 heterozygous (Cx43⫹/⫺) mice. (A) In Cx40-null hearts, atrioventricular and intraventricular conductions are slower with characteristic longer PR and QRS intervals; partial conduction blockage through the His–Purkinje system leads to uncoordinated ventricular activation with a high incidence of split QRS complex (Simon et al., 1998, with permission from Elsevier Science). (B) Types of arrhythmias in hearts of Cx40⫺/⫺ mice: (1) sinus arrhythmia, (2) atrial ectopia (third P wave of the tracing), (3) sinus arrhythmia or sino-atrial block, (4) total AV block, (5) total AV block with ventricular ectopic beat, and (6) intraatrial reentrant tachycardia. From Kirchhoff et al. (1998), with permission from Elsevier Science. (C and D) Delayed intraventricular conduction and prolonged QRS interval are observed in Cx43⫹/⫺ hearts, but other ECG parameters are similar to those recorded from wild-type mice. From Thomas et al. (1998).

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VI. SUMMARY Gap junctions are major components of intercalated disks, which are prominent structural features connecting cardiac myocytes. High-resolution images indicate that gap junction channels are formed of hexamers of connexin proteins contributed by each cell. Each connexin is a tetraspan membrane protein, with at least one and perhaps more of the transmembrane stretches contributing to the channel pore. Three of the 15 known connexins are prominent in cardiac tissue (Cx40, Cx43, and Cx45). Each displays characteristic regional and temporal expression patterns, with Cx43 the major gap junction protein in ventricles and Cx40 most abundant in atria and conduction system. Gap junction proteins (connexins) exhibit very short life spans, during which they progress to the junctional membrane through the endoplasmic reticulum and the Golgi apparatus, where they are assembled into connexons. Death of junctional proteins may involve internalization and breakdown through both proteasomal and lysosomal pathways. Basal promotor mapping has been performed for two of the cardiac connexin genes, indicating the existence of an estrogen response element for Cx43. Gap junction channels are gated by a variety of stimuli, including transjunctional voltage, intracellular pH (and Ca2⫹), phosphorylating agents, and lipophilic molecules. Conditions prevailing under ischemic conditions (exaggerated voltage gradients, acidosis, fatty acid generation, reduced phosphorylation) are all predicted to decrease junctional conductance. Gap junction expression and/or distribution is altered in postinfarct myocardium and in animal models of chagasic cardiomyopathy. Moreover, mice engineered to express reduced Cx43 or Cx40 display slowed ventricular or atrial Purkinje conduction. Although no human genetic disease has been linked conclusively to connexin mutations, these studies on diseased human heart and mouse models strongly suggest that high incidence conduction disorders may be due to alterations in intercellular communication through gap junction channels.

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9 Electrogenesis of the Resting Potential NICHOLAS SPERELAKIS, MASANORI SUNAGAWA, and MARIKO NAKAMURA Department of Molecular and Cellular Physiology University of Cincinnati Medical Center Cincinnati, Ohio 45267

I. INTRODUCTION

understanding is to examine the electrical properties of myocardial cells at rest, including the origin of the resting membrane potential (resting Em). The resting Em results from special properties of the cell membrane. Resting potentials of the cells of the various tissues of the heart are summarized in Table I.

The purpose of this chapter is (a) to provide the reader with an understanding of the basis for the resting potential of the cells of the heart and vascular smooth muscle of the coronary vessels and (b) to set the background for a number of subsequent chapters. For example, the resting potential has very important effects on the action potential (rate of rise and duration), propagation of excitation, and automaticity. Not only does this chapter discuss the genesis of the resting potential, it also provides the basis for several other chapters, including those on the Na/K pump (Chapter 21), Na/Ca exchange (Chapter 22), Ca pump (Chapter 25), and electrogenesis of the action potential (Chapter 10). In addition, this chapter presents basic information about the cell membrane, diffusion, permeability, and ion fluxes. The present chapter is an update, revision, and reorganization of the previous chapter in the third edition of this book (Sperelakis, 1995a). The cell membrane exerts tight control over the contractile machinery during the process of excitation– contraction (E-C) (electromechanical) coupling. Some drugs and toxins exert primary or secondary effects on the electrical properties of the cell membrane, thereby exerting effects on automaticity, arrhythmias, and force of contraction. Therefore, for an understanding of the mode of action of cardioactive and cardiotoxic agents, neurotransmitters, hormones, and plasma electrolytes on the electrical and mechanical activity of the heart, it is necessary to understand the electrical properties and behavior of the myocardial cell membrane at rest and during excitation. The first step in gaining such an

Heart Physiology and Pathophysiology, Fourth Edition

II. PASSIVE ELECTRICAL PROPERTIES A. Membrane Structure and Composition The cell membrane is composed of a bimolecular leaflet of phospholipid molecules (e.g., phosphatidylcholine and phosphatidylethanolamine) with protein molecules floating in the lipid bilayer (Singer and Nicolson, 1972). The nonpolar hydrophobic ends of the phospholipid molecules project toward the middle of the membrane, and the polar hydrophilic ends project toward the edges of the membrane bordering on the water phases (Fig. 1). This orientation is thermodynamically ˚ favorable. The lipid bilayer membrane is about 50–70A thick, and the phospholipid molecules are about the ˚ ) to stretch across half of the memright length (30–40 A brane thickness. Cholesterol molecules are in high concentration in the cell membrane (of animal cells), giving a phospholipid:cholesterol ratio of about 1.0; they are inserted between the phospholipid molecules. Some of the large protein molecules inserted in the lipid bilayer matrix protrude through the entire membrane thickness, e.g., the Na, K-ATPase, Ca-ATPase, and the various ion channel proteins. These proteins ‘‘float’’ in the lipid bilayer matrix, and the membrane has

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TABLE I Comparison of Resting Potentials (RP) of Cells in Different Regions of the Mammalian Cardiovascular System Type of cell

RP (mV)

Ventricular cell Atrial cell SA nodal cell AV nodal cell Purkinje fiber Vascular smooth muscle

⫺78–85 ⫺78–85 ⫺50–60 ⫺60–70 ⫺90–95 ca. ⫺55

fluidity (reciprocal of microviscosity), such that the protein molecules can move around laterally in the plane of the membrane. The outer surface of the cell membrane is lined with strands of mucopolysaccharides (the cell coat or glycocalyx), which endow the cell with immunochemical properties. The cell coat is negatively charged and therefore can bind cations, such as Ca2⫹ ion. Neuraminidase removes sialic acid residues and destroys the cell coat.

B. Membrane Capacitance and Resistivity Lipid bilayer membranes made artificially have a specific membrane capacitance (Cm) of 0.4–1.0 애F/cm2, which is close to the value for biological membranes. The capacitance of cell membranes is due to the lipid bilayer matrix. Calculation of membrane thickness (웃)

from the following equation for capacitance, assuming a measured membrane capacitance (Cm) of 0.7 pF/cm2 ˚: and a dielectric constant (⑀) of 5, gives 63 A Cm ⫽

⑀Am 1 웃 4앟k

(1)

where Am is the membrane area (in cm2) and k is a constant (9.0 ⫻ 1011 cm/F). Most oils have dielectric constants of 3–5. The more dipolar the material, the greater the dielectric constant (e.g., water, which is very dipolar, has a value of 81). The dielectric property of the cell membrane is very good. For a resting Em of ˚ , the voltage about 80 mV and a thickness of about 60 A gradient sustained across the membrane is about 133,000 V/cm. Thus, the cell membrane tolerates an enormous voltage gradient. The artificial lipid bilayer membrane, however, has an exceedingly high specific resistance (Rm) of 106 –109 ⍀-cm2, which is several orders of magnitude higher than the biological cell membrane (about 103 ⍀-cm2). Rm is greatly lowered, however, if the bilayer is doped with certain proteins or substances, such as macrocyclic-polypeptide antibiotics (ionophores). The added ionophores may be of the ion-carrier type, such as valinomycin, or of the channel-former type, such as gramicidin. Therefore, the presence of proteins that span across the thickness of the cell membrane must account for the relatively low resistance (high conductance) of the cell membrane. These proteins include those associated with the voltage-dependent gated ion channels of the excitable membrane. In summary, the capacitance is due to the lipid bilayer matrix, and the conductance is due to proteins inserted in the lipid bilayer.

FIGURE 1 Diagrammatic illustration of cell membrane substructure showing the lipid bilayer. Nonpolar hydrophobic tail ends of the phospholipid molecules project toward the middle of the membrane, and polar hydrophilic heads border on the water phase at each side of the membrane. The lipid bilayer is about 50–70 ˚ thick. For simplicity, the cholesterol molecules are not shown. Large protein molecules protrude through A the entire membrane thickness, as illustrated. These proteins include various pump enzymes associated with the cell membrane as well as ionic channels. Membrane has fluidity so that the protein and lipid molecules can move around in the plane of the membrane and fluorescent probe molecules inserted into the hydrophobic region of the membrane have freedom to rotate.

9. Electrogenesis of the Resting Potential

C. Membrane Fluidity Electrical properties and ion transport properties of the cell membrane are determined by the molecular composition of the membrane. The lipid bilayer matrix even influences the function of the membrane proteins, e.g., Na,K-ATPase activity is affected by the surrounding lipid. A high cholesterol content lowers the fluidity of the membrane. The polar portion of cholesterol lodges in the hydrophilic part of the membrane, and the nonpolar part of the planar cholesterol molecule is wedged between the fatty acid tails, thus restricting their motion and lowering fluidity. A high degree of unsaturation and branching of the tails of phospholipid molecules raise the fluidity; the chain length of the lipids also affects fluidity. Phospholipids with unsaturated and branched-chain fatty acids cannot be packed tightly because of steric hindrance due to their greater rigidity; hence such phospholipids increase membrane fluidity. Low temperature decreases membrane fluidity, as expected. Ca2⫹ and Mg2⫹ may diminish the charge repulsion between the phospholipid head groups; this allows the bilayer molecules to pack more tightly, thereby constraining the motion of the tails and reducing fluidity. ˚ 2, and Each phospholipid tail occupies about 20–30 A 2 ˚ each head group about 60 A (Jain, 1972). Membrane fluidity changes occur in muscle development and in certain muscle diseases. The hydrophobic portion of local anesthetic molecules may interpose between the lipid molecules. This separates the acyl chain tails of the phospholipid molecules further, reducing the Van der Waals forces of interaction between adjacent tails, and so increasing the membrane fluidity. Local anesthetics also produce a nonselective depression of most conductances of the resting and the excited membrane. At least part of this depression could come about indirectly by the anesthetics’ effect on the fluidity of the lipid matrix. At the concentration of a local anesthetic required to completely block excitability, its estimated concentration in the lipid bilayer is more than 100,000/애m2. Part of the depression of the Na,K-ATPase activity by local anesthetics (Henn and Sperelakis, 1968) could be explained by an effect on the fluidity, although a direct effect on the protein enzyme may also occur.

D. Potential Profile across Membrane The cell membrane has fixed negative charges at its outer and inner surfaces. The charges are presumably due to acidic phospholipids in the bilayer and to protein molecules, either embedded in the membrane (islands floating in the lipid bilayer matrix) or tightly adsorbed to the surface of the membrane. Most proteins have an

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acid isoelectric point, so that at a pH near 7.0, they possess a net negative charge. The charge at the outer surface of the cell membrane, with respect to the solution bathing the cell, is known as the zeta potential. This charge is responsible for the electrophoresis of cells in an electric field, with the cells moving toward the anode (positive electrode) because unlike charges attract. The surface charge affects the true potential difference (PD) across the membrane, as illustrated in Fig. 2. At each surface, the fixed charge produces an electric field that extends a short distance into the solution and causes each surface of the membrane to be slightly more negative (by a few millivolts) than the extracellular and intracellular solutions. The potential theoretically recorded by an ideal electrode, as the electrode is driven through the solution perpendicular to the membrane surface, should become negative as the electrode approaches within a few angstrom units of the surface. The potential difference between the membrane surface and the solution declines exponentially as a function of distance

FIGURE 2 Potential profile across the cell membrane. Because of fixed negative charges (at pH 7.4) at the outer and inner surfaces of the membrane, there is a negative potential that extends from the edge of the membrane into the bathing solution on both sides of the membrane. This surface potential falls off exponentially with distance into the solution. Magnitude of the surface potential is a function of the charge density. ⌿o is the electrical potential of the outside solution, ⌿i is that of the inside solution, and membrane potential (Em) is the voltage difference (⌿i ⫺ ⌿o). Em is determined by the equilibrium potentials and relative conductances. The profile of the potential through the membrane is shown as linear (the constant-field assumption), although this need not be true for the present purpose. If the outer surface potential is exactly equal to that in the inner surface, then the true transmembrane potential (Em) is exactly equal to the (microelectrode) measured membrane potential (Em). If the outer surface potential is different from the inner potential, for example, by elevating the extracellular Ca2⫹ concentration or lowering the pH to bind Ca2⫹ or H⫹ to more of the negative charges, then the E⬘m is greater than the measured Em . Diminution of the inner surface charge decreases E⬘m. The membrane ion channels are controlled by E⬘m .

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from the surface, with the length constant being a function of the ionic strength (or resistivity) of the solution. The lower the ionic strength, the greater the length constant. The magnitude of the PD depends on the density of the charge sites (number per unit membrane area); the number of charges is also affected by ionic strength and pH. In Fig. 2, the membrane potential measured by an intracellular microelectrode (Em) is the potential of the inner solution (⌿i , the active microelectrode) minus the potential of the outer solution (⌿o , the reference electrode): E m ⫽ ⌿i ⫺ ⌿o

(2)

The true PD across the membrane (E⬘m), however, is really that PD directly across the membrane, as shown in Fig. 2. If the surface charges at each surface of the membrane are equal, then E⬘m ⫽ Em . If the outer surface charge is decreased to zero by extra binding of protons or cations (such as Ca2⫹), then the membrane becomes slightly hyperpolarized (E⬘m ⬎ Em), although this is not measurable by the intracellular microelectrode, which measures the PD between the two solutions (Fig. 2, dotted arrow). Conversely, if the inner surface charge were to be neutralized, then the membrane would become slightly depolarized (E⬘m ⬍ Em). Because the membrane ionic conductances are controlled by the PD directly across the membrane (i.e., by E⬘m and not by Em), changes in the surface charges (e.g., by drugs, ionic strength, or pH) can lead to apparent shifts in the threshold potential, activation curve, and inactivation curve. For example, elevated [Ca]o is known to raise the threshold potential (i.e., the critical depolarization required to reach electrical threshold), as expected from the small increase in E⬘m that should occur. The apparent mechanical threshold (the Em value at which contraction of muscle just begins) can also be shifted by a similar mechanism.

time crossing the plane from the side of higher concentration to the side of lower concentration than there are crossing in the opposite direction. Thus, there is a flux or movement of molecules in both directions (unidirectional fluxes), but the net flux is from the side of higher concentration to the side of lower concentration. Now if the imaginary plane were replaced with a thin membrane permeable to the molecules, then the same situation would apply; namely, the particles would diffuse from the side of higher concentration to the side of lower concentration across the membrane. We will assume for simplicity that the solutions on either side are well stirred, i.e., that there are no concentration gradients within the bulk solution on either side of the membrane (although there probably are unstirred layers near the membrane). We confine ourselves here to the diffusion of small molecules or ions across membranes. Fick’s diffusion equation states that the rate of transfer of uncharged molecules across a membrane is equal to the concentration gradient (dC/dx) times the area of the membrane (Am) times a coefficient of diffusion (D), expressed as dQ dC ⫽ ⫺DAm dt dx

where Q is the amount of substance (in moles) transported, t is the time, C is the molecular concentration, and x is the distance. Although concentration is used throughout this chapter for clarity, activity is actually meant (i.e., the thermodynamically active concentration), which is the concentration times the activity coefficient. For a thin membrane, when the solutions are well stirred on both sides, the concentration gradient in the steady state is equal to the difference in the molecular concentration (⌬C) of the solute on both sides of the membrane divided by the thickness of the membrane (⌬x), J⬘ ⫽ ⫺DAm

III. DIFFUSION, PERMEABILITY, AND FLUXES A. Diffusion and Diffusion Coefficient Any substance in solution tends to move from regions of higher concentration to regions of lower concentration until the substance is distributed uniformly. Once distributed uniformly (i.e., at equilibrium), the molecules of the substance continue to move about, but the net movement is zero. Diffusion occurs because of the random thermal motion of the molecules. If a region of high solute concentration is adjacent to one of low solute concentration, separated by an imaginary plane, it is probable that there will be more molecules per unit of

(3)

⌬C ⌬x

(4)

where J⬘ ⫽ dQ/dt and is the rate of movement (or flux) of the substance across the membrane in mol/sec. Now we can transpose Am from the right side of the equation to the left side, and letting J⬘/Am ⫽ J, where J is the flux density in mol/sec ⭈ cm2, J ⫽ ⫺D

⌬C ⌬x

mol cm2 mol/cm3 2⫽ sec · cm sec cm

(5)

Thus, the net flux is equal to the concentration gradient times the diffusion coefficient. The diffusion coefficient is a constant for a given substance and membrane under

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9. Electrogenesis of the Resting Potential

a given set of conditions, and the coefficient is given in cm2 /sec. The negative sign in Eq. (3) refers to the direction of the net flow, namely, down the concentration gradient. The diffusion coefficient of substances in free solution is dependent on the molecular size (and shape, for large molecules). For ions, the smaller the unhydrated radius, the greater the charge density, which means that more water molecules are held in the hydration shells (larger hydrated radius); the larger water shell causes diffusion to be slower. The time required for diffusion to become 50, 63, or 90% complete varies directly with the square of the distance, and inversely with the diffusion coefficient. Thus, diffusion is extremely fast over short distances (e.g., 10–1000 nm), but exceedingly slow over long distances (e.g., 1 cm). For a small particle like K⫹, with a D value of about 1 ⫻ 10⫺5 cm2 /sec, the time required for 90% equilibration to be reached for a distance of 1 애m is about 1 msec. Because the time required is a function of distance, there is a critical thickness (e.g., 0.5–1.0 mm) of a muscle bundle in a bath that allows adequate diffusion of oxygen to the cells within the core of the strip, no matter how vigorously the bath is oxygenated. The diffusion coefficient across cell membranes for various substances is generally greater when the molecular size of the substance is small and when the lipid solubility is high, i.e., small molecules of high lipid solubility (i.e., less polar and nonpolar molecules) penetrate the fastest through the membrane. Most nonpolar molecules pass through the lipid bilayer matrix of the membrane rather than through special sites (e.g., water-filled pores or channels). Small charged ions (e.g., Na⫹, K⫹, Cl⫺, Ca2⫹) apparently pass through water-filled channels, some of which have a voltage-dependent gating mechanism. Such channels usually exhibit a high degree of selectivity for specific ions; the selectivity orders are not solely based on their hydrated or unhydrated sizes.

B. Permeability Coefficient Returning to Eq. (3), we can make one final combination. Because the cell membrane is relatively fixed in thickness, we can combine ⌬x and D into a new coefficient, the permeability coefficient (P): P ⫽ D/⌬x

(6)

Substitution into Eq. (3) gives J ⫽ ⫺P⌬C ⫽ ⫺P(Co ⫺ Ci) mol cm mol 2⫽ sec · cm sec cm3

(7) (7a)

where Ci is the internal concentration and Co is the external concentration. The unit of the permeability coefficient is cm/sec, i.e., the unit of velocity. Hence, the net flux of a nonelectrolyte across a membrane is equal to the permeability coefficient times the difference in concentration across the membrane. The permeability coefficient for K⫹ (PK) across resting striated muscle membrane is about 1 ⫻ 10⫺6 cm/sec. Note that although diffusion coefficients apply to diffusion in free solution or in membranes, permeability coefficients only apply to membranes. The higher the diffusion coefficient for movement of a substance across a membrane, the higher the permeability coefficient. Unidirectional fluxes are given by Ji ⫽ ⫺PCo

(8)

Jo ⫽ ⫹PCi

(9)

where Ji is the influx of the substance and Jo is the outflux (efflux). Thus, for the net flux: J ⫽ Ji ⫺ Jo

(10)

⫽ ⫺PCo ⫺ (⫹PCi) J ⫽ ⫺P(Co ⫺ Ci)

(10a)

Equations (8) and (9) show that the influx of a substance is equal to its P value times the external concentration, whereas the efflux is equal to the P value times the internal concentration. Conversely, the permeability is equal to the ratio of flux to concentration. Equations (7–9) apply only to uncharged molecules. If there is a net charge on the molecule, then the unidirectional and net fluxes are also influenced by any electrical field that may exist across the membrane. The permeability coefficient (for a charged ion) can be better understood by considering the relationship: 웁 RT 웃 F cm cm/sec 1 V ⫽ sec V/cm cm 1 P⫽U

(11)

where U is the mobility of the ion through the membrane and has units of a velocity per unit driving (electrophoresing) voltage gradient (cm/sec per V/cm), 웁 is the partition coefficient for the ion between the bulk solution and the edge of the membrane and is dimensionless, 웃 is the membrane thickness (in cm), and RT/F ⫽ 0.026 V at 37⬚C. Equation (11) indicates that the permeability coefficient is a direct function of the mobility of the ion through the membrane, i.e., it is a velocity in a unit electrical field. The electrochemical potential (애) is a measure of the useful energy, and its units are in joules/mole (just as voltage is in joules/coulomb). The electrochemical po-

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II. Cellular Electrophysiology

tential is composed of a chemical part (애c) and an electrical part (애e). The net flux is from the side of greater electrochemical potential to the side of lesser electrochemical potential. The potential difference across the membrane (Em) equals ⌿i ⫺ ⌿o , where ⌿i is the inside potential and ⌿o is the outside potential (see Fig. 2). The electrical current (i, in amp) is equal to the flux (J⬘, in mol/sec) times zF or i ⫽ J⬘zF

(12)

or the current density (I ): I ⫽ JzF amp mol coul ⫽ cm2 sec · cm2 mol

(13)

where I is in amp/cm2 and J is the flux density in mol/sec ⭈ cm2.

As pointed out earlier unidirectional fluxes are influenced by the permeability coefficient. Ussing (1949) developed the so-called flux ratio equation, in which the ratio of influx to efflux is used and permeability cancels out. Specifically, for the ratio of Na⫹ fluxes (by simple electrodiffusion), the following applies: (14)

Because the EmF/RT term is dimensionless [EF is the electrical energy, RT is the thermal energy, and F/RT ⫽ (0.026 V)⫺1 at 37⬚C], the term exp EmF/RT is also dimensionless. Thus, for Na⫹, the ratio of influx:efflux (passive) in a resting membrane (assuming a resting potential of ⫺80 mV) is J iNa J oNa

150 mM ⫹80 mV/26 mV ⫽ e 15 mM ⫽ 10 ⫻ e3.08

(14a)

⫽ 217 Thus, the passive influx of Na⫹ should be 217 times greater than the passive efflux because of the large electrochemical gradient directed inward. The flux ratio for K⫹ would be J iK 4 mM ⫹80 mV/26 mV ⫽ e J oK 150 mM ⫽

1 3.07 e 37.5

⫽ 0.574

Ji ⫽ e⫺(Em⫺Ei)F/RT Jo

(15)

where Ei is the equilibrium potential for the cation in question, and Ji and Jo are inward and outward fluxes, respectively. The newer sign convention refers inside solution to outside solution. Thus, for Na⫹ we have J iNa ⫽ e⫺[⫺80 mV⫺(⫹60 mV)]/26 mV J oNa ⫽ e⫹5.38

(15a)

⫽ 217

C. Ussing Flux Ratio Equation

J iNa [Na⫹]o ⫺E F/RT ⫽ e m J oNa [Na⫹]i

Thus, the passive influx should be 0.57 times the passive efflux. Because the K⫹ equilibrium potential (EK) is only slightly greater (more negative by about 14 mV) than the resting potential, it is expected that the passive flux ratio should be close to 1. A modified form of Eq. (14) can be obtained by substituting the ratio of ions with e EiF/RT (derived from the Nernst equation), giving

(14b)

This value of 217 obtained for the flux ratio is identical to that obtained from Eq. (14). One advantage of Eq. (15) is that it is obvious at a glance that when Ei ⫽ Em , the flux ratio is exactly 1.0, because e 0 ⫽ 1, where e is the base of the natural logarithm. Thus, if Cl⫺ is passively distributed so that ECI ⫽ Em , its flux ratio should be 1.0, i.e., influx equals efflux, so there is no net flux. The larger the difference between Ei and Em , i.e., the farther the ion is off equilibrium, the greater the flux ratio. Sometimes Eqs. (14) and (15) do not fit the experimental facts. Data are better fitted if the exponential term contains another factor (N), which is an empirical factor: Ji ⫽ e⫺(Em⫺Ei)(F/RT)N Jo

(16)

The best fit of data is when N has a value of 2.5 to 4.0, depending on the membrane under investigation. The interpretation given to N is that if the length of the water-filled pore that the ion must traverse across the membrane is much longer than the ion diameter, as is likely, then for so-called single-file diffusion, N hits on the same side are required for the ion to complete its journey across the membrane. One could consider, for example, that there are three potential energy wells, or chain of reactive sites, along the length of the pore, and the only way for the ion to escape the well is to receive a kinetic bump from an adjacent ion in the file. Complete permeation of an ion through the pore is more likely to happen if the ion is moving in the same direction as the majority, i.e., down the electrochemical gradient.

9. Electrogenesis of the Resting Potential

Therefore, this factor makes the flux ratio much greater than would otherwise be predicted.

IV. ION DISTRIBUTIONS AND MAINTENANCE A. Resting Potentials and Ion Distributions The transmembrane potential in resting atrial and ventricular myocardial cells is about ⫺80 mV (Table I). The resting Em or maximum diastolic potential in Purkinje fibers is somewhat greater (about ⫺90 mV), whereas that in nodal cells is lower (about ⫺60 mV). The ionic composition of the extracellular fluid bathing the heart cells is similar to that of the blood plasma. It is high in Na⫹ (about 145 mM) and Cl⫺ (about 103 mM), but low in K⫹ (about 4.5 mM). The Ca2⫹ concentration is about 2 mM. In contrast, the intracellular fluid has a low concentration of Na⫹ (about 15 mM or less) and Cl⫺ (about 5–8 mM), but a high concentration of K⫹ (about 150–170 mM). The free intracellular Ca2⫹ concentration ([Ca]i) is about 10⫺7 M, but during contraction it may rise as high as 10⫺5M. The total intracellular Ca2⫹ is much higher (about 2 mM/kg), but most of this is bound to molecules such as proteins or is sequestered into compartments such as mitochondria and the sarcoplasmic reticulum (SR). Most of the intracellular K⫹ is

181

free and has a diffusion coefficient only slightly less than K⫹ in free solution. Thus, under normal conditions the myocardial cell maintains an internal ion concentration markedly different from that in the medium bathing the cells, and it is these ion concentration differences that underlie the resting potential and excitability. Ion distributions and related pumps and exchange reactions are depicted in Fig. 3. Intracellular ion concentrations are maintained differently from those in the extracellular fluid by active ion transport mechanisms that expend metabolic energy to push specific ions against their concentration or electrochemical gradients. These ion pumps are located in the cell membrane at the cell surface and probably also in the transverse tubular membrane. The major ion pump is the NaK-linked pump, which pumps Na⫹ out of the cell against its electrochemical gradient, while simultaneously pumping K⫹ in against its electrochemical gradient (Fig. 3). The coupling of Na⫹ and K⫹ pumping is obligatory, as in zero [K]o the Na⫹ can no longer be pumped out, i.e., a coupling ratio of 3 Na⫹:0 K⫹ is not possible. The coupling ratio of Na⫹ pumped out to K⫹ pumped is generally 3:2. If the ratio were 3:3, the pump would be electrically neutral or nonelectrogenic because the pump would pull in three positive charges (K⫹) for every three positive charges (Na⫹) it pushed out. When the ratio is 3:2, the pump is electrogenic and

FIGURE 3 Intracellular and extracellular ion distributions in a myocardial cell. Also given are the polarity and magnitude of the resting potential. Arrows give direction of the net electrochemical gradients. The Na⫹ –K⫹ pump is located in the cell surface membrane and in the transverse (T)tubule membrane. A Ca-ATPase/Ca pump, similar to that in the SR, is located in the cell membrane, as is a Ca–Na exchange carrier.

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II. Cellular Electrophysiology

directly produces a potential difference that causes the membrane potential (Em) to be greater (more negative) than it would be otherwise, namely, on the basis of the ion concentration gradients and relative permeabilities or net diffusion potential (Ediff) alone. Under normal steady-state conditions, the contribution of the Na⫹ –K⫹ electrogenic pump potential to Em (⌬Vp) in myocardial cells is only a few millivolts (see Section VIII). The driving mechanism for the Na–K pump is a membrane ATPase, the Na,K-ATPase, which spans across the membrane and requires both Na⫹ and K⫹ ions for activation. This enzyme requires Mg2⫹ for activity. ATP, Mg2⫹, and Na⫹ are thus required at the inner surface of the membrane, and K⫹ is required at the outer surface. A phosphorylated intermediate of the Na,K-ATPase occurs in the transport cycle, with its phosphorylation being Na⫹ dependent and its dephosphorylation being K⫹ dependent [for references, see Sperelakis (1995b, 1998) and Chapter 21]. The pump enzyme usually drives three Na⫹ ions in and two K⫹ ions out for each ATP molecule hydrolyzed. The Na,K-ATPase is specifically inhibited by the vanadate ion and by cardiac glycosides (digitalis drugs) acting on the outer surface. The pump enzyme is also inhibited by sulfhydryl reagents (such as N-ethylmaleimide, mercurial diuretics, and ethacrynic acid), thus indicating that the SH groups are crucial for activity. Blockade of the Na–K pump produces only a small immediate effect on the resting Em: a small depolarization of about 2–5 mV, representing the contribution of Ip to Em (⌬Vp). Because excitability and generation of APs are almost unaffected at short times, excitability is independent of active ion transport. However, over a period of many minutes, depending on the ratio of surface area to volume of the cell, the resting Em slowly declines because of gradual dissipation of the ionic gradients. The progressive depolarization depresses the rate of rise of the AP, and hence the propagation velocity, and eventually all excitability is lost. Thus, a large resting potential and excitability, although not immediately dependent on the Na–K pump, are ultimately dependent on it. The rate of Na–K pumping in myocardial cells must change with the heart rate in order to maintain the intracellular ion concentrations relatively constant. A higher frequency of APs results in a greater overall movement of ions down their electrochemical gradients, and these ions must be repumped. For example, the cells tend to gain Na⫹, Cl⫺, and Ca2⫹ and to lose K⫹. Factors that control the rate of Na–K pumping include [Na]i and [K]o . In cells that have a large surface area to volume ratio (such as small-diameter nonmyelinated neurons), [Na]i may increase by a relatively large percentage during a train of APs, which would stimulate the

pumping rate. Likewise, an accumulation of K⫹ occurs externally and also stimulates the pump (the Km value for K⫹, i.e., the concentration for half-maximal rate, is about 2 mM). It has been shown that [K]o is increased significantly during the AP in cardiac muscle (Kline and Morad, 1976).

B. Cl⫺ Distribution In some invertebrate and vertebrate nerve and muscle cells, the Cl⫺ ion does not appear to be actively transported, i.e., there is no Cl⫺ ion pump. In such cases, Cl⫺ is often distributed passively (no energy used) in accordance with Em . In such a case, ECl is equal to Em in a resting cell. In mammalian myocardial cells, Cl⫺ also seems to be close to passive distribution because [Cl]i is at, or only slightly above, the value predicted by the Nernst equation from the resting Em (for references, see Sperelakis, 1995b, 1998). When distributed passively, [Cl]i is low because the negative potential inside the cell (the resting potential) pushes out the negatively charged Cl⫺ ion (like charges repel) until the Cl⫺ distribution is at equilibrium with the resting Em . Hence, for a resting Em of ⫺80 mV, and taking [Cl]o to be 103 mM, [Cl]i calculated from the Nernst equation (see Section V) would be at 5.0 mM: Em ⫽ ⫹61 mV log

[Cl]i [Cl]o

⫽ ⫺61 mV log

[Cl]o [Cl]i

⫺80 mV 103 mM ⫽ log ⫺61 mV [Cl]i

(17)

103 mM ⫺80 mV ⫽ antilog ⫽ antilog 1.31 ⫽ 20.4 [Cl]i ⫺61 mV [Cl]i ⫽

103 mM ⫽ 5.0 mM 20.4

During the AP, the inside of the cell goes in a positive direction, and a net Cl⫺ influx (outward Cl⫺ current, ICl) will occur and thus increase [Cl]i . The magnitude of the Cl⫺ influx depends on the Cl⫺ conductance (gCl) of the membrane: ICl ⫽ gCl(Em ⫺ ECl)

(18)

This equation is discussed later in Section VI. Thus the average level of [Cl]i in myocardial cells of the beating heart should depend on the frequency and duration of the AP, i.e., on the mean Em averaged over many AP cycles.

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9. Electrogenesis of the Resting Potential

C. Ca2⫹ Distribution 1. Need for Calcium Pumps For the positively charged Ca2⫹ ion, there must be some mechanism for removing Ca2⫹ from the myoplasm (see Chapter 22). Otherwise, the myocardial cell would continue to gain Ca2⫹ until there was no electrochemical gradient for net influx of Ca2⫹. Ca2⫹ loading would occur until the free [Ca]i in the myoplasm was even greater than that outside (ca. 2 mM) because of the negative potential inside the cell. Therefore, there must be one or more Ca2⫹ pumps in operation. The SR membrane contains a Ca2⫹-activated ATPase (which also requires Mg2⫹) that actively pumps two Ca2⫹ ions from the myoplasm into the SR lumen at the expense of one ATP. This pump ATPase is capable of pumping down the Ca2⫹ to less than 10⫺7 M. The Ca-ATPase of the SR is regulated by an associated low molecular weight protein, phospholamban. Phospholamban is phosphorylated by cyclic AMP-dependent protein kinase (PKA) and, when phosphorylated, stimulates the Ca-ATPase and Ca2⫹ pumping by derepression. The sequestration of Ca2⫹ by the SR is essential for muscle relaxation. The mitochondria can also actively take up Ca2⫹ almost to the same degree as the SR, but this Ca2⫹ pool does not play a significant role in normal E-C coupling processes. However, the resting Ca2⫹ influx and the extra Ca2⫹ influx that enters with each AP must be returned to the interstitial fluid. This occurs by several mechanisms: (a) A Ca-ATPase, similar to that in the SR, is present in the sarcolemma and (b) a Ca–Na exchange occurs across the cell membrane. There is a Ca-ATPase in the sarcolemma of myocardial cells (Dhalla et al., 1977; Jones et al., 1980) and smooth muscle (Daniel et al., 1977) that actively transports two Ca2⫹ outward against an electrochemical gradient, utilizing one ATP in the process. Phospholamban is not associated with the sarcolemmal Ca-ATPase. 2. Ca/Na Exchange Reaction The Cai /Nao exchange reaction ( forward mode) exchanges one internal Ca2⫹ ion for three external Na⫹ ions via a membrane carrier molecule [for references, see Sperelakis (1995b, 1998) and Chapter 22; Fig. 3]. This reaction is facilitated by ATP, but ATP is not hydrolyzed (consumed) in the reaction. Instead, the energy for the pumping of Ca2⫹ against its large electrochemical gradient comes from the Na⫹ electrochemical gradient, i.e., the uphill transport of Ca2⫹ is coupled to the downhill movement of Na⫹. Effectively, the energy required for this Ca2⫹ movement is derived from the Na,K-ATPase. Thus, the Na–K pump, which uses ATP to maintain the Na⫹ electrochemical gradient, indirectly

helps maintain the Ca2⫹ electrochemical gradient. Hence, the inward Na⫹ leak is greater than it would be otherwise. A complete discussion of the Ca/Na exchanger is given in Chapter 22. The energy cost (⌬GCa , in joules/mole) for pumping out Ca2⫹ ion is directly proportional to its electrochemical gradient. These energetic equations are as follows (where ⌬G is the change in free energy): ⌬GCa ⫽ zF(Em ⫺ ECa)

(19)

The energy available from the Na⫹ distribution is directly proportional to its electrochemical gradient: ⌬GNa ⫽ zF(Em ⫺ ENa)

(20)

Depending on the exact values of [Na]i and [Ca]i at rest in a cardiac cell, the energetics would be about adequate for an exchange ratio of 3 Na⫹:1 Ca2⫹. An exchange ratio of 3:1 would produce a small depolarization due to a net inward flow of current (3 Na⫹ in to 1 Ca2⫹ out) via this electrogenic Cai /Nao exchanger. This net exchanger current can be measured in whole cell voltage clamp studies when all ionic currents and Na/K pump current are blocked. The exchange reaction depends on relative concentrations of Ca2⫹ and Na⫹ on each side of the membrane and on relative affinities of the binding sites to Ca2⫹ and Na⫹. Because of this Cai /Nao exchange reaction, whenever the cell gains Na⫹, it will also gain Ca2⫹ because the Na⫹ electrochemical gradient is reduced and the exchange reaction becomes slowed. The Ca/Na exchange process has been proposed as the mechanism of the positive inotropic action resulting from cardiac glycoside inhibition of the Na–K pump. In addition, when the membrane is depolarized during the AP plateau, the exchange carriers will exchange the ions in reverse (reverse mode,) namely, internal Na⫹ for external Ca2⫹, and thus increase Ca2⫹ influx. The net effect of this mechanism is to elevate [Ca]i . Such reversed Cao /Nai exchange appears to be a significant source of Ca2⫹ for contraction in cardiac muscle of some species. The ratio of free energy changes for Na⫹ to Ca2⫹ was calculated for a coupling ratio of 3 Na⫹:1 Ca2⫹ (3⌬GNa / ⌬GCa) and a [Na]i of 15 mM and was plotted as a function of membrane potential for different [Ca]i levels (Fig. 4A). This plot allows a simple assessment of how the directionality of the exchanger is affected by Em , i.e., forward mode versus reverse mode of operation, and how the reversal potential of the exchanger is shifted by [Ca]i . When the ratio is 1.0, 3⌬GNa ⫽ ⫺⌬GCa , and the sum equals zero: 3⌬GNa ⫹ ⫺⌬GCa ⫽ 0. Therefore, the exchanger would be at equilibrium. For ⌬G ratios ⬎1.0, there is a net inward current carried by Na⫹ ion,

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II. Cellular Electrophysiology

FIGURE 4 Electrogenic Na/Ca exchange for [Na]i of 15 mM. (A) Calculated ratio of the free energies for Na⫹ versus Ca2⫹ (3 ⌬GNa /⌬GCa) plotted as a function of membrane potentials (Em) for coupling the ratio of 3 Na⫹:1 Ca2⫹ for four different intracellular Ca2⫹ concentrations ([Ca]i). At a ratio of 1.0, 3 ⌬GNa ⫽ ⫺⌬GCa and the sum equals zero: 3⌬GNa ⫹ ⌬GCa ⫽ 0. Therefore, the exchange would be at equilibrium at the Em value at which each [Ca]i curve crosses the ratio of 1.0 line, i.e., this gives the value of the exchanger equilibrium potential, ENa/Ca . At ⌬G ratios ⬎1.0, there is net inward current carried by Na⫹ ion, coupled with net Ca2⫹ efflux from the cell, i.e., this represents the forward mode of operation of the exchanger. At ⌬G ratios ⬍1.0, there is a net outward current carried by Na⫹ ion, coupled with net Ca2⫹ influx into the cell, reflecting the reverse mode of operation of the exchanger. Note that at higher [Ca]i levels, there is a rightward shift of ENa/Ca . (B) The reversal potential (ENa/Ca) for the Na/Ca exchanger, assuming a coupling ratio of 3 Na⫹:1 Ca2⫹, is plotted on the ordinate as a function of [Ca]i on the abscissa. The family of curves is for three different [Na]i levels.

185

9. Electrogenesis of the Resting Potential

coupled with net Ca2⫹ efflux from the cell. This represents forward mode of operation of the exchanger. The equilibrium potential or reversal potential for the Ca/Na exchanger (ENa/Ca), for an exchange ratio of 3 Na⫹:1 Ca2⫹, is ENa/Ca ⫽ 3 ENa ⫺ 2 ECa

(21)

where ENa and ECa are the equilibrium potentials for Na⫹ and Ca2⫹, respectively, as calculated from the Nernst equation. Thus, the ENa/Ca varies with the [Na]i level and during changes in [Ca]i levels that occur with contraction. ENa/Ca is less negative (more positive) when [Ca]i is elevated and shifts to more negative potentials when [Na]i is elevated (Fig. 4B). Therefore, when a myocardial cell changes from the resting potential (ca. ⫺80 mV) to the AP plateau (ca. 20 mV), simultaneous with [Ca]i being elevated from about 0.1 to 3 애M, the exchanger switches to the reverse mode of operation, with Ca2⫹ influx. As stated previously, this Ca2⫹ influx can be a significant source of the total Ca2⫹ influx during E-C coupling. If the [Na]i level were 25 mM, the exchanger would almost always operate in the reverse mode at the physiological Em levels.

V. EQUILIBRIUM POTENTIALS For each ionic species distributed unequally across the cell membrane, an equilibrium potential (Ei) or battery can be calculated for that ion from the Nernst equation (for 37⬚C): Ei ⫽

Ci ⫺61 mV log z Co

(22)

where Ci is the internal concentration of the ion, Co is the extracellular concentration, and z is the valence (with sign). The ⫺61-mV constant (2.303 RT/F) becomes ⫺59 mV at 22⬚C. R is the gas constant (8.3 joules/ mol · ⬚K), T is the absolute temperature (⬚K ⫽ 273 ⫹ ⬚C), F is the Faraday constant (96,500 coul/equiv), zF ⫽ coul/mol, and 2.303 is the conversion factor for natural log to log10 . The Nernst equation gives the potential difference (electrical force) that would exactly oppose the concentration gradient (diffusion force). Only very small charge separation (Q. in coulombs) is required to build up a very large PD: Em ⫽

Q Cm

(23)

where Cm is the membrane capacitance. For the ion distributions given previously, the approximate equilibrium potentials are [from Eq. (22)]

ENa ⫽

⫺61 mV 15 mM log ⫽ 60 mV ⫹1 145 mM

ECa ⫽

⫺61 mV 1.0 ⫻ 10⫺4 mM log ⫹2 1.80 mM

⫽ (⫺30.5)(⫺4.26) ⫽ 130 mV EK ⫽

⫺61 mV 155 mM log ⫽ ⫺94 mV ⫹1 4.5 mM

ECl ⫽

⫺61 mV 5 mM log ⫽ ⫺80 mV ⫺1 103 mM

The sign of the equilibrium potential is for the inside with reference to the outside of the cell (Fig. 5). In a concentration cell, the side of higher concentration becomes negative for positive ions (cations) and positive for negative ions (anions). Any ion whose equilibrium potential is different from the resting potential (e.g., ⫺80 mV) is off equilibrium, and therefore must effectively be pumped at the expense of energy. In the myocardial cell, only Cl⫺ ion appears to be at or near equilibrium, whereas Na⫹, Ca2⫹, and K⫹ are actively transported. Even H⫹ ion is off equilibrium, with EH being close to zero potential (e.g., ⫺25 mV if pHi ⫽ 7.0 and ⫺30 mV if pHi ⫽ 6.9). Extensive discussion of concentration cells and diffusion is given by Sperelakis (1998), and the mechanisms for development of the equilibrium potential are depicted in Fig. 6 and discussed in its legend. In brief, when a membrane separates two compartments containing ions at different concentrations, then an equilibrium potential can be calculated for each ion from the Nernst equation. For example, if the membrane contains water-filled pores that are negatively charged, as depicted in Fig. 6, then the positively charged K⫹ ion can permeate through the membrane, whereas the negatively charged Cl⫺ ion cannot. If the concentration ratio for K⫹ is 10 (e.g., 0.1 M on side No. 1 and 0.01 M on side No. 2), then the voltage developed across this membrane (i.e., the voltage between the two solutions) would be ⫺59 mV (at 20⬚C), with side No. 1 being negative with respect to side No. 2. As depicted in the lower part of Fig. 6, there are about three negatively charged binding sites within the pore itself that the K⫹ ion binds to on its journey through the pore (channel). The significance of this was discussed in Section III,C on the Ussing flux ratio equation.

VI. ELECTROCHEMICAL DRIVING FORCES AND MEMBRANE IONIC CURRENTS The electrochemical driving force for each species of ion is the algebraic difference between its equilibrium

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II. Cellular Electrophysiology

FIGURE 5 Electrical equivalent circuits for a myocardial cell membrane at rest. (A) Membrane as a parallel resistance–capacitance circuit, with the membrane resistance (Rm) being in parallel with the membrane capacitance (Cm). Resting potential (Em) is represented by an 80-mV battery in series with the membrane resistance, with the negative pole facing inward. (B) Membrane resistance is divided into its four component parts, one for each of the four major ions of importance: K⫹, Cl⫺, Na⫹, and Ca2⫹. Resistances for these ions (RK , RCl , RNa , and RCa) are parallel to one another and represent totally separate and independent pathways for permeation of each ion through the resting membrane. These ion resistances are depicted as their reciprocals, namely, ion conductances (gK , gCl , gNa , and gCa). The equilibrium potential for each ion (e.g., EK), determined solely by the ion distribution in the steady state and calculated from the Nernst equation, is shown in series with the conductance path for that ion. The resting potential of ⫺80 mV is determined by the equilibrium potentials and by the relative conductances. There are at least two separate K⫹ conductance pathways [labeled here gK1 and gK(del)]. Arrowheads in series with the K⫹ conductances represent rectifiers, with the arrowhead pointing in the direction of least resistance to current flow. Thus gK(del) allows K⫹ flux to occur more readily in the outward direction (outwardly directed rectification), whereas gK1 allows K⫹ flux to occur more readily in the inward direction (inwardly directed rectification).

potential, Ei , and the membrane potential, Em . The total driving force is the sum of two forces: an electrical force (the negative potential inside a cell at rest tends to pull in positively charged ions because unlike charges attract) and a diffusion force (based on the concentration gradient; Fig. 7). Thus, in a resting cell, the driving force for Na⫹ is (Em ⫺ ENa) ⫽ ⫺80 mV ⫺ (⫺60 mV) ⫽ ⫺140 mV (24) The negative sign means that the driving force is directed to bring about net movement of Na⫹ inward. The driving force for Ca2⫹ is (Em ⫺ ECa) ⫽ ⫺80 mV ⫺ (⫹130 mV) ⫽ ⫺210 mV (25) The driving force for K⫹ is (Em ⫺ EK) ⫽ ⫺80 mV ⫺ (⫺94 mV) ⫽ 14 mV

force for inward Cl⫺ movement (Cl⫺ influx), thus given an outward Cl⫺ current. Similarly, the driving force for K⫹ outward movement increases during the AP, whereas those for Na⫹ and Ca2⫹ decrease. The net current for each ionic species (Ii) is equal to its driving force times its conductance (gi , reciprocal of the resistance) through the membrane: I⫽

V ⫽ g·V R

(28)

This is essentially Ohm’s law, modified for the fact that in an electrolytic system the total force tending to drive net movement of a charged particle must take into account both the electrical force and the concentration (or chemical) force. Thus, for the four ions, the net current can be expressed as

(26)

INa ⫽ gNa(Em ⫺ ENa)

(29)

Hence, the driving force for K is small and directed outward. The driving force for Cl⫺, if distributed passively, is nearly zero for a cell at rest:

ICa ⫽ gCa(Em ⫺ ECa)

(30)

IK ⫽ gK(Em ⫺ EK)

(31)

(Em ⫺ ECl) ⫽ ⫺80 mV ⫺ (⫺80 mV) ⫽ 0 mV

ICI ⫽ gCl(Em ⫺ ECl)

(32)

⫹

(27)

However, during the AP, when Em is changing, the driving force for Cl⫺ becomes large and there is a net driving

In a resting cell, because Cl⫺ and Ca2⫹ essentials can be neglected, the Na⫹ current (inward) must be equal

9. Electrogenesis of the Resting Potential

187

known as the IK1 channel, allows K⫹ ion to pass more readily inward (against the usual net electrochemical gradient for K⫹) than outward, and is known as the inward-going rectifier or anomalous rectification. This channel is responsible for the rapid decrease in K⫹ conductance on depolarization and increases with repolarization, and helps set the resting potential and to bring about the terminal repolarization (phase 3) of the AP. A second type of voltage-dependent K⫹ channel, known as the delayed rectifier [IK or IK(del)], is similar to the usual K⫹ channel found in other excitable membranes, which opens (increasing total gK) on depolarization. This channel allows K⫹ to pass more readily outward (down the usual electrochemical gradient for K⫹) than inward, and so is also known as the outward-going rectifier. This delayed rectifier channel in myocardial

FIGURE 6 (Top) Diagram of concentration cell diffusion potential developed across an artificial membrane containing negatively charged pores. The membrane is impermeable to Cl⫺ ions, but is permeable to cations such as K⫹. The concentration gradient for K⫹ causes a potential to be generated, with the side of higher K⫹ concentration becoming negative. (Bottom) Expanded view of a water-filled pore in the membrane, showing the permeability to K⫹ ions, but the lack of penetration of Cl⫺ ions. Potential difference is generated by charge separation, with a slight excess of K⫹ ions being held close to the right-hand surface of the membrane; a slight excess of Cl⫺ ions is plastered up close to the left surface.

and opposite to the K⫹ current (outward) in order to maintain a steady resting potential: IK ⫽ ⫺INa

(33)

Thus, although in the resting membrane the driving force for Na⫹ is much greater than that for K⫹, gK is much larger than gNa , so the currents are equal. Hence, there is a continual leak of Na⫹ inward and K⫹ outward, even in a resting cell, and the system would run down if active pumping were blocked. Because the ratio of the Na⫹ /K⫹ driving forces (⫺140 mV/⫺14 mV) is about 10, the ratio of conductances (gNa /gK) will be about 1:10. The fact that gK is much greater than gNa accounts for the resting potential being close to EK and far from ENa . The myocardial membrane has at least five separate voltage-dependent K⫹ channels (Fig. 5C). One of them,

FIGURE 7 Representation of the electrochemical driving forces for Na⫹, Ca2⫹, K⫹, and Cl⫺. Equilibrium potentials for each ion (e.g., ENa) are positioned vertically according to their magnitude and sign; they were calculated from the Nernst equation for a given set of extracellular and intracellular ion concentrations. The measured resting potential is assumed to be ⫺80 mV. Electrochemical driving force for an ion is the difference between its equilibrium potential (Ei) and the membrane potential (Em), i.e., (Em ⫺ Ei). Thus at rest the driving force for Na⫹ is the difference between ENa and the resting Em ; if ENa is 60 mV and resting Em is ⫺80 mV, the driving force is 140 mV, i.e., the driving force is the algebraic sum of the diffusion force and the electrical force and is represented by the length of the arrows in the diagram. The driving force for Ca2⫹ (about 210 mV) is even greater than that for Na⫹, whereas that for K⫹ is much less (about 14 mV). Direction of the arrows indicates the direction of the net electrochemical driving force, namely, the direction for K⫹ is outward, whereas that for Na⫹ and Ca2⫹ is inward. If Cl⫺ is distributed passively, then its distribution across the cell membrane can only be determined by the net membrane potential; for a cell sitting a long time at rest, ECl ⫽ Em and there is no net driving force.

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II. Cellular Electrophysiology

cells turns on much more slowly than in nerve, skeletal muscle, or smooth muscle. The activation of this channel produces the increase in total gK that terminates the cardiac AP plateau (phase 3 repolarization). A third type of K⫹ channel is kinetically fast [compared to IK(del)] and provides a rapid outward K⫹ current that produces a small amount of initial repolarization, known as phase 1 repolarization. This occurs immediately following the rapidly rising spike portion of the AP and is known as the transient outward current (Ito). In some cells, the Cl⫺ current may contribute to Ito (Cl⫺ influx provides an outward ICl). A fourth type of K⫹ channel is activated by the elevation of [Ca]i and is therefore known as the Ca2⫹activated K⫹(KCa) channel or IK(Ca). With Ca2⫹ influx and internal release from the SR during the AP and contraction, IK(Ca) channels are activated and help IK(del) channels repolarize the AP. These KCa channels also help protect the myocardial cells against Ca overload. A fifth type of K⫹ channel is regulated by ATP and provides a current known as IK(ATP) . In normal myocardial cells, this KATP channel is inhibited or masked or silent due to the high [ATP]i . However, in ischemic or hypoxic conditions, when the ATP level is lowered, IK(ATP) channels become unmasked and provide a large outward IK that prematurely shortens the cardiac AP. This KATP channel provides a protection mechanism for the heart, namely, the ischemic region of the heart develops very abbreviated APs, and hence contraction is greatly depressed. This acts to conserve ATP in afflicted cells, enabling full recovery if the blood flow returns to normal after a short time period. Thus, KCa and KATP channels both act to protect ischemic myocardial cells. These two mechanisms are in addition to the important mechanisms provided by three special properties of the CaL channels (reviewed in Sperelakis, 1994, 1995c). (1) CaL channels are rapidly (and reversibly) inhibited by acidosis, being much more sensitive to acidosis than Naf channels and K⫹ channels (Vogel and Sperelakis, 1977; Irisawa and Sato, 1986). Therefore, the rapid acidosis (intracellular and extracellular) that accompanies ischemia inhibits the CaL channels quickly, thereby decreasing Ca⫹ influx and contraction. (2) CaL channels require binding of internal ATP in order to exhibit activity (Sperelakis and Schneider, 1976; O’Rourke et al., 1992; Yokoshiki et al., 1997). Therefore, when [ATP]i is lowered in ischemic cells, CaL channels are inhibited, thereby decreasing Ca2⫹ influx and contraction. (3) The activity of CaL channels is markedly stimulated by phosphorylation by PKA (and by other protein kinases, such as PKC and Tyr-PK), which requires ATP. So if ATP is sufficiently lowered during ischemia, phosphorylation will be compromised, thereby decreasing CaL channel activity, Ca⫹ influx, and

contraction. The most important of these three mechanisms is probably the first one (inhibition by acidosis) (Sperelakis, 1995a). The most important ion channel for protecting heart cells during ischemia is probably the CaL channel (compared to KATP and KCa channels).

VII. DETERMINATION OF RESTING POTENTIAL AND NET DIFFUSION POTENTIAL (Ediff) For given ion distributions, which normally remain nearly constant under usual steady-state conditions, the resting potential is determined by the relative membrane conductances (g) or permeabilities (P) for Na⫹ and K⫹ ions, i.e., the resting potential (of about ⫺80 mV) is close to EK (about ⫺94 mV) because gK Ⰷ gNa or PK Ⰷ PNa . (There is a direct proportionality between P and g at constant Em and concentrations.) From simple circuit analysis (using Ohm’s law and Kirchhoff’s laws), one can prove that this should be true. Therefore, the membrane potential will always be closer to the battery (equilibrium potential) having the lowest resistance (highest conductance) in series with it (Figs. 5 and 7). In the resting membrane, this battery is EK , whereas in the excited membrane it will be ENa (or ECa) because there is a large increase in gNa (and gCa) during the AP. Any ion that is distributed passively cannot determine the resting potential; instead, the resting potential determines the distribution of that ion. Therefore, Cl⫺ drops out of consideration for myocardial cells because it seems to be distributed nearly passively. However, transient net movements of Cl⫺ across the membrane do influence Em , e.g., washout of Cl⫺ (in Cl⫺-free solution) produces a transient depolarization, and reintroduction of Cl⫺ produces a transient hyperpolarization until redistribution occurs. Because of its relatively low concentraion, coupled with its relatively low resting conductance, the Ca2⫹ distribution has only a relatively small effect on the resting Em and so it can be ignored. Therefore, a simplified version of the Goldman– Hodgkin–Katz constant-field equation can be given (for 37⬚C): PNa [Na]i PK Em ⫽ ⫺61 mV log PNa [K]o ⫹ [Na]o PK [K]i ⫹

(34)

This equation shows that for a given ion distribution, the resting Em is determined by the PNa /PK ratio, the relative permeability of the membrane to Na⫹ and K⫹. For myocardial cells, the PNa /PK ratio is about 0.04, whereas for nodal cells this ratio is closer to 0.10. Inspection of the constant-field equation shows that

9. Electrogenesis of the Resting Potential

the numerator of the log term will be dominated by the [K]i term [as the (PNa /PK) [Na]i term will be very small], whereas the denominator will be affected by both [K]o and (PNa /PK) [Na]o terms. This relationship thus accounts for the deviation of the Em versus log [K]o curve from a straight line (having a slope of 61 mV/decade) in normal Ringer solution (Fig. 8). When [K]o is elevated ([Na]o reduced by an equimolar amount), the denominator becomes more and more dominated by the [K]o term and less and less by the (PNa /PK) [Na]o term. Therefore, in bathing solutions containing high K⫹, the constantfield equation approaches the simple Nernst equation for K⫹, and Em approaches EK . As [K]o is raised stepwise, EK becomes correspondingly reduced, as [K]i stays relatively constant; therefore, the membrane becomes more and more depolarized (Fig. 8). An alternative method of approximating the resting potenial is by the chord-conductance equation: Em ⫽

gK gNa EK ⫹ ENa gK ⫹ gNa gK ⫹ gNa

(35)

This equation can be derived simply from Ohm’s law and circuit analysis for the condition when net current is zero (INa ⫹ IK ⫽ 0). The chord-conductance equation again illustrates the important fact that the gK /gNa ratio

189

determines the resting potential. When gK Ⰷ gNa , then Em is close to EK ; conversely, when gNa Ⰷ gK (as during the spike part of the cardiac AP), Em shifts to close to ENa . When [K]o is elevated (e.g., to 8 mM) in some cells, a hyperpolarization of up to about 10 mV may be produced. Such behavior is often observed in cells with a high PNa /PK ratio (due to low PK) and therefore a low resting Em , such as in young embryonic hearts. This hyperpolarization could be explained by several factors: (a) stimulation of the electrogenic Na⫹ pump, (b) an increase in PK (and therefore gK) due to the [K]o effect on PK , and (c) an increase in gK (but not PK) due to the concentration effect. A similar explanation may apply to the fallover in the Em versus log [K]o curve, hence depolarizing the cells, when [K]o is lowered to 1 mM and less; this effect is prominent in rat skeletal muscle, for example. Inhibition of the Na–K pump will gradually run down the ion concentration gradients. The cells lose K⫹ and gain Na⫹, and therefore EK and ENa become smaller. The cells thus become depolarized (even if the relative permeabilities are unaffected), which causes them to gain Cl⫺ (because [Cl]i was held low by the large resting potential) and therefore also water (cells swell). In summary, in the presence of ouabain (short-term exposure only) to inhibit the electrogenic Na–K pump, the resting potential or net diffusion potential Ediff is determined by the ion concentration gradients for K and Na⫹ and by the relative permeability for K⫹ and Na⫹. When the Na–K pump is operating, there is normally a small additional contribution of the electrogenic pump to the resting Em of about 2–8 mV in myocardial cells (discussed in the following section).

VIII. ELECTROGENIC SODIUM PUMP POTENTIALS

FIGURE 8 Theoretical curves calculated from the Goldman constant-field equation for resting potential (Em) as a function of [K]o . The family of curves is given for various PNa /PK ratios (0.001, 0.01, 0.05, 0.1, and 0.2). The K⫹ equilibrium potential (EK) was calculated from the Nernst equation (broken straight line). Curves were calculated for a [K]i of 150 mM and a [Na]i of 15 mM. Calculations were made holding [K]o ⫹ [Na]o constant at 154 mM, i.e., as [K]o was elevated, [Na]o was lowered by an equimolar amount. Change in PK as a function of [K]o was not taken into account for these calculations. The point at which Em is zero gives [K]i . The potential reverses in sign when [K]o exceeds [K]i .

As discussed earlier, the Na–K pump is responsible for maintaining cation concentration gradients. The equilibrium potentials for K⫹ (EK) and Na⫹ (ENa) are about ⫺94 and ⫹60 mV, respectively. The resting potential value is usually near EK because the K⫹ permeability (PK) is much greater than PNa in a resting membrane. If there were no elecrogenic pump potential contribution to the resting potential, Em would equal Ediff . However, a direct contribution of the pump to the resting Em can be demonstrated. For example, if the Na–K pump is blocked by the addition of ouabain, there usually is an immediate depolarization of 2–8 mV, depending on the type of heart cell, i.e., the direct contribution of the electrogenic Na⫹ –K⫹ pump to the measured resting Em is small under physiological conditions, but very important.

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II. Cellular Electrophysiology

However, under conditions in which the pump is stimulated to pump at a high rate, e.g., when [Na]i or [K]o is abnormally high, the direct electrogenic contribution of the pump to the resting potential can be much greater, and Em can actually exceed EK by as much as 20 mV or more. For example, if he ionic concentration gradients are allowed to run down (e.g., by storing the tissues in zero [K]o and at low temperatures for several hours), then after allowing the tissues to restart pumping, the measured Em can exceed the calculated EK (e.g., by 10–20 mV) for a period of time (Fig. 9). The Na⫹ loading of the cells occurs in zero [K]o because external K⫹ is necessary for the Na–K pump to operate; Km of the

FIGURE 9 Diagrammatic representation of an electrogenic sodium pump potential. (A) Muscle cell in which the net ionic diffusion potential (Ediff , function of ion equilibrium potentials and relative conductances) is ⫺80 mV, yet exhibits a measured membrane resting potential (Em) that is greater (i.e., more negative). Difference between Em and Ediff represents the contribution of the electrogenic pump to the resting potential. The usual direct contribution of the pump is only a few millivolts and can be measured by the amount of depolarization produced immediately after complete inhibition of the Na,K-ATPase by cardiac glycosides. The contribution of the electrogenic pump potential to the resting potential (Em ⫺ Ediff) is equal to ⌬Vp . (B) A cell that was run down (Na loaded, K depleted) over several hours by inhibition of Na-K pumping, resulting in a low resting potential. Returning the muscle cell to a pumping solution allows the resting Em to build back up as a function of time. Buildup in Em occurs faster than buildup in EK , as illustrated. Whenever Em is greater (more negative) than the K⫹ equilibrium potential (EK), the difference (⌬Vp) must reflect the contribution of the sodium pump potential.

Na,K-ATPase for K⫹ is about 2 mM. After a period, the internal concentrations of Na⫹, K⫹, and Cl⫺ approach the concentrations in the bathing Ringer solution, and the resting potential is very low (⬍⫺30 mV). When the tissue is transferred to a pumping solution, the pump turns over at a maximal rate because the major control over the pump rate is [Na]i and [K]o The low initial Em also stimulates the pump rate because the energy required to pump out Na⫹ is less. The measured Em of such Na⫹ preloaded cells increases more rapidly than EK , as shown in Fig. 7. After this transient phase, however, a crossover of the two curves occurs, so that EK again exceeds Em , as in the physiological condition. Cardiac glycosides prevent or reverse the transient hyperpolarization beyond EK (Glitsch, 1972). The possibility that ionic conductance changes (e.g., an increase in gK or a decrease in gNa) can account for the observed hyperpolarization can be ruled out whenever Em exceeds (is more negative than) EK . Rewarming cardiac muscles that were previously cooled leads to the rapid restoration of the normal resting potential (within 10 min), whereas recovery of intracellular Na⫹ and K⫹ concentrations is slower (Page and Storm, 1965). During prolonged hypoxia, the resting potential decreases less than EK decreases (a difference of about 25 mV) (McDonald and MacLeod, 1971). In such a situation, the electrogenic pump appears to act in an attempt to hold the resting potential constant, despite dissipating ionic gradients. Another method used to demonstrate that the pump is electrogenic is to inject Na⫹ ions into the cell through a microelectrode. This procedure rapidly produces a small transient hyperpolarization, which is immediately abolished or prevented by ouabain. The pump current and the rate of Na⫹ extrusion increase in proportion to the amount of Na⫹ injected. To prove that the pump is electrogenic, it must be demonstrated that the hyperpolarization is not the result of enhanced pumping of an electroneutral pump that could cause depletion of external K⫹ in a restricted diffusion space just outside the cell membrane; such a depletion would lead to a larger EK and thereby hyperpolarize. Depletion could occur if the Na–K pump pumped in K⫹ faster than it could be replenished by diffusion from the bulk interstitial fluid. It has been suggested that the electrogenic Na⫹ pump may be influenced by the membrane potential. From energetic considerations, depolarization should enhance the electrogenic Na⫹ pump, whereas hyperpolarization should inhibit it. This is because depolarization reduces the electrochemical gradient (and hence the energy requirements) against which Na⫹ must be extruded, whereas hyperpolarization increases the gradient. There should be a distinct potential, more negative than EK , at which Na⫹ pumping is prevented (e.g., a pump equilib-

191

9. Electrogenesis of the Resting Potential

Em ⫽ Ediff ⫹ RmIp ⫽ Ediff ⫹ ⌬Vp

FIGURE 10 Hypothetical electrical equivalent circuit for an electrogenic sodium pump. The model consists of a pump current generator pathway in parallel with the membrane resistance (Rm) pathway [and the membrane capacitance (Cm) pathway]. This model fits the evidence that the pump is independent of short-range membrane excitability and that the pump proteins and channel proteins are embedded in the lipid bilayer as parallel elements. A net diffusion potential (Ediff , determined by the ion equilibrium potentials and relative permeabilities) of ⫺80 mV is depicted in series with Rm . The pump leg is assumed to consist of a battery in series with a fixed resistor (pump resistance, Rp , assumed to be at least 10 times greater than Rm) that does not change with changes in Rm and whose value is 10-fold higher than Rm . The net electrogenic pump current is developed by the pumping in of only two K⫹ ions for every three Na⫹ ions pumped out. For the values given (namely, Rm of 1000 ⍀, Ediff of ⫺80 mV), if ⌬Vp is ⫺1.8 mV, it may be calculated that Ip is 1.8 애A: Ip ⫽ ⌬Vp / Rm ⫽ 1.8 ⫻ 10⫺3 V/1.0 ⫻ 103 ⍀ ⫽ 1.8 ⫻ 10⫺6 amp.

rium potential). A value close to ⫺140 mV was reported for cardiac cells (Gadsby and Nakao, 1989). Any method used to increase membrane resistance increases the contribution of the pump to the resting potential (Fig. 10), i.e., the electrogenic Na⫹ pump contribution is augmented under conditions that increase membrane resistance. The contribution of the pump potential to the measured Em is the difference in Em when the pump is operating versus immediately after the pump has been stopped by the addition of ouabain or zero [K]o . Consequently, it appears as though the contribution from the electrogenic pump potential (⌬Vp) was in series with the net cationic diffusion potential (Ediff):

(36)

where Ip is the electrogenic component of the pump current and Ediff is the Em that would exist solely on the basis of the ionic gradients and relative permeabilities in the absence of an electrogenic pump potential (as calculated from the constant-field equation). Equation (22) states that Em is the sum of Ediff and a voltage (IR) drop produced by the electrogenic pump current generator. Because the density of pump sites is more than 1000-fold greater than that of Na⫹ and K⫹ channels in resting membrane, there is no relation between the pump pathway (the active flux path) and Rm (the passive flux paths), i.e., the pump path and the passive conductance paths are in parallel. One possible equivalent circuit for an electrogenic Na⫹ pump, which takes into account some of the known facts, is given in Fig. 10. The pump resistance (Rp) is estimated to be at least 10-fold higher than Rm . If so, the pump resistance acts to minimize a short-circuit path to Ediff when the pump is inhibited. If the pump is stopped by ouabain, Ip goes to zero. Using circuit analysis for the values of the parameters given in Fig. 8, Em would be ⫺81.8 mV, moderately close to Ediff (⫺80 mV). If Rm is raised 2-fold (to 2000 ⍀), Em would become ⫺83.6 mV. In vascular smooth muscle, the contribution of the pump potential to Em is greater. In general, Cl⫺ ions are known to have a short-circuiting effect on the electrogenic Na⫹ pump potential. For example, if the external Cl⫺ is replaced by less permeant anions, the magnitude of the hyperpolarization produced by the electrogenic Na⫹ pump is increased substantially. This Cl⫺ effect could be caused by the raising of membrane resistance in the absence of Cl⫺. The greater Rm is, the greater the contribution of the electrogenic pump potential to resting Em [see Fig. 10 and Eq. (22)]. The density of Na–K pump sites, estimated by specific binding of [3H] ouabain, is usually about 700– 1000/애m2. The turnover rate of the pump is generally estimated to be 20–100/sec. The pump current (Ip) has been estimated as IP ⫽

⌬VP Rm

(37)

where ⌬Vp is the pump potential contribution. Values of about 20 pmol/cm2-sec were obtained. A density of 1000 sites/mm2 (1011 sites/cm2) times a turnover rate of 40/sec gives 4 ⫻ 1012 turnovers/cm2-sec. If 3 Na⫹ are pumped with each turnover, this gives 12 ⫻ 1012 Na⫹ ions/cm2-sec; dividing by Avogadro’s number (6.02 ⫻ 1023 ions/mol) yields 20 ⫻ 10⫺12 mol/cm2-sec, which is the same value as the 20 pmol/cm2-sec measured. The net pump current would be less, depending on the

192

II. Cellular Electrophysiology

amount of K⫹ pumped in the opposite direction, i.e., on the coupling ratio (e.g., 3 Na⫹:2K⫹). Whenever the Na–K pump is stimulated to turn over faster, e.g., by increasing [Na]i or [K]o , the electrogenic pump potential contribution to Em becomes larger. In skeletal muscle, insulin has been reported to increase the number of Na/K pump sites in the sarcolemma by increasing the rate of translocation from an internal pool. Ion flux ( J) be converted to current (I) by the following relationship: I ⫽ J · zF A mol coul ⫽ cm2 sec cm2 mol

(38)

Thus, a flux of 20 pmol/cm2-sec is equal to approximately 2 애A/cm2 (20 ⫻ 10⫺12 mol/sec-cm2 ⫻ 0.965 ⫻ 105 coul/mol). Because ⌬Vp ⫽ ip ⫻ Rm , if Rm were 1000 ⍀-cm2 and ip were 2 애A/cm2, the electrogenic pump contribution to Em would be 2 mV (Em ⫽ Ediff ⫹ IpRm). Because 3 Na⫹ are known to be transported per molecule of ATP spent, it is assumed that the pump molecule has a net negative charge. The pump at the inner surface of the membrane may have a much higher affinity for Na⫹ than for K⫹, whereas the converse may be true at the outer surface. It is assumed that the pump cannot cycle unless fully loaded with 3 Na⫹ ions. The electrogenic pump potential has physiological importance in heart cells. Although small, the electrogenic pump potential contribution to the resting potential could have significant effects on the level of inactivation of the fast Na⫹ channels, and hence on propagation velocity. Further, an electrogenic pump potential could act to delay depolarization under adverse conditions (e.g., ischemia and hypoxia) and would act to speed repolarization to the normal resting potential during recovery from adverse conditions. It is crucial that the excitable cell maintain its normal resting potential as much as possible because of the effect on the AP rate of rise and conduction velocity with small depolarizations, and complete loss of excitability with larger depolarizations. The rate of firing of pacemaker nodal cells is affected significantly by very small potential changes. In cells in which there are lower resting potentials (e.g., vascular smooth muscle cells and cardiac nodal cell; Table I), the electrogenic pump potential contribution can be considerably larger. Sinusoidal oscillations in the Na–K pumping rate could produce oscillations in Em , which could exert important control over the spontaneous firing of the cell. The period of enhanced pumping hyperpolarizes the cell and suppresses automaticity, whereas slowing of the pump leads to depolarization and consequently to triggering of APs. Oscillation of the pump would be brought about by oscillating

changes in [Na]i . For example, the firing of several APs should raise [Na]i (nodal cells have a small volume/ surface area ratio) and stimulate the electrogenic pump. The increased pumping rate, in turn, hyperpolarizes and suppresses firing, thus allowing [Na]i to become lower again and removing the stimulation of the pump; the latter depolarizes and triggers spikes, and the cycle repeated. Noma and Irisawa (1974) concluded that, in rabbit sinoatrial nodal cells, the electrogenic Na⫹ pump might be one factor that modulates the heart rate under physiologic conditions. When stimulated at a high rate, cardiac Purkinje fibers and nodal cells undergo a transient period of inhibition of automaticity after cessation of the stimulation, known as overdrive suppression of automaticity. Stimulation of the electrogenic pump due to elevation in [Na]i is the major cause of this phenomenon (Vassalle, 1970; Pelleg et al., 1980).

IX. PACEMAKER POTENTIALS AND AUTOMATICITY In order to maintain a steady resting potential, the net outward current must equal the net inward current: Iout ⫽ Iin

(39)

Assuming Cl⫺ is distributed passively, the outward K current must be equal and opposite to the inward Na⫹ ⫹ Ca2⫹ current. If the inward current exceeds the outward current, then the membrane will depolarize along a certain time course (i.e., slope of the pacemaker potential or diastolic or phase-four depolarization), depending on the excess (or net) inward current. The inward leak of Na⫹ and Ca2⫹ currents is often called the background inward current. For the inward current to exceed the outward current (i.e., for a net inward current), either the inward current can be increased or the outward current IK can be decreased. Both of these mechanisms are used for genesis of pacemaker potentials (automaticity). For example, if a time-dependent decrease (decay) in gK occurs following an AP and hyperpolarizing afterpotential, then IK decreases and the membrane depolarizes (gNa /gK progressively increases). Conversely, if an agent such as acetylcholine (ACh) were to increase the resting gK , then the outward IK is increased, the membrane hyperpolarizes, and the slope of the pacemaker potential decreases, thus reducing the frequency of firing. Some agents, such as norepinephrine, increase the background inward current, thereby increasing the slope of the pacemaker potential. A prerequisite for automaticity is that the cells must have a relatively low Cl⫺ conductance (gCl). This condition holds true for all or most cell types in the heart. A high gCl acts to clamp Em , making it difficult for a ⫹

9. Electrogenesis of the Resting Potential

pacemaker potential to be developed. For example, the addition of Ba2⫹ (0.5 mM) to frog sartorius muscle fibers, which have a high gCl /gK ratio of about 4.0, has very little immediate effect. However, when the fibers are first equilibrated in Cl⫺-free solution to reduce gCl to zero, Ba2⫹ produces a prompt depolarization, an increase in Rm , and automaticity. Another way to view this effect of Cl⫺ is by Cole’s (1968) parallel capacitance–inductance (CmLm) circuit for an excitable membrane that tends to oscillate spontaneously when the RCl shunt resistance (and RNa) is very high. The apparent inductance Lm arises because of the peculiar behavior of the K⫹ resistance, namely, anomalous rectification. The rapid turnoff of this inwardly rectifying K⫹ channel causes a very fast decrease in gK with depolarization. During the time course of the pacemaker potential, Rm increases progressively due to a decrease in gK (Sperelakis and Lehmkuhl, 1964; Trautwein and Kassebaum, 1961). The progressive turnoff of the inwardly rectifying K⫹ channels (IK1 current) causes Rm to increase progressively. In addition, there is a progressive turnoff of the gK increase (delayed rectification) responsible for the rapid repolarizing phase of the AP and the subsequent hyperpolarizing (positive) afterpotential usually exhibited by pacemaker cells. The decreasing gK helps produce the depolarization. The pacemaker depolarization in heart cells is usually linear (or a ramp). Myocardial cells can be made to exhibit abnormal automaticity of another type under pathophysiological conditions. A large depolarizing afterpotential arises from the hyperpolarizing afterpotential and triggers the subsequent spike once the threshold potential (Vth) is reached. In this type of automaticity, each AP is triggered by the preceding one, and the abnormal automaticity of this type is known as triggered automaticity. Thus a train of spikes can be turned off by simply stopping one spike in the train from developing (e.g., by a brief hyperpolarizing current pulse). The depolarizing afterpotential is depressed by verapamil and is facilitated by cardiac glycosides and 웁-adrenergic agonists (Ferrier and Moe, 1973; Kass et al., 1978), with the latter agents tending to produce ectopic pacemaker activity. They do so by increasing Ca2⫹ influx, and hence the degree of Ca2⫹ loading in the SR. Spontaneous release of Ca2⫹ from the overloaded SR activates a Ca2⫹-regulated mixed Na⫹ –K⫹ conductance [gNa,K(Ca)] or transient inward current (Iti). Turn on of these nonselective channels produces the observed depolarization: delayed afterdepolarizations (DADs) or oscillatory afterpotentials (OAPs) (Kojima and Sperelakis, 1984). Calcium antagonist drugs block DADs/OAPs by depressing Ca2⫹ influx, and thus preventing the Ca2⫹ overload of the SR. All heart cells are capable of exhibiting automaticity under certain conditions. For example, ventricular cells

193

placed into cell culture can develop automaticity. Myocardial cells exposed to Ba2⫹ to decrease their gK and depolarize them develop automaticity (Hermsmeyer and Sperelakis, 1970; Sperelakis and Lehmkuhl, 1964; Sperelakis, 1972). Ventricular muscle depolarized by the application of current also fires spontaneously during the current pulse (Imanishi and Surawicz, 1976; Katzung, 1975; Reuter and Scholz, 1977; Sperelakis and Lehmkuhl, 1964), i.e., when Em is brought into the voltage region that can develop pacemaker potentials, automaticity occurs. In any cardiac pacemaker cell, if the membrane potential is hyperpolarized by a current pulse, the frequency of spontaneous firing is slowed and stopped, i.e., automaticity is suppressed at high resting potentials (Sperelakis and Lehmkuhl, 1964). Conversely, the application of depolarizing current increases the frequency of discharge. Thus, the slope of the pacemaker potential is exquisitely sensitive to small changes in Em . The further Em is above EK (within limits), the greater the degree of automaticity. A low gK , which also means a relatively lower resting Em , facilitates automaticity. Elevations of [K]o , which increases gK , suppress automaticity despite depolarizing (Vassalle, 1965; Kojima and Sperelakis, 1984). Under normal circumstances in the heart, the hierarchy of automaticity capability is SA nodal cells ⬎ AV nodal cells ⬎ Purkinje fibers. Ventricular or atrial myocardial cells develop automaticity only under pathologic conditions such as regional ischemia. The genesis of automaticity in Purkinje fibers is somewhat different from that in nodal cells (Carmeliet and Vereecke, 1979; Irisawa, 1978; Noble, 1975). Normally the automaticity of cells lower in the hierarchy (e.g., the Purkinje cells) is latent (latent pacemakers) because the cells are driven at higher rates by the primary pacemaker (SA node). Because of the phenomenon of overdrive suppression of automaticity (fast drives of a pacemaker cell tend to hyperpolarize the cell and cause a pause in automaticity after the drive is terminated), the latent pacemakers normally may be chronically in a state of overdrive suppression. Thus a pacemaker potential may be seen during diastole. One factor in the production of overdrive suppression is stimulation of the electrogenic Na⫹ pump by the increase in [Na]i accompanying a high rate of driving (Vassalle, 1970; Pelleg et al., 1980). In addition, K⫹ tends to accumulate extracellularly during the fast drive. One characteristic of a pacemaker cell is that accommodation does not occur; the cell fires no matter how slowly Em is brought to the threshold potential Vth . Automaticity of the heart (nodal cells) is normally under control of the autonomic nerves. The release of ACh from parasympathetic nerves increases gK , and thereby hyperpolarizes (toward EK) and depresses auto-

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maticity. ACh also depresses the inward slow Ca2⫹ current, which also would tend to depress automaticity (for references, see Josephson and Sperelakis, 1982). The release of norepinephrine from sympathetic nerves tends to increase the inward slow Ca2⫹ current and to decrease gK (kinetics of turn on), both of which tend to enhance automaticity. For a complete discussion of the electrogenesis of pacemaker potentials, the reader is referred to Chapter 18.

X. EFFECT OF RESTING POTENTIAL ON ACTION POTENTIAL Any agent that affects the resting potential (e.g., depolarizes) will have important repercussions on the cardiac AP. Depolarization reduces the rate of rise of the AP, thereby also slowing its velocity of propagation. A slow spread of excitation throughout the heart will interfere with the heart’s ability to act as an efficient blood pump. This effect is progressive as a function of the degree of depolarization. If the myocardial cells and Purkinje fibers are depolarized to about ⫺50 mV, then the rate of rise goes to zero and all excitability (and contraction) is lost, leading to cardiac arrest. Hyperpolarization usually produces only a small in-

crease in the rate of rise, and large hyperpolarizations may actually slow the propagation velocity (because the critical depolarization required to bring the membrane to threshold is increased) or cause propagation block. The explanation of the effect of resting Em (or takeoff potential) on the maximum rate of rise (dV/dt max) of the AP is based on the sigmoidal h앝 versus Em curve. In Hodgkin–Huxley notation, h is the inactivation variable for the fast Na⫹ conductance of the cell; it is a probability factor that deals with open (h ⫽ 1.0) versus closed (h ⫽ 0) positions of the inactivation (I) gate of each channel (Figs. 11 and 12). The value of h is a function of Em and time (t), and h앝 is the h value at steady state or infinite time (practically, t ⬎ 20 msec). h앝 is 0.9–1.0 at the normal resting potential (⫺80 mV) and diminishes with depolarization, becoming nearly zero at about ⫺50 mV. The I gates are open in a resting membrane and close slowly (time constant of several milliseconds) on depolarization, thus inactivating the fast Na⫹ conductance (Figs. 11 and 12). The slow channels in myocardial cells are similar to the fast Na⫹ channels, except that their A and I gates appear to operate much more slowly kinetically on a population basis, i.e., the slow conductance turns on (activates) more slowly, turns off (inactivates) more slowly, and recovers more slowly (Figs. 11 and 12). In

FIGURE 11 Models for a fast Na⫹ channel (left) and for a Ca2⫹ slow channel (right) in a myocardial cell membrane. As depicted, two gates are associated with each type of channel: activation (A) and inactivation (I). Gates are presumably charged positively so that they can sense membrane potential. The I gate moves more slowly than the A gate. Gates of the slow channel are kinetically much slower than those of the fast Na⫹ channel. Both channels are depicted in the resting state (A gate closed, I gate open) and just beginning the process of activation. Depolarization causes the A gate to open quickly so that the channel becomes conducting (active state). However, the I gate closes slowly during depolarization and inactivates the channel (inactive state). During recovery on repolarization, the A gate closes and the I gate opens (returns to resting state). Not depicted is the hypothesis that the protein constituent of the slow channel must be phosphorylated in order for the channel to be in a functional state available for voltage activation. Tetrodotoxin (TTX) blocks the fast Na⫹ channel from the outside, presumably by binding in the channel mouth. Ca antagonists and Mn2⫹ block the slow CaL channel.

9. Electrogenesis of the Resting Potential

195

FIGURE 12 Illustration of the hypothetical states of the fast Na⫹ channel. The three states patterned after the Hodgkin–Huxley view were modified to reflect the fact that there is evidence for three closed states. As depicted, in the most closed state (C3), all three m gates (or particles) are in the closed configuration. In the mid-closed state (C2), two m gates are closed and one is open. In the least closed state (C1), one gate is closed and two are open. In the resting state, the activation gate (A) is closed and the inactivation gate (I) is open: m ⫽ 0, h ⫽ 1. Depolarization to the threshold activates the channel to the active state, the A gate opening rapidly and the I gate still being open: m ⫽ 1, h ⫽ 1. The activated channel spontaneously inactivates to the inactive state due to closure of the I gate: m ⫽ 1, h ⫽ 0. The recovery process on repolarization returns the channel from the inactive state back to the resting state, thus making the channel again available for reactivation. The Na⫹ ion is depicted as being bound to the outer mouth of the channel and poised for entry down its electrochemical gradient when both gates are in the open configuration. The reaction between the resting state and the active state is readily reversible, and there is some reversibility of the other reactions. The fast Na⫹ channel is blocked by tetrodotoxin binding to the outer mouth and plugging it.

addition, the voltage inactivation curve for the slow Ca2⫹ conductance is shifted to the right so that inactivation begins at about ⫺45 mV and is not complete until about 0 mV. The slow channels also have a lower activation (threshold) potential of about ⫺35 mV (compared to about ⫺55 mV for the fast Na⫹ channels). When the fast Na⫹ channels are either blocked by tetrodotoxin (TTX) or voltage inactivated in elevated [K]o (25 mM; Em of about ⫺40 mV), positive inotropic agents, such as catecholamines or histamine, restore excitability in the form of slow-rising APs by increasing the number of slow Ca2⫹ channels available for voltage activation. The rate of rise of the slow AP is also dependent on the h앝 variable (often termed the f앝 variable for myocardial slow channels), with depression of dV/dt max begin-

ning at ⫺45 mV and going to zero at about ⫺20 mV. These relationships are depicted in Fig. 13. The resting potential also affects the duration of the cardiac AP. With polarizing current, depolarization lengthens the AP, whereas hyperpolarization shortens it. In contrast, when elevated [K]o is used to depolarize the cells, the AP is usually shortened. [Under special conditions, such as Cl⫺-free solution, the AP may be prolonged with elevation of [K]o (Carmeliet and Vereecke, 1979).] One important determinant of the AP duration is the K⫹ conduction (gK). Agents or conditions that increase gK , such as elevation of [K]o , tend to shorten the duration. In contrast, agents that decrease gK or slow the activation of gK , such as Ba2⫹ ion or tetraethylammonium ion (TEA⫹), tend to lengthen the

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shortens at high heart rates. Three factors could contribute toward this effect: (a) the increase in [Ca]i (resulting from increase in [Na]i) produces an increase in resting gK and enhances gK activation kinetics by the activation of a Ca2⫹-regulated K⫹ channel [gK(Ca) ; the GardosMeech effect] (Meech, 1972; Isenberg, 1975); (b) K⫹ accumulates outside the cell membrane, thereby increasing gK ; and (c) recovery of the slow channels is incomplete, thereby depressing Isi

XI. SUMMARY

FIGURE 13 Graphic representation of differences in behavior, with respect to voltage inactivation, of fast Na⫹ channels and slow CaL channels. h앝 inactivation factor of Hodgkin–Huxley (gNa ⫽ gNa m3h, where gNa is the Na⫹ conductance, m and h앝 are probability variables, and the overbar means maximal) for fast Na⫹ channels and for slow CaL channels as a function of resting potential (Em). The h앝 , represents h at t ⫽ infinity or steady state (practically, after 20 msec for fast channels and after 1 sec for slow channels). This graph illustrates that fast Na⫹ channels of myocardial cells begin to inactivate at about ⫺90 mV, and complete inactivation occurs at about ⫺50 mV (h ⫽ 0). In contrast, slow channels inactivate between about ⫺50 and 0 mV. The relative maximal rate of rise of the action potential (max dV/dt or ⫹ ˙ max) parallels the h앝 curve over most of its range (except at the V ˙ max is a measure of the inward current more depolarized regions). ⫹V intensity (everything else, such as membrane capacitance, held constant), which, in turn, is dependent on the number of channels available for activation. The curve for fast Na channels was taken from data in Sada et al. (1995) and that for CaL channels from Kusaka et al. (1995).

AP duration. Because of anomalous rectification (i.e., a decrease in gK with depolarization), depolarization by current prolongs the AP and hyperpolarization shortens it. Other factors are also important in determining the AP duration. For example, agents that slow the closing of I gates of fast Na⫹ channels, such as veratridine, prolong the AP. Prolongation or stimulation of the inward slow current (Isi) tends to prolong the AP; conversely, agents that depress Isi , such as verapamil or Mn2⫹, slightly shorten the AP. Conditions that depress metabolism of the heart and lower ATP, such as ischemia or hypoxia, greatly depress Isi and also act to turn on gK earlier by removing ATP inhibition of an ATPregulated K⫹ channel [gK(ATP)] (Noma and Matsuda, 1987) and so greatly abbreviate the cardiac AP (Vasalle, 1965; McDonald and MacLeod, 1973; Schneider and Sperelakis, 1974; Vleugels et al., 1976). The cardiac AP

Most of the factors that determine or influence the resting Em of heart cells were discussed in this chapter. The structural and chemical composition of the cell membrane was briefly examined and correlated with the membrane’s resistive and capacitative properties. Factors that determine intracellular ion concentrations in myocardial cells were examined. These factors include the Na–K-coupled pump, the Ca–Na exchange reaction, and a sarcolemmal Ca pump. The Na–K pump enzyme, the Na,K-ATPase, requires both Na⫹ and K⫹ for activity and transport 3 Na⫹ ions outward and usually 2 K⫹ ions inward per ATP hydrolyzed. Cardiac glycosides are specific blockers of this transport ATPase. The Na–K pump is not directly related to excitability, but only indirectly related by its role in maintaining Na⫹ and K⫹ concentration gradients. The carrier-mediated Ca/Na exchange reaction may be driven by the Na⫹ electrochemical gradient; i.e., the energy for transporting out internal Ca2⫹ by this mechanism comes from the Na,K-ATPase. The Ca/Na exchange reaction exchanges one internal Ca2⫹ ion for three external Na⫹ ions when working in the forward mode in cells at rest. During the AP depolarization, the energetics cause the Ca/Na exchanger to operate in reverse mode, allowing Ca2⫹ influx. The mechanism whereby ionic distributions give rise to diffusion potentials was discussed, as were the factors that determine the magnitude and polarity of each ionic equilibrium potential. The equilibrium potential for any ion and the transmembrane potential determine the total electrochemical driving force for that ion, and the product of this driving force and membrane conductance for that ion determine the net ionic current or flux. The net ionic movement can be inward or outward across the membrane, depending on the direction of the electrochemical gradient. The key factor that determines the resting Em —in the absence of any electrogenic pump potential contributions and for fixed ionic distributions—is the relative permeability of the various ions, particularly of K⫹ and Na⫹, i.e., the PNa /PK ratio (or gNa /gK ratio), as calculated

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from the Goldman constant-field equation. The major physiologic ions that have some effect on the resting Em or on the APs are K⫹, Na⫹, Ca2⫹, and Cl⫺. The Ca2⫹ electrochemical gradient has only a small direct effect on the resting Em , although low external Ca2⫹ can affect the permeabilities and conductances for the other ions such as Na⫹ and K⫹. Elevation of internal Ca2⫹ can increase the permeability to K⫹ by activating Ca2⫹-operated K⫹-selective IK(Ca) channels. Cl⫺ ion is distributed passively according to the membrane potential, i.e., not actively transported. However, some evidence indicates that [Cl]i may be somewhat higher than that predicted from Em in some cells. Before one can conclude that there is a Cl⫺ pump directed inward, however, the calculated ECl (concentrations corrected for activity coefficients) must be proven to be significantly more positive than the mean resting Em of the cell averaged over a period of time, i.e., for example, any spontaneous APs must be taken into account. If Cl⫺ is distributed passively, it cannot determine the resting Em . However, transient net movements of Cl⫺ ions, e.g., during the AP, can and do affect the Em , particularly when gCl is high. Elevation of [K]o to more than the normal concentration of about 4.5 mM decreases the K⫹ equilibrium potential (EK), as predicted from the Nernst equation ([K]i about constant), and depolarization is produced. Sometimes, however, some hyperpolarization is produced at a [K]o level between 5 and 9 mM. In addition, lowering [K]o to 0.1 mM often produces a prominent depolarization. These effects are usually explained on the basis that (a) PK is lowered in low [K]o and elevated in higher [K]o and (b) an electrogenic Na–K pump potential is inhibited at a low [K]o (Km of about 2 mM). The resting Em not only is the potential energy storehouse that is drawn upon for propagation of the APs, but because the membrane voltage-dependent cationic channels are inactivated with sustained depolarization, the rate of rise of the AP, and hence propagation velocity, are critically dependent on the level of the resting Em . For example, a relatively small elevation of K⫹ concentration in the blood has dire consequences for functioning of the heart. The contribution of the Na–K pump to the resting Em depends on (a) the coupling ratio of Na⫹ pumped out to K⫹ pumped in, (b) the turnover rate of the pump, (c) the number of pumps, and (d) the magnitude of the membrane resistance. The electrogenic pump potential is in parallel to the net ionic diffusion potential (Ediff), determined by the ionic equilibrium potentials and by the relative permeabilities. The contribution of the electrogenic pump potential to the measured resting Em of myocardial cells is generally small (only a few millivolts) so that the immediate depolarization produced by com-

plete Na–K pump stoppage with cardiac glycosides is only a few millivolts. Of course, long-term pump inhibition produces a larger and larger depolarization as the ionic gradients are dissipated. The rate of Na–K pumping, and hence the magnitude of the electrogenic pump contribution to Em , is controlled primarily by [Na]i and by [K]o . The electrogenic pump potential might be physiologically important to the heart under certain conditions that tend to depolarize the cells, such as transient ischemia or hypoxia. In such cases, the actual depolarization produced may be less because of a relatively constant pump potential in parallel with a diminishing Ediff . The electrogenic pump potential may also affect automaticity of the nodal cells.

Bibliography Carmeliet, E., and Vereecke, J. (1979). Electrogenesis of the action potential and automaticity. In ‘‘Handbook of Physiology’’ R. M. Berne and N. Sperelakis, (eds.), pp. 269–334. American Physiological Society, Bethesda, MD. Cole, K. S. (1968). ‘‘Membranes, Ions and Impulses: A Chapter of Classical Biophysics.’’ University of California, Berkeley, CA. Daniel, E. E., Kwan, C. Y., Matlib, M. A., Crankshaw, D., and Kidwai, A. (1977). Characterization and Ca2⫹-accumulation by membrane fractions from myometrium and artery. In ‘‘Excitafion– Contraction Coupling in Smooth Muscle’’ (R. Casteels, T. Godfraind, and J. C. Ruegg, eds.), pp. 181–188. Elsevier/North-Holland, Amsterdam. Dhalla, N. S., Ziegelhoffer, A., and Hazzow, J. A. (1977). Regulatory role of membrane systems in heart function. Can. J. Physiol. Pharmacol. 55,1211–1234. Ferrier, G. R., and Moe, G. K. (1973). Effects of calcium on acetylstrophanthidin-induced transient depolarizations in canine Purkinje tissue. Circ. Res. 33, 508–515. Gadsby, D. C., and Nakao, M. (1989). Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes. J. Gen. physiol. 94, 511–537. Glitsch, H. G. (1972). Activation of the electrogenic sodium pump in guinea-pig auricles by internal sodium ions. J. Physiol. (Lond.) 220, 565–582. Henn, F. A., and Sperelakis, N. (1968). Stimulative and protective action of Sr2⫹ and Ba2⫹ on (Na⫹,K⫹)-ATPase from cultured heart cells. Biochim. Biophys. Acta 163, 415–417. Hermsmeyer, K., and Sperelakis, N. (1970). Decrease in K⫹ conductance and depolarization of frog cardiac muscle produced by Ba2⫹. Am. J. Physiol. 219, 1108–1114. Imanishi, S., and Surawicz, B. (1976). Automatic activity in depolarized guinea pig ventricular myocardium. Circ. Res. 39, 751–759. Irisawa, H. (1978). Comparative physiology of the cardiac pacemaker mechanism. Physiol. Rev. 58, 461–498. Irisawa, H., and Sato, R. (1986). Intra- and extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells. Circ. Res. 59, 348–355. Isenberg, G. (1975). Is potassium conductance of cardiac Purkinje fibres controlled by [Ca2⫹]i ? Nature 253, 273–274. Jain, M. K. (1972). ‘‘The Bimolecular Lipid Membrane: A System.’’ Van Nostrand, New York. Jones, L. R., Maddock, S. W., and Besch, H. R., Jr. (1980). Unmasking effect of alamethucin on the (Na⫹, K⫹)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein

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kinase activities of cardiac sarcolemmal vesicles. J. Biol. Chem. 255, 9971–9980. Josephson, I., and Sperelakis, N. (1982). On the ionic mechanism underlying adrenergic-cholinergic antagonism in ventricular muscle. J. Gen. Physiol. 79, 69–86. Kass, R. S., Tsien, R. S., and Weingart, R. (1978). Ionic basis of transient inward current induced by strophanthidin in cardiac Purkinje fibres. J. Physiol. (Lond.) 281, 209–226. Katzung, B. G. (1975). Effects of extracellular calcium and sodium on depolarization-induced automaticity in guinea papillary muscle. Circ. Res. 37, 118–127. Kline, R., and Morad, M. (1976). Potasium efflux and accumulation in heart muscle. Biophys. J. 16, 367–372. Kojima, M., and Sperelakis, N. (1984). Properties of oscillatory afterpotentials in young embryonic chick hearts. Circ. Res. 55, 497–503. Kusaka, M., and Sperelakis, N. (1996). Genistein inhibition of fast Na⫹ current in uterine leiomyosarcoma cells is independent of tyrosine kinase inhibition. Biochim. Biophys. Acta. 1278, 1–4. McDonald, T. F., and MacLeod, D. P. (1971). Maintenance of resting potential in anoxic guinea pig ventricular muscle: Electrogenic sodium pumping. Science 172, 570–572. McDonald, T. F., and MacLeod, D. P. (1973). Metabolism and the electrical activity of anoxic ventricular muscle. J. Physiol. 229, 559–582. Meech, R. W. (1972). Intracellular calcium injection causes increased potassium conductance in Aplysia nerve cells. Comp. Biochem. Physiol. A 42, 493–499. Noble, D. (1975). ‘‘Initiation of the Heartbeat.’’ Clarendon, Oxford. Noma, A., and Irisawa, H. (1974). Electrogenic sodium pump in rabbit sinoatrial node cell. Pfu¨g. Arch. 351, 177–182. Noma, A., and Matsuda, H. (1987). Potassium channels identified with single channel recordings and their role in cardiac excitation. In ‘‘Heart Funcfion and Metabolism’’ (N. S. Dhalla, G. N. Pierce, and R. D. Beamish, eds.), pp. 67–78. International Society for Heart Research, Winnipeg, Canada. O’Rourke, B., Backx, P. H., and Marban, E. (1992). Phosphorylationindependent modulation of L-type calcium channels by magnesium-nucleotide complexes. Science 257, 245–248. Page, E., and Storm, S. R. (1965). Cat heart muscle in vitro. VIII. Active transport of sodium in papillary muscles. J. Gen. Physiol. 48, 957–972. Pelleg, A., Vogel, S., Belardinelli, L., and Sperelakis, N. (1980). Overdrive suppression of automaticity in cultured chick myocardial cells. Am. J. Physiol. 238, H24–H30. Reuter, H., and Scholz, H. (1977). The regulation of the calcium conductance of cardiac muscle by adrenaline. J. Physiol. (Lond.) 264, 49–62. Sada, H., Ban, T., and Sperelakis, N. (1995). Oxime depression of the fast sodium current in myocardial cells. Arch. Int. Pharmacodyn. Ther. 330, 319–331. Schneider, J. A., and Sperelakis, N. (1974). The demonstration of energy dependence of isoproterenol-induced transcellular Ca2⫹

current in isolated perfused guinea pig hearts: An explanation for mechanical failure of ischemic myocardium. J. Surg. Res. 16, 389–403. Singer, S. J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720–731. Sperelakis, N. (1972). (Na⫹, K⫹)-ATPase activity of embryonic chick heart and skeletal muscles as a function of age. Biochim. Biophys. Acta 266, 230–237. Sperelakis, N. (1980). Changes in membrane electrical properties during development of the heart. In ‘‘The Slow Inward Current and Cardiac Arrhythmias’’ (D. P. Zipes, J. C. Bailey, and V. Elharrar, eds.), pp. 221–262. Martinus Nijhoff, The Hague. Sperelakis, N. (1994). Regulation of calcium slow channels of heart by cyclic nucleotides and effects of ischemia. Adv. Pharmacol. 31, 1–24. Sperelakis, N. (1995a). Basis of the cardiac resting potential. In ‘‘Physiology and Pathophysiology of the Heart,’’ 3rd Ed., Chapter 3. Kluwer, Boston. Sperelakis, N. (1995b). ‘‘Electrogenesis of Biopotentials in the Cardiovascular System.’’ Kluwer, Boston. Sperelakis, N. (1995c). Defining ischemia: When cells start screaming for help. Br. Med. J. 311, 890–891. Sperelakis, N. (1998). Diffusion and permeability. In ‘‘Cell Physiology Source Book’’ (N. Sperelakis, ed.), 2nd Ed., Chapter 12. Academic Press, San Diego. Sperelakis, N., and Lehmkuhl, D. (1964). Effect of current on transmembrane potentials in cultured chick heart cells. J. Gen. Physiol. 47, 895–927. Sperelakis, N., and Lehmkuhl, D. (1966). Ionic interconversion of pacemaker and nonpacemaker cultured chick heart cells. J. Gen. Physiol. 49, 867–895. Sperelakis, N., and Schneider, J. A. (1976). A metabolic control mechanisms for calcium ion influxes that may protect the ventricular myocardial cell. Am. J. Cardiol. 37, 1079–1085. Sperelakis, N., Schneider, M., and Harris, E. J. (1967). Decreased K⫹ conductance produced by Ba2⫹ in frog sartorius fibers. J. Gen. Physiol. 50, 1565–1583. Trautwein, W., and Kassebaum, D. G. (1961). On the mechanism of spontaneous impoise generation in the pacemaker of the heart. J. Gen. Physiol. 45, 317–330. Vassalle, M. (1965). Cardiac pacemaker potentials at different extraand intracellular K concentrations. Am. J. Physiol. 208, 770–775. Vassalle, M. (1970). Electrogenic suppression of automaticity in sheep and dog Purkinje fibers. Circ. Res. 27, 361–377. Vleugels, A., Carmeliet, E., Bosteels, S., and Zaman, M. (1976). Differential effects of hypoxia with age on the chick embryonic heart: Changes in membrane potential, intracellular K and Na, K efflux and glycogen. Pflu¨g. Arch. 365, 159–166. Vogel, S., and Sperelakis, N. (1977). Blockade of myocardial slow inward current at low pH. Am. J. Physiol. 233, C99–C103. Yokoshiki, H., Katsube, Y., and Sperelakis, N. (1997). Regulation of Ca2⫹ channel currents by intracellular ATP in smooth muscle cells of rat mesenteric artery. Am. J. Physiol. 272, H814–H819.

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10 Cardiac Action Potentials GORDON M. WAHLER Department of Physiology Midwestern University Downers Grove, Illinois 60515

I. INTRODUCTION

of the information on cardiac ion currents has been obtained from cardiac cells isolated from experimental animals, the small number of studies on human cardiac cells (e.g., Coraboeuf and Nargeot, 1993; Ravens et al., 1996) suggests that the currents in the human heart are generally similar to those observed in other animals, especially other mammals.

Heart cells are similar to other cell types in that their internal ionic composition is quite different from the extracellular ionic environment. For example, measurement of the intracellular versus extracellular ionic composition shows that the intracellular ionic composition is low in sodium ions (Na⫹) and high in potassium ions (K⫹), while the reverse is true of the extracellular ionic composition. These concentration differences, together with the selective permeability characteristics of the cell membrane, generate a potential difference of between 80 and 90 mV across the cell membrane of the resting cardiac cell, with the inside being negative with respect to the outside. Additionally, heart cells are excitable cells, i.e., they are capable of generating all-or-none electrical responses known as action potentials (APs). The cardiac AP is caused by the complex interaction of a number of different ionic currents. This chapter reviews the physiological characteristics of the cardiac AP, with emphasis on the major currents responsible for the various components or phases of the APs, as well as some pathophysiology of cardiac APs. The electrical activity of cardiac cells has been studied for several decades by impaling the cells with highresistance microelectrodes. The more recent developments of methods to isolate viable single adult cardiac cells (e.g., Powell et al., 1980), together with the development of the patch clamp technique (Hamill et al., 1981) for recording single-channel (microscopic) currents and whole cell (macroscopic) currents from single cells, has led to an explosion of information on the currents responsible for generation of the cardiac AP. While most

Heart Physiology and Pathophysiology, Fourth Edition

II. CURRENTS DURING PHASES OF THE ACTION POTENTIAL A. Overview Cardiac cells have certain properties in common with other excitable cells, such as nerve and skeletal muscle cells. However, the behavior of cardiac cells also differs from the behavior of nerve and skeletal muscle cells in some important respects. Nerve and skeletal muscle APs are relatively brief, consisting primarily of a rapid depolarization phase (when the inside of the cell becomes more positive) followed immediately by a rapid repolarization phase (when the inside of the cell returns to a more negative potential). The depolarization phase of nerve and skeletal muscle cells is caused by the rapid influx of positive sodium ions into the cell, down the electrochemical gradient for Na⫹, which makes the cell interior more positive. The subsequent repolarization is caused by the efflux of positive potassium ions from the cell, down the electrochemical gradient for K⫹, which returns the cell interior to its original more negative resting potential. The entire process of the nerve or skeletal muscle APs is largely complete within a few

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milliseconds. In cardiac cells, APs are more complex and generally much longer in duration. Thus, for example, a typical cardiac ventricular AP may be at least 100–200 msec in duration (Fig. 1). Additionally, unlike for nerve and skeletal muscle cells, APs from different regions of the heart vary substantially in shape. There are two primary types of cardiac cells. One cell type (fast response) is found in the working cells of the atria, ventricles, and the specialized conduction cells of the His–Purkinje network. These cells have a high resting K⫹ permeability between APs. APs in these cells are generated by a fast Na⫹ current and are known as fast APs. The prolonged duration of the AP in ventricular cells and Purkinje fibers is caused by an approximate balance of an inward Ca2⫹ current and outward K⫹ currents, which results in a prominent plateau phase. Atrial cells have a less prominent plateau. The second type of cardiac cells (slow response) is found in sinoatrial and atrioventricular nodes. These cells have a low K⫹ permeability between APs and are automatic (i.e., spontaneously active). The upstroke of these APs is generated by a Ca2⫹ current, which is smaller and slower than the fast Na⫹ current. Because of this, and the comparatively low density of Ca2⫹ channels, conduction in nodal cells is much slower than conduction in regions having Na⫹-dependent APs. These APs are known as slow APs. Slow APs (slow responses) may sometimes also occur under pathological conditions (e.g., ischemia) in cells that normally have fast APs. The configuration of the fast cardiac AP can be divided into several phases. The following description describes the five phases of the AP found in ventricular cells (phases 0 through 4) and indicates the current or

FIGURE 1 Ventricular action potential. This is a diagrammatic representation of a typical adult mammalian ventricular AP. The five phases of the ventricular AP are labeled (0–4).

TABLE I Phases of the Ventricular Action Potential and Primary Currents Responsible a Phase Phase Phase Phase Phase Phase

0 1 2 3 4

Current INa Ito ICa , IK , Ito IK (IKs , IKr ), IKur ?, IK1 IK1

a INa and ICa are inward currents; potassium currents (Ito , IK , IK1 , etc) are outward currents.

currents that are primarily responsible for each phase. The phases and primary currents in the ventricle are summarized in Table I. Phase 0 is the upstroke of the AP, phase 1 is the early repolarization phase, phase 2 is the plateau phase, phase 3 is the primary repolarization phase, and phase 4 is the resting potential phase of the ventricular AP. Additional information about regional differences in AP phases and currents will be also presented for sinoatrial nodal cells, atrial cells, atrioventricular nodal cells, and Purkinje fibers in Section IV.

B. Phase 0—Sodium Current Phase 0 is the rapid upstroke of the AP. In ventricular cells, as well as in cells of the atria and His–Purkinje network, this upstroke is dependent on a fast Na⫹ current quite similar to the current responsible for the upstroke of the nerve or skeletal muscle APs. This current is designated as fast because it exhibits very rapid activation and inactivation kinetics, in contrast to other currents. Because of the fast upstroke and fast conduction of ventricular APs, APs of these cells (and those of atrial and Purkinje fiber cells) are referred to as ‘‘fast APs,’’ as noted earlier. Figure 2 illustrates the kinetics and voltage dependence of the Na⫹ current. Following a large depolarization, INa reaches a peak in less than 1 msec. Following this peak activation of INa , the amplitude of this current decreases spontaneously. This decay of INa is due to closure (inactivation) of fast Na⫹ channels; thus INa is nearly zero after only a few milliseconds. The fast activation and large magnitude of INa has caused difficulties in recording this current accurately in voltage-clamped cardiac cells. Thus, INa can generally only be recorded in adult cardiac cells at reduced [Na⫹]o levels and/or at low temperatures. Alternatively, INa can be studied in very small cells, such as the embryonic chick ventricular cell (e.g., Fig. 2), because INa (and other currents) are at least an order of magnitude smaller than in adult cells.

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As the cell begins to depolarize further beyond the threshold, INa increases as more Na⫹ channels are activated. Eventually, with even greater depolarization, INa begins to decline as the membrane potential approaches ENa . Thus, peak INa occurs between ⫺30 and ⫺20 mV. The membrane potential never reaches ENa for several reasons: (1) As the membrane potential gets closer to ENa , the driving force for Na⫹ influx is diminished. (2) The Na⫹ channels close shortly after opening (beginning after about 1 msec); thus some Na⫹ channels are already closing during the latter part of the upstroke. (3) Repolarizing currents are beginning to activate during the latter portion of the upstroke. Thus, the maximum positive membrane potential normally attained is approximately 35 mV (Fig. 1). Nevertheless, the upstroke causes a substantial voltage change (110–120 mV) within 1–2 msec in fast cardiac cells.

C. Phase 1—Transient Outward Current

FIGURE 2 Sodium current (INa) recorded from a small embryonic chick ventricular cell. INa in these cells is much smaller than in adult cells due to the very small size of these cells. Additionally, the small spherical shape of these cells and lack of T tubules also contribute to better voltage control. (A) Original INa currents obtained upon stepwise depolarization from ⫺100 mV to the indicated voltages. The current peaks in less than 1 msec at very depolarized potentials and then inactivates rapidly. (B) Current–voltage curve of the peak current at each voltage. Note that the threshold for INa is approximately ⫺70 mV and the current peaks at approximately ⫺25 mV.

For the rapid upstroke of phase 0 to occur, the cell needs to be depolarized to the voltage necessary to open some of the fast Na⫹ channels (approximately ⫺70 mV). When the Na⫹ channels begin to open, Na⫹ flows down the electrochemical gradient into the cell. This causes the cell to depolarize further (i.e., the inside becomes more positive), which opens additional Na⫹ channels. Therefore, once sufficient Na⫹ channels open, the process becomes self-perpetuating, resulting in rapid depolarization (i.e., the membrane potential moves rapidly toward the equilibrium potential for Na⫹). The voltage at which a sufficient number of Na⫹ channels open to initiate the AP is the threshold for firing of the AP.

Phase 1 of the cardiac AP is the transient and relatively small repolarization phase that immediately follows the upstroke of the AP. The size of phase 1 repolarization varies between species and also between different regions of the heart within a given species. Thus, APs recorded from the outer (epicardial) layer of ventricular cells display a more prominent phase 1, whereas APs recorded from the inner (endocardial) layer of ventricular cells display a small phase 1 repolarization (Liu et al., 1993). Phase 1 is also very large in Purkinje fibers and in atrial cells, but is largely absent in nodal cells. Identification of the specific current or currents responsible for phase 1 repolarization has been controversial. It is now clear that the primary ion responsible for most of the phase 1 repolarization is K⫹ (Kenyon and Gibbons, 1979), although a Cl⫺ component does appear to contribute to phase 1 repolarization (Bouron et al., 1991; see later). Phase 1 repolarization is largely due to a transient outward current (Ito). Ito turns on rapidly with depolarization (i.e., beginning during the final portion of the AP upstroke) and is only active at very depolarized potentials; the threshold for activation is approximately ⫺30 mV (Fig. 3). Thus, Ito has a characteristic transient shape—the rapid activation of this current is followed by inactivation during the AP plateau. Because of its voltage dependence and time course, Ito significantly overlaps (and opposes) the inward Ca2⫹ current (which is the primary depolarizing current during the plateau phase, see later). This current (Ito) is actually composed of at least two separate currents (Ito1 and Ito2), which are carried through two physically distinct channels (Tseng and

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component (Ito2) may become more important when intracellular levels of Ca2⫹ become too high. Thus, under Ca2⫹ overload conditions, Ito2 would be activated and shorten the AP duration, thereby indirectly abbreviating the duration of Ca2⫹ current, resulting in a reduced Ca2⫹ influx. Thus, activation of Ito2 by intracellular Ca2⫹ likely acts as a negative feedback mechanism to reduce calcium overload.

D. Phase 2—Calcium Current

FIGURE 3 Transient outward current (Ito) recorded from a 21-day rat ventricular cell. (A) Original currents obtained upon stepwise depolarization from ⫺80 mV. Ito activates rapidly and then inactivates. Currents were recorded in the presence of tetrodotoxin to eliminate INa and cadmium to eliminate overlapping ICa . (B) The current– voltage curve shows increasing activation of this current at voltages above approximately ⫺30 mV (䊊). This current is the Ca2⫹-independent, 4-aminopyridine-sensitive component of Ito (i.e., Ito1) and is therefore blocked readily by 4-aminopyridine (4-AP, 䊉).

Hoffman, 1989). One of the currents (Ito1) is a K⫹ current that is independent of the internal Ca2⫹ concentration ([Ca2⫹]i) and is sensitive to the K⫹ channel blocker 4aminopyridine (4-AP). This component of Ito is very similar to the IA current recorded in nerve fibers. The second component of Ito (Ito2) is Ca2⫹ dependent and less sensitive to 4-AP, but is more sensitive to another K⫹ channel blocker, tetraethylammonium ion (TEA⫹). It is thought that Ito2 is, at least in part, a Ca2⫹-activated Cl⫺ channel (Harvey, 1996). Under physiological conditions, the first Ito component, Ito1 , is by far the larger of the two components. Thus, irrespective of the exact nature of Ito2 , the efflux of K⫹ (through Ito1 channels) appears responsible for the vast majority of phase 1 repolarization. The second

Phase 2 is commonly called the plateau phase. It follows the early repolarization phase (phase 1) and is a period of time in which the membrane potential remains relatively constant, i.e., it does not repolarize rapidly. The presence of this prominent plateau phase is responsible for the long AP duration in cardiac cells, which is the major difference between cardiac cells and nerve or skeletal muscle fibers. The plateau is caused by an approximate balance of positive inward current (which would tend to cause depolarization) and positive outward currents (which would tend to cause repolarization). The primary inward current is a Ca2⫹ current. The primary outward K⫹ current during the plateau phase of ventricular cells (particularly in latter part of the plateau) is the slowly activating K⫹ current known as the delayed rectifier, which is described in greater detail under phase 3 repolarization. Additionally, Ito contributes to the early plateau phase in those cells that have a substantial Ito . In addition to Ca2⫹ and K⫹ currents, there is a small contribution of the fast Na⫹ current to the plateau. Thus, while INa largely inactivates within a few milliseconds after depolarization, a very small fraction of the INa inactivates slowly (see Fig. 1), causing a late sodium current known as the ‘‘sodium window current,’’ which is sufficient to affect the plateau of the AP and hence AP duration (Attwell et al., 1979). The inward Ca2⫹ current (ICa) exhibits activation and inactivation much like INa , but on a slower time scale (Fig. 4). This second inward current is carried by Ca2⫹ and peaks within a few milliseconds, but requires a few hundred milliseconds to completely inactivate. ICa is at least one order of magnitude smaller than INa in a given cell. The threshold for activation of ICa is approximately ⫺40 mV, the current is maximal near 0 mV, and the ECa is around 100 mV. Thus, ICa is active over a more positive (depolarized) potential range than INa . This classical Ca2⫹ current is now commonly referred to as the ‘‘L-type calcium current’’ [ICa(L)] to distinguish it from the novel transient ‘‘T-type’’ Ca2⫹ current [ICa(T)] described later. The L-type Ca2⫹ current is central to many aspects of cardiac function. For example, it is the primary link in excitation–contraction coupling in the heart. Thus,

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very important in automaticity and conduction due to the Ca2⫹-dependent nature of nodal APs. ICa(L) is a major regulatory site in control of cardiac electrical activity and contraction by neurotransmitters, hormones, intracellular ions, and so on. Perhaps the most important regulator of ICa(L) in the heart is the autonomic nervous system. Thus, release of norepinephrine from cardiac sympathetic nerves or release of epinephrine from the adrenal gland stimulates the 웁-adrenergic receptors of the cardiac cell. A cascade of events ensues, which involves the production of cyclic AMP (adenosine 3⬘:5⬘ cyclic monophosphate) and stimulation of the cyclic AMP-dependent protein kinase. This protein kinase directly phosphorylates the Ca2⫹ channel, thereby enhancing the channel activity. The net result of this cascade is that ICa(L) , and thereby force of contraction, is stimulated. For a detailed review of this process, see the chapter on phosphorylation (Chapter 20). The parasympathetic neurotransmitter, acetylcholine (ACh), inhibits cyclic AMP formation. Additionally, ACh stimulates cyclic GMP (guanosine 3⬘ : 5⬘ cyclic monophosphate) production. Cyclic GMP is a second messenger similar to cyclic AMP, but which reduces ICa(L) (see Wahler and Dollinger, 1995).

E. Phase 3—Delayed Rectifier Current

FIGURE 4 L-type calcium current [ICa(L)] recorded from an adult guinea pig ventricular cell. (A) Original currents obtained upon stepwise depolarization from ⫺80 mV to the indicated voltages. INa was inactivated by briefly pulsing to ⫺40 mV prior to applying the indicated test pulse. Ca2⫹ currents show the same general shape as Na⫹ currents (Fig. 2.), i.e., they activate upon depolarizing steps and inactivate during the voltage pulse. However, both activation and inactivation are much slower than for INa (note difference in time scale). (B) The current–voltage curve shows a similar shape as the INa current–voltage relationship; however, both the threshold voltage and the peak voltage are shifted approximately 30 mV in the depolarizing direction. ICa(L) is roughly the same size as INa shown in Fig. 2 only because this cell is so much larger than the embryonic chick cell used in Fig. 2. INa in this guinea pig cell would be approximately 20 nA or more and could not be voltage clamped under these conditions.

the influx of Ca2⫹ ions during the plateau of the AP is what links the electrical events of the AP to the mechanical events, namely contraction. Ca2⫹ influx via ICa(L) stimulates Ca2⫹ release from internal stores (which is the major source of contractile Ca2⫹ in most adult mammalian hearts), replenishes the internal Ca2⫹ stores available for subsequent release, and, to some extent, directly activates the contractile proteins. ICa(L) is also

Phase 3 is the late or final repolarization phase following the AP plateau. It is similar to the repolarization observed in nerve and skeletal muscle cells. It is primarily caused by the unbalance of the currents that were relatively balanced during phase 2. ICa(L) decreases with time (due to inactivation) and the delayed rectifier current (IK) increases (due to slow activation). This eventually leads to the outward current (IK) overwhelming the inward current [ICa(L)]. IK is the primary repolarizing current in most ventricular preparations. The classical delayed rectifier activates slowly, compared to most other currents, and does not inactivate with time. Thus, IK increases gradually during sustained depolarization at voltages around the plateau level. This current is similar to the delayed rectifier in nerve cells, although slower. IK can be clearly distinguished from Ito by slow activation, lack of inactivation, and different pharmacology. Due to technical reasons, IK is often greatly underestimated in patch clamp recordings. For example, in canine atria, IK was found in 4% of cells isolated using a ‘‘chunk’’ method vs 99% of cells isolated using a ‘‘perfusion’’ method (Yue et al., 1996). There are several components of IK ; thus many investigators divide IK into a very slowly activating component (IKs) and a more rapidly activating component (IKr) (Sanguinetti and Jurkiewicz, 1990; Lindblad et al., 1996). The sensitivity of the two IK components to pharmaco-

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logical agents differs, as does the shape of their current– voltage curves and their activation threshold voltage, suggesting that these two components of IK are carried through distinct channels. The 웁-adrenergic–cyclic AMP cascade can stimulate IK , similar to its ability to stimulate ICa(L) (e.g., Walsh and Kass, 1988), which would tend to shorten the AP. Thus, 웁-adrenergic stimulation tends to lengthen the AP duration by enhancing ICa and at the same time tends to shorten AP duration by enhancing IK . Thus the overall effect of 웁-adrenergic stimulation on AP duration is determined by the relative contribution of the changes in these two currents (and perhaps also the contribution of ICl , see later). The slower component of IK , IKs , is the component of IK that is enhanced in conditions of 웁-adrenergic stimulation (Sanguinetti and Jurkiewicz, 1990). Another potentially important repolarizing current has been demonstrated in human atrial myocytes. This ‘‘ultrarapid-activating’’ delayed rectifier K⫹ current (IKur) shows slow deactivation and is insensitive to TEA, Ba2⫹, and dendrotoxin, but is sensitive to 4-AP (Feng et al., 1998). Thus, IKur is a K⫹ current that can be distinguished from both Ito1 (from which it differs by voltage dependence) and the classical IK (from which it differs by sensitivity to 4-AP). Some ventricular preparations also exhibit a Ca2⫹-dependent IK (Tohse, 1990). This component may help shorten the AP duration similar to the effect of the Ca2⫹-dependent Ito described earlier. However, the relative magnitude and importance of these currents are still unclear. As the membrane potential continues to repolarize and approaches the resting membrane potential, largely due to the effects of IK , the inwardly rectifying K⫹ current (IK1 , which is the primary determinant of the resting potential) also begins to contribute to repolarization. Thus, it is clear that a number of potassium currents may contribute to phase 3 of the action potential. Interestingly, in general, APs are longer in females than males, resulting in a greater Q-T interval. This suggests that the repolarizing currents may be smaller in females than males, which may explain the greater incidence of Torsades de Pointes arrhythmias in females (Vizgirda, 1999) (see Section VI, B, for discussion of the role of channel mutations in the development of long Q-T syndrome and Torsades de Pointes).

F. Phase 4—Inward Rectifier Current In ventricular cells, and most other cardiac cells, phase 4 is the resting potential. The resting potential is defined as the stable, negative potential that occurs between APs in nonspontaneous cells. The resulting negative resting potential is maintained by the Na⫹ –K⫹

pump. It is very near EK due to a relatively high permeability of the resting ventricular cell membrane to K⫹ ions, and a very low permeability to other ions. Thus, the resting potential of nonautomatic cardiac cells is similar to the resting potential in skeletal muscle cells. The resting potential is determined largely by a K⫹ current known as the inward (or anomalous) rectifier, IK1 . The most notable characteristic of IK1 is that it displays inward rectification. Thus, it passes current more readily in the inward direction than in the outward direction. This characteristic is evident in Fig. 5. This inward rectification of the IK1 channel current is thought to be

FIGURE 5 Inwardly rectifying K⫹ current (IK1) recorded from an adult guinea pig ventricular cell. (A) Original barium-subtracted currents obtained upon stepwise hyperpolarization and depolarization from a holding potential of ⫺40 mV. The current displays the typical inward rectification, i.e., the current is smaller in the outward (physiological) direction than in the inward direction. (B) The current– voltage curve for IK1 . The current also displays a negative slope region between approximately ⫺60 and ⫺30 mV. Thus, larger depolarizations actually result in a decrease in outward K⫹ flux during this voltage range, such that the current at ⫺30 mV is less than half as large as at ⫺60 mV. The net result is that during repolarization (phase 3) the K⫹ current increases as the membrane potential approaches the resting potential.

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due to blockade of the channels by intracellular Mg2⫹ (Matsuda et al., 1987) and polyamines (Lopatin et al., 1994). Of course, the outward K⫹ current is the only one occurring physiologically, as the membrane potential does not normally hyperpolarize beyond EK . In most preparations, IK1 also demonstrates a negative slope region in the outward direction. That is, as the cell is depolarized from near its resting potential to progressively more depolarized potentials, the outward current first increases and then decreases with further depolarization, with the outward current dropping to very low levels at voltages positive to ⫺40 mV. Thus, the contribution of IK1 to repolarization tends to be less at potentials near the plateau and greater as the membrane potential approaches the resting potential, i.e., at the time that IK is declining.

III. ADDITIONAL CURRENTS CONTRIBUTING TO THE ACTION POTENTIAL A. Pump Current ⫹

⫹

The Na –K ATPase, or pump, is the primary transport system that maintains the ionic imbalance between the cell exterior and interior. That is, each pump cycle extrudes three Na⫹ ions out of the cell and transports two K⫹ ions into the cell, thus building up [K]i and reducing [Na]i . Because of the exchange of three positive for two positive ions, each pump cycle generates a net loss of one positive charge, which generates a pump current (Ipump). At the resting potential, Ipump is an outward current, which may hyperpolarize the membrane potential slightly. In addition, Ipump can cause considerable shortening of the action potential when [Na]i increases pathologically (Gadsby, 1984). Additionally, blockade of the pump with toxic concentrations of digitalis may lead to a significant increase in intracellular [Na⫹], which may in turn activate a Na⫹-dependent K⫹ current [IK(Na)] (Luk and Carmeliet, 1990). However, the primary role of the Na⫹ –K⫹ ATPase is to set up and maintain the ionic gradients that generate the electrochemical driving forces for the currents responsible for the action potential.

B. Na–Ca Exchange Current A Na/Ca exchanger also exists in the cardiac sarcolemma. Working in the ‘‘normal mode’’ the Na/Ca exchanger exchanges intracellular Ca2⫹ for extracellular Na⫹; thus the exchanger is an important mechanism whereby Ca2⫹ is removed from the cytoplasm. The exchanger may also work in ‘‘the reverse’’ mode, exchang-

ing intracellular Na⫹ for extracellular Ca2⫹. Under these conditions, the exchanger can contribute Ca2⫹ influx for excitation–contraction coupling. The exchanger transports three Na⫹ for each Ca2⫹ under most conditions, leading to the net movement of one positive charge. Thus, the exchanger generates a current that can also contribute to the action potential. The equilibrium potential for Na/Ca exchange current is generally slightly negative to 0 mV; therefore near the resting potential the Na/Ca exchanger works in the normal mode and generates an inward current. During the initial portion of the plateau, the Na/Ca exchanger transiently works in the reverse mode and briefly generates an outward current prior to returning to the normal mode. Thus, the Na/Ca exchange current may contribute to the shape of the AP (for review, see Janvier and Boyett, 1996).

C. Cyclic AMP-Stimulated Chloride Current Under basal conditions, the Cl⫺ current (ICl) in the heart is relatively small and probably does not contribute a great deal to the configuration of the AP. However, when cyclic AMP levels are stimulated (as with sympathetic nerve stimulation), a significant time-independent Cl⫺ current develops. Activation of this current by 웁-adrenergic stimulation can cause a small depolarization of the resting potential and significant shortening of the action potential (Harvey et al., 1990).

IV. REGIONAL DIFFERENCES IN ACTION POTENTIALS A. Overview The normal pathway for electrical activation of the heart is sinoatrial (SA) node, atria, atrioventricular (AV) node, bundle of His, Purkinje fibers, ventricles. APs differ from region to region, reflecting the different roles played by the different cell types. The following description characterizes AP potentials in each region and indicates how each differs from the ventricular AP.

B. Sinoatrial Node The SA node contains specialized cells that generate APs, which are quite different from the ventricular APs described previously (Fig. 6). Unlike ventricular APs, these cells do not have a true resting potential, i.e., the membrane potential between APs is not stable, but rather exhibits a slow spontaneous depolarization known as phase 4 depolarization or the pacemaker potential. Because there is no resting potential in these

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of approximately ⫺55 to ⫺70 mV, the lack of IK1 in these cells does not significantly slow repolarization because IK1 contributes to repolarization primarily at potentials nearer to EK .

C. Atria

FIGURE 6 Sinoatrial node AP. This is a diagrammatic representation of a typical sinoatrial node AP in the mammalian heart. Note the pacemaker potential and the slower upstroke compared to the fast AP of ventricular cell (Fig. 1).

cells, the most negative potential the cell reaches between APs is called the maximum diastolic potential. This potential is less negative than the resting potential of ventricular cells due to a lower K⫹ permeability (caused by a lack of IK1 in these cells). The maximum diastolic potential ranges from approximately ⫺55 mV for true primary pacemaker cells to approximately ⫺70 mV for transitional cells on the border between the SA node and atria. The pacemaker potential takes the nodal cell from the maximum diastolic potential to the threshold for generating an AP in these cells (approximately ⫺40 mV). Thus, these cells are spontaneously active, and the slope of phase 4 depolarization is an important determinant of the rate of AP generation and thereby heart rate. Once the phase 4 depolarization brings the cell to the threshold for AP firing, an AP occurs, as in ventricular cells. However, in nodal cells, the upstroke (phase 0) is quite different from the upstroke in ventricular (and nerve or skeletal muscle) cells: it is a much slower upstroke and is Ca2⫹ dependent rather than Na⫹ dependent, i.e., INa is negligible in SA node cells and does not contribute significantly to phase 0 (Irisawa et al., 1993). Thus, the upstroke in nodal cells is generated by the inward Ca2⫹ current, ICa(L) . Because the speed of the upstroke largely determines the speed of conduction, this slow phase 0 is very important in cardiac function, as it results in a slow conduction in nodal cells. There is generally no phase 1 and a brief plateau (phase 2) in nodal cells. Phase 3 repolarization returns the cell to the maximum diastolic depolarization. Because nodal cells only repolarize to the maximum diastolic potential

Atrial cells have APs that are similar in many aspects to the ventricular APs described earlier. Thus, the resting potential (phase 4) is approximately ⫺85 mV, and there is a fast upstroke (phase 0) generated by INa . The most distinguishing feature of the atrial AP is that it has a more triangular appearance than the ventricular AP. This more triangular appearance seems to be due to a prominent phase 1 in atrial cells. Thus, in atrial cells, phases 1, 2, and 3 tend to run together, resulting in a triangular shape, with a distinct plateau not always apparent. This is likely due to a large Ito in atrial cells.

D. Atrioventricular Node Cells of the AV node generate APs that are quite similar to the APs of the SA node. Thus, these cells fire Ca2⫹-dependent APs and also display spontaneous phase 4 depolarization (i.e., automaticity). However, the rate of phase 4 depolarization in AV nodal cells is much slower than the rate of phase 4 depolarization in SA nodal cells. Thus, SA node cells fire APs before AV node cells fire, which is why SA node cells are the normal pacemaker cells of the heart.

E. Purkinje Fibers Purkinje APs are in most respects similar to ventricular APs. Thus, these cells have a negative resting potential between APs (phase 4) and a very rapid upstroke (phase 0) generated by INa . Purkinje fibers differ from ventricular cells in that they have a more prominent phase 1 repolarization and a longer plateau (phase 2). The plateau is followed by a phase 3 repolarization that is virtually identical to phase 3 in ventricular cells. Cells in the bundle of His appear to have APs similar to those in Purkinje fibers; however, in general, the bundle of His cells have not been studied in detail. Additionally, Purkinje cells may exhibit automaticity, especially when the extracellular K⫹ concentration is low. Thus, under some conditions, they exhibit phase 4 depolarization. Purkinje cells have all the currents found in ventricular cells, described previously (INa , Ito , ICa , IK , IK1). Purkinje cells also have some additional currents, which are absent or very small in ventricular cells, which

10. Cardiac Action Potentials

are related to the latent pacemaker function of these cells. These additional currents are described in the following section.

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SA node cells using various mathematical models. There are limitations to this approach, as there are considerable differences between automatic cell types. Thus, for instance, the pacemaker potential range is much more depolarized in SA node cells compared to Purkinje cells, which is bound to influence the relative contribution of various currents.

A. Overview Automaticity refers to the ability of some cardiac cells to depolarize and fire repetitive APs spontaneously. Thus, as noted earlier, automatic cells (e.g., in the SA node) do not have a stable resting potential between APs, but rather have a maximum diastolic potential followed by a spontaneous phase 4 depolarization known as the pacemaker potential. The spontaneous depolarization to threshold generates the AP in that cell type. Following repolarization of the AP, the spontaneous phase 4 depolarization occurs again. The slope of the pacemaker potential (i.e., rate of spontaneous phase 4 depolarization) largely determines the rate of AP firing. Because the rate of firing of APs is normally fastest in the primary pacemaker cells of the SA node, this region acts as the normal pacemaker of the heart. Once this region fires an AP, the wave of depolarization is propagated to other regions of the heart, ultimately leading to contraction of the heart. Automatic cells in other regions of the heart (e.g., AV node) normally do not have the opportunity to spontaneously fire before the wave of depolarization arising from the SA node drives them to threshold.

B. Mechanisms of Automaticity Automaticity is a property of several cell types in the heart under physiological conditions. Under pathophysiological conditions, even normally nonspontaneous cells (e.g., ventricular cells) may exhibit automaticity. Physiologically, the most important pacemaker potential is that of normal pacemaker cells in the SA node. However, the very small size of the SA node cells makes all the currents difficult to measure accurately; additionally, the very high input resistance (due to lack of IK1 in the SA node) means that extremely small currents may be significant contributors to the pacemaker potential in SA node cells. These technical difficulties have limited our understanding of the pacemaker process in SA node cells. Therefore, considerable controversy exists regarding the precise mechanism of automaticity in SA node cells (for reviews, see Baumgarten and Fozzard, 1992; Campbell et al., 1992). Because of these limitations, much of what we know about automaticity is derived experimentally from other automatic cell types (e.g., Purkinje cells) and then extrapolated to

1. Automaticity in Purkinje Fibers Under physiological conditions, Purkinje fibers can have an extremely slow phase 4 pacemaker potential. In contrast to SA node cells, the mechanisms for automaticity in Purkinje cells are fairly well understood. As noted earlier, Purkinje cells have all the currents that ventricular cells have, plus some additional currents not found to any significant degree in adult ventricular cells. The large IK1 current in Purkinje cells tends to clamp the membrane potential near EK and, therefore, a large depolarizing current is needed to overcome this clamping effect. The primary current responsible for the pacemaker potential in Purkinje has some unusual properties, earning it the designation as the ‘‘funny current’’ (If). If is a slowly activating inward depolarizing current activated by hyperpolarization, which is present in automatic cells. If is largely absent in adult ventricular cells (or it may be present but nonfunctional in ventricular cells due to voltage inactivation; see Yu et al., 1993). It is a nonselective cation current, i.e., it is carried by a mixture of both Na⫹ and K⫹ ions. In Purkinje cells, If is responsible for most of the depolarizing current that generates the pacemaker potential. In addition to If , there may be a small contribution of the ‘‘T-type’’ Ca2⫹ current to the latter stages of the pacemaker potential in Purkinje cells (see description of automaticity in nodal cells), as well as a small contribution of INa to the final portion of the pacemaker potential in these cells. Once the Purkinje cell is depolarized to the threshold for INa (approximately ⫺70 mV) Na⫹ channels will open. The inward flux of Na⫹ may contribute to the final phase of the pacemaker potential, as the membrane potential approaches the threshold for AP generation. Thus, the Purkinje fiber will fire an AP, which is generated by INa , as described earlier for ventricular cells. The remainder of the AP is generated by essentially the same mechanisms as described previously for ventricular cells. 2. Automaticity in Nodal Cells Several factors contribute to the pacemaker potential in nodal cells. Because of the very low density of IK1 channels in nodal cells, the resting K⫹ permeability is

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much lower in nodal cells than in ventricular cells. The large resting K⫹ permeability in ventricular cells generated by IK1 tends to keep the interior of the cells negative, opposing depolarization of the cell toward threshold by ‘‘clamping’’ the membrane potential near EK . A much smaller current is sufficient to depolarize the nodal cells due to this much lower resting K⫹ permeability. Thus, currents that may be too small to measure accurately using present electrophysiological techniques (small background currents or currents produced by various electrogenic transport mechanisms) could produce sufficient current to affect the pacemaker potential. Because of this limitation, the analysis of the relative contribution of various currents to the pacemaker potential in nodal cells is much less clear than for Purkinje cells. Therefore, investigators often use mathematical models of nodal electrophysiology, which are largely based on the characteristics of various currents measured in other automatic cell types (e.g., Purkinje fibers, sinus-venosus of amphibian hearts) in which the measurements are made more readily. However, the mathematical models of nodal cells at present are inadequate to accurately describe changes in automaticity under a variety of physiological conditions (see Dokos et al., 1996). The major depolarizing current during the pacemaker potential of Purkinje cells, If , is also present in nodal cells and probably contributes somewhat to the pacemaker potential in the SA node. As noted earlier, If is an unusual depolarizing current in that it is activated by hyperpolarization (DiFrancesco, 1993). Thus, it is likely to be quantitatively less important to normal pacemaker activity in nodal cells as in comparison to Purkinje cells, as nodal cells normally operate at much more depolarized voltages. In fact, in studies on SA node cells using perforated patch recording, in which the intracellular environment should be more physiological, blockade of If had only a little effect on SA node automaticity (Liu et al., 1998). It may be that that If may not be very important for normal automaticity, but may be a protective mechanism, maintaining automaticity when the maximum diastolic potential becomes much more hyperpolarized [e.g., with strong vagal stimulation, resulting in opening of many IK(ACh) channels and considerable hyperpolarization; see later]. In addition to If , the interaction between a depolarizing background current (Ib) and the decay of the major repolarizing current, the delayed rectifier (IK) may also provide a depolarizing current capable of contributing to the pacemaker potential in nodal cells. Because the relative contribution of these currents to the pacemaker potential in nodal cells cannot be determined accurately experimentally, there has been considerable contro-

versy over which depolarizing current (If or Ib) plays the greater role in determining the slope of the pacemaker potential in these cells. This small inward background current (Ib), present in nodal cells, is a cation current carried primarily by Na⫹ ions (Hagiwara et al., 1992). Ib contributes a constant small depolarizing current. Due to the small size of this current, relatively little is known about its magnitude and characteristics in mammalian SA node cells; however, indirect evidence suggests that it may be a very important component in determining automaticity (see Campbell et al., 1992; Dokos et al., 1996). The role of a constant Ib in generating a variable pacemaker potential likely stems from the interaction of Ib with a variable IK . IK is the primary current responsible for repolarization in nodal cells, as in other cardiac cells. As in ventricular cells, IK has also been shown to consist of at least two components (IKs and IKr) (see Dokos et al., 1996). IK displays essentially no inactivation during a prolonged depolarizing pulse, but displays a slow decay upon repolarization toward EK . The time course of the IK decay is very slow at membrane potentials in the voltage range of the pacemaker potential in nodal cells. The depolarizing action of Ib is opposed by IK . Thus, the depolarization due to a constant background current Ib increases progressively with time due to a gradual reduction of opposing repolarizing current, IK . This is thought to be a significant factor in development of the pacemaker potential in nodal cells. Other small currents may also contribute to and/or modulate the pacemaker potential in nodal cells, e.g., the electrogenic sodium–potassium ATPase pump current, Ipump , and/or the electrogenic Na–Ca exchange current (Noble, 1984; Dokos et al., 1996). As noted earlier, the upstroke (phase 0) of nodal cells is generated by an L-type Ca2⫹ current rather than a Na⫹ current. This Ca2⫹ current appears identical to the classic long-lasting L-type Ca2⫹ current [ICa(L)], which is largely responsible for the plateau in both nodal and working cardiac cells. In addition to ICa(L) , nodal cells (and other pacemaker cells) have a second type of Ca2⫹ current, which is activated at more negative potentials and has a much more rapid inactivation (Hagiwara et al., 1988). This second, rapid type of Ca2⫹ current has been named the transient, or ‘‘T-type’’ Ca2⫹ current [ICa(T)], in contrast to the classic, slowly inactivating, long-lasting, or ‘‘L-type’’, Ca2⫹ current [ICa(L)]. This T-type Ca2⫹ current is small to nonexistent in adult ventricular cells. It appears that both T- and L-type Ca2⫹ currents may contribute to the latter part of the pacemaker potential in nodal cells (Doerr et al., 1989). Because the T-type channels are active at more negative potentials than the L-type channels, the presence of

10. Cardiac Action Potentials

T channels effectively lowers the threshold for ICa(L) . That is, the T-type current may not only contribute significantly to the pacemaker potential, but opening of T-type channels can depolarize the cells further toward the threshold for L-type channels.

C. Modulation of Automaticity If and ICa(L) are both enhanced by the sympathetic neurotransmitter, norepinephrine (NE), and inhibited by the parasympathetic neurotransmitter, acetylcholine. Thus, NE increases the slope of the phase 4 depolarization, the threshold is reached sooner, and heart rate increases. In contrast, ACh decreases the slope of the phase 4 depolarization, the threshold is reached more slowly, and heart rate decreases. In addition to the effect on If and ICa(L) , ACh also activates another specific K⫹ current, IK(ACh) , which hyperpolarizes the cell, i.e., driving the maximum diastolic potential further from threshold. Thus, when IK(ACh) is activated, it takes a longer time to reach threshold and, also, the rate of phase 4 depolarization (slope) is decreased; thus the heart rate is decreased. The pacemaker cells of the SA node are richly innervated by sympathetic and parasympathetic nerves. These actions of NE and ACh on If , ICa(L) , and IK(ACh) in SA node cells are the basis of the stimulating and inhibiting effect on the heart rate of sympathetic or parasympathetic nerve stimulation.

VI. PATHOPHYSIOLOGY OF CARDIAC ACTION POTENTIALS

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of intracellular ATP (Fig. 7) and thus appears to contribute little to the AP configuration under conditions of adequate oxygenation. However, during inadequate oxygenation, the AP is shortened as IK(ATP) replaces IK1 . This is in large part because IK(ATP) displays less inward rectification than IK1 and thus has a greater effect on repolarization. The physical characteristics of the IK(ATP) channel (e.g., ATP sensitivity and/or degree of rectification) are altered in some pathophysiological states, such as hypertrophy (Cameron et al., 1988) or diabetic cardiomyopathy (Smith and Wahler, 1996; Shimoni et al., 1998). This may be an important factor in the abnormal responses to ischemia in these conditions. In addition to the direct effects of opening of IK(ATP) on the AP, K⫹ often accumulates extracellularly during ischemia due to the opening of IK(ATP) channels and concomitant zero or low flow conditions. This accumulation of extracellular K⫹ causes depolarization of the cell. The depolarization will lead to voltage inactivation of sodium channels. Thus, APs in the ischemic area will have a reduced INa , leading to slow APs, which have a slower upstroke, which is mediated by either a combination of INa and ICa for small depolarizations, or largely by ICa if the depolarization is greater. This slowed upstroke will lead to slow conduction through the region and enhanced likelihood of arrhythmias. The intracellular ATP levels reached during acute ischemia generally remain higher than required to open a substantial number of IK(ATP) channels. However, the decreasing intracellular pH and increasing lactate accumulation can also promote IK(ATP) channel opening (Fan and Makielski, 1993), in addition to a fall in intracellular

A. Ischemia When the oxygen supply declines, as occurs during ischemia, AP duration shortens. The shortening of the AP duration accelerates inactivation of ICa , thereby reducing contractility. The reduced contractility decreases the energy demands of the cell greatly, thereby sparing ATP. This mechanism contributes to the survival of the myocardial cell during temporary ischemia. However, in addition to this beneficial effect, regional shortening of the AP can also lead to arrhythmias due to the dispersion of refractory periods. The shortening of the AP during ischemia is caused, in large part, by activation of a unique outward K⫹ current, which is inhibited by intracellular ATP. The decreased oxygen supply reduces ATP levels in the cell and IK1 is inhibited. At the same time, another K⫹ current [IK(ATP)] is activated (Noma, 1983). This ATPsensitive K⫹ current is inhibited by physiological levels

FIGURE 7 ATP-sensitive K⫹ current [IK(ATP)] recorded from an inside-out patch from an adult rat ventricular cell. Single-channel currents shown were recorded in the absence of ATP (top) and in the presence of physiological levels of ATP (3 mM, bottom). Channel openings are downward. In the absence of ATP, channel activity is high. Thus, up to two channels are open simultaneously (number of open channel levels are indicated at the side). In the presence of 3 mM ATP, the channels are rarely open. In the presence of ATP, the openings are extremely brief, such that they do not appear to reach the normal open level (1).

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ATP levels. Furthermore, opening of only a small fraction of IK(ATP) channels may be sufficient to shorten the AP substantially (Faivre and Findlay, 1990). Other channels may also be activated by ischemia, in addition to IK(ATP) channels, e.g., lysophosphatidylcholine and long-chain acylcarnitine resulting from membrane phospholipid metabolism accumulate rapidly in early ischemia and may activate the arachidonic acid-activated K⫹ channel [IK(AA)] and the phosphatidylcholine-activated K⫹ channel [IK(PC)] (Monsuez, 1997a).

B. Cardiac Channelopathies— Long Q-T Syndrome Advances in molecular biology have discovered mutations in several genes encoding a number of different ion channels in the heart (or channel subunits) that appear to be responsible for specific electrophysiological abnormalities. The best understood of these electrophysiological abnormalities is the long Q-T syndrome. The long Q-T syndrome is a condition that involves slowed repolarization and results in an increased risk of sudden death from Torsades de Pointes arrhythmias (Veldkamp, 1998; Vizgirda, 1999). There are several forms of long Q-T syndrome, which have been attributed to mutations in several genes encoding several potassium channel subunits (KVLQT1, IsK, HERG) and at least one mutation in a gene encoding a subunit of the fast sodium channel (SCN5A) (Lehman-Horn and Rudel, 1997; Barhanin et al., 1998).

C. Development of New Antiarrhythmic Agents A large number of drugs have been used in the treatment of a variety of arrhythmias. In the past, many of the antiarrhythmic drugs were relatively nonspecific, i.e., they had effects on a large number of different ion channels. In recent years, significant advances in electrophysiological and molecular biology have led not only to the identification of different subclasses of ion channels (especially potassium channels), but to the development of more channel-specific antiarrhythmic agents. For example, D,L-Sotalol has been used for many years as a class III antiarrhythmic agent. It is a relatively nonselective potassium channel blocker in that it has been shown to block IKr , IK1 , Ito1 , and IK(ACh) (the latter being due to D,L-Sotalol’s action as a muscarinic receptor antagonist). However, D-Sotalol, E-4031, and a number of other new agents have been shown to be more specific IKr blockers (Monsuez, 1997b). Thus, the possibility exists, if not in the present—then in the not too distant future, of treating arrhythmias caused by muta-

tions in a specific ion channel with drugs targeting that specific channel.

VII. SUMMARY APs in the heart are generated by the complex timedependent interaction of several currents carried primarily by Na⫹, K⫹, and Ca2⫹ ions. Some cardiac cells display automaticity due to the presence of a cyclical spontaneous depolarization called the pacemaker potential. Cells of the sinoatrial node normally exhibit the fastest spontaneous depolarization of automatic cells in the heart and are therefore the normal pacemaker cells. Automaticity in nodal cells is caused by the interaction of several currents, including a hyperpolarizationactivated cation current (the ‘‘funny current’’), a small background Na⫹ current, the delayed rectifier K⫹ current, and Ca2⫹ currents. The sympathetic and parasympathetic nerves play important (and opposite) roles in regulating the force of myocardial contraction and heart rate. Thus, sympathetic nerves stimulate the Ca2⫹ current, which enhances the force of contraction of the heart. Parasympathetic nerves inhibit the Ca2⫹ current, which decreases the force of contraction of the heart. Sympathetic nerves enhance the rate of spontaneous phase 4 depolarization in the sinoatrial node, resulting in an increased heart rate. Parasympathetic nerves decrease the rate of phase 4 depolarization, and also hyperpolarize sinoatrial node cells, resulting in a decreased heart rate. Thus, actions of neurotransmitters on cardiac ionic currents are a major site for modulating cardiac function. In addition to the physiological modulation of ion currents in the heart, a number of pathological conditions can alter the relative contributions of the various currents to the overall AP. Examples discussed include ischemia and genetic mutations in ion channel subunits, which affect the overall electrophysiology of the heart due to their effects on one or more specific ionic currents.

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10. Cardiac Action Potentials ment of a chloride conductance in the transient outward current of whole-cell voltage-clamped ferret ventricular myocytes. Pflug. Arch. 419, 534–536. Cameron, J. S., Kimura, S., Jackson-Burns, D. A., Smith, D. B., and Bassett, A. L. (1988). ATP-sensitive K⫹ channels are altered in hypertrophied ventricular myocytes. Am. J. Physiol. 255, H1254– H1258. Campbell, D. L., Rasmussen, R. L., and Strauss, H. C. (1992). Ionic current mechanisms generating vertebrate primary cardiac pacemaker activity at the single cell level: An integrative view. Annu. Rev. Physiol. 54, 279–302. Coraboeuf, E., and Nargeot, J. (1993). Electrophysiology of human cardiac cells. Cardiovasc. Res. 27, 1713–1725. DiFrancesco, D. (1993). Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55, 455–472. Doerr, T., Denger, R., and Trautwein, W. (1989). Calcium currents in single SA nodal cells of the rabbit heart studied with the action potential clamp. Pflug. Arch. 413, 599–603. Dokos, S., Celler, B., and Lovell, N. (1996). Ion currents underlying sinoatrial node pacemaker activity: a new single cell mathematical model. J. Theor. Biol. 181, 245–272. Fan, Z., and Makielski, J. C. (1993). Intracellular H⫹ and Ca⫹⫹ modulation of trypsin modified ATP sensitive K⫹ channels in rabbit ventricular myocytes. Circ. Res. 72, 715–722. Faivre, J.-F., and Findlay, I. (1990). Action potential duration and activation of ATP-sensitive potassium current in isolated guineapig ventricular myocytes. Biochim. Biophys. Acta 1029, 167–172. Feng, J., Xu, D., Wang, Z., and Nattel, S. (1998). Utrarapid delayed rectifier current inactivation in human atrial myocytes: properties and consequences. Am. J. Physiol. 275, H1717–1725. Gadsby, D. C. (1984). The Na⫹ /K⫹ pump of cardiac cells. Annu. Rev. Biophys. Bioeng. 13, 373–378. Hagiwara, N., Irisawa, H., and Kameyama, M. (1988). Contribution of two types of calcium currents to the pacemaker potential of rabbit sino-atrial node cells. J. Physiol. 395, 233–254. Hagiwara, N., Irisawa, H., Kasanuki, H., and Hosoda, S. (1992). Background current in sino-atrial node cells of the rabbit heart. J. Physiol. 448, 53–72. Hamill, O. P., Marty, A., Neher, A., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflug. Arch. 391, 85–100. Harvey, R. D. (1996). Cardiac chloride currents. N.I.P.S. 11, 175–181. Harvey, R. D., Clark, C. D., and Hume, J. R. (1990). Chloride current in mammalian cardiac myocytes: Novel mechanism for autonomic regulation of action potential duration and resting membrane potential. J. Gen. Physiol. 95, 1077–1102. Irisawa, H., Brown, H. F., and Giles, W. (1993). Cardiac pacemaking in the sinoatrial node. Physiol. Rev. 73, 197–227. Janvier, N. C., and Boyett, M. R. (1996). The role of Na-Ca exchange current in the cardiac action potential. Cardiovasc. Res. 32, 69–84. Kenyon, J. L., and Gibbons, W. R. (1979). Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers. J. Gen. Physiol. 73, 117–138. Lehman-Horn, F., and Rudel, R. (1997). Channelopathies: Their contribution to our knowledge about voltage-gated ion channels. News Physiol. Sci. 12, 105–112. Lindblad, D. S. Murphey, C. R., Clark, J. W., and Giles, W. R. (1996). A model of the action potential and underlying membrane currents in a rabbit atrial cell. Am. J. Physiol. 271, H1666–H1696. Liu, D. W., Gintant, G. A., and Antzelevitch, C. (1993). Ionic bases

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for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ. Res. 72, 671–687. Liu, Y. M., Yu, H., Li, C. Z., Cohen, I. S., and Vassalle, M. (1998). Cesium effects on if and IK in rabbit sinoatrial node myocytes: Implications for SA node automaticity. J. Cardiovasc. Pharmacol. 32, 783–790. Lopatin, A. N., Makhina, E. N., and Nichols, CG. (1994). Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 372, 368–369. Luk, H. N., and Carmeliet, E. (1990). Na⫹ activated K⫹ current in cardiac cells: Rectification, open probability block and role in digitalis toxicity. Pflug. Arch. 416, 766–768. Matsuda, H., Saigusa, A., and Irasawa, H. (1987). Ohmic conductance through the inwardly rectifying K channel and blocking by the integral Mg⫹⫹. Nature 325, 156–159. Monsuez, J. J. (1997a). Cardiac potassium currents and channels. I. Basic science aspects. Int. J. Cardiol. 61, 209–219. Monsuez, J. J. (1997b). Cardiac potassium current and channels. II. Implications for clinical practice and therapy. Int. J. Cardiol. 62, 1–12. Noble, D. (1984). The surprising heart: a review of recent progress in cardiac electrophysiology. J. Physiol. (London) 353, 1–50. Noma, A. (1983). ATP-regulated K⫹ channels in cardiac muscle. Nature 305, 147–148. Powell, T., Terrar, D. A., and Twist, V. W. (1980). Electrical properties of individual cells isolated from adult rat ventricular myocardium. J. Physiol. (Lond.) 302, 131–153. Ravens, U., Wettwer, E., Ohler, A., Amos, G. J., and Mewes, T. (1996). Electrophysiology of ion channels of the heart. Fundam. Clin. Pharmacol. 10, 321–328. Sanguinetti, M. C., and Jurkiewicz, N. K. (1990). Two components of cardiac delayed rectifier K⫹ current: Differential sensitivity to block by class III antiarrhythmic agents. J. Gen. Physiol. 96, 195–215. Shimoni, Y., Light, P. E., and French, R. J. (1998). Altered ATP sensitivity of ATP-dependent K⫹ channels in diabetic rat hearts. Am. J. Physiol. 275, E568–E576. Smith, J. M., and Wahler, G. M. (1996). ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats. Mol. Cell. Biochem. 158, 43–51. Tohse, N. (1990). Calcium-sensitive delayed rectifier potassium current in guinea pig ventricular cells. Am. J. Physiol. 258, H1200– H1207. Tseng, G. N., and Hoffman, B. F. (1989). Two components of transient outward current in canine ventricular myocytes. Circ. Res. 64, 633–647. Veldkamp, M. W. (1998). Is the slowly activating component of the delayed rectifier, Iks, absent from undiseased human ventricular myocardium? Cardiovasc. Res. 40, 433–435. Vizgirda, V. M. (1999). The genetic basics for cardiac dysrhythmias and the long QT syndrome. J. Cardiovasc. Nurs. 13, 34–45. Wahler, G. M., and Dollinger, S. J. (1995). Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMPdependent protein kinase. Am. J. Physiol. 268, C45–C54 Walsh, K. B., and Kass, R. S. (1988). Regulation of a heart potassium channel by protein kinase A and C. Science 242, 67–69. Yu, H., Chang, F., and Cohen, I. S. (1993). Pacemaker current exists in ventricular myocytes. Circ. Res. 72, 232–236. Yue, L., Feng, J., Li, G. R., and Nattel, S. (1996). Transient outward and delayed rectifier currents in canine atrium: Properties and role of isolation methods. Am. J. Physiol. 270, H2157–H2168.

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11 Electrophysiology of Vascular Smooth Muscle JANE A. MADDEN

NANCY J. RUSCH

Department of Neurology Medical College of Wisconsin Zablocki Veterans Administration Medical Center Milwaukee, Wisconsin 53295

Department of Pharmacology and Toxicology Medical College of Wisconsin Milwaukee, Wisconsin 53226

I. INTRODUCTION

cal signaling of vascular tissue. Ultimately, this complex system enables the vasculature to provide appropriate and rapid changes in systemic or vascular tone to optimize blood pressure in the host animal, while concurrently adapting levels of local blood flow to support organ and tissue integrity. Since the mid-1980s, our knowledge of the cellular basis of electrical signaling in vascular smooth muscle has been revolutionized by the definition of ion channel structure by molecular biology, by protein crystallography, and by the direct study of ion channel function using patch-clamp techniques. Many of the genes encoding the pore-forming subunits of ion channels have been cloned, and the function of these subunits has been characterized alone or in combination with regulatory proteins in heterologous expression systems. The purpose of this chapter is to briefly summarize the basic principles of electrical signaling, review the structure and function of several ion channel families that regulate electrical excitability in vascular smooth muscle cells, and outline the ionic pathways by which physiological stimuli regulate Em in arteries and arterioles.

The plasma membrane of the vascular smooth muscle cell expresses a unique population of ion channels, and the ionic currents generated by these channels provide the electrical signals that regulate electromechanical coupling in the blood vessel wall. Under resting conditions, K⫹ efflux across the plasma membrane is the primary driving force that confers a negative level of membrane potential (Em) to the vascular smooth muscle cell. This negative level of Em minimizes voltage-gated Ca2⫹ influx and helps maintain the vascular smooth muscle cells in a relaxed state such that distal tissues are perfused according to their metabolic needs. However, changing systemic and local blood flow requirements can result in a variable input of neuronal, chemical, and mechanical stimuli to the vascular smooth muscle cell. This regulatory input may either activate membrane K⫹ channels for additional hyperpolarization and vasodilation or produce an inhibition of these channels, which results in depolarization. In the latter condition, the resulting influx of Ca2⫹ through voltage-gated Ca2⫹ channels will lead to vasoconstriction. To add to the complexity, ligand binding to receptors on the smooth muscle membrane is linked to the activation of ligand-gated cation channels, which may further enable smooth muscle contraction by permitting the influx of Na⫹ and Ca2⫹ into the vascular smooth muscle cell independent of changes in Em level. Thus, the diverse population of ion channels expressed in the vascular smooth muscle membrane, coupled to their regulation by a wide variety of stimuli, provides for the electrophysiological circuitry that mediates the graded and controlled electri-

Heart Physiology and Pathophysiology, Fourth Edition

II. MEMBRANE AND ELECTRICAL PROPERTIES OF VASCULAR SMOOTH MUSCLE A. Principles of Electrical Signaling Electrical signaling across excitable cell membranes relies on the phospholipid bilayer that forms the plasma

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TABLE I Intracellular and Extracellular Concentrations of Some Ion Species Constituent

Intracellular concentration

Extracellular concentration

Na⫹ K⫹ Ca2⫹ Cl⫺ Osmolarity pH

14 mEq/liter 140 mEq/liter 10⫺7 M (ionized) 30 mEq/liter 295 mOsm/liter 7.1

140 mEq/liter 4 mEq/liter 2 mEq/liter 140 mEq/liter 295 mOsm/liter 7.4

membrane. This bilayer is impermeant to the negatively charged proteins and phosphorylated compounds found in the cell cytoplasm, so that sequestration of these substances within the cell confers a negative voltage to the cell interior called the cell membrane potential (Em). As will be discussed briefly, this negative Em is maintained between ⫺35 and ⫺50 mV in vascular smooth muscle cells by the opening of K⫹ channels and by the electrogenic Na⫹,K⫹ pump. Major ion species are distributed between intracellular and extracellular spaces according to their propensity for membrane permeation and their electrochemical gradients. Typical estimates for the ionic concentrations of major intracellular and extracellular components are shown in Table I. Table I shows that the extracellular space contains high concentrations of Na⫹ and Cl⫺ ions similar to those in the bloodstream, whereas K⫹ is conserved in high concentrations inside the cell. Notably, the concentration of Ca2⫹ is more than 104-fold higher outside than inside the cell, providing a steep chemical gradient for Ca2⫹ influx when voltage-gated Ca2⫹ channels are activated during cell excitation. Against this background, a change in the permeability of the plasma membrane to specific ion species generates the electrical signal that alters vascular smooth muscle cell Em. Notably, the direction and the amount of an ion that crosses the cell membrane are determined by two factors: the electrochemical driving force and the permeability coefficient of the membrane for that ion. Whereas the transmembrane gradient of an ion determines its chemical driving force (Table I), its electrical driving force is proportional to the difference between its equilibrium potential and the cell Em level. The equilibrium potential (Eion) represents the Em level at which an ion is passively distributed across the cell membrane. In practical terms, it can be considered as the level of Em that would be generated if the cell membrane was selectively and completely permeable only to a specific ion species. The Eion for a specific ion species is predicted from the Nernst equation, which states that

Eion ⫽ RT/FZ ln

Ci Co

where Eion is the equilibrium potential in millivolts, R is the gas constant (2 cal/mol/⬚K), T is the absolute temperature (⬚K), F is Faraday’s constant (2.3 ⫻ 104 cal/V/mol), Z is the valence of the ion, ln is the logarithm to the base e, and Ci and Co are the inside and outside concentrations (mEq) of a positively charged ion. The numerator and denominator of the Ci /Co ratio are reversed to calculate the equilibrium potential for an ion that is negatively charged. For example, by replacing the constants in the Nernst equation with their numerical values and converting from the natural log to the base 10 log, the following estimated values would represent the Eion for K⫹, Na⫹, Ca2⫹, and Cl⫺: EK ⫽ ⫺60 log [140]/[4] 앒 ⫺90 mV ENa ⫽ ⫺60 log [14]/[140] 앒 60 mV ECa ⫽ ⫺60 log [0.0001]/[2] ⱖ 200 mV ECl ⫽ ⫺60 log [30]/[140] 앒 ⫺40 mV Thus, for example, if a vascular smooth muscle cell with a resting Em of ⫺40 mV permitted K⫹ to equilibrate across its plasma membrane, K⫹ would diffuse out of the cell down its electrochemical gradient. This would hyperpolarize the smooth muscle cell membrane until its Em level approached the EK value of ⫺90 mV. Indeed, the physiological correlate of this event is observed when dilating factors released from the vascular endothelium activate smooth muscle K⫹ channels to mediate membrane hyperpolarization. Conversely, at typical resting Em levels found in vascular smooth muscle cells, the opening of voltage-gated Ca2⫹ channels by depolarizing stimuli permits Ca2⫹ to flow into the cell down its electrochemical gradient. The partial equilibration of Ca2⫹ across the plasma membrane promotes the depolarization process, but its impact on Em is much less than that of K⫹ because the intracellular and extracellular Ca2⫹ concentrations are lower. Regardless, the subsequent rise in the cytosolic-free Ca2⫹ concentration ([Ca]i) has the potential to trigger vascular contraction and thereby couple Em changes to vascular tone. As will be discussed briefly later, Na⫹ and Cl⫺ channels appear to contribute less than K⫹ and Ca2⫹ channels to the regulation of Em in vascular smooth muscle cells. Indeed, the interaction between K⫹ and Ca2⫹ channels is thought to be the primary determinant of excitation– contraction coupling. Clearly, the final level of Em depends on the concentration gradients of all of the major ions and the relative permeability of the membrane to each of these ion species. The Goldman–Hodgkin–Katz constant-field equa-

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tion can be used to predict the contribution of different ionic permeabilities to the Em, according to V ⫽ RT/F ln

(PK[Ki ] ⫹ PNa[Nai ] ⫹ PCl[Clo ] ⫹ PX [X]) (PK[Ko ] ⫹ PNa[Nao ] ⫹ PCl[Cli ] ⫹ PX [X])

where V is the membrane potential in millivolts, R is the gas constant, T is the absolute temperature, F is the Faraday constant, Px is the permeability of the membrane to ion x, and [X] is the concentration (in mEq) of ion x on the inside or outside of the cell membrane. Notably, the resting Em in vascular smooth muscle cells ranges between ⫺35 and ⫺50 mV, relatively close to the negative EK value of ⫺90 mV as compared to the positive values for ENa and ECa . This fact, coupled with the observation that pharmacological blockers of K⫹ channels depolarize resting smooth muscle cells, suggests that the plasma membrane is highly permeable to K⫹ compared to other ions under resting conditions and that smooth muscle cells rely on a high membrane K⫹ conductance to maintain a negative Em level. Presumably, the high permeability of the vascular smooth muscle to K⫹ is due to its dense expression of K⫹ channels, which show an adequate open-state probability at negative Em levels to permit significant K⫹ efflux. The reader is referred to other references (Hermsmeyer, 1982; Sperelakis, 1998) for a more detailed discussion of the physiochemical basis of the resting Em .

B. Role of Ion Exchangers Ultimately, ionic gradients across the cell membrane are maintained by populations of membrane-bound ion exchangers that restore specific ion species to their original side of the membrane. These ion exchangers are diverse families of membrane-spanning proteins representing complex tertiary structures that bind ion ligands to induce conformational changes of the protein molecule. Only two of these exchangers will be mentioned here because they are closely involved with the regulation of vascular smooth muscle Em and the electromechanical coupling process. The Na⫹,K⫹ (ATPase) pump is a large polypeptide that spans the cell membrane and uses energy derived from ATP to pump Na⫹ and K⫹ against their concentration gradients. Traditionally, it is thought that three Na⫹ ions are pumped out of the cell and two K⫹ ions are pumped in. This unequal exchange results in the generation of a hyperpolarizing current that contributes to the negativity of the resting Em. Each exchange cycle requires one molecule of ATP. The Na⫹,K⫹ (ATPase) pump is composed of two subunits: a main functional 움-subunit protein that contains the binding sites for Na⫹,K⫹ and ATP and a regulatory 웁 subunit that influ-

ences the expression and activity of the 움-subunit protein. The contribution of the Na⫹,K⫹ (ATPase) pump to resting Em is between 5 and 20% of the total voltage in vascular smooth muscle cells. Thus, its hyperpolarizing influence reinforces the primary contribution of membrane K⫹ conductance to negative Em levels. The Na⫹,K⫹ (ATPase) pump performs another important function of establishing the transmembrane chemical gradient for Na⫹ required for proper functioning of the Na⫹,Ca2⫹ exchanger. This pump relies on the high transmembrane (inward driven) Na⫹ gradient established by the Na⫹,K⫹ (ATPase) pump to transport Ca2⫹ out of the cell by secondary active transport. Although the Na⫹,Ca2⫹ exchanger, like most other transport exchangers and channels, can transport ions bidirectionally, it generally extrudes Ca2⫹ from the cell. This occurs because the Na⫹ concentration is 10-fold higher at the outer surface of the plasma membrane; thus, according to the principles of mass action, Na⫹ will bind more often to the external than the internal face of the transport protein. Subsequently, the cobinding of Ca2⫹ to the internal face results in a conformational change, and protein sidedness reverses within the membrane so that Na⫹ is translocated into the cell and Ca2⫹ is extruded. The stochiometry of the Na⫹,Ca2⫹ exchanger in vascular smooth muscle is unclear, but may involve the exchange of extracellular Na⫹ for intracellular Ca2⫹ on a two-for-one basis (electrically neutral) or a three-to-one basis (electrogenic). Regardless, the subsequent decrease in [Ca]i will inhibit Ca2⫹-dependent excitation–contraction coupling in the smooth muscle cell. In addition, the cell membrane and the sarcoplasmic reticulum network express other Ca2⫹ transport proteins such as the Ca2⫹ (ATPase) pump. At a given level of Em, the activity of these Ca2⫹ transport proteins will modulate the amount of [Ca]i available to activate the contractile proteins to modulate vascular tone.

C. Unique Electrical Profile of Vascular Smooth Muscle Vascular smooth muscle cells from different vascular beds show distinct ion channel distributions and electrophysiological profiles. In general, however, several unique characteristics distinguish the electrical properties of vascular smooth muscle cells from those of neuronal cells or cardiac myocytes. First, vascular smooth muscle membranes generally show an absence or scarcity of voltage-gated ‘‘fast’’ Na⫹ channels, whereas these Na⫹ channels are critical mediators of the upstroke of the action potential in neurons and in cardiac ventricular cells. In the absence of significant Na⫹ channel expression, vascular smooth muscle cells rely predominantly on the inhibition of resting K⫹ efflux to depolarize

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the membrane. Second, the resting Em level of ⫺35 to ⫺50 mV in vascular smooth muscle cells is considerably less negative than the Em range of ⫺70 to ⫺90 mV in neuronal or cardiac cells. In effect, the more positive Em level provides for the steady-state activation of voltagegated Ca2⫹ channels, which provides a sufficient [Ca]i to mediate Ca2⫹-dependent vascular tone in small arteries. Third, whereas neurons and cardiac myocytes rely on spontaneously electrical activity for the repetitive secretion of neurotransmitters and cardiac pacemaker activity, respectively, most types of vascular smooth muscle cells do not demonstrate spontaneous electrical activity under normal conditions. Rather, they rely on graded changes in Em to provide activator Ca2⫹ for tonic contraction. In the systemic circulation, this tonic activation of vascular smooth stabilizes vascular resistance to prevent rapid fluctuations in blood pressure. At the local level, it provides for graded changes in blood flow so that tissues and organs are perfused commensurate with their metabolic state.

III. EXPRESSION OF ION CHANNELS A. General Structure of Ion Channels Ion channels are specialized proteins in the plasma membrane that provide a passageway through which charged ions can cross the plasma membrane down their electrochemical gradient. The resulting ionic current, generated by the movement of charged ions through membrane channels, can be measured by patch-clamp methods. Most ion channels are heteromultimer complexes composed of one to four pore-forming 움 subunits arranged around a central membrane-spanning shaft. Smaller regulatory subunits (움2, 웁, 웃, ␭) may influence the gating behavior of the channel and modify its expression level in the plasma membrane. The pores of most ion channels have a selectivity filter, which permits the channel to conduct only a single type of ion. However, ligand-gated ion channels that are coupled to membrane receptors often permit the passage of multiple ion species, including Na⫹ and Ca2⫹.

B. Potassium Channels Potassium channels are the primary determinants of resting Em , and their protein subunits emanate from many genes. In vascular smooth muscle cells, at least four K⫹ channel superfamilies may interact to regulate electrical excitability. Voltage-gated K⫹ (KV) channels have been described in most types of vascular smooth muscle cells, and they participate in the setting of the resting Em and in the regulation of vascular tone. Di-

verse KV channels corresponding to multiple gene families (KV1 to KV9) appear to be highly expressed throughout the vasculature, and selective transcription or translation of specific KV channel subtypes is proposed as a possible source of regional diversity. Structurally, KV 움 subunits contain six hydrophobic transmembrane segments (S1–S6) flanked by hydrophilic amino- and carboxy-teminal sequences located in the cell interior (Fig. 1A). Domain S4 appears to possess an intrinsic voltage sensor, which is associated with channel gating. Because the channel pore represents a heterotetrameric 움-subunit complex (Fig. 1A, inset), which may be composed of similar or different 움 subunits originating from the same Kv gene family, the ‘‘mixing and matching’’ of KV 움 subunits provides for a heterogeneous population of channels. Indeed, single-channel recordings in isolated membrane patches of vascular smooth muscle cells show variable unitary conductances between 8 and 50 pS for KV channels. An additional level of complexity is introduced by KV 웁 subunits, which may interact with the cytoplasmic domains of the 움 subunit to enhance channel inactivation or modify cell surface expression. Not surprisingly, a complex pattern of site-specific expression and coassembly of KV channels is thought to confer electrical heterogeneity within and between different circulatory beds (Archer et al., 1996). High-conductance, Ca2⫹ -activated K⫹ channels, called BKCa channels because of their big (B) unitary conductance (150 to 300 pS), are densely expressed in most vascular smooth muscle membranes. Similar to KV channels, they contribute to the resting Em level and also buffer depolarizing responses and vascular constriction. Unlike KV channels, which originate from multiple gene families, BKCa channel 움 subunits arise from a single gene family, but phenotypic diversity may be generated by a high level of alternative splicing of the common primary transcript. Notably, the N-terminal region of the pore-forming 움 subunit of the BKCa channel shares partial homology with KV channels, showing six transmembrane domains (S1–S6) and a highly conserved pore region between S5 and S6 (Fig. 1B). The Ca2⫹ sensitivity of the BKCa channel appears to be conferred by extra four transmembrane domains (S7–S10) at the C-terminal region of the 움 subunit and by close association with a regulatory 웁 subunit (Tanaka et al., 1997). Additionally, inward rectifier K⫹ (KIR) channels and adenosine 5⬘-triphosphate-sensitive K⫹ (KATP) channels may also contribute to the resting Em of vascular smooth muscle cells (Quayle et al., 1997). The pore-forming 움 subunits of KIR and KATP channels are both subtypes of the inward rectifier K⫹ channel family (Kir) that show the common property of inward rectification in that they conduct inward K⫹ current more readily than outward current. The unitary conductance of KIR and KATP

11. Vascular Smooth Muscle

217

FIGURE 1 Proposed topology of K⫹ channels expressed in vascular smooth muscle membranes, including (A) voltage-gated K⫹ channels, (B) high conductance Ca2⫹-activated K⫹ channels, (C) inward rectifier K⫹ channels, and (D) ATP-sensitive K⫹ channels. The inset in A indicates the assembly of four 움 subunits to form the pore of the KV channel.

channels varies widely in vascular smooth muscle (10 to 260 pS), implying a high level of structural heterogeneity (Quayle et al., 1997). The Kir channel family also shows a tetrameric 움-subunit structure, but has a unique membrane topology of only two transmembrane regions (M1 and M2), leading to cytoplasmic amino and carboxy termini, and a central pore-forming loop (Fig. 1C). In addition, KATP channels, which are inhibited by intracellular ATP, are thought to represent a channel complex composed of a KIR channel subtype coupled to a sulphonylurea receptor (SUR) that possesses a ‘‘ATP-binding cassette’’ (Fig. 1D). In vascular smooth muscle, KATP channels are thought to open when cytosolic ATP levels become suppressed during conditions of metabolic stress, thereby mediating hyperpolarization and vasodilation to enhance blood flow to compromised tissues.

C. Ca2⫹ Channels Vascular smooth muscle cells express at least two types of voltage-activated Ca2⫹channels. The transient

(‘‘T-type’’) Ca2⫹ channels are activated and inactivated at negative potentials near or negative to the resting Em level of vascular smooth muscle and mediate a short burst of Ca2⫹ influx. Although T-type Ca2⫹ channels appear to contribute significantly to pacemaker activity in the sinoatrial node of the heart, and hence can affect cardiovascular dynamics by modulating heart rate, their role in the direct regulation of electrical excitability in the circulation appears to be minimal. This may relate to the more positive resting Em level in vascular smooth muscle cells compared to cardiac myocytes, which may at least partially inactivate these channels to preclude their participation in Em regulation (Rusch and Hermsmeyer, 1994). In contrast, the long-lasting (‘‘L-type’’) Ca2⫹ channels activate and inactivate at more positive membrane potentials and show sustained activity at the Em levels found in vascular smooth muscle cells. These channels represent the major pathway by which sustained Ca2⫹ influx enters the vascular smooth muscle cell during membrane depolarization. In vascular smooth muscle,

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FIGURE 2 Proposed topology of the L-type Ca2⫹ channel showing its subunit components.

the L-type Ca2⫹ channel minimally represents a poreforming 움1C subunit associated with regulatory 움2웃 and 웁 subunits (Fig. 2). In contrast to the heteromultimer structure of K⫹ channel pores, the pore-forming structure of the L-type Ca2⫹ channel is a single polypeptide consisting of four repeat domains (I–IV), each containing six transmembrane-spanning segments (S1–S6). Pore formation and voltage sensing are conferred by this central subunit, and the 움1 subunit also contains the binding sites for the Ca2⫹ channel-blocking drugs that are used clinically to treat hypertension, angina, and vasospastic diseases. The 움1C subunit may function independently as a voltage-sensing channel, showing a unitary conductance of 10 to 25 pS, but its behavior is highly modulated by its ancillary subunits. The gene for the 움1 subunit in vascular smooth muscle, which was cloned from aorta (Koch et al., 1990), is now recognized as a splice variant of the 움1C gene of cardiac muscle.

D. Chloride Channels Whereas the structures of the L-type Ca2⫹ channel and many types of K⫹ channels are partially delineated, less is known about the structure or functional role of other types of potentially important channels expressed in vascular smooth muscle. For example, recent findings suggest that volume- or Ca2⫹- sensitive Cl⫺ channels, which are activated by stretch or increases in [Ca]i during cell excitation, may contribute to the depolarization of vascular smooth muscle cells. Indeed, two distinct genes corresponding to volume- and Ca2⫹- sensitive Cl⫺ channels have been identified in vascular smooth muscle, and Cl⫺ selective channels have been detected in several types of vascular smooth muscle cells by patchclamp methods (Pusch and Jentsch, 1994; Yamazaki et al., 1998). At least one of these Cl⫺ channels (CIC) appears to have 12 transmembrane-spanning regions,

suggesting a double-barreled structure that is unique among ion channel pores, and results in a low singlechannel conductance. The calculated equilibrium potential (ECl) for Cl⫺ of about ⫺40 mV is comparable to or slightly more positive than the resting Em of most smooth muscle cells. Thus, the opening of Cl⫺ channels may mediate Cl⫺ efflux and membrane depolarization of most smooth muscle cells. Indeed, the pharmacological block of Cl⫺ channels reportedly depolarizes and contracts smooth muscle cells, but the blocking drugs that are used to inhibit Cl⫺ flux also may affect other transport processes. Thus, the structure of vascular Cl⫺ channels and their functional role with respect to electromechanical coupling in vascular smooth muscle cells requires further clarification.

E. Nonselective Cation Channels Another type of ion channel family that is the focus of recent attention in vascular smooth muscle is nonselective cation channels. The hallmark feature of these channels is their distinct but variable permeability to at least two cations, most notably Na⫹ and Ca2⫹. Ligand binding to G-protein-coupled receptors in the smooth muscle membrane is thought to induce a conformation change in the channel that permits Na⫹ and Ca2⫹ ions to enter the vascular smooth muscle cells according to their electrochemical gradients. Under some conditions, this influx is thought to provide the initial depolarizing signal that activates L-type Ca2⫹ channels to trigger vasoconstriction. The molecular structure of nonselective channels in vascular tissue remains unclear, but the channel complex is thought to consist of both receptor and channel domains and may have only two transmembrane-spanning segments, similar to the KIR channel. Attempts to characterize these channels by patch-clamp methods have revealed a diverse group of low conductance (5 to 25 pS) channels that show at least a mild

11. Vascular Smooth Muscle

level of voltage sensitivity. Indeed, nonselective cation channels appear to arise from multiple gene families and show variable ion selectivity, voltage sensitivity, and regulatory profiles in vascular smooth muscle.

IV. REGULATION OF VASCULAR TONE BY ION CHANNELS AND MEMBRANE POTENTIAL A. Role of K⫹ Channels The level of resting Em in vascular smooth muscle cells is the fundamental determinant of arterial diameter. Diseases such as pulmonary and systemic hypertension, in which the affected vascular smooth muscle cells show depolarized Em levels, also show anomalous levels of vasoconstriction (Berger and Rusch, 1999). Thus, the tight regulation of resting Em levels is required for normal vascular function, and smooth muscle K⫹ channels play the critical role of maintaining normal Em levels. It is likely that parallel and redundant pathways for K⫹ efflux enabled by at least four K⫹ channel superfamilies provide the basis for Em regulation in vascular smooth muscle. In particular, the coexpression of KV channels and BKCa channels in vascular smooth muscle membranes appears to provide a fundamental mechanism for regulating the Em and tone of resistance arteries (for review, see Berger and Rusch, 1999). Their presence has been noted in small arteries from cerebral, coronary, mesenteric, and pulmonary vascular beds. For example, the

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resting Em and diameter of isolated, pressurized rabbit cerebral arteries are regulated by both KV and BKCa channels. Pharmacological block of KV channels depolarizes the vascular smooth muscle cells in these cerebral arteries by 19 mV and initiates vasoconstriction. In the same preparation, a block of BKCa channels also triggers a 7-mV depolarization and causes vascular activation (Knot and Nelson, 1995; Brayden and Nelson, 1992). Similarly, a pharmacological block of either KV or BKCa channels in small human coronary arteries depolarizes the vascular smooth muscle cells and reduces vessel diameter at physiological intraluminal pressures (Koch et al., 2000). Finally, a pharmacological block of KV or BKCa channels in isolated, perfused rat lungs enhances pulmonary arterial pressures, indicating that both KV and BKCa channels may contribute to the resting membrane K⫹ conductance and the regulation of resting vascular tone in pulmonary resistance vessels (Hasunuma et al., 1991; Post et al., 1992) In parallel studies, patch-clamp experiments in numerous types of vascular smooth muscle have confirmed the presence of both KV and BKCa channels in isolated membrane patches (Fig. 3). This expression profile appears to vary between different vascular tissues. For example, not all blood vessels appear to contain the same transcript or protein subunits corresponding to distinct KV channel types (Berger and Rusch, 1999). Similarly, the density and properties of BKCa channels appear to vary between arterial muscle membranes from different vascular origins. In this regard, transcript and patch-clamp analyses indicate that a single vascular smooth muscle cell may express several isoforms of the

FIGURE 3 Single-channel recording of low-amplitude KV currents (A) and high-amplitude BKCa currents (B) in inside-out membrane patches from rat mesenteric arterial cells. Recordings were obtained at a depolarized patch potential (Em) of 40 mV to activate both channel types. (A) The external patch surface was exposed to 100 nmol/liter iberiotoxin to inhibit BKCa channels and permit the recording of KV channels in relative isolation. In both sets of traces, horizontal sweeps were obtained as [Ca]i was increased from 10 to 1000 nmol/ liter. This increase in [Ca]i inhibited KV channels (A), but activated BKCa channels (B). Li and Rusch (unpublished recordings).

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BKCa channel, which vary in their level of Ca2⫹ sensitivity. Also, the Ca2⫹ sensitivity of BKCa channels expressed in small resistance arteries or arterioles appears to be less than that of apparently similar channels expressed in the smooth muscle membranes of larger vessels. Variation in the expression level of 움 and 웁 subunits, and their level of coassembly within the vascular smooth muscle membrane, may underly these differences. Thus, under normal circumstances, each type of blood vessel may express its own unique population of K⫹ channels, which in turn provides the appropriate level of resting Em for optimal tissue perfusion. It is thought that in response to initial depolarizing stimuli, the opening of KV channels provides the initial negative feedback to attenuate Ca2⫹ entry and buffer vasoconstriction. As [Ca]i rises, however, the KV channels are inhibited (Fig. 3A). In contrast, both membrane depolarization and rises in [Ca]i act synergistically to enhance the open-state probability of the BKCa channels (Fig. 3B). The small size and high input resistance of vascular smooth muscle cells, coupled to the large unitary amplitude of BKCa channel currents, provide the BKCa channel with a highly favorable environment in which to exert a hyperpolarizing influence. In this manner, the opening of BKCa channels during smooth muscle activation effectively limits the final level of arterial constriction. In addition to KV and BKCa channel families, KIR channels, which conduct K⫹ current preferentially at negative Em levels, may also contribute to the resting Em in resistance arteries and arterioles. However, in the absence of selective pharmacological blockers of this channel, its role in regulating excitability in the vasculature has been difficult to ascertain. In contrast, KATP channels, which are blocked by glibenclamide, appear to contribute to the maintenance of resting Em in cerebral and skeletal arterioles. In addition, the activation of KATP channels appears to contribute to the hyperpolarizing and vasodilator responses to hypercapnic acidosis in small coronary and cerebral arteries, and mediates, at least in part, the vasodilator response to hypoxia possibly as a result of decreasing cytosolic ATP levels. The exact nature of the role of KATP channels in the vasodilator response to hypoxia is uncertain. However, it has been postulated that hypoxia may activate KATP channels directly. These channels may also be activated indirectly through the release of endotheliumderived dilating factors, such as nitric oxide and prostacyclin (PGI2), and possibly endothelium-derived hyperpolarizing factors (EDHF) or adenosine released from extravascular tissue. Thus, KATP channels appear to mediate several critical vasodilator responses to metabolic challenges (Quayle et al., 1997).

B. Role of L-Type Ca2⫹ Channels Depolarization of the plasma membrane in vascular smooth muscle cells activates voltage-gated, L-type Ca2⫹ channels, leading to an influx of Ca2⫹, which binds to calmodulin. This, in turn, leads to the activation of myosin light chain kinase, phosphorylation of the regulatory light chains of the myosin molecule, and crossbridge cycling to produce vessel contraction. In contrast, hyperpolarization of the smooth muscle membrane closes voltage-gated Ca2⫹ channels, resulting in the reversal of these processes and vasodilation. Indeed, in the intact organism, normal levels of vascular tone appear to rely on the tonic activation of L-type Ca2⫹ channels and the subsequent intracellular signaling cascade that mediates smooth muscle contraction. For example, pharmacological blockers of L-type Ca2⫹ channels reduce vascular resistance in most circulatory beds in vivo and profoundly lower blood pressure in the absence of neural compensatory mechanisms. Thus, L-type Ca2⫹ channels appear to represent functional membrane pathways that permit Ca2⫹ influx under physiological conditions. Patch-clamp studies in vascular smooth muscle cells indicate that the biophysical properties of L-type Ca2⫹ channels are consistent with their critical role in cell excitation. These channels are expressed ubiquitously in vascular smooth muscle membranes and show lowconductance (10 to 25 pS) unitary currents that would permit the fine regulation of Ca2⫹ influx during membrane depolarization (Fig. 4). Modeling of L-type Ca2⫹ current kinetics and densities in arterial muscle cells suggests that at least several thousand pore-forming Ca2⫹ channel subunits are expressed in a single cell (Nelson et al., 1990). Because L-type Ca2⫹ channels only slowly inactivate during sustained depolarization, voltage-gated Ca2⫹ influx permitted by a small fraction of these channels is sufficient for tonic constriction. Finally, as mentioned earlier, L-type Ca2⫹ channels are highly susceptible to pharmacological block by 1,4-dihydrophyridine antagonists and other clinical families of Ca2⫹ channel blockers. Therapeutic targeting by these blockers causes vasodilation and lowers blood pressure in human subjects. Notably, in addition to sustaining voltage-gated Ca2⫹ influx into the smooth muscle cells to maintain contraction, L-type Ca2⫹ channels may also provide an excitatory template upon which endogenous vasoactive substances can act to modulate arterial diameter further (Nelson et al., 1988). For example, the activation of single L-type Ca2⫹ channels by norepinephrine and other vasoconstrictor substances has been observed in isolated patches of arterial smooth muscle membranes (Fig. 4), but the transducing mechanism that links recep-

11. Vascular Smooth Muscle

FIGURE 4 Single-channel recording of voltage-dependent Ca2⫹ currents in a cell-attached membrane patch of rabbit mesenteric artery. The membrane potential was stepped from a holding potential of ⫺95 to ⫺20 mV to elicit the currents. The addition of norepinephrine to the bathing solution increased the open-state probability of the Ca2⫹ channels. Adapted from Nelson et al. (1988), with permission from Macmillan Magazines Ltd.

tor activation to channel opening is unclear. Thus, although the open-state probability of L-type Ca2⫹ channels is fundamentally regulated by the level of Em in vascular smooth muscle cells, these channels may also contribute by a voltage-independent mechanism to the dynamic responses of the vasculature to vasoactive stimuli.

C. Electromechanical Coupling in Vascular Smooth Muscle Patch-clamp and current-clamp techniques have been used extensively to predict the relative contributions of different ion channels to the resting Em of isolated vascular smooth muscle cells. However, the contribution of ion channels to electromechanical coupling in vascular smooth muscle cells can also be studied under more physiological conditions by measuring Em and vessel diameter in isolated, cannulated arteries. Under these conditions, small arteries that are perfused at a normal intraluminal pressure of 앒80 mmHg depolarize, develop spontaneous Ca2⫹-dependent tone, and show higher [Ca]i than single smooth muscle cells isolated enzymatically from the same blood vessel for patchclamp measurements (Davis and Hill, 1999). Thus, important functional roles for ion channels may be un-

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covered in microelectrode studies of Em in pressurized arteries that would not be detected in unstretched smooth muscle cells or unpressurized vascular segments. Under these conditions, the profile of electromechanical coupling in pressurized arteries has been delineated during the past two decades by correlating Em levels to diameter changes in small vessels. As reviewed previously (Rusch and Hermsmeyer, 1994), resting Em levels measured in isolated, pressurized arteries are clustered closely between ⫺35 and ⫺50 mV. Notably, these values are similar to resting Em levels measured in small arteries in situ, which range between ⫺35 and ⫺45 mV. Interestingly, no obvious differences in resting Em levels have been observed between arteries from different species or even between different vascular beds from the same species, although the pattern of electrical excitability in response to constrictor and dilating stimuli varies greatly. This consensus of resting Em levels suggests that multiple, redundant pathways have evolved that tightly maintain resting Em at a voltage that provides for the optimal regulation of vascular tone. Indeed, the relationship between Em and arterial diameter is steep, which is thought to reflect a tight coupling among Em , activation of L-type Ca2⫹ channels, and vascular tone. For example, depolarizing responses of only 15 to 20 mV elicited by norepinephrine or other vasoconstrictor substances induce maximal contractile responses of most arteries. Within this range, small changes in Em affect the activity of L-type Ca2⫹ channels profoundly, thereby altering the level of contraction in vascular smooth muscle cells.

V. EFFECT OF PHYSIOLOGICAL FACTORS ON MEMBRANE POTENTIAL As mentioned earlier, neuronal, chemical, and mechanical stimuli can alter the activity of K⫹ channels in the vascular smooth muscle cell membrane. In the presence of vasoconstrictor substances, K⫹ channels may be inhibited so that the membrane depolarizes, L-type Ca2⫹ channels are activated, and the vessel constricts. Alternatively, if vasodilator substances are present, they may activate K⫹ channels to hyperpolarize the membrane, inhibit L-type Ca2⫹ channels, and induce vasodilation. These responses, which can be modulated further by factors released from the endothelium, are necessary to control blood pressure and to regulate blood flow to the distal tissues under a variety of circumstances. The additional regulation of other ion channel types, including receptor-coupled nonselective cation channels and Cl⫺ channels, may determine the final pattern of Em response to a given pattern of vasoactive

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stimuli. The following section considers some of the more common physiological influences on Em in vascular smooth muscle cells and their consequences on vascular tone.

A. Norepinephrine and Other Vasoconstrictor Substances The release of excitatory neurotransmitters such as norepinephrine, as well as other vasoconstrictor agents, occurs in response to a variety of stimuli to mediate vasoconstriction and maintain normal blood pressure levels. The constrictor response of a vessel to these agents is generally preceded by depolarization, which is initiated by the binding of a ligand molecule to its corresponding receptor on the plasma membrane. Two representative studies have demonstrated that norepinephrine released from sympathetic nerve endings in vivo exerts a tonic depolarizing influence on the resting Em of vascular smooth muscle cells. For example, the smooth muscle cells of rat cremaster arterioles studied in situ show a resting Em of ⫺43 mV. Local chemical sympathectomy with 6-hydroxydopamine or block of 움-adrenoceptors on vascular muscle cells by phentolamine hyperpolarize the arteriolar smooth muscle cells to ⫺51 mV (Stekiel et al., 1993). The direct effect of norepinephrine on Em and vessel diameter in vivo has also been studied in the hamster cheek pouch. In this preparation, microiontophoric injection of norepinephrine depolarized arterioles from ⫺35 mV to about ⫺20 mV, and the depolarization spread along the smooth muscle cells of the vessel. Approximately 2 sec after depolarization occurred, the vessel diameter decreased by over 50%. The magnitude of the vasoconstriction increased with the magnitude of the electrical response, indicating the presence of electromechanical coupling in these small arteries (Welsh and Segal, 1998). The ionic signal that links 움-adrenoceptor stimulation to membrane depolarization remains controversial, but at least three candidate pathways have been proposed, and these may represent complementary mechanisms. First, nonselective cation channels linked to G-protein-coupled 움-adrenoceptors on the smooth muscle membrane may undergo a conformational change in response to receptor occupation, thereby permitting Na⫹ and Ca2⫹ influx. Second, norepinephrine may also directly activate L-type Ca2⫹ channels in the vascular smooth muscle membrane to enhance Ca2⫹ influx (Fig. 4), and further support depolarization and vasoconstriction. Finally, as depolarization ensues and [Ca]i rises, the Ca2⫹-dependent block of KV channels may provide a permissive environment for continuing depolarization. Faced with these depolarizing stimuli, the

hyperpolarizing influence of BKCa channels and other types of K⫹ channels may not be adequate to return smooth muscle Em to its resting level, thereby allowing vascular contraction to be maintained. Another vasoconstrictor substance that has been the focus of attention is endothelin (ET-1), which is the most potent vasoconstrictor discovered to date. Similar to norepinephrine, this endothelium-derived peptide appears to exert its contractile effect by depolarizing the smooth muscle cell membrane to permit Ca2⫹ influx through voltage-gated L-type Ca2⫹ channels (Salter and Kozlowski, 1998). It appears that endothelin evokes this depolarization by inhibiting K⫹ efflux, but the specific K⫹ channels involved seem to vary depending on the blood vessel studied, and more than a single K⫹ channel type may be affected in a given artery. For example, in rat pulmonary arteries, endothelin is thought to inhibit both KV channels and BKCa channels to mediate its depolarizing effect (Shimoda et al., 1998; Peng et al., 1998). However, in human pulmonary arteries, endothelin is reported to activate BKCa channels at low concentrations, but inhibit channel activity at higher concentrations (Peng et al., 1998). From these findings, it has been proposed that the elevation of circulating levels of endothelin during cardiovascular disease states may result in endothelin-induced block of BKCa channels, smooth muscle depolarization, and the elevation of vascular tone. Similarly, the endothelin-induced inhibition of BKCa current is postulated to contribute to the increased vascular tone observed in chronic, hypoxiainduced pulmonary hypertension.

B. Endothelium-Derived Dilating Factors The level of arterial tone in vivo is thought to be dynamically modulated by factors released from the vascular endothelium. Indeed, endothelial cells are thought to regulate vascular smooth muscle tone primarily by releasing nitric oxide, EDHF, and/or prostacyclin. From a wealth of studies, it is generally believed that nitric oxide is a major chemical mediator of endothelium-derived relaxation in both conduit and resistance arteries, whereas EDHF may be more involved in the relaxation of resistance arteries. For example, acetylcholine-induced relaxation of large arteries may be mediated entirely by the release of nitric oxide, and exposing large arteries to nitric oxide synthase inhibitors eliminates acetylcholine-induced relaxation. The vasodilating action of nitric oxide and other nitro-containing compounds may rely on the activation of BKCa channels and possibly KATP channels to hyperpolarize the smooth muscle membrane. In instances in which nitric oxide has been shown to induce membrane hyperpolarization of large arteries, BKCa channels appear to be the pri-

11. Vascular Smooth Muscle

mary target of activation (Bolotina et al., 1994; Vanheel and Van de Voorde, 1997). However, in smaller arteries, acetylcholine and bradykinin appear to initiate relaxation through the production of EDHF, a metabolite of arachidonic acid derived from the cytochrome P450-dependent monooxygenase pathway (Fig. 5). The release of EDHF reportedly activates BKCa channels, KV channels, and also small-conductance, Ca2⫹-activated K⫹ channels in coronary resistance arteries. For example, in guinea pig and human coronary arteries, the hyperpolarizing and vasodilator responses to bradykinin appear to be mediated by a medley of endothelium-derived factors that act on at least two types of K⫹ channels to induce smooth muscle hyperpolarization (Nishiyama et al., 1998; Miura et al., 1999). PGI2 may also be an important hyperpolarizing factor for mediating endothelium-derived vasodilation of arterial smooth muscle cells in some vascular beds, including coronary and cerebral circulations. Its vasodilating action is thought to be mediated by a cyclic adenosine monophosphate (cAMP)-induced activation of KATP channels. Overall, considering the critical role that endothelium-dependent vasodilation plays in the regulation of vascular tone, the finding that multiple endothelium-derived vasodilating substances act on a diverse population of K⫹ channels in most vascular beds to provide redundant pathways for vasodilation is not unexpected.

C. Intraluminal Pressure In small, myogenically active arteries, stepwise increases in intraluminal pressure induce a progressive depolarization of smooth muscle cells in the arterial wall (Harder, 1984; Davis and Hill, 1999). For example, small feline middle cerebral arteries show a stepwise depolarization of smooth muscle cells and vessel constriction in response to increases in intraluminal pressure from 40 to 90 and 140 mm Hg (Fig. 6). The same arteries

FIGURE 5 Intracellular recording of Em showing endothelin-induced depolarization followed by bradykinin-induced hyperpolarization in cannulated, pressurized human coronary arterioles. Numbers under arrows indicate the values for Em and vessel diameter under control conditions, and after endothelin and bradykinin. Adapted from Miura et al. (1999), with permission.

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constrict in response to these increases in intraluminal pressure. Depolarization in response to pressure is also observed in smaller feline cerebral arterioles as well, although the resting Em in these smaller vessels is more depolarized than in larger arteries from the same vascular bed (⫺38 mV versus ⫺50 mV) (Dietrich and Dacey, 1994). These depolarizing and contractile responses induced by pressure, termed the ‘‘myogenic response,’’ have been observed in resistance-sized arteries from most other vascular beds, including coronary, renal, and skeletal muscle circulations. Neither the characteristic depolarization nor constriction depends on endothelial or neurally derived factors, but rather appear to be inherent properties of the vascular smooth muscle cell. Interestingly, changing Em while holding transmural pressure constant can also produce changes in arterial diameter; hyperpolarization results in vasodilation, whereas depolarization causes constriction. Extrapolated to the intact organism, this finding implies that the diameter of small arteries is a function of the local perfusion pressure, but it is further modulated by regulatory factors that alter smooth muscle Em independent of the pressure stimulus. Ion channels responsible for the depolarizing response to pressure have not been clearly identified, although voltage-gated Ca2⫹ influx appears to be intimately involved. Indeed, the pharmacologic block of L-type Ca2⫹ channels eliminates much of the myogenic response in isolated, cannulated small arteries. The development of new technologies, such as fluorescent imaging to measure changes in [Ca]i in vascular smooth muscle cells, has further implicated voltage-dependent Ca2⫹ channels as mediators of the myogenic response in small arteries. For example, in cannulated, pressurized cerebral arteries in which Em is measured using microelectrodes and [Ca]i is monitored using the fluoroprobe fura-2, increasing intraluminal pressure triggers depolarization of the cerebral vessels concurrent with rises in [Ca]i (Knot and Nelson, 1998). The rise in [Ca]i is attributed to Ca2⫹ influx through dihydropyridinesensitive, voltage-gated, L-type Ca2⫹ channels. These findings suggest that pressure-induced depolarization, in the absence of outside vasocactive factors, can increase the activity of L-type Ca2⫹ channels to decrease arterial diameter. In addition to L-type Ca2⫹ channels, the activation of other ion channels by the pressure stimulus may contribute to the myogenic response of small arteries (for review, see Davis and Hill, 1999). Theoretically, the inhibition of K⫹ channels, or the activation of Cl⫺ channels or nonselective cation channels, also represents potential mechanisms for pressure-induced depolarization. Stretch-activated nonselective ion channels and mechanosensitive Cl⫺ channels have been described in several

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II. Cellular Electrophysiology

FIGURE 6 (Top) Photomicrograph of the pressure-induced decrease in the diameter of a cannulated, pressurized middle cerebral artery of a cat. (Bottom) Intracellular recording of Em showing depolarization in response to increased intraluminal pressure. Adapted from Harder (1984), with permission.

cell types and participate in many aspects of cell function, including volume regulation. Similarly, the presence of stretch-activated currents in vascular smooth muscle cells has been confirmed by patch-clamp studies, in which a patch pipette is used to apply suction to the outside surface of a channel-containing membrane patch. These studies indicate the presence of a mechanosensitive nonselective cation channel in mesenteric, coronary, and pulmonary smooth muscle membranes that is permeable to Na⫹ and Ca2⫹ and characterized by a unitary conductance of 앒 40 pS. The activation of these channels is proposed to initiate the myogenic response by depolarizing the smooth muscle membrane past the threshold for activation of L-type Ca2⫹ channels. Subsequently, L-type Ca2⫹ channels may provide the prominent pathway for Ca2⫹ influx to maintain the pressureinduced constrictor response.

D. Intraluminal Flow Although the effect of intraluminal flow rate on the reactivity of arteries and veins has been studied both

in vivo and in vitro, little is known about the mechanisms by which flow affects smooth muscle cell Em. To date, only two studies have investigated these mechanisms by measuring Em as a function of different flow rates in isolated, perfused cerebral arteries. In an initial study, ring segments of rabbit middle cerebral artery were mounted on a wire myograph and subjected to flows of 20 애1/min delivered from a cannula inserted into the lumen of the stretched ring (Bevan and Wellman, 1993). The application of flow depolarized arteries that showed smooth muscle cell Em levels more negative than ⫺58 mV, whereas arteries with more positive Em values tended to hyperpolarize with flow. More recent experiments in cannulated, pressurized feline cerebral arteries have confirmed that flow, as an isolated stimulus, can modulate the level of Em in vascular smooth muscle cells (Madden and Christman, 1999). In these studies, arterial Em and diameter were monitored continuously during stepwise increases in flow between 0 and 4.0 ml/min, which were used to generated shear stresses similar to those encountered in vivo. As flow was increased at a constant transmural pressure of

11. Vascular Smooth Muscle

FIGURE 7 Effect of intraluminal flow on vessel diameter and Em in a cannulated, pressurized middle cerebral artery of a cat. Adapted from Madden and Christman (1999), with permission.

100 mm Hg, the smooth muscle cells depolarized concurrent with a reduction in arterial diameter (Fig. 7). The vessels relaxed and smooth muscle Em values returned to control values when flow was decreased or halted. Flow-induced constriction was dependent on the presence of extracellular Ca2⫹, but was independent of a functional endothelium. Based on pharmacological findings, it was further determined that the flow stimulus, transduced by integrin signaling and generation of superoxide radicals and tyrosine kinase, culminates in constriction by the activation of L-type Ca2⫹ channels. Integrins are membrane-spanning heterodimeric proteins that transduce mechanical forces across the cell membrane. Hence, increases in intraluminal flow may act to depolarize and constrict vascular smooth muscle cells as a negative feedback mechanism to maintain constant blood flow to regulatory beds.

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ated by the prostacyclin-induced activation of KATP channels (Lombard et al., 1999). However, K⫹ channels may not be the only ion channels involved in the dilator response to hypoxia. Evidence from studies in rabbit systemic and main pulmonary arteries suggests that hypoxia inhibits an O2- sensitive Ca2⫹ channel to induce hypoxia-induced dilation (Franco-Obregon and LopezBorneo, 1996). In contrast to the dilation to hypoxia exhibited by most arteries, pulmonary resistance arteries constrict when exposed to low PO2, a mechanism that helps match regional levels of pulmonary perfusion to pulmonary ventilation. In these arteries, hypoxia depolarizes the smooth muscle cells from ⫺51 to ⫺37 mV (Madden et al., 1985; Harder et al., 1985), and this depolarization is accompanied by the generation of Ca2⫹-dependent action potentials, which are dependent on Ca2⫹ influx through L-type Ca2⫹ channels (Fig. 8). Thus, multiple O2-sensing mechanisms appear to exist in the vasculature, and various ion channels have been proposed as the sensors. Which ion channel subunits, if any, respond directly to changes in O2 levels remains to be determined, but there is evidence that the closure of KV channels may be involved in the depolarizing and contractile response of pulmonary smooth muscle cells to a hypoxic environment (Post et al., 1995). Alternatively, it has been proposed that this response may be mediated by a sustained increase in [Ca]i resulting from the activation of Ca2⫹-activated Cl⫺ channels by hypoxia (Yuan, 1997).

E. Oxygen, Carbon Dioxide, and pH It is well recognized that changes in the availability of oxygen contribute to the local regulation of blood flow, and this response is particularly critical in the cerebral and pulmonary circulations. In the cerebral circulation, arteries dilate when PO2 is reduced, but whether this dilation results from a direct action of PO2 on smooth muscle cells or is caused by the release of endothelium-derived vasodilator factors is not universally agreed upon. For example, cannulated, pressurized feline cerebral arteries hyperpolarize and dilate in response to low PO2, and spontaneous electrical spike activity is inhibited under these conditions (Lombard et al., 1986). Similarly, rat cerebral arteries also hyperpolarize and dilate in response to low PO2, and this endothelium-dependent response appears to be medi-

FIGURE 8 Microelectrode recordings showing the effect of hypoxia on the resting Em of a cannulated, pressurized pulmonary resistance artery of a cat. Hypoxia-induced depolarization and generation of action potentials were eliminated by verapamil. Adapted from Harder et al. (1985), with permission.

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In addition to the PO2 level, the level of PCO2 sensed by the vascular smooth muscle cell is a potent regulator of arterial Em and diameter. Elevating PCO2, which concurrently may reduce intracellular pH, generally hyperpolarizes and dilates the smooth muscle cells of small arteries, whereas reducing PCO2 induces depolarization and constriction. The sensitivity of the resting Em of vascular smooth muscle cells to PCO2 and pH is particularly evident and important in the cerebral circulation. For example, elevated PCO2 levels or decreased pH both markedly hyperpolarize and dilate cannulated, pressurized rat cerebral arteries (Harder, 1982). Indeed, changes in intracellular pH independent of changing PCO2 may directly affect the level of resting Em in vascular smooth muscle cells. In rat cerebral arterioles, alkalosis (pH 7.65) depolarizes smooth muscle cells from ⫺37 to ⫺31 mV (Harder and Madden, 1985), whereas acidosis (pH 6.8) induces a profound hyperpolarizing response, resulting in a final Em level of ⫺58 mV (Dietrich and Dacey, 1994). Notably, KV channels may also be instrumental in monitoring the local metabolic milieu, and changes in pH may differentially modulate the activity of KV channels in different vascular beds. For example, intracellular acidosis acutely suppresses the level of KV channel current in vascular smooth muscle cells from small pulmonary arteries, but under the same conditions enhances KV current in coronary smooth muscle cells (Berger et al., 1998). The opposing effect of acidosis on KV channel activity in these two vascular beds is postulated to contribute to the contrasting vasoconstrictor and vasodilator responses of the small pulmonary and coronary arteries to acidosis, respectively. Thus, the different vasoactive responses of blood vessels to metabolic signals may reflect the existence of sitespecific populations of K⫹ channels expressed by different regions of the circulation.

VI. SUMMARY The development of microelectrodes to characterize the properties and modulation of Em levels provided the first tool for understanding the basis of electrical excitability in vascular smooth muscle cells. This technique established that the resting Em of vascular smooth muscle cells depends primarily on the distribution and transport of K⫹ ions across the cell membrane, although the permeability of the membrance to other ions also contributes to the level of Em in varying degrees. More recent advancements, including a variety of patch-clamp methods and molecular biology approaches, have resulted in the discovery and characterization of diverse populations of ion channels in vascular smooth muscle

membranes that allow ions to move back and forth across the cell membrane in response to neuronal, chemical, and mechanical stimuli. Fluorescent imaging, which facilitates the direct measurement of changes in [Ca2⫹]i , has also clarified the link among Em, L-type Ca2⫹ channels, and excitation–contraction coupling in vascular smooth muscle cells. However, much work remains to be done to clarify the role of ion channels in vascular excitability. Little is known about the different protein subunits that coassemble to confer normal function to ion channels in native tissue or how endogenous substances alter the interaction of these proteins to regulate channel activity. Also, the critical mechanisms that permit the diverse expression and functional profiles of ion channel populations in the various vascular beds, which ultimately appear to reflect the demands of the distal tissue, remain to be discovered. This knowledge will also help clarify the role of ion channels and Em in the normal development of the vascular smooth muscle and will shed light on the pathways by which the abnormal expression of ion channels contributes to the pathogenesis of vasoconstrictor diseases, including coronary spasm, and pulmonary and systemic hypertension.

Bibliography Archer, S. L., Huang, J. M. C., Reeve, H. L., Hampl, V., Tolarova, S., Michelakis, E., and Weir, E. K. (1996). Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ. Res. 78, 431–442. Berger, M. G., and Rusch, N. J. (1999). Voltage and calcium-gated potassium channels: Functional expression and therapeutic potential in the vasculature. In ‘‘Animal Toxins and Potassium Channels’’ (H. Darbon and J.-M. Sabatier, eds.), pp. 313–332. Kluwer, Dordrecht, The Netherlands. Berger, M. G., Vandier, C., Bonnet, P., Jackson, W. F., and Rusch, N. J. (1998). Intracellular acidosis differentially regulates Kv channels in coronary and pulmonary vascular muscle. Am. J. Physiol. 275, H1351–H1359. Bevan, J. A., and Wellman, G. C. (1993). Intraluminal flow-initiated hyperpolarization and depolarization shift the membrane potential of arterial smooth muscle toward an intermediate level. Circ. Res. 73, 1188–1192. Bolotina, V. M., Najibi, S., Palacino, J. J., Pagano, P. J., and Cohen, R. A. (1994). Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368, 850–853. Brayden, J. E., and Nelson, M. T. (1992). Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 356, 532–535. Davis, M. J., and Hill, M. A. (1999). Signaling mechanisms underlying the vascular myogenic response. Physiol. Rev. 79, 387–423. Dietrich, H. H., and Dacey, R. G. (1994). Effects of extravascular acidification and extravascular alkalinization on constriction and depolarization in rat cerebral arterioles in vitro. J. Neurosurg. 81, 437–442. Franco-Obregon, A., and Lopez-Borneo, J. (1996). Low PO2 inhibits

11. Vascular Smooth Muscle calcium channel activity in arterial smooth muscle cells. Am. J. Physiol. 40, H2290–H2299. Harder, D. R. (1982). Effect of H⫹ and elevated PCO2 on membrane electrical properties of rat cerebral arteries. Pflug. Arch. 394, 182–185. Harder, D. R. (1984). Pressure-dependent membrane depolarization in cat middle cerebral artery. Circ. Res. 55, 197–202. Harder, D. R., and Madden, J. A. (1985). Cellular mechanism of force development in cat middle cerebral artery by reduced PCO2. Pflug. Arch. 403, 402–404. Harder, D. R., Madden, J. A., and Dawson, C. A. (1985). Hypoxic induction of Ca2⫹-dependent action potentials in small pulmonary arteries of the cat. J. Appl. Physiol. 59, 113–118. Hasunuma, K., Rodman, D. M., and McMurty, I. F. (1991). Effects of K⫹ channel blockers on vascular tone in the perfused rat lung. Am. Rev. Respir. Dis. 144, 884–887. Hermsmeyer, K. (1982). Electrogenic ion pumps and other determinants of membrane potential in vascular muscle. Physiologist 25, 454–465. Knot, H. J., and Nelson, M. T. (1995). Regulation of membrane potential and diameter by voltage-dependent K⫹ channels in rabbit myogenic cerebral arteries. Am. J. Physiol. 269, H348–H355. Knot, H. J., and Nelson, M. T. (1998). Regulation of arterial diameter and wall [Ca2⫹] in cerebral arteries of rat by membrane potential and intravascular pressure. J. Physiol. 508, 199–209. Koch, W. J., Ellinor, P. T., and Schwartz, A. (1990). cDNA cloning of a dihydropyridine-sensitive calcium channel from rat aorta. J. Biol. Chem. 265, 17786–17791. Koch, R. O., Treib, M., Koschak, A., Wanner, S. G., Gauthier, K. M., Rusch, N. J., and Knaus, H.-G. (2000). Design and use of antibodies for mapping K⫹ channel expression in the cardiovascular system. In ‘‘Potassium Channels in Cardiovascular Biology’’ (S. A. Archer and N. J. Rusch, eds.), Plenum, New York. Lombard, J. H., Smeda, J., Madden, J. A., and Harder, D. R. (1986). Effect of reduced oxygen availability upon myogenic depolarization and contraction of cat middle cerebral artery. Circ. Res. 58, 565-569. Madden, J. A., and Christman, N. T. (1999). Integrin signaling, free radicals and tyrosine kinase mediate flow constriction in isolated cerebral arteries. Am. J. Physiol. 277, H2264–H2271. Madden, J. A., Dawson, C. A., and Harder, D. R. (1985). Hypoxiainduced activation in small isolated pulmonary arteries of the cat. J. Appl. Physiol. 59, 113–118. Miura, H. Y., Liu, Y., and Gutterman, D. D. (1999). Human coronary arteriolar dilation to bradykinin depends on membrane hyperpolarization. Circulation 99, 3132–3138. Nelson, M. T., Patlak, J. B., Worley, J. F., and Standen, N. B. (1990). Calcium channels, potassium channels, and voltage-dependence of arterial smooth muscle tone. Am. J. Physiol. 259, C3–C18. Nelson, M. T., Standen, N. B., Brayden, J. E., and Worley, J. F. (1988). Noradrenaline contracts arteries by activating voltage-dependent calcium channels. Nature 336, 382–385.

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Nishiyama, M., Hashitani, H., Fukuta, H., Yamamoto, Y., and Suzuki, Y. (1998). Potassium channels activated in the endothelium-dependent hyperpolarization in guinea-pig coronary artery. J. Physiol. 510, 455–465. Peng, W., Michael, J. R., Hoidal, J. R., Karwande, S. V., and Farrukh, I. S. (1998). ET-1 modulates KCa-channel activity and arterial tension in normoxic and hypoxic human pulmonary vasculature. Am. J. Physiol. 275, L729–L739. Post, J. M., Gelband, C. H., and Hume, J. R. (1995). [Ca2⫹] inhibition of K⫹ channels in canine pulmonary artery: Novel mechanism for hypoxia-induced membrane depolarization. Circ. Res. 77, 131–139. Post, J. M., Hume, J. R., Archer, S. A., and Weir, E. K. (1992). Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am. J. Physiol. 262, C882–C890. Pusch, M., and Jentsch, T. J. (1994). Molecular physiology of voltagegated chloride channels. Physiol. Rev. 74, 813–827. Quayle, J. M., Nelson, M. T., and Standen, N. B. (1997). ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol. Rev. 77, 1165–1231. Rusch, N. J., and Hermsmeyer, K. (1994). Calcium channels in hypertension. In ‘‘Handbook on Ionic Transport in Hypertension: New Perspectives’’ (A. Coca, ed.), Chapter 10. CRC Press, Boca Raton, FL. Salter, K. J., and Kozlowski, R. Z. (1998). Differential electrophysiological actions of endothelin-1 on Cl⫺ and K⫹ currents in myocytes isolated from aorta, basilar and pulmonary artery. JPET 284, 1122– 1131. Shimoda, L. A., Sylvester, J. T., and Sham, J. S. (1998). Inhibition of voltage-gated K⫹ current in rat intrapulmonary arterial myocytes by endothelin-1. Am. J. Physiol. 273, L842–L853. Sperelakis, N. (1998). Diffusion and permeability, and origin of resting membrane potentials, In ‘‘Cell Physiology Source Book’’ (N. Sperelakis, ed.), 2nd Ed., pp. 171–201. Academic Press, New York. Stekiel, W. J., Contney, S. J., and Rusch, N. J. (1993). Altered 웁-receptor control of in situ membrane potential in hypertensive rats. Hypertension 21, 1005–1009. Tanaka, Y., Meera, P., Song, M., Knaus, H.-G., and Toro, L. (1997). Molecular constituents of maxi KCa channels in human coronary smooth muscle: Predominant 움 ⫹ 웁 subunit complexes. J. Physiol. 502, 545–557. Vanheel, B., and Van de Voorde, J. (1997). Nitric oxide induced membrane hyperpolarization in the rat aorta is not mediated by glibenclamide-sensitive potassium channels. Can. J. Physiol. Pharmacol. 75, 1387–1392. Welsh, D. G., and Segal, S. S. (1998). Endothelial and smooth muscle cell conduction in arterioles controlling blood flow. Am. J. Physiol. 274, H178–H186. Yuan, X.-J. (1997). Role of calcium-activated chloride current in regulating pulmonary vasomotor tone. Am. J. Physiol. 272, L959–L968. Yamazaki, J., Duan, D., Janiak, R., Kuenzli, K., Horowitz, B., and Hume, J. R. (1998). Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells. J. Physiol. 507, 729–736.

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12 Sodium Channels KATSUSHIGE ONO and MAKOTO ARITA Department of Physiology Oita Medical University Oita 879-5593, Japan

I. INTRODUCTION

II. MOLECULAR DETERMINATIONS OF Na⫹ CHANNEL FUNCTION

Many ions carry transsarcolemmal currents that contribute importantly to electrical activity in cardiac myocytes, where the influx of Na⫹ ions plays an especially unique and critical role. The sodium current (INa) is not only responsible for initial cardiac action-potential depolarization, but also the influx of Na⫹ ions into the cytoplasm, which helps generate the prolonged depolarization characteristics of the action-potential plateau. The magnitude and kinetics of INa are controlled in a complex manner by both membrane potential and intracellular metabolites. Such Na⫹-preferring voltagedependent channels are present in a wide variety of electrically excitable membranes of cardiac and skeletal muscles, nerves, and other tissues. Because the voltagegated cardiac Na⫹ channel has been studied extensively by electrophysiological approaches, we have accumulated a large amount of information on the biophysical properties of this channel since the early 1980s. Considerable attention has been placed on the molecular function of the Na⫹ channel. Mutations of the human voltage-gated cardiac Na⫹ channel gene that cause several forms of inherited diseases have been identified. The underlying defects in the Na⫹ channel-related disease state include aberrant regulation of channel function, resulting in lethal ventricular tachyarrhythmias and so on. This chapter reviews certain aspects involved in the structure and regulation of the cardiac sarcolemmal voltage-gated Na⫹ channel in physiological and particularly in pathophysiological conditions.

Heart Physiology and Pathophysiology, Fourth Edition

A. Structural Features In 1984, in order to clarify the primary structure of the Na⫹ channel, Numa and colleagues used partial sequences from purified eel electroplax Na⫹ channels to clone the cDNA for the eel Na⫹ channel 움 subunit (1). Using this probe, they cloned three Na⫹ channel 움 subunits from rat brain (1). The rat heart isoform (rHt) was cloned by Rogart et al. (2) and its human counterpart (hHt) by Gellens et al. (3). The 움 subunit of the cardiac Na⫹ channel has been reported to be 240 kDa. A schematic diagram of the proposed membrane topology for the 움 subunit of the cardiac Na⫹ channel is shown in Fig. 1. The 움 subunit consists of four large homologous membrane domains (I–IV) (4). Each domain comprises six putative 움 helices (S1–S6). The pore of the channel is assumed to extend between segments S5 and S6 (5). Studies with molecular–biological techniques, particularly site-directed mutagenesis of Na⫹ channel proteins and functional expression mutant channels, have pinpointed regions that are involved in different functions of the channel; these studies were reviewed previously in detail by Fozzard and Hanck (6). Between each of the six transmembrane segments and particularly between each of the four larger domains are extensive regions that fold out from the membrane. Highly charged S4 segments of each domain transduce the transmembrane electrical voltage by moving across

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FIGURE 1 Molecular structure of the human cardiac Na⫹ channel (hH1) and locations of mutations causing the chromosome-3-linked long QT syndrome and the Brugada syndrome. The SCN5A gene encodes the Na⫹ channel 움 subunit, a protein 2016 amino acids long. The 움 subunit consists of four putative transmembrane domains (I–IV), with each domain containing six transmembrane segments (SI–S6). Each of the four S4 segments consists of a charged residue at every third position. This structure is likely responsible for the voltagesensing mechanism. Long QT syndrome-associated mutations are shown by pentagons, and Brugada syndrome mutations are shown by diamonds. The protein kinase C (PKC) site is denoted by the boxed S. The putative protein kinase A (PKA) sites are denoted by the circled S, constitute of R R/K X S/T or R X X S/T or R X S/T, where X designates any amino acid. Also shown are sites of glycosylation (␺) and amino acid residues that form the inactivation particle IFM by an oval.

the transmembrane electrical field. This movement causes conformational changes that result in an opening or gating of the Na⫹-conducting pathway. The opening of the channel, also called ‘‘activation,’’ allows Na⫹ ions to passively enter the cell down the Na⫹ concentration gradient to depolarize the membrane. N and C termini, as well as linkers between segments, alternate between intracellular and extracellular surfaces. Potential glycosylation sites are all found in putative extracellular loops (Fig. 1). In addition, several protein kinase A (PKA) or protein kinase C (PKC) consensus phosphorylation sites are present on the intracellular linkers of the 움 subunit of the cardiac Na⫹ channel. The existence of these sites does not imply that they are functionally phosphorylatable. Moreover, other serine or threonine residues that do not reside in typical consensus phosphorylation sites could be phosphorylated by protein kinases (7). Therefore, it is essential to measure the incorporation of phosphate groups in Na⫹ channels induced by protein kinase stimulation. Only a limited number of studies, however, have provided this essential information. Initial work from Catterall’s laboratory has determined that 3–4 mol of phosphate are incorporated in 1 mol of purified rat brain Na⫹ channel 움 subunit following the activation of

PKA under basal conditions (8). It is known that four serine residues located in that linker can be phosphorylated by PKA in vitro (9). A fifth serine residue located at position 554 in linker I-II might be endogenously phosphorylated (Fig. 1). Regions between the S4 and the S5 segments in all four domains fold so as to form part of the extracellular mouth of the channel, and the S6 segments line the ionconducting pore. Mutations or chimeras formed from specific regions of Na⫹ channels alter channel properties such as Ca2⫹ and Na⫹ ion selectivity (10). Although the three-dimensional structure of Na⫹ channels is not known, most researchers (6, 11–13) assume that the transmembrane segments of each of the four domains fold similarly to models of voltage-dependent K⫹ channel proteins. K⫹ channels exist as tetramers of identical subunits (14, 15) rather than single proteins with four domains. Each of the four subunits of the K⫹ channel is thought to exist as a closely packed group of transmembrane helices arranged in a rosette around the central ion-conducting pore (15). Presumably, the four homologous domains of Na⫹ channels act much like the four identical subunits of a K⫹ channel. In addition to the main 움 subunit of Na⫹ channels, there are two auxiliary subunits that copurify with the

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움 protein: the 웁1 subunit (36 kDa) and the 웁2 subunit (33 kDa). The functions of 웁 subunits appear to be subsidiary to the 움-subunit functions of Na⫹ channels because the basic physiology and the action of several toxins and drugs with isolated 움 subunits are roughly comparable to those of native Na⫹ channels (16, 17). Na⫹ channel 웁1 subunits are found in association with cardiac and skeletal muscle, and brain Na⫹ channels, whereas 웁2 subunits are found only with brain Na⫹ channels.

B. Channel Kinetics ˙ max in the Study of Naⴙ Current 1. Role of V In most parts of the heart, the sodium channel current (INa) is the principal current responsible for normal excitation and conduction. INa is also thought to play a major role in the slow conduction and block that lead to some arrthymias in certain pathophysiological conditions, and the Na⫹ channel is a target for many antiarrhythmic drugs. For the studies of normal and abnormal behavior of cardiac INa, the patch-clamp technique has been applied since the early 1980s for both whole cell (macroscopic) current and single-channel current recordings by the methods described by Hamill et al. (18). At that time and even presently in certain conditions, in place of the quantitative measurement of INa by the voltageclamp method, many cardiac investigators have used the maximal depolarization rate of the action potential ˙ max) as a semiquantitative index of cardiac INa . It (V is therefore important to address the question of the ˙ max as a measure of INa . The action potenaccuracy of V tial in cardiac myocytes is a sudden regenerative depolarization of the transmembrane potential and subsequent repolarization back to the resting potential, which results from an increase in membrane permeability to Na⫹ resulting in INa . According to calculations of the ˙ max compared to the fractional peak INa obfractional V tained form Na⫹ channel availability plots (Fig. 2) (19), ˙ max consistently overestimated the availability of Na⫹ V current as measured by peak INa during voltage clamp. Therefore, in short, it resulted that the physiological ˙ max are different. processes underlying the peak INa and V ⫹ ˙ max , In terms of single Na channel events underlying V ˙ it has been pointed out that Vmax is usually achieved long before many channels can open. Therefore, the ˙ max is much less than fraction of channels open at V ˙ max those open at peak INa . Moreover, the fraction at V depends on the speed of depolarization of the action potential. Thus it may be important to recognize that ˙ max is an indirect parameter of INa, which only reflects V changes in INa due to a combination of factors related to the voltage dependence and kinetics of the channel.

FIGURE 2 Fractional V˙max comparaed to fractional peak INa obtained from Na⫹ channel availability plots. The experimental plots ˙ max overestideviated from an identity line, indicating that fractional V mates INa (19).

2. Macroscopic Naⴙ Current The classical description of ionic currents in the squid giant axon by Hodgkin and Huxley (20, 21) has been widely used for other electrically excitable membranes. In the Hodgkin–Huxley equations for the Na⫹ current, the term m3 ⭈ h is considered to be the probability of an open sodium channel. The symbol m denotes the activation variable, h the inactivation variable, and the kinetics of m and h are described by first-order differential equations. Therefore, the current relaxation at a given membrane potential is specified by the parameters m앝 (steady-state activation variable), h앝 (steady-state inactivation variable), ␶m (time constant of Na⫹ activation), and ␶h (time constant of Na⫹ inactivation). The steady-state properties of activation and inactivation have been discussed for a long time and can be summarized as follows. Hodgkin and Huxley observed the onset of the Na⫹ current as a sigmoidal form after a voltage step, so that they postulated that activation was a multistep process or the result of three independent events with the probability m, yielding the probability of opening of m3. Therefore the voltage dependence of the activation process itself would be the cubic root of the peak conductance. The decay of INa represented the accumulation of channels in the inactivation state. Because they observed that INa decayed with a single time constant, they described inactivation as only a single transition with the probability of (1 ⫺ h). These parameters of activation (m3) and inactivation (h) in cardiac myocytes are plotted as a function of membrane

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potential (Fig. 3) (22). Overlap between the activation and the inactivation curve is independent of the assumed resting potential. The ‘‘window’’ between the m3앝 and h앝 curves (shaded area in Fig. 3) is of particular interest because it may reflect the potential range in which steady-state Na⫹ currents can be observed. The amplitude of these stationary currents may be proportional to m3앝 ⭈ h앝. In the classical Hodgkin–Huxley equations described earlier, the probability of an open Na⫹ channel was formulated by the product of the probabilities of a channel being activated (m3) and not being inactivated (h): the transient Na⫹ current was proportional to m3 ⭈ h. Therefore, it was assumed that the two gating processes of Na⫹ activation and inactivation proceed independently of each other. This concept has long been supported by experiments in which strong modification of Na⫹ inactivation had only a small effect on Na⫹ activation. However, some experiments have provided evidence that activation and inactivation are functionally coupled (23, 24). This bold hypothesis has been confirmed by mutagenesis studies involving mutations in the outer S4 segment of domain IV. Replacing charged arginine residues with cysteines markedly slows the rate of fast inactivation during depolarization in the Na⫹ channel in skeletal muscle. In the cardiac counterpart, naturally occurring mutations in the domain IV-S4 segment are implicated in both skeletal muscle myopathies (25) and the long QT syndrome (described later) (26). It is therefore concluded that the domain IV-S4 charged segment serves as the voltage sensor for both activation and inactivation and that outward motion of this segment is a prerequisite to normal inactivation gating. Studies also suggest that the charged S4 segments in all four domains contribute to both activation and inactiva-

tion (26, 27), indicating that the classical Hodgkin– Huxley model is probably wrong and a reinterpretation of the activation/inactivation gate is required based on the molecular function (28). 3. Single-Channel Conductance An early estimation of the single Na⫹ channel conductance (웂) was obtained from the analysis of macroscopic Na⫹ current fluctuations (29). The first measurements of current through single Na⫹ channels in rat heart myocytes established a range of 15–20 pS for the conductance (30). Yamamoto et al. (31) observed a rapid and voltage-dependent block of single sodium channels in neuroblastoma cells caused by extracellular Ca2⫹ ions, resulting in a decrease in the apparent channel conductance at increased [Ca2⫹]o concentrations. A similar voltage-dependent block of open Na⫹ channels caused by extracellular Ca2⫹ ions has been observed in single canine cardiac Purkinje cells (32). It appears that the different single-channel conductance values in cardiac Na⫹ channels under physiological conditions can be attributed to differences in the ionic compositions of the extra- and intracellular solutions. Lower conductances in Na⫹ channels are not only observed with increased Ca2⫹ concentrations in the solutions or after treatment with alkaloids, but they are also found in physiological solutions, even under unmodified conditions. These lowconductance Na⫹ channels frequently appear in preparations from cardiac muscles together with openings of normal conductance (33–35). A typical single-channel record with multilevel openings shows currents in a normal and a low-conductance Na⫹ channel of myocytes from rat ventricle. In this study, two populations of Na⫹ channels produce different peaks in the amplitude histogram, but there are no apparent differences in their gating kinetics, consistent with data from canine Purkinje cells. However, as reported in a study with rat cardiac myocytes, low-conductance Na⫹ channels exhibited slower inactivation, a longer and voltage-insensitive open time, and sometimes even an altered ionic selectivity in comparison to normal Na⫹ channels (35). 4. Single-Channel Kinetics

FIGURE 3 Steady-state parameters m앝, m3앝, and h앝 of Na⫹ current activation and inactivation in a single myocardial mouse cell (22).

Activation of Na⫹ channels is thought to result from a voltage-driven conformational change that opens a transmembrane pore through the protein. Channel opening is triggered by membrane depolarization, exerting an electrical force on the voltage sensor located within the transmembrane electrical field of the S4 segment in each domain. The movement of the gating charges through the membrane has been directly measured as an outward gating current (36). Because Na⫹

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FIGURE 4 Open-channel histogram in cell-attached patch recordings at ⫺41 mV (A) and waiting times (latencies) for the two potentials (B and C). (A) A single exponential fit is shown with a ␶ of 1.3 msec. (B) Waiting times for all openings. The peak probability of opening was quite slow and openings are spread over 20 msec. (C) Waiting time to the first openings. Significant numbers of reopenings occurred at the test potentials (71).

channel currents can be blocked by toxin without affecting these gating currents, it is suggested that elements of the channel protein involved in voltage-dependent activation are separate from those that form the pore. Similarly, mutations of the P region (see Fig. 1) that alter ion conductance have no effect on gating currents (37). Na⫹ channel behavior appears to be stochastic in terms of the single-channel characteristics (34). Voltage steps alter the probability that channels will make a transition between conformational states such as the closed state (C) to the open state (O), i.e., C 씮 O. This probabilistic behavior can be described from an examination of a collection of many channel openings, and the general voltage-dependent characteristics can be seen by comparison of behavior from a number of activating voltage steps (34). In this context, the channel opens with a variable delay after an activating voltage step, and this characteristic can be summarized by a histogram of the latencies, or the waiting time from the depolarization to the first opening, which shows a clear rising phase that is dependent on the potential (Fig. 4). A histogram of open times describes the probable lifetime of the open state (34). In the case of burst openings, the channel may open, close, and reopen be-

fore closing permanently (35). Infrequently, a channel never opens even though the voltage is stepped into the full activation range. The average current from many openings by one channel is called the ensemble. It closely resembles the macroscopic current in term of the current structure, and in that it requires several closed steps before opening. Combined with gating current measurements, single-channel analyses have led to the development of sequential state models of the Na⫹ channel that appear to be superior to the Hodgkin– Huxley model in fitting experimental data (38). Moreover, they allow predictions on the transitions in the actual molecular structure of the channel in response to function.

III. MODULATION BY PHOSPHORYLATION A. Biological Insights on Phosphorylation Modulation Voltage-dependent Na⫹ channels, which are responsible for the generation of action potentials in heart, are phosphorylated by adenosine 3⬘,5⬘-cyclic monophos-

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phate (cAMP)-dependent protein kinase (PKA). The combination of molecular biology to create channels with point mutations and electrophysiological techniques to study function encourages the idea that predictions of structures responsible for 웁-adrenergic modulation are possible. With the goal of understanding the properties of cAMP-dependent modification of the cardiac Na⫹ channel, this chapter introduces what is known about the effects of cAMP and 웁-adrenergic agonists and considers the relationship of the molecular function to the phosphorylation-dependent modification of the cardiac Na⫹ channel. The voltage-dependent sodium current (INa) is the basis of excitability in the heart responsible for the upstroke of the action potential in atrial, ventricular, and His–Purkinje cells and, consequently, for rapid conduction in these tissues. Na⫹ channels open or activate in response to membrane depolarization and then, within a few milliseconds, inactivate. The molecule responsible for these activities must contain a highly selective ion pore as well as appropriate machinery for gating the flow of sodium ions through the channel. Pharmacological studies have shown that 웁-adrenergic stimulation is assumed to modify these gating properties of Na⫹ channels in cardiac muscle. In addition, biochemical studies revealed that Na⫹ channels are good substrates for phosphorylation. Moreover, advances in molecular– biological techniques revealed that the cardiac Na⫹ channel isoform contains putative phosphorylation sites for serine/threonine kinase or cAMP-dependent protein phosphorylation kinase. Cardiac and brain Na⫹ channels are good substrates for phosphorylation in vivo and in vitro by cAMPdependent protein kinase (39–42). Sites for phosphorylation by cAMP-dependent protein kinase have been identified using a combination of protease digestion, two-dimensional phosphopeptide mapping of the resulting peptides, immunoprecipitation of the phosphopeptides with site-directed antibodies, and phosphopeptide microsequencing (9). The effects of cAMP are believed to emerge from the phosphorylation of some specific sites located in the intracellular domain connecting repeats I and II (I-II linker) of the 움 subunit of the Na⫹ channel protein (4). Four serines in the I-II linker of rat nerve Na⫹ channels were reported to be phosphorylated in vitro or in vivo by PKA (9), and serine-1506 in the III-IV linker is phosphorylated by PKC (42) (see Fig. 1). In order to address the functional phosphorylation sites of the Na⫹ channel, five threonine or serine residues residing in PKA consensus sites located in the amino terminus, I-II, and II-III linkers of the 움 subunit of rat heart Na⫹ channels were replaced with different amino acids (43). When this mutant channel with disrupted

PKA consensus site was expressed in oocytes, intracellular injection of cAMP enhanced Na⫹ currents, thereby suggesting that either an unknown phosphorylated protein is modulating Na⫹ channels and/or phosphorylation of Na⫹ channels is occuring at other threonine and/or serine residues. The fundamental effect of increased [cAMP]i and subsequent PKA activation on nerve Na⫹ channels expressed in oocytes or in mammalian cell lines was typically the reduction of INa . There are minimal changes in the voltage dependence of the kinetics due to PKA phosphorylation (44, 45), implying that the mechanism is a reduction in channel open probability. However, Schreibmayer et al. (43) found a shift of the availability curve in the hyperpolarizing direction in the cardiac Na⫹ channel coexpressed with the 웁2-adrenergic receptor in oocytes and stimulated with a catecholamine. They reported that isoproterenol enhanced the Na⫹ current in rat heart Na⫹ channels (skM2 or rH1) via PKA activation. It is of interest to note that PKA attenuated Na⫹ currents in rat brain channels and augmented Na⫹ currents in rat heart (skM2) channels (44). Such divergent effects of PKA stimulation may be reconciled in part by referring to a fascinating finding reported by Li et al. (46). These authors claimed that prior PKC stimulation was necessary for the inhibitory effect of PKA activation to appear. Such dependence of the PKA effect on PKC could be abolished when serine in the PKC site was replaced with an unphosphorylatable amino acid, alanine. These data suggest that the mechanism probably represents simply the effect of a negative charge at this site, because replacing the serine of the PKC site with glutamic acid, of which the isoelectric point is 3.22, simulated the effect of PKC-dependent phosphorylation. This idea may not apply to the cardiac isoform. Namely, the mutation of serine-1504 in cardiac Na⫹ channels expressed in Xenopus oocytes did not alter the current response to cAMP (47). However, one report found that phosphorylation of serine-1505 is required for both the negative shift of the availability curve and the reduction of the Na⫹ current by PKC in cardiac Na⫹ channels (rH1) (48). Furthermore, the mutation of the PKC site to make the site suitable for phosphorylation by PKA resulted in kinetic-effect characteristics of PKC. Presumably, multiple modulatory steps exist in a complex cascade of phosphorylation modification of channel events.

B. Effects of PKA ˙ max of Action Potentials 1. PKA on V The action potential in atrial, ventricular, and Purkinje fibers produces a rapid increase in membrane per-

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meability to Na⫹ ions and induces INa . Most cardiac investigators have used the maximal depolarization rate ˙ max) as a semiquantitative meaof the action potential (V ˙ max is not a linear paramesure of cardiac INa, although V ter of INa (6, 19) as described in the previous section. Adrenergic regulation appears to be an important feature of Ca2⫹ channels and of some K⫹ channels; however, it has not been known until recently that Na⫹ channels in the heart could also be adrenergically regulated. The first indication of this was reported as a reduc˙ max by catecholamines during the recording of tion of V action potentials in ventricular muscle fibers (49). This ˙ max was seen only when the preparations reduction of V were partially depolarized, indicating a shift of the voltage dependence of inactivation in the hyperpolarized direction. Similar evidence has been obtained in a variety of preparations such as in guinea pigs (50–52) and ˙ max as an index of INa . These studies dogs (53) using V reported that catecholamines, membrane-permeant ˙ max cAMP, or phosphodiesterase inhibitor suppressed V of action potentials in cardiac muscles in a voltagedependent manner. They concluded that 웁-adrenergic agents exerted these effects via phosphorylation(s) of the Na⫹ channel secondary to the activation of cAMPdependent protein kinase, which is consistent with the biochemical findings that the cardiac Na⫹ channel can be phosphorylated by PKA in vitro (54, 55) (Fig. 1). ˙ max was The depressant effects of isoproterenol on V reversed when the activity of adenylate cyclase (to produce cAMP) was depressed via the stimulation of Gi proteins by acetylcholine, an agonist for the muscarinic receptor (52). The effect of 웁-adrenergic stimulation on the upstroke of ventricular action potentials is quite clear. However, one study (53) that compared the effect of 웁-adrenergic agonists on cardiac myocytes and tissues reported that isoproterenol showed no effect on the upstroke velocity of intact cardiac muscles regardless of the resting potential, whereas in isolated myocytes isoproterenol increased the upstroke velocity at the normal resting potentials and decreased it when the cells were depolarized. Hisatome and Arita (56) found that the depression of the action potential upstroke by catecholamines was found under the condition of either normoxia or hypoxia in guinea pig ventricular muscles.

Na⫹ channel was performed in 1989 using guinea pig cardiac ventricular myocytes under the configuration of whole cell current recording (57). With the holding potential at ⫺80 mV, isoproterenol (1 애M) reversibly decreased the Na⫹ current by 50%. The effect was not observed when the myocytes were pretreated by atenolol, an agonist of the 웁1-adrenergic receptor. A similar inhibition of the Na⫹ current was observed either when the membrane-permeant form of cAMP (dibutyryl cAMP) was applied or when forskolin, a stimulator of adenylate cyclase, was applied. One study of Kirstein et al. (58) reported the similar shift of the availability curve (h앝 vs membrane potential) and a significant shortening of the time to peak INa in the presence of isoproterenol, thereby suggesting that both activation and inactivation parameters could have been modified by 웁-adrenergic agonists. Moreover, several studies under different experimental conditions using partially depolarized myocardium, i.e., with depressed Na⫹ current, have led to the circumstantial conclusion that the cardiac Na⫹ channel can be depressed by cAMP-dependent mechanisms (59–62). In addition, Sunami et al. (63) reported that the catalytic subunit of PKA decreased INa in guinea pig ventricular myocytes. However, Matsuda et al. (64) claimed that INa in rabbit cardiac myocytes is not decreased, but is instead increased by isoproterenol in a manner depending on the holding potential. Subsequently, several conflicting reports have appeared regarding 웁-adrenergic effects on cardiac Na⫹ channels (6, 58, 65). Ono et al. (66) carefully examined the effect of PKA activation under voltage-clamp conditions using cell-attached macropatches at room temperature, where the intracellular environment was kept ‘‘physiologic.’’ They found a phosphorylation-dependent negative shift (toward the voltage axis) of both availability and activation parameters, so that, depending on the holding and/or test potentials (Fig. 5), the channel phosphorylation by PKA could either increase or decrease INa (Fig. 6), thereby explaining most of the controversial effects of PKA reported so far, such as a hyperpolarizing shift of the availability curves with an increase in conductance under conditions of full channel availability (50–52, 56, 58, 67).

2. PKA on Whole Cell Naⴙ Current

3. PKA and Single Naⴙ Channels

˙ max contributed to Although the measurements of V ⫹ our understanding of Na current behavior during 웁-adrenergic stimulation, the exact mechanism of the Na⫹ current block by 웁-adrenergic agonists should be investigated using a voltage-clamp study. The first study of the functional effect of increased [cAMP]i and subsequent PKA activation by catecholamines on the cardiac

Most investigators studying Na⫹ channels may agree that the shift in both the activation and the availability parameters is de facto evidence for the effect of PKA on the Na⫹ current, but not all the studies of PKAdependent phosphorylation on single Na⫹ channel kinetics are associated with changes in the voltage dependency of these kinetics. Herzig and Kohlhardt (59)

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FIGURE 5 Time-dependent and cAMP-dependent changes in the kinetics of INa . (A) Time-dependent negative shift of fractional conductance curves (inverted S shaped) and voltage-dependent availability curves (S shaped) recorded from two different patches on the same cell. The conductance curve shifted 2.2 mV and the voltage-dependent availability curve shifted 2.4 mV in the hyperpolarized direction over 11 min (a and b). However, a second patch made on the same cell showed conductance and voltagedependent availability characteristics (c) (dotted curve) similar to the early determination in the first patch (a). (B) Fractional conductance and voltage-dependent availability shifted (5–10 mVz) in the hyperpolarized direction after exposure to the cAMP analogue, CPTcAMP (b). Unlike the case where the fractional conductance and the voltage-dependent availability shifted over time, these characteristics remained shifted in a second patch (c) (dotted curve) (66).

found that cAMP in inside-out patches reduced INa under cell-free conditions, mostly accompanied by a decrease in the number of channel openings. Schubert et al. (61, 62) proposed that 웁-adrenergic inhibition of single cardiac Na⫹ channels was voltage dependent, being more pronounced at depolarized potentials. The voltage dependency of the Na⫹ channel behavior was found from the voltage-dependent stochastic process at the single-channel level. Voltage steps alter the probability that the channel will make a transition between conformation states such as C 씮 O. On the basis of the voltage dependency of the open-channel characteristics described earlier; one study (64) reported an increase in the first latency of the single Na⫹ channel by application of the catalytic subunit of the cAMP-dependent protein kinase in the inside-out configuration, although the unitary current amplitude and mean open time were not affected (Fig. 7). The most consistent single-channel study, with the macroscopic Na⫹ current modulation by PKA, reported a change in the voltage dependency of single-channel open durations (68). For cardiac Na⫹ channels, unlike neuronal Na⫹ channels, evidence of voltage dependency for the transition from the open

state to the inactivation state (O씮I) comes from the finding that the mean channel open time has a biphasic bell-shaped dependence on voltage, with the longest open times occuring near -40 mV (69–72). In the case of PKA stimulation, Ono et al. (68) found that the voltage dependency of the mean open time of the channels was shifted in the hyperpolarizing direction, a consistent finding when the hyperpolarizing shift of the fractional conductance of INa activation was measured using whole cell current (Fig. 8). The effect of phosphorylation was clearly not the result of addition of a negatively charged phosphate to the inside face of the channel because an additional negative charge to the cytosolic side of the membrane was expected to further bias the voltage sensors of the ion channels, resulting in shifts of both parameters in the depolarizing direction. Therefore, cAMP-dependent phosphorylation conceivably affects voltage-dependent kinetics, as expected, by an electrostatic interaction with the voltage sensor. With regard to the shift of the activation and availability curves, it is important to recognize that Na⫹ currents exhibit a spontaneous hyperpolarizing shift with time under a variety of experimental conditions, and

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FIGURE 7 Effect of the catalytic (C) subunit of cAMP-dependent protein kinase on the single Na⫹ channel kinetics of guinea pig cardiac myocytes. (A) Open-time histograms. Mean open times were not changed by the C subunit. (B) First-latency histograms. A function composed of multiexponentials derived from a three-state model with one open state and two closed states was fitted to the first latency cumulative histogram. The time derivative was used as a quantitative description of the first-latency distribution. After application of the C subunit, both time constants reversibly increased (63).

4. Membrane-Delimited Effect of ␤ Agonist FIGURE 6 The cyclic-AMP analogue (CPTcAMP) either incresased or decreased Na⫹ channel current depending on the voltage protocol for the current measurement (A), and the effect was blocked by a specific cAMP-dependent protein kinase inhibitor (B). (A) Protocol used to evaluate the shift of fractional conductance (activation curve) (P1) and voltage-dependent availability (inactivation curve) (P2), simultaneously. (B) Peak INa recorded before and during exposure to the cAMP analogue. The current from the hyperpolarized (⫺150 mV) holding potential, IP1 (䊊), was increased, and the current from the depolarized holding potential (⫺90 mV), IP2 (䊐), was decreased by cAMP. (C) INa changes used by cAMP were prevented when the cell was pretreated with 1 애M H-89 (a specific cAMP-dependent protein kinase inhibitor) for 60 min (66).

this shift could easily be confused with a phosphorylation effect. This shift occurs under conditions of whole cell (61, 73) and isolated patch recordings in mammalian cells (38, 74, 75). Although the rate of shift is not well characterized, it is usually as slow as less than 1 mV/min and is often less than 0.5 mV/min (66). In fact, one experiment clearly provided evidence that the cAMP-dependent change in the kinetics is distinct from time-dependent changes in that the background change in the kinetics was restricted to the membrane area under the patch and could be added to the cell-wide changes induced by [cAMP]i (Fig. 5). Also, the shift of the kinetics parameters by phosphorylation cannot be examined by superimposing an additionally charged PO4 , which would increase the internal negative surface charge, as this would produce a shift of parameters in the opposite direction.

There is growing evidence that G-proteins, in addition to their regulation of second messenger systems, may also regulate ionic channels in a more direct fashion. For instance, for muscarinic modulation of a potassium-ACh channel (IK ⭈ Ach), only three macromolecules are used in the signaling cascade: receptor, G-protein,

FIGURE 8 Mean open time–voltage realtionship obtained from a singe-channel recording of the cardiac Na⫹ channel. After application of the cAMP analogue (CPTcAMP), the relationships was shifted in the hyperpolarizing direction by 10 mV, which is consistent with a shift of the fractional conductance in the same direction (68).

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and channel. These three components remain within the membrane throughout, the activated G-protein interacts directly with the channel, and no cytoplasmic second messenger is involved. In cardiac myocytes, the search for the direct regulation of Na⫹ channel activity by G-proteins has been led by Brown and colleagues (61, 62). In their initial study on membrane patches excised from rat ventricular myocytes, Na⫹ channel activity was somewhat decreased by isoproterenol and GTP in the absence of cytosolic-free Mg2⫹, whereas it was decreased markedly by further addition of GTP웂S plus Mg2⫹. The Gs purified from human erythrocytes and activated by GTP웂S (Gs*) reproduced the same reduction, as did the activated subunit Gs움*, but not their unactivated counterparts or preactivated Gi protein. However, Matsuda et al. (64) found an opposite membrane-delimited effect of Gs activation on INa in rabbit cardiac myocytes. They reported an enhancement of Na⫹ channel current by 웁-adrenergic stimulation through a direct G-protein pathway, using the Gsactivating GTP analogue, GTP웂S, and the stimulatory G-protein subunit, Gs움. This is an interesting field of investigation, but at present the results seem rather confusing. Careful studies are needed to clarify the presence or the absence of the membrane-delimited control of INa by catecholamines and to resolve these apparently opposite effects of G proteins on INa .

C. Effects of PKC Protein kinase C is a Ca2⫹ /phospholipid-dependent regulatory enzyme activated by diacylglycerol. Its activation is believed to involve a translation to cell membranes, followed by activation, after which substrate phosphorylation takes place. The finding that 움 subunits of purified Na⫹ channels from rat brain are phosphorylated by PKC was the first report to suggest that the Na⫹ current may be modulated by the Ca2⫹-dependent diacylglycerol signaling pathway (76). PKC phosphorylates a single serine residue (serine-1506) of the 움 subunit of the rat brain Na⫹ channel under physiological conditions. Serine-1506 is located in the inactivation gate formed by the short intracellular loop between domains III and IV (III-IV linker), which is highly conserved in cardiac as well as skeletal muscle and brain Na⫹ channels (42). Actually it has been reported by Qu et al. (48) that phosphorylation of serine-1505 in the cardiac Na⫹ channel inactivation gate is required for modulation by PKC, which is consistent with the structural homogeneity of the III-IV linker of the 움 subunit. Electrophysiological studies of wild type and the S1505A/1506A mutant showed that the phosphorylation of S1505/1506 has common and divergent effects in brain and cardiac Na⫹ channels (48, 77–80). Phosphorylation of this site by

PKC is required for reduction of the peak Na⫹ current in both brain and cardiac Na⫹ channels, but phosphorylation of S1506 in brain Na⫹ channels slowed the inactivation (Fig. 9). The phosphorylation of S1505 in cardiac, but not S1506 in brain, Na⫹ channels causes a negative shift in the inactivation curve. Taken together, it is implicated that the interaction of the phosphorylated S1505/1506 in the cardiac Na⫹ channel with other regions of the channel protein may differ for different genotypes. Phosphorylation of S1505 by PKC alone is sufficient to slow inactivation in brain Na⫹ channels, which is consistent with the location of this residue in a protein segment essential to inactivation gating. However, the reduction of peak Na⫹ currents requires the PKC phosphorylation of both S1506 in the III-IV linker and S610 in the I-II linker (41). However, as proposed by Li et al. (46), the reduction of peak Na⫹ currents by the cAMP-dependent phosphorylation pathway only occurs when S1506 is phosphorylated by PKC in brain Na⫹ channels, as has been mentioned in the previous section. Because S1505 and adjacent amino acids of the PKC phosphorylation site are conserved in Na⫹ channel 움 subunits from brain, heart, and skeletal muscle (1, 2, 81–83), it is suggested that the Na⫹ channel function may be modulated by the activation of PKA dependent on the phosphorylation condition of S1506 by PKC not only in neuronal Na⫹ channels, but also in a wide range of excitable tissues, including cardiac myocytes. In conclusion, although there are some discrepancies arising from differences in preparations and PKC activations, the phosphorylation of the Na⫹ channel by PKC is likely to play a specific role in controlling the activation and inactivation status in cardiac Na⫹ channels.

FIGURE 9 Effects of a PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, on wild-type (WT) and S1505A mutant Na⫹ current. The currents were recorded in response to test depolarizations to ⫺24 mV from the indicated holding potentials. Calibration bars: 12 pA, 4 msec for WT, and 24 pA, 4 msec for S1505A mutant (48).

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IV. SODIUM CHANNELOPATHIES The human genome contains a number of almost identical genes coding for slightly different voltagegated Na⫹ channels. The different channel genes are specific for expression in the various tissues and this specialization may help restrict the consequence of a mutation to a single type of cell or tissue. The various isoforms of voltage-gated Na⫹ channels are heterometric proteins containing a large, heavily glycosylated 움 subunit and one or two small 웁 subunits. In heart and skeletal muscle, only the 웁1 subunit has been identified, whereas in the brain, the 웁1 and a disulfide-linked 웁2 subunit copurify with the 움 subunit. Eight different genes (SCN1A–SCN8A) are known so far to encode 움 subunits, most of them being expressed in heart, brain, muscle, and peripheral nerve (84). Among them, SCN5A, located on human chromosome 3p21 (85), is expressed in adult cardiac muscle (2, 3) and in fetal skeletal muscle (skM2), and its gene product is characterized by low tetrodotoxin sensitivity. A single gene on human chromosome 19q13.1, SCN1B, encodes the 웁1 subunit expressed in heart, brain, and skeletal muscle (86–88).

A. Long QT Syndrome In the early 1960s, Romano et al. and Ward independently described families with QT prolongation, syncope, and sudden death, inherited in an autosomal dominant fashion (89, 90). They speculated that the mechanism of sudden death was asystole. In this congenital disorder of long QT syndrome (LQT), the cardiac action potential is prolonged, as clinically evidenced by a prolonged QT interval of the electrocardiogram. Patients are predisposed to syncope and sudden death because of polymorphic ventricular tachycardia or ‘‘torsades de points.’’ Linkage studies have demonstrated genetic heterogeneity for the disease by the identification so far of five loci: LQT1 on chromosome 11p15.5, LQT2 on 7q35-36, LQT4 on 4q25-27, LQT5 on 21q22.1-2, and LQT3 on 3p21-24 (91). While no candidate gene is known for LQT4, mutations in potassium channels have been claimed to cause LQT1, LQT2, and LQT5 (92). LQT3 was shown to cosegregate with polymorphisms within SCN5A, the gene encoding the 움 subunit of the major cardiac sodium channel (93). A study using a technique of site-directed mutagenesis found that the three-residue hydrophobic sequence IFM in positions 1488–1490 in the cytoplasmic III-IV linker is critical for the fast inactivation. Replacement of these amino acids by glutamine (Q) (IFM씮QQQ) abolished inactivation or macroscopic current decay (Fig. 10) (94). Because an initially identified form of the LQT3 mutant, ⌬KPQ, had

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3 defective amino acids that are only 17 amino acids away from the key structure of the channel inactivation particle (IFM), a potential role for the Na⫹ channel abnormality caused by the ⌬KPQ mutation was suggested: modification of inactivation gating (95, 96). This hypothesis was examined for the first time electrophysiologically by Bennett et al., who demonstrated a noninactivating component of the Na⫹ channel current at the single-channel level in Xenopus oocytes injected with mRNA encoding wild-type and ⌬KPQ mutant channels (Fig. 11). The ⌬KPQ and other mutations in LQT3 reduce the stability of inactivation, generating a small noninactivating plateau of inward current during depolarization. This defect causes persistent inward current during action-potential repolarization, prolonging the QT interval and setting the stage for fatal ventricular arrhythmias. Five SCN5A mutations, including the ⌬KPQ mutant (Fig. 1), have been identified so far in DNA from affected members of LQT families: ⌬KPQ (a deletion of residues 1505 to 1507), R1644H (an arginine-to-histidine substitution at position 1644), N1325S (an aspartate-toserine substitution at position 1325), R1623Q (an arginine-to-glutamine substitution at position 1623), and D1790G (an aspartate-to-glycine substitution at position 1790). The conditions are transmitted as autosomal dominant traits in all of these cases except for the R1623Q mutant. The sporadic R1623Q missense mutant was found in 1997 in a Japanese girl, but was not identified in the unaffected biological parents or brother of the patient (97, 98). Studies have revealed in detail a biological phenotype for ⌬KPQ, R1644H, and N1325S by means of single-channel experiments. In the mutations of ⌬KPQ, R1644H, and N1325S, a late phase of tetrodotoxin-sensitive inward current was potentiated. According to a pharmacological evaluation of the three LQT3 mutant Na⫹ channels (⌬KPQ, N1325S, R1644H), and in comparison with the normal human Na⫹ channel (hH1), the inhibition of the LQT3 characteristic late-opening channels by a local anesthetic antiarrhythmic agent (mexiletine) was much greater than the inhibition of the peak current in both wild-type and mutant channels (99). Because a dominant feature of LQT3 channels is their propensity to more frequently reenter the open state(s), it is possible that the mutant channels enter a conformation during the altered gating mode that has a higher affinity for local anesthetics (100). Wang et al. (99) speculated that the charged form of local anesthetics opportunistically interacts and inhibits the late openings. Therefore, those agents that favor open channels could selectively inhibit late openings without greatly modifying the initial peak opening of Na⫹ channels. In these mutations, two types of singlechannel activity were responsible for the late current:

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FIGURE 10 Effects of mutations of a cluster of hydrophobic residues within the III-IV linker on current inactivation. The whole cell Na⫹ current recorded in Xenopus oocytes injected with mRNA encoding either wild-type 움 subunit (A) or mutant 움 subunits a indicated: Phe-1483 (F1483Q) or the contiguous hydrophobic residues Ile-1488, Phe-1489, and Met-1490(IFM-QQQ) were replaced with glutamine. The currents were elicited by step depolarizations from a holding potential of ⫺100 mV to test potenitals of ⫺50 to 10 mV in 10-mV increments (94).

isolated brief openings and long-lasting burst openings (Fig. 12). Dumaine et al. (101) stated that both mechanisms contributed to the phenotype of ⌬KPQ and made it the most severe of those studied, whereas the milder R1644H and N1325S phenotypes only showed increased numbers of isolated openings (Fig. 13). Chandra et al. (102) studied the single-channel properties of the late current and clearly distinguished burst openings from isolated openings in terms of open-time durations (Fig. 14). Whereas mean open times for isolated openings showed a weak voltage dependence, those during the burst openings increased progressively with membrane depolarization, providing insight into the openings that were of the same duration as the open times of the events that make up the early transient current. It is therefore likely that those isolated openings result from an increase in the reversibility of fast inactivation (101). Because normal cardiac Na⫹ channels open once at most depolarization potentials, the fast inactivated state could be an absorbing state: O u I. Therefore, the isolated openings in ⌬KPQ mutant Na⫹ channels may recover from the fast inactivated state at a slow but significant rate: O s I. In contrast to isolated openings, the mean open times of the bursts were strongly voltage dependent. In wildtype cardiac Na⫹ channels, Patlak and Ortiz proposed that bursts result from a transient failure of fast inactiva-

tion: Cn s O ⭈ ⭈ ⭈ I (103, 104). However, Bennett et al. (105) proposed that late openings result from a modal shift to a single bursting state. Pharmacological evaluations of ⌬KPQ late openings support the former estimation of two modes of gating, demonstrating that local anesthetic open-channel blockers favorably inhibit burst openings with little affinity for isolated openings (106, 107). Over all, the ⌬KPQ mutation has a wide range of effects on both the activation and the inactivation of the Na⫹ channel, including a transient change of an open state to and from a closed state and an additional open state(s), as proposed earlier. Makita et al. (97) and Kambouris et al. (98) characterized a novel missense mutation (R1623Q) identified in an infant Japanese girl with a severe form of LQT3. The R1623Q mutant differs substantially from other previously studied LQT3 mutations (⌬KPQ, R1644H, N1325S) in terms of the presence of a significantly slower macroscopic inactivation. The former three LQT3 mutations exhibit similar biophysical abnormalities that are characterized by near-normal macroscopic activation and inactivation, but small persistent currents were evident during long depolarizations (less than 5% of the peak current) (105, 108). Moreover, single-channel analyses demonstrated that the mean open time of ⌬KPQ in the transient initial opening was comparable to that of the wild-type Na⫹ channel (105). However,

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the mechanism of impaired inactivation of the R1623Q mutation may be a functional defect in the activation– inactivation coupling of the channel, as has been postulated by Chahine et al. (109) in paramyotonia congenita mutations R1448H/C in Na⫹ channels of the skeletal muscle. The other Na⫹ channel mutant discovered in an extended LQT family (D1790G) has a unique kinetic behavior in the channel: it has no sustained inward current (110). Although the D1790G mutant shows a steady-state inactivation curve shifted by 16 mV in the hyperpolarized direction, this property could not be fully responsible for the prolongation of ventricular excitation or QT prolongation. It was suggested that the D1790G mutant induced changes in 움 and 웁1 interaction, but other unknown mechanisms may be involved in this LQT3 mutant, producing the apparent QT prolongation in the ECG.

B. Brugada Syndrome

FIGURE 11 Single-channel recordings from Na⫹ channels in insideout membrane patches excised from Xenopus oocytes injected with either wild-type (WT) or ⌬KPQ mutant Na⫹ channel mRNA. Selected recordings of unitary WT (a) and ⌬KPQ channel currents (b) in response to a 50-msec depolarization pulse to 0 mV from a holding potential of ⫺120 mV. Records during 200-msec voltage steps from WT (c) and ⌬KPQ channels (d). Ensemble average of WT (e) and ⌬KPQ channels (f). The WT channel opens only once and very briefly at the beginning of the depolarization pulse, whereas the ⌬KPQ channel occasionally exhibited multiple openings during the voltage pulse, giving rise to a sustained inward current, as shown in the ensemble average (105).

the R1623Q mutant exhibited both a normal gating mode and a noninactivating burst mode; the mean open time of the R1623Q mutant was approximately three times longer than the wild type as well as the ⌬KPQ mutant (105). Because the R1623Q and R1644H mutations are located in the S4 segment of domain IV of the Na⫹ channel, and their functional disturbances are mainly in macroscopic inactivation but not in activation,

The Brugada syndrome was originally described by Pedro and Josep Brugada as ‘‘right bundle branch block and persistent ST-segment elevation in V1-V3’’ in patients with no apparent structural heart disease (111, 112). According to recent consensus on the disease, the Brugada syndrome is characterized by (1) a peculiar (coved or saddle-back shape) ST segment elevation in the right precordial leads often accompanied by apparent conduction block in the right ventricle, (2) a structurally normal heart, and (3) a propensity for life-threatening ventricular tachyarrhythmias (113, 114). The first gene to be linked to the Brugada syndrome was reported by Chen et al. (115), who found mutations in the cardiac Na⫹ channel gene, SCN5A, to be responsible. The mutations were at sites other than those known to contribute to the LQT3 (115). This finding provides direct evidence in support of the hypothesis that the Brugada syndrome is a primary electrical disease. This finding is in accord with the demonstration that inhibition of the Na⫹ channel with a class Ic antiarrhythmic agent could induce ST segment elevation in isolated tissue preparations under certain conditions (116). Biophysical analysis of the two missenses in SCN5A of Brugada patients showed a shift in the voltage dependence of steady-state inactivation toward more positive potentials, which is associated with a 25–30% acceleration in recovery time from inactivation at potentials near ⫺80 mV. The gene defects caused either an acceleration of the recovery of the Na⫹ channel (missense mutation) or a nonfunctional Na⫹ channel (insertion and deletions) (115). Therefore, it is likely that a reduction in the number of functional Na⫹ channels promotes the development of reentrant arrhythmias in Brugada

FIGURE 12 Single Na⫹ channel current recorded from cells expressing wild-type (A) and ⌬KPQ mutant Na⫹ channels (B). Currents elicited duirng 10 consecutive depolarizing pulses are shown; the averaged current is shown in the lowest trace. The dashed line indicates the zero current level in the averaged current traces. Onsets of depolarization are marked. Membrane currents were obtained in the cell-attached configuration with 200-msec depolarization pulses from ⫺100 to ⫺40 mV. Note that isolated late openings and burst openings are occasionally shown in the ⌬KPQ mutant (102).

FIGURE 13 The relative amount of late current (A) and the occurrence of traces containing burst (B) and isolated openings (C) in wild-type (WT), R1644H, N1325S, and ⌬KPQ mutant channels. The average persistent current was normalized by dividing by the peak current in the 0- to 25-msec range after the start to the pulse and is expressed as a percentage. A burst index (B) was calculated from the ratio of traces containing bursts to the total number of traces. Traces containing isolated openings were identified by the criterion that an isolated opening was a single open event separated from its neighbors by a closed interval longer than 0.6 msec (101).

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FIGURE 14 Voltage dependency of mean open times of isolated openings (filled circles) and burst openings (open circles) in the ⌬KPQ mutant channel (102).

patients. However, the mechanism responsible for the ST segment elevation and the genesis of VT/VF in this syndrome are not fully understood. Available data at least indicate that Na⫹ channel blockers can be used to unmask the Brugada syndrome and should be avoided by Brugada patients. Importantly, other Brugada patients were found not to be linked to SCN5A, suggesting a genetic heterogeneity of the Brugada syndrome.

V. SUMMARY This chapter presented the function of the voltagegated Na⫹ channel and its metabolic regulation, at concentrated on modulation and defects in molecular function. Studies of the cardiac Na⫹ channel at the molecular level utilizing the patch-clamp technique and a recombinant expression system shed light on our understanding of how the Na⫹ channel behaves in terms of molecular function in physiological and pathophysiological situations. The discovery of the Na⫹ channel gene defects that cause LQT and Brugada syndromes represents a major advancement in the field. Clearly, the powerful combination of molecular genetics with detailed studies of the structure and function of recombinant Na⫹ channels not only will continue to provide insight into the mechanistic factors that cause the clinical phenotypes of many inherited diseases related to the Na⫹ channel,

but also offers the possibility of developing specific therapeutic approaches to disease management that are based on the specific functional properties of the Na⫹ channel in physiological and pathophysiological conditions.

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13 Voltage-Dependent Calcium Channels ´ AND FRANZ HOFMANN LUBICA LACINOVA Institut fu¨r Pharmakologie und Toxikologie Technische Universita¨t Mu¨nchen D-80802 Mu¨nchen, Germany

concentrates mainly on the L-type 움1C calcium channel with some introduction to the LVA channels.

I. INTRODUCTION Calcium channels are membrane-spanning proteins that regulate the intracellular concentration of calcium ions (Ca2⫹). After entering the cell, Ca2⫹ activates specific calcium receptor proteins, e.g., calmodulin, troponin-C, or calcium-activated calcium, potassium, and chloride channels. In the healthy heart, voltageactivated calcium channels are essential for the generation of normal cardiac rythm, for the propagation of excitation through the atrioventricular node, and for contraction in atrial and ventricular myocytes. In diseased myocardium, calcium channels can contribute to abnormal impulse generation and cardiac arrhythmias. In blood vessels, they provide calcium, which controls smooth muscle contraction and vascular tone. Two types of voltage-activated calcium channels have been identified in cardiac and vascular smooth muscle by electrophysiological properties and cDNA cloning. These channels were classified according to the membrane potential at which they are activated as lowvoltage activated (LVA) and high-voltage activated (HVA) calcium channels (Fig. 1). Molecular cloning has identified three members of the LVA channels encoded by the 움1G , 움1H , and 움1I subunit and two distinct classes of HVA channels. One class of HVA channels belongs to the L (long-lasting)-type calcium channels encoded by the 움1C , 움1D , 움1F , and 움1S subunit, whereas the other class contains the neuronal P (Purkinje)-type, N (neither L nor T channel)-type, and R (remaining)-type calcium channels encoded by the 움1A , 움1B , and 움1E subunit (for further details, see Hofmann et al., 1999). This chapter

Heart Physiology and Pathophysiology, Fourth Edition

II. HIGH VOLTAGE-ACTIVATED CALCIUM CHANNELS A. Cardiac L-Type Calcium Channel The most abundant HVA Ca2⫹ channel of the heart and vascular smooth muscle is the L-type 움1C calcium channel, which provides an essential part of the Ca2⫹ necessary for contraction. In addition, the L-type Ca2⫹ current contributes to the plateau of the cardiac action potential. Under physiological conditions, the threshold membrane potential for the activation of macroscopic L-type Ca2⫹ current is above ⫺40 mV with the maximum current around 10 mV. Its unitary conductance is 25 pS in the presence of 100 mM Ba2⫹ (for review, see McDonald et al., 1994). The relative high unitary conductance, together with the long duration of the current, increases the subsarcolemmal Ca2⫹ from submicromolar to above micromolar concentrations, which are necessary to trigger Ca2⫹ release from the sarcoplasmic reticulum. To prevent Ca2⫹ overload, these channels are inactivated by the inflowing Ca2⫹ and by the depolarized membrane potential (Fig. 2). Pharmacologically, the L-type Ca2⫹ current is blocked by three classes of organic channels blockers: dihydropyridines (DHPs), phenylalkylamines (PAAs), and benzothiazepines (BTZs). The cardiac L-type calcium channel is the product of four genes that code for the 움1 subunit and the auxilliary

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FIGURE 1 Phylogenetic tree of the family of voltage-activated calcium channels. Comparison of the 움1 subunits was based on the primary structure alignment of the membrane-spanning regions. The matching percentage was calculated using the program CLUSTAL. HVA and LVA are acronyms for high and low voltage-activated calcium channels, respectively. New names for the 움1 subunits as recommended by the Nomenclature Committee of the International Union of Pharmacology are given in parentheses.

움2웃, 웁, and 웂 subunit. The prominent features of the calcium channel complex can be assigned to the 움1C subunit. The 움1C subunit contains the ion-conducting pore, the ion selectivity filter, the voltage sensor, and the interaction sites for the auxiliary 웁 and 움2웃 subunits, for calmodulin, and for the calcium channel blockers and activators. The cardiac 움1C subunit, which has a predicted molecular mass of 242,500 Da, belongs to the dihydropyridine (DHP)-sensitive L-type Ca2⫹ channels. The biophysical and pharmacological properties of the cardiac 웁1C subunit are modified by the auxiliary cardiac 웁2a-c , 움2웃-1, or 움2웃-2 and eventually by 웂 subunits (Fig. 3). A second HVA Ca2⫹ channel detected in heart is the non-L-type 움1E channel, which is widely distributed in the brain and has been identified in cardiac endocrine cells. This channel is not blocked by organic channel blockers. The cardiac location of the 움1E channel suggests that non-L-type calcium currents may be involved in the secretion of atrionatriuretic and other cardiovascular active peptides.

five to six positively charged amino acids (arginine or lysine), which sense the membrane potential. At positive membrane potential it moves outward and thereby initiates channel opening. The pore region (P region) is formed by the linker connecting the S5 and S6 segments in repeats I to IV. These linkers contribute to the outer vestibule of the channel pore and span the outer half of the membrane. In analogy to the recently obtained crystal structure of the Streptomyces lividans potassium channel (Doyle et al., 1998), the calcium channel pore can be envisioned to have the structure of an inverted teepee with the vertex inside the cell. The helices of the four S6 segments would form the poles of this teepee, which are widely separated near the outer membrane surface and converge toward a narrow zone at the inner surface. This outer structure would stabilize an inner ring formed by the four P regions, which control the speed of permeation and the ion selectivity. The ion selectivity of the calcium channel is determined by the four glutamic acid residues E413, E731, E1140, and E1441 (amino acid numbering is according to the 움1C-b sequence, Biel et al., 1990) in the P region of repeats I, II, III, and IV. Equivalent glutamates are present in all HVA calcium channels. LVA channels have aspartates instead of glutamates in the P region of repeats III and IV (Fig. 4). This difference may be the cause of their distinct ion selectivity. The four glutamates form a single, high-affinity Ca2⫹-binding site within the pore of the HVA channels. The glutamic acid residues of each repeat contribute differently to the Ca2⫹ affinity, selectivity, and speed of permeation. Mutation of E1140 in repeat III has a much greater effect on ion selectivity and permeation than comparable mutations in the other three repeats. Comparison between the

B. Subunits of the L-Type Calcium Channel 1. ␣1C Subunit The human gene for the 움1C subunit is localized to the distal region of chromosome 12p13. The gene spans about 150 kb and is composed of 44 invariant and over 6 alternative exons. The primary structure of the 움1 subunit shows four repeats with six transmembrane segments (Fig. 4). The S4 segment of each repeat contains

FIGURE 2 Comparison of L-type and T-type calcium and barium currents. The L-type current was measured in a HEK cell transfected with 움1C , cardiac 웁2a , and 움2웃-1 subunits. The T-type current was measured in a HEK cell transfected with the 움1G subunit. Barium (20 mM) and calcium (20 mM) currents were determined in the same cell by alternative perfusion of the extracellular ion solution. For better comparison, the amplitudes were normalized. The lower part shows the voltage protocol.

FIGURE 3 Composition of a L-type calcium channel. Extracellular and intracellular spaces are above and below the black lines, respectively. Roman numbers above the 움1 subunit indicate the homologous repeats I, II, III, and IV of this subunit. S or S-S stands for a sulfhydryl bond between the 움2 and the 웃 proteins of the 움2웃 subunit. Rods indicate 움 helices.

FIGURE 4 Identified structural domains of the 움1 subunit. (Top) Domains of the L-type 움1C subunit. (Bottom) Domains of the T-type 움1G,H,I subunit. I, II, III, and IV, respective repeats of the 움1 subunit. E, glutamate; D, aspartate; P, phosphorylation site; 웁, 웁 subunit interaction site; CaM, calmodulin interaction site; e-c coupling, the II-III loop is responsible for the electrochemical coupling.

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skeletal muscle 움1S and the cardiac 움1C L-type calcium channel suggested that the higher unitary current of the cardiac 움1C channel is caused by the higher negative net charge (⫺5) of the cardiac IS5-IS6 linker than that of the skeletal muscle homologue (⫺2). It is plausible that the more negatively charged vestibule of the cardiac than skeletal muscle channel increases the conduction by electrostatic attraction of Ca2⫹ ions to the channel pore. Glutamates of the P region also play a role in the reaction of the cell to extracellular acidification. A decreased extracellular pH, which occurs during episodes of ischemia, inhibits the L-type Ca2⫹ current strongly. Analysis of the mutated 움1C subunit localized the proposed single H⫹ site to the glutamates of the pore region. Controversial data have been published suggesting that H⫹ binding requires either only E1140 in repeat III or E413 and E1140 in repeats I and III. The two glutamate model may explain better the unusual high pKa (pH ⬎ 8) of the protonated site than the single glutamate model. The interpretation of these results is further complicated by the observation that the removal of protons increases L-type current only when the 움1C subunit is expressed together with the cardiac 웁2a subunit (for references, see Hofmann et al., 1999). Several properties of the 움1C subunit, e.g., voltage dependence, kinetics, and magnitude of the calcium channel current, are modulated by coexpression of a 웁 subunit. The interaction site of the 웁 subunit with the 움1 subunit was located at the cytoplasmic linker between domain I and II of the 움1 subunit (Fig. 4). A detailed analysis of different 움1 subunits revealed that a highly conserved sequence motif, called AID for alpha subunit interaction domain, is responsible for this specific interaction, i.e., 428QQ-E–L-GY–WI—E445 (amino acid numbering is according to the 움1C-b sequence), positioned 24 amino acids from the IS6 transmembrane domain in each 움1 subunit. Further mutations showed that only the sequence ⫺437Y–WI441- is essential for highaffinity binding of the 웁 subunits. Mutation of the tyrosine to a serine (-Y–WI- to -S–WI-) reduced the affinity of the AID for 웁 subunits dramatically. This mutation abolishes the stimulation of peak currents, the change in the inactivation kinetics, and the voltage dependence of activation by the 웁 subunit (reviewed in Walker and De Waard, 1998). 2. ␤ Subunit 웁 subunits are intracellularly located proteins ranging from 50 to 72 kDa (Fig. 3). Four genes—웁1 , 웁2 , 웁3 , and 웁4 —have been identified that give rise to several splice variants. The 웁1 subunit is expressed in skeletal muscle, brain, and spleen. The 웁2 gene is expressed abundantly in heart and to a lower degree in aorta, trachea, lung,

and brain, whereas 웁3-specific mRNA is detectable in brain and different smooth muscle tissues. The 웁4 subunit is expressed in brain. A primary structure alignment of 웁 subunits revealed that all share a common central core, whereas their N and C termini and a part of the central region differ significantly (for references, see Hofmann et al., 1999) Coexpression of a 웁 subunit with various 움1 subunits increases peak current most likely by increasing the number of functional surface membrane channels and by facilitating opening of the channel pore. With the exception of rat brain 웁2a , which is palmitoylated at the N-terminal cysteines 3 and 4, all other 웁 subunits accelerate channel activation and inactivation and shift the steady-state inactivation curve to hyperpolarized membrane voltages. The differences in reported effects most likely depend on the particular combination of both subunits and splice variants. These modulatory effects are the consequence of conformational changes in the quarternary structure resulting from the specific interaction of subunit surfaces. In contrast to neuronal calcium channels, which associate apparently with different 웁 subunits, the cardiac L-type calcium channel is associated exclusively with the cardiac 웁2a subunit. Because all four 웁 subunits can modulate the kinetics and voltage dependence of the 움1 subunit and bind to the AID, it was likely that 웁 subunits contain a conserved motif, which binds to AIDs. A 30 amino acid domain of the 웁 subunit (amino acid 215–245 of 웁1b) is sufficient to induce all the modulatory effects of this subunit. This sequence stretch is located at the amino terminus of the second region of high conservation among all four 웁 subunits. Modifications in this region changed or abolished the stimulation of calcium currents by the 웁 subunit and the binding to the 움1 subunit. 3. ␣2␦ Subunit The 움2웃 subunit is a highly glycosylated membrane protein of 125 kDa. Three genes coding for the 움2웃-1, 움2웃-2, and 움2웃-3 subunit have been identified that share between 30 and 50% identical amino acids. The 움2웃 protein is posttranslationally cleaved to yield disulfidelinked 움2 and 웃 proteins (for review, see Walker and De Waard, 1998). The 웃 part anchors the 움2 protein to the 움1 subunit via a single transmembrane segment, whereas the 움2 protein is localized extracellularly (Fig. 3). Northern blot analysis indicates that 움2웃-3 is expressed exclusively in brain, whereas 움2웃-2 is found in several tissues, including heart, and 움2웃-1 is expressed ubiquitously. Extensive splicing of the 움2웃-1 subunit results in at least five different isoforms, which are expressed in a tissue-specific manner. Generally, coexpression of the 움2웃 subunit with 움1 and 웁 subunits shifts the

13. Voltage-Dependent Calcium Channels

voltage dependence of channel activation and inactivation in a hyperpolarizing direction, accelerates the kinetics of current activation and inactivation, and increases the current amplitude. The increase in current density can be partly accounted for by the improved targeting of the expressed 움1 subunit to the cell membrane. Effects of the coexpression of the 움2웃 subunit on time course and/or voltage dependence of current activation and inactivation also suggest a specific modulation of channel gating. At present it is not clear if the cardiac and vascular smooth muscle 움1C subunit is complexed with the 움2웃-1 or 움2웃-2 protein (for references, see Hofmann et al., 1999). 4. ␥ Subunit Five genes code for the 웂 subunit. The 웂1 subunit is an integral membrane protein consisting of 222 amino acids with a predicted molecular mass of 25 kDa, which is expressed exclusively in skeletal muscle and regulates the skeletal muscle 움1S L-type calcium channel. The 웂2 subunit, which has 25% identity with 웂1 , has been identified in brain (Letts et al. 1998). The 웂3 and 웂4 subunits are highly homologous to the 웂2 subunit and are expressed in different brain regions. The 웂5 sequence has 25% identity with the 웂1 and 웂2 subunits and is present in muscular and other nonneuronal tissues. The human 웂1 and 웂2 subunits are encoded on chromosome 17q23 and 22q12-13, respectively. Hydrophobicity analysis revealed the existence of four putative transmembrane helices with intracellular located amino and carboxy termini (Fig. 3). The presence of two extracellular potential N-glycosylation sites is consistent with the observed strong glycosylation of these subunits. Coexpression of a 웂 subunit together with 움1 , 움2웃, and 웁 subunits shifts the steady-state inactivation curve to hyperpolarized membrane potentials. The 웂5 subunit could be part of the cardiac L-type or T-type calcium channel.

III. REGULATION OF L-TYPE CALCIUM CHANNELS BY VOLTAGE, CALCIUM, AND CHANNEL BLOCKERS

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of the S4 segment of each repeat of the human skeletal muscle sodium channel (Cha et al., 1999). Fluorescence changes indicated that the S4 segments of repeat I and II move during channel activation, whereas the S4 segments of repeat III and IV move during voltagedependent inactivation of the channel. 1. Voltage-Dependent Activation A role of the charged residues in the S4 segments in L-type calcium channel gating was demonstrated by mutating individual S4 arginines. Mutations in repeats I and III of a skeletal/cardiac 움1 chimera affected the midpoint and time constant of activation, whereas those of repeats II and IV were without effect. In addition to the S4 segment of repeat I, other sequences of repeat I contribute to the speed of channel activation. The fivefold difference in the speed of activation between skeletal 움1S (slow) and cardiac 움1C (fast) L-type calcium channels is affected by the IS3 segment and the linker between IS3 and IS4 (Fig. 4) and by the sequence between IIIS5 and IVS6. The speed of activation is modulated further by 웁 and 움2웃 subunits (for references, see Hofmann et al., 1999). 2. Voltage-Dependent Inactivation L-type calcium channels show two types of inactivation: slow and fast. Slow inactivation is voltage dependent, whereas fast inactivation is caused by the permeating calcium ion. The kinetics of voltage-dependent/slow inactivation, which is common to all HVA calcium channels, differs considerably among the various types of calcium channels and determines the amount of calcium entering during electrical activity. The IS6 segment and its flanking regions are critical for voltage-dependent inactivation of the 움1C channel (Fig. 4). In addition, inactivation of the 움1C channel is controlled by amino acids located in the intracellular carboxy terminus. Removal of the 움1C carboxy-terminal sequence beyond amino acid 1733 increased the current without increasing the charge moved or the density of dihydropyridine (DHP) binding sites.

A. Regulation by Voltage

B. Calcium-Dependent Inactivation

Voltage-activated calcium channels—as implied already in their name—are regulated by a change in the membrane potential. Together with voltage-gated K⫹ and Na⫹ channels, they belong to the class of so-called S4 channels. Mutational analysis suggested that the positive charges of the S4 segment in each repeat function as a voltage sensor. This concept was strenghtened further by a study using site-directed fluorescent labeling

Calcium-dependent inactivation of calcium channels is a negative biological feedback mechanism by which the increase of the intracellular calcium concentration speeds up channel inactivation and prevents calcium overload of the cell. This inactivation type is important in the cardiac and the smooth muscle and is mediated by the 움1C subunit. Calcium-dependent inactivation requires a carboxy-terminal sequence containing an IQ

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motif (Fig. 5). The underlying mechanism appears to be that calmodulin (CaM) binds to the IQ motif in the carboxy terminus of the 움1C subunit and mediates calcium-dependent inactivation of the channel (Peterson et al., 1999; Zu¨hlke et al., 1999). According to these authors, CaM is tethered by the 움1C subunit in a calcium-independent manner at a site different from the IQ motif. Binding of calcium to CaM then allows interaction of CaM with the IQ motif followed by rapid inactivation of the channel. The basis of the constitutive interaction of CaM with the 움1C channel, as well as the role of the EF hand motif located in the intracellular sequence directly after the last transmembrane segment IVS6, remains to be elucidated.

C. Regulation of Calcium Channels by Antagonist and Agonists The L-type calcium channel is the target of a large number of clinically important drugs. The major classes of these drugs are DHP, phenylalkylamines (PAA), and benzothiazepines (BTZ). Photoaffinity labeling and peptide mapping studies of the skeletal muscle channel revealed that all three classes bind to the transmembrane region of repeat IV of the 움1 subunit with additional sites on repeat III and repeat I for DHPs (reviewed in Striessnig et al., 1998; Zahradnikova´ et al.,

1998). As shown in Fig. 6, three to four amino acids of the IVS6 segment interact with the various calcium channel blockers. Additional amino acids are required for high-affinity interaction of the calcium channel blockers with the resting, open, and inactivated state of the channel. Due to the overlap of the interacting site chains, it is difficult to reconcile new structural data with the previously described allosteric modulation of DHP binding by diltiazem or PAA. 1. The Dihydropyridine-Binding Site DHPs block the native and the expressed cardiac L-type calcium channels, with nanomolar affinity. The affinity for inhibitory DHPs increases at depolarized membrane potential, indicating that the voltage-inactivated channel contains the conformation of the highaffinity binding site. This high-affinity binding site is formed by eight L-type calcium channel-specific amino acids and two conserved amino acids in repeats III and IV (Fig. 6): Thr1061 and Gln1065 of IIIS5, Ile1175, Ile1178, Met1183, and the conserved Tyr1174 of IIIS6 and Tyr1485, Met1486, Ile1493, and the conserved Asn1494 of IVS6 (amino acid numbering is according to the 움1C-b sequence). The largest effects are observed with the mutation of Thr1061 to Tyr, which virtually abolishes channel block. Mutation of the other amino

FIGURE 5 Model of calcium-dependent inactivation of the L-type 움1C channel. (Top) Current traces of an expressed 움1C channel with barium or calcium as charge carrier. The amplitude has been normalized. (Bottom, from left to right) Closed, open, and calciuminactivated channels. The location of the EF and IQ motif and the potential (?) binding sites for calmodulin (CaM) are shown.

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FIGURE 6 Calcium channel blocker binding sites. Amino acids interacting with dihydropyridines (DHP; black circles or rods), phenylalkylamines (PAA, open squares), dihydropyridines and phenylalkylamines (DHP⫹PAA, gray circles or rods), phenylalkylamines and benzothiazepines (PAA⫹BTZ, dashed squares), and all three blocker types (DHP⫹PAA⫹BTZ, dashed circle or rod) are shown. Amino acids conserved between DHPsensitive, L-type calcium channels and DHP-insensitive, non-L-type calcium channels are marked by an arrowhead. The IS6 segment contributes significantly to the high-affinity binding of dihydropyridines to the smooth muscle L-type calcium channel.

acids increased the IC50 value for isradipine 3- to 200fold in a partially additive manner. High-affinity binding of DHPs to the native channel requires the coordination of Ca2⫹ to the glutamates of pore region III and IV, presumably because coordination of Ca2⫹ by these glutamates is required for an optimal conformation of the high-affinity DHP-binding site. Mutation of the highaffinity site showed that the DHP isradipine interacts with the open state of the 움1C channel with low affinity (IC50 앒 1 애M), indicating that DHPs can interact with multiple structures of the 움1C subunit, including the pore region. Less amino acids are required to abolish the stimulatory effect of the DHP agonists Bay K 8644 or (⫹)S-202-791: Thr1061 in IIIS5, Tyr1174 in IIIS6, and Tyr1485, Met1486 in IVS6 (for references, see Hofmann et al., 1999). The successful reconstruction of the DHPbinding site in DHP-insensitive 움1A or 움1E channels confirmed this concept. The transfer of nine L-type specific nonconserved amino acids (see earlier discussion) introduced a high-affinity block by the DHP antagonist isradipine and allowed stimulation of these channels by the DHP agonist Bay K 8644. The reconstructed DHP site interacted stereospecifically with DHPs but failed to

transfer the voltage/inactivation-dependent increase in affinity. 2. The Phenylalkylamine- and Benzothiazepine-Binding Site Phenylalkylamines such as verapamil, gallopamil, or devapamil block the L-type calcium current in an usedependent manner from the intracellular side of the membrane. In contrast to PAAs, benzothiazepines access the channel from the extracellular side. It has been shown that the PAA devapamil and the 1,4-BTZ semotiadil labeled a short sequence of the IVS6 segment. Mutation of the L-type calcium channel-specific IVS6 amino acids Tyr1485, Ala1489, and Ile1492 decreased the affinity for PAAs and BTZs (Fig. 6). Highaffinity interaction of PAAs required additional amino acids located at the IIIS6 segment (the L-type calcium channel-specific Ile1175 and the conserved Tyr1174, Phe1186, and Val1187) and the two glutamates (Glu1140 and Glu1441) in the pore region of repeats III and IV. Mutational analysis also identified components of the state-dependent block of the calcium channel by PAAs and BTZ. In addition to the three amino acids

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Tyr1485, Ala1489, and Ile1492 in IVS6, amino acids in the IVS5 segment proximal to the cytoplasmic mouth of the channel (Ile1392, Val1393, Met1394, Leu1395, Phe1396) and close to the extracellular mouth of the channel (Val1401, Ile1402, Gln1405, Val1406) participate in a state-dependent block of the channel by PAAs and BTZs, respectively (Motoike et al., 1999). These amino acids probably are not involved directly in drug binding, but guard the access path to the receptor site.

pressing the 움1C-a and cardiac 웁2a subunits together with the 움2웃-1 or 움2웃-3 subunit, in line with the known effect of the 움2웃 subunit on the gating of the channel. These results demonstrate clearly that facilitation of the cardiac L-type current can be observed with channels that do not contain the established cAMP kinase phosphorylation site at Ser1928. Further experiments will be required to solve the mechanism underlying cAMPdependent stimulation of the cardiac L-type calcium current.

IV. HORMONAL REGULATION OF THE CARDIAC CALCIUM CHANNEL

B. Modulation by Protein Kinase C-Dependent Phosphorylation

A. Modulation by cAMP-Dependent Phosphorylation

L-type calcium channels are tightly regulated by hormonal and neuronal signals. Protein kinase C (PKC) is one such regulator, which increases cardiac, smooth muscle, and neuronal L-type current by an increase in the open probability of the channel. The response to PKC activators is usually biphasic, with an increase followed by a later decrease. The biphasic response to PKC stimulators was fully reconstituted when the 움1C-a subunit was expressed in Xenopus oocytes. Only a decrease in current was observed with the human 움1C-c splice from, which has the amino terminus of the 움1C-b subunit (Fig. 7), suggesting that PKC-dependent regulation may be controlled by the different amino termini of the two splice variants. This prediction was confirmed (Shistik et al., 1998; see also for further references). Deletion of amino acids 2–46 in the amino terminus of the 움1C-a subunit prevented a PKC-dependent current increase. The effects of PKC activation were larger in the presence of the 움1C-a and 움2웃-1 subunits and were decreased by the coexpression of the cardiac 웁2a subunit. Upregulation of the current was not affected by truncation of the 움1C-a subunit at residue 1665 or mutation of a proposed PKC phosphorylation site at Ser533 in the I-II linker. Upregulation depended on the splice variation of the amino terminus and was not observed with the amino terminus of the 움1C-b subunit. These studies show that, depending on the splice variant, the amino terminus affects channel gating and mediates PKC-dependent upregulation.

The positive inotropic action of catecholamines is mainly caused by an increased calcium influx through L-type calcium channels. Isoproterenol, a 웁-adrenoceptor agonist, enhances the cardiac calcium current threeto seven-fold by cAMP-dependent phosphorylation of the 움1C subunit or an associated protein. cAMPdependent phosphorylation increases the availability of the channel to open upon depolarization. Cardiac calcium channels also show facilitation of the current amplitude during high-frequency stimulation or after strong depolarization. The precise mechanism by which phosphorylation affects the cardiac L-type calcium channel is not fully understood. Partially purified rabbit cardiac sarcolemma contains a large 240-kDa and a small 210-kDa form of the 움1C subunit. The small 210-kDa form is truncated at residue 1870 in the carboxy-terminal sequence. The expressed full-length 240-kDa 움1C subunit is phosphorylated in vivo in CHO cells and HEK 293 cells at Ser1928, which correlates with an increase in current amplitude. According to Gao and co-workers (1997), cAMP kinase-dependent stimulation of barium current required the coexpression of the cAMP kinase anchoring protein AKAP 79, 움1C-a , and neuronal 웁2a subunit in HEK 293 cells. AKAP 79 anchors the kinase at the plasma membrane. These authors reported that phosphorylation of Ser1928 was required for the cAMPdependent stimulation of barium currents. However, a careful reexamination of these results using overexpression of AKAP 79—cloned from HEK 293 cells and identical to that used by Gao and co-workers (1997)— failed to reproduce a cAMP kinase-dependent increase in current amplitude or facilitation of the current by strong depolarization (Dai et al., 1999). In contrast, cAMP-independent facilitation was observed when 움1C-a and cardiac 웁2a , or 움1C-a truncated at residue 1733 were used. Prepulse facilitation was prevented by ex-

V. THE VASCULAR SMOOTH MUSCLE L-TYPE CALCIUM CHANNEL The cardiac and the vascular smooth muscle L-type calcium channels are splice variants of the 움1C gene. The 움1 subunit of the cardiac (움1C-a ; Mikami et al., 1989) and smooth muscle (움1C-b ; Biel et al. 1990) calcium channels differs only at four sites (Fig. 7). The biophysical and pharmacological properties of the vascular smooth

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FIGURE 7 Structural differences between cardiac and vascular smooth muscle L-type calcium channels. (Top) The suggested structure of the 움1C subunit with the four different splice sites (a, b, c, and d) marked by dashed blocks. (Bottom) Amino acids of the splice variants 움1C-a (cardiac) and 움1C-b (smooth muscle). Identical amino acids are blocked. Gaps are indicated by dashes.

muscle 움1C-b subunit are modified by the auxiliary 웁3 , 움2웃-1, or 움2웃-2 and eventually by a 웂 subunit. In addition, molecular analysis showed that the alternatively spliced exon 8, which codes for the IS6 segment, is expressed differentially in cardiac and vascular smooth muscle and is responsible in part for the different DHP sensitivity of the cardiac and vascular smooth muscle L-type currents (Welling et al. 1997). The cardiac 움1C-a channel, which contains the segment IS6a, is blocked at higher DHP concentrations than the vascular smooth muscle 움1C-b channel, which contains the segment IS6b (Fig. 6). The higher affinity of the 움1C-b channel is caused by differences in the DHP-binding site rather than by a difference in inactivation kinetics of the two splice variants. Transfer of four amino acids of the IS6 segment from the 움1C-b sequence to the 움1E channel containing the reconstructed DHP site (see earlier discussion) increased the affinity of the chimeric 움1E channel and proved that the IS6 segment contributes directly to the DHP-binding pocket. The cardiac and the vascular smooth muscle L-type calcium channels are regulated differently by hormones. The cardiac channel is regulated by cAMP-dependent phosphorylation, whereas the same regulation has not been observed in vascular smooth muscle. This different regulation by cAMP kinase suggests that cAMP kinasedependent phosphorylation of the 움1C subunit itself cannot be the only mechanism underlying 웁-adrenergic

regulation of the cardiac L-type calcium channel. As the cardiac channel, the smooth muscle channel may be regulated by protein kinase C. PKC only decreased the current of an 움1C channel containing the amino terminus of the smooth muscle 움1C-b channel, suggesting that PKC-dependent regulation is controlled by the different amino termini (see earlier discussion). The significance of the other two splice sites is unclear. The insert observed in the I-II loop of the 움1C-b subunit may affect the binding of additional proteins, possibly the 웁웂 subunit of G-proteins, which does not bind to the cardiac 움1C-a subunit.

VI. LOW VOLTAGE-ACTIVATED CALCIUM CHANNELS A. The T-Type Calcium Channel Low-voltage activated calcium channels are expressed predominantly in the cardiac atria and carry a tiny and transient (T-type) current. T-type channels may contribute to the generation of the action potential in the sinus node, to cell depolarization, and support the propagation of the atrioventricular calcium action potential. These currents were also identified in vascular smooth muscle cells. Their unitary conductance is threefold smaller than that of the L-type Ca2⫹ channel being

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8 pS with 100 mM Ba2⫹ as a charge carrier. The transient character of the current is caused by fast inactivation (Fig. 2). The threshold membrane potential for the activation of macroscopic T-type Ca2⫹ current is around ⫺60 mV and the maximal current amplitude is reached around ⫺20 mV. The identity of the 움1 subunit and the subunit composition of the cardiac and vascular smooth muscle T-type channel are unclear at present. Three 움1 subunits (움1G , 움1H , and 움1I) have been identified (Fig. 1) that induce large T-type current in the absence of additional subunits after expression in Xenopus oocytes and in HEK cells (Cribbs et al., 1998; Klugbauer et al., 1999). Northern analysis suggests that the 움1I protein is expressed in brain, the 움1G protein predominantly in brain and less abundantly in heart, and the 움1H protein abundantly in heart, kidney, and liver (Cribbs et al., 1998). Lowvoltage activated channels may be composed of a single 움1 subunit protein that contains the voltage sensor, the selectivity filter, the ion-conducting pore, and binding sites for the T-type channel blockers mibefradil and kurtoxin. Elimination of the four known 웁 subunits by transfection of neuronal cells with antisense oligonucleotides or overexpression of the neuronal 웁2a subunit did not affect the size or voltage dependence of native T-type current. Coexpression of the 움1G subunit with the 움2웃-1 or 움2웃-3 subunit did not or only minimally modulated the T-type current. Some expression studies support the notion that the 웂4 and 웂5 subunits can interact with the 움1G channel. These results do not rule out that the native T-type calcium channels are composed of an 움1 subunit and so far unidentified auxiliary subunits.

B. Regulation of T-Type Calcium Channels by Voltage The kinetics of T-type calcium channel activation, inactivation, and deactivation is a property of the 움1 subunit and lacks modulation by the currently known 웁, 움2웃, and 웂 subunits. The gating of T-type calcium channels differs in most aspects from L-type channels. In comparison with L-type channels, the voltage dependence of T channel activation is shifted by 20–30 mV in hyperpolarized direction, inactivation is rapid and not intrinsically voltage dependent, deactivation is slow, and unitary conductance is threefold lower compared to that of the L channel. Unlike any of the HVA calcium channels, T-type channels inactivate faster with Ba2⫹ than Ca2⫹ as the charge carrier (Klugbauer et al., 1999). All three identified T-type calcium channels possess charged S4 segments, which do not differ significantly from those of HVA channels. In contrast to HVA channels, T-type channels are not selective for Ba2⫹ over Ca2⫹ and completely lack a Ca2⫹-dependent inactivation

mechanism (Fig. 2). Instead of the highly conserved glutamates, they have glutamates in pore regions I and II but aspartates in pore regions III and IV (Fig. 4). Indirect evidence suggests that aspartates of pore regions III and IV control the relative low unitary conductance. Further structural determinants of the T-type channel regulation remain to be elucidated.

C. Pharmacology of T-Type Calcium Channels Previous studies on the pharmacology of T-type calcium channels were hampered by a lack of cloned members of this channel family. In all tissues, including myocardium, T-type currents are masked to a considerable extent by HVA calcium currents and could be analyzed only after subtraction of the overlapping part of the L-type current using pharmacological and/or biophysical techniques. Published results showed considerable variability, which was attributed to the putative existence of multiple channel types. The molecular diversity of T-type calcium channels was confirmed by the isolation of three genes: 움1G , 움1H , and 움1I . It has been suggested that the 움1H and, to a lesser extent, the 움1G subunits are responsible for cardiac T-type calcium current. Both subunits are relatively insensitive to the common DHP channel blockers and activators. The 움1H current, but not the 움1G current, is blocked by submillimolar concentrations of amlodipine. The calcium channel blocker mibefradil has about a 10-fold higher affinity for T-type than L-type calcium channels and inhibits 움1H and 움1G currents with IC50 values of 1.2 and 0.4 애M, respectively. The recently identified 움-scorpion toxin kurtoxin (Chuang et al., 1999) blocked the 움1G channel with an IC50 of 15 nM by interfering with its activation gate. Ni2⫹ ions inhibited the 움1H and 움1G channels with IC50 values of 10 애M and 1 mM, respectively, disqualifying a Ni2⫹ block as a reliable test for T-type current. The 움1G channel is blocked to a small extent by therapeutical concentrations of ethosuximide and phenytoin.

VII. SUMMARY Calcium ions enter myocardial cells during the action potential through voltage-activated calcium channels. Two classes of voltage-activated calcium channels are present in the heart: high voltage-activated L-type calcium channels and low-voltage activated T-type calcium channels. Genes encoding the principal 움1 subunit of both channel types have been identified. The 움1 subunit containes the pore, major structural determinants for channel activation, inactivation, ion selectivity, and the binding site for channel activators and blockers. Volt-

13. Voltage-Dependent Calcium Channels

age-dependent gating of the 움1C subunit of the L-type calcium channel is modified further by the auxiliary 웁2 , 움2웃-2, and, perhaps, a 웂 subunit. Interaction of the T-type 움1H subunit with modulatory subunits has not been identified yet. The clinically important L-type calcium channel blockers, i.e., dihydropyridines, phenylalkylamines and benzothiazepines, interact with amino acids of the IIIS5, IIIS6, and IVS6 segments (DHPs) or IIIS6 and IVS6 segments (PAAs and BTZs). The high affinity of the vascular smooth muscle L-type 움1C calcium channel for dihydropyridines is caused in part by an alternatively spliced IS6 segment. Amino acids of IIIS6 and IVS5 segments control channel transition into the inactivated state, enabling a use-dependent block by PAAs and BTZs. No clinically relevant high-affinity inhibitors of cardiac T-type calcium channel are available so far. The cardiac but not the vascular smooth muscle L-type 움1C calcium channel is regulated by cAMP- and PKC-dependent phosphorylation. Phosphorylation by these protein kinases increases calcium influx. The mechanism underlying the phosphorylation-dependent increase in available channels is controversial and may be a property of the 움1C subunit, but may be modulated also by other proteins, e.g., the 웁 or 웂 subunit and the cAMP anchoring protein AKAP 79.

Bibliography Biel, M., Ruth, P., Bosse, E., Hullin, R., Stu¨hmer, W., Flockerzi, V., and Hofmann, F. (1990). Primary structure and functional expression of a high voltage activated calcium channel from rabbit lung. FEBS Lett. 269, 409–412. Cha, A., Ruben, P. C., George, A. L., Fujimoto, E., and Bezanilla, F. (1999). Voltage sensors in domains III and IV, but not I and II, are immobilized by Na⫹ channel fast inactivation. Neuron 22, 73–87. Chuang, R. S. I., Jaffe, H., Cribbs, L., Perez-Reyes, E., and Swartz, K. J. (1998). Inhibition of T-type voltage-gated calcium channel by a new scorpion toxin. Nat. Neurosci. 1, 668–674. Cribbs, L. L., Lee, J.-H., J., Satin, J., Zhang, Y., Daud, A., Barclay, J., Williamson, M. P., Fox, M., Rees, M., and Perez-Reyes, E. (1998). Cloning and characterization of 움1H from human heart, a member of the T-type Ca2⫹ channel gene family. Circ. Res. 83, 103–109. Dai, S., Klugbauer, N., Zong, X., Seisenberger, C., and Hofmann, F. (1999). The role of subunit composition on prepulse facilitation of the cardiac L-type calcium channel. FEBS Lett. 442, 70–74.

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Doyle, D. A., Cabral, J. M., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998). The structure of the potassium channel: Molecular basis of K⫹ conduction and selectivity. Science 280, 69–77. Gao, T., Yatani, A., Dell’Acqua, M. L., Sako, H., Green, S. A., Dascal, N., Scott, J. D., and Hosey, M. M. (1997). cAMP-dependent regulation of cardiac L-type Ca2⫹ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19, 185-196. Hofmann, F., Lacinova´, L., and Klugbauer, N. (1999). Voltagedependent calcium channels: From structure to function. Rev. Physiol. Biochem. Pharmacol. 139, 35–87. Klugbauer, N., Marais, E., Lacinova´, L., and Hofmann, F. (1999). A T-type calcium channel from brain. Pflu¨g. Arch. 437, 710– 715. Letts, V. A., Felix, R., Biddlecome, G. H., Arikkath, J., Mahaffey, C. L., Valenzuela, A., Bartlett, F. S., Mori, Y., Campbell, K. P., and Frankel, W. N. (1998). The mouse stargazer gene encodes a neuronal Ca2⫹-channel 웂 subunit. Nat. Gen. 19, 340–347. McDonald, T. F., Pelzer, S., Trautwein, W., and Pelzer, D. P. (1994). Regulation and modulation of calcium channels in cardiac, skeletal and smooth muscle cells. Physiol. Rev. 74, 365–507. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989). Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 340, 230–233. Motoike, H. K., Bodi, I., Nakayama, H., Schwartz, A., and Varadi, G. (1999). A region in IVS5 of the human cardiac L-type calcium channel is required for the use-dependent block by phenylalkylamines and benzothiazepines. J. Biol. Chem. 274, 9409–9420. Peterson, B. Z., DeMaria, C. D., and Yue, D. T. (1999). Calmodulin is the Ca2⫹ sensor for Ca2⫹-dependent inactivation of L-type calcium channels. Neuron 22, 549–558. Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998). Crucial role of N terminus in function of cardiac L-type Ca2⫹ channel and its modulation by protein kinase C. J. Biol. Chem. 273, 17901– 17909. Striessnig, J., Grabner, M., Mitterdorfer, J., Hering, S., Sinneger, M., and Glossmann, H. (1998). Structural basis of drug binding to L type Ca2⫹ channels. Trends Pharmacol. Sci. 19, 108–115. Walker, D., and De Waard, M. (1998). Subunit interaction sites in voltage-dependent Ca2⫹ channels: role in channel function. Trends Neurosci. 21, 148–154. Welling, A., Ludwig, A., Zimmer, S., Klugbauer, N., Flockerzi, V., and Hofmann, F. (1997). Alternatively spliced IS6 segments of the 움1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type calcium channels. Circ. Res. 81, 526–532. Zahradnikova´, A., and Lacinova´, L. (1998). Molecular determinants of the interaction of calcium channels with calcium channel drugs. Exp. Clin. Cardiol. 3, 121–127. Zu¨hlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W., and Reuter, H. (1999). Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399, 159–162.

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14 Voltage-Dependent K⫹ Channels HAROLD C. STRAUSS, MICHAEL J. MORALES, SHIMIN WANG, MULUGU V. BRAHMAJOTHI, and DONALD L. CAMPBELL Department of Physiology and Biophysics University at Buffalo State University of New York Buffalo, New York 14214

II. OVERVIEW OF K⫹ CURRENTS IN CARDIAC MYOCYTES

I. INTRODUCTION Voltage-dependent K⫹ currents play an essential role in repolarization of cardiac myocytes (1–9). Our present understanding of the role played by the different K⫹ currents in cardiac repolarization has been derived from the identification and biophysical characterization of native K⫹ currents in isolated cardiac myocytes using a combination of the patch-clamp and pharmacological techniques applied to enzymatically isolated cardiac myocytes. Biophysical data on the individual K⫹ currents have in turn been used in computational models to determine their role in cardiac repolarization. These studies on myocytes have also been complemented by recent cellular and molecular biological studies. These different approaches have led to significant advances in the field of cardiac electrophysiology, which include demonstration of the following: the role played by the different currents in the repolarization process; the molecular bases of many of the native K⫹ currents; the complex signaling cascades that modulate these currents; the mutations in the genes that encode for voltagedependent K⫹ channels and cause familial long QT syndrome and cardiac arrhythmias; and the remarkable heterogeneity of K⫹ channel function among different regions and myocyte types in the heart. This chapter reviews voltage-gated K⫹ channels and emphasizes selected topics to illustrate some of the important recent advances in the field.

Heart Physiology and Pathophysiology, Fourth Edition

Macroscopic patch clamp studies on atrial and ventricular myocytes have suggested a minimum of five distinct K⫹ currents (Figs. 1 and 2) in these cells. Briefly, these different currents are as follows. 1. The calcium-independent transient outward K⫹ current (Ito1) is prominent in many cardiac ventricular and atrial myocyte types (vide infra) (Fig. 2A). When present, Ito1 is a major contributor to phase 1 and the early part of phase 2 of the action potential (1, 10–15). The magnitude of Ito1 determines the magnitude of repolarization during phase 1 and, as a result, the subsequent contribution of other voltage-gated currents during the remainder of the action potential. Because of its overlap with the L-type Ca2⫹ current, Ito1 can also modulate excitation–contraction coupling in myocytes in addition to its role in early repolarization. An additional calcium-dependent transient outward K⫹ current (Ito2) has also been putatively identified in some studies (1, 10–15). However, concerns about the exact nature of this putative current linger because of the limitations of the voltage-clamp protocols employed and the use of nonselective, frequency- and voltage-dependent blocking agents (e.g., reverse use-dependent block by 4-aminopyridine) (1, 132). 2. In most species, the macroscopic delayed recti-

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gated K⫹ current (IK,ACh.) (30–33). Because this chapter is devoted to voltage-activated K⫹ channels, we will not discuss the properties of these inward rectifiers further (see Chapter 15).

FIGURE 1 A simplified scheme of a myocardial cell is superimposed on a representative transmembrane action potential. The cell is connected to two neighboring cells via gap junction channels (Igj). Contribution of the different inward (INa , ICa,L) and outward currents (Ito . IKur , IKr , IKs , IK1) to the transmembrane action potential is approximated by the arrows. The time course of the different currents is discussed in further detail in text. IKur is enclosed in parentheses because the current has not been detected in human ventricular cells. INa ,Na⫹ current; ICa,L ,L-type Ca2⫹ current; Ito , transient outward K⫹ current; IKur , ultrarapid component of the delayed rectifier K⫹ current; IKr , rapid component of the delayed rectifier K⫹ current; IKs , slow component of the delayed rectifier K⫹ current; IK1 , inward rectifier K⫹ current. Reproduced with permission from Jongsma Current Biology 8, R568–R571 (1998).

fier K⫹ current consists of three components; an ultrarapid (IKur), a rapid (IKr) and a slow (IKs) component (Fig. 2). IKur (Fig. 2B), which is identified readily in atrial myocytes of many mammalian hearts, including humans, begins in phase 1 and contributes to the remainder of the action potential plateau (16–21). While IKur has not been identified in human ventricular myocytes, it has been identified in ventricular myocytes of other species (18, 20, 21). IKr and the slower IKs components (Figs. 2C and 2D) of the delayed rectifier K⫹ current make an increasing contribution throughout phase 2 and the early part of phase 3 (22–26). It should be noted that the designation ‘‘rapid component of the delayed rectifier current’’ is somewhat of a misnomer, as the time constants for IKr activation are relatively slow, being on the order of many tens of milliseconds (26). 3. The inward rectifier K⫹ current (IK1) produces the terminal part of repolarization and is a major determinant of the resting potential in phase 4 in working atrial and ventricular myocytes (25). It is most prominent in ventricular myocytes, relatively less prominent in atrial myocytes, and is essentially absent in primary sinoatrial nodal pacemaker cells (17, 25, 28, 29, 29a, 29b). Two related inward rectifier K⫹ currents are the ligand-gated ATP-sensitive K⫹ current (IK,ATP) and the muscarinic-

In summary, compelling experimental evidence indicates the existence of at least five functionally distinct K⫹ currents in cardiac myocytes isolated from different mammalian species. However, as will be shown later, the establishment of the molecular bases of these distinct K⫹ channel phenotypes has proven to be challenging, as the number of compatible genes that have been identified exceeds the number of functional K⫹ current phenotypes. Furthermore, data clearly indicate that the distribution of these different K⫹ currents between different regions of the heart and between myocyte types is quite heterogeneous, indicating that the molecular correlates may vary markedly between different regions of the heart.

III. VOLTAGE-DEPENDENT K CHANNEL STRUCTURE AND FUNCTION Progress toward understanding the molecular basis of voltage-dependent potassium currents began with the cloning of members of the voltage-dependent Na⫹, Ca2⫹, and K⫹ channel superfamily (34–36). Each voltagegated K⫹ channel consists of a symmetrical arrangement of four 움 subunits, with each subunit consisting of six transmembrane-spanning segments arranged around a central pore (Fig. 3A). Interaction between Kv움 subunits appears to occur between the highly conserved NAB domain in the N terminus of the 움 subunits, just upstream from the first membrane-spanning segment (81,83,84,93). One segment from each monomer, the P loop, located between S5 and S6, folds back into the membrane to partially span it. Each P loop contains a highly conserved glycine–tyrosine–glycine sequence that forms the K⫹ selectivity filter. This sequence forms the narrowest part of the pore and is located near the extracellular surface. The inner pore region consists of amino acids from the interior portion of the sixth membrane-spanning segment (37–39). The highly conserved pore region in voltagedependent K⫹ (Kv) channels is also seen in a two transmembrane-spanning segment pH-dependent K⫹ channel from the bacterium Streptomyces lividans, KcsA (39). This channel has been crystallized, and its structure ˚ resolution) has provided significant new insights (3.2 A into the K⫹ channel pore region. These data are of particular interest as previous studies have suggested that different K⫹ channels behave as multi-ion channels, i.e., multiple K⫹ ions occupy the pore as they transverse

14. Voltage-Dependent K⫹ Channels

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FIGURE 2 Four representative voltage-dependent K⫹ currents obtained from isolated cardiac myocytes. (A) Ito1 in a ferret right ventricular myocyte. HP ⫽ ⫺70 mV, 500-msec clamp pulses from 10 to 100 mV. From Campbell et al., J. Gen. Physiol. 101, 571–601, (1993). (B) IKur in a human atrial myocyte. HP ⫽ ⫺50 mV, 50-msec clamp pulses from ⫺40 to 50 mV. Reprinted with permission from Fedida et al., Circ. Res. 73, 210 (1993). (C) IKr in a neonatal mouse ventricular myocyte. Reproduced with permission from Wang et al., Am. J. Physiol. 270, H2088, (1996). IKr is elicited by depolarizing pulses to potentials ranging between ⫺20 to 40 mV followed by a return to ⫺40 mV. Note that the magnitude of IKr decreases with depolarizations positive to 0 mV. Tail currents are larger than the currents elicited by the depolarizing pulses because of the rapid inactivation that occurs during depolarization. (D) IKs in a guinea pig ventricular myocyte at 35⬚C. Reproduced with permission from Sanguinetti and Jurkiewicz, Am. J. Physiol. 259, H1881 (1990). Currents were elicited by 7.5-sec depolarizing pulses ranging between ⫺20 and 60 mV followed by repolarization to ⫺50 mV. Note the slow activation and deactivation of IKs .

the permeation path in single file. Crystallographic data show that the channel is a tetramer, with four of the second transmembrane 움 helices (S6 equivalents) forming an inverted cone or ‘‘teepee.’’ The net length of the ˚ , whereas its diameter varies from channel pore is 45 A a very narrow selectivity filter (only wide enough to ˚ allow an unhydrated K⫹ ion to pass) to a large (앑10 A wide) cavity isolated appropriately in the middle of the membrane (Fig. 3B). The selectivity filter, which is only ˚ long and resides in a narrow region between the 12 A outer mouth of the pore and the central cavity, is lined by the carbonyl oxygen atoms of the GYG sequence; however, the wall lining the internal pore and cavity is predominantly hydrophobic. Presumably, when the channel opens, K⫹ ions enter the inner mouth of the pore in their hydrated form, pass through the long vesti-

bule to the central cavity, and are stripped of their water molecules to pass through the narrow selectivity region. ˚ The selectivity filter contains two K⫹ ions (앑7.5 A apart); it is believed that this configuration promotes conduction through electrostatic repulsive forces. This repulsion overcomes the strong interaction between ion and carbonyl oxygen atoms, thereby enabling rapid conduction to occur in the setting of high selectivity. Be˚ cavity large neath this narrow selectivity region is a 10-A enough to contain several free water molecules, which subsequently help overcome electrostatic destabilization within the interior of the lipid membrane. Part of the P segment is 움 helical; these four helices point their electronegative carboxy-terminals toward the central cavity and generate a negative field, which helps neutralize the charge of the cations. The relatively large electro-

FIGURE 3 (A) Kv channel membrane topology. N and C termini are intracellular. A hypothetical model of the tetrameric structure as viewed ‘‘looking down’’ from the extracellular surface. The region designated by the heavy curved line around the four S5, S6, and P domains has been modeled to scale from a ‘‘slice’’ (at ˚ diameter. the level of external head groups) through the KcsA channel. Circles represent helices of 11.8 A Unlabeled helices are proposed hypothetical positions of S1–S4. (B) Schematic model of the pore of the KscA ˚ resolution by Doyle et al. (39). Two fully dehydrated ions sit within K⫹ channel crystallized and solved to 3.2 A the ‘‘selectivity filter’’ and another partially or fully hydrated ion sits outside the selectivity filter in the central ˚. cavity or long vestibule. Note that the pore only interacts with fully dehydrated K⫹ ions in its outer 12 A ˚ distance, 앑80% of the transmembrane potential is imposed; the rate-limiting steps for K⫹ flux Over this 12 A through the channel are effectively limited to only a very short distance within the pore. (A) Reproduced with permission from Hong and Miller, J. Gen. Physiol. 115, 51–58, (2000) and (B) reproduced with permission from McCleskey, J. Gen. Physiol. 113, 765–772 (1999).

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static effect of the pore helices (39a) combined with ion self-energy is responsible for the selectivity for monovalent cations in the water-filled cavity. Based on both sequence similarity and conservation of scorpion toxinbinding sites in the pore, these motifs in the bacterial K⫹ channel are also likely to be present in voltagesensitive cardiac K⫹ channels. In contrast to the pore region, there is at present no direct structural information on the voltage-sensitive domains responsible for channel gating. Nonetheless, a general picture has emerged wherein transmembrane segments S1–S4 are believed to form a domain-like voltage-sensing structure. In particular, each voltagegated K⫹ channel (Kv) monomer contains a membranespanning segment (S4) that is enriched with positive charges, usually every third residue in the 움 helix, which serves as part of the voltage sensor of the channel (Fig. 3A). Initial proposals suggested that depolarization caused outward movement of S4. A variety of approaches, including analysis of gating currents, examination of the effects of charge neutralization and reversal in S4 and adjacent S2 and S3 membrane spanning segments, and analysis of changes in fluorescent signals resulting from the movement of attached fluorescent indicators, have been used to refine this model (40–47). These studies have confirmed the role of the charged residues in S4 in channel gating. Additionally, they have shown that neutral amino acids and interactions among S4, S2, and S3 also play an important role in the conformational changes that occur during depolarization. Biophysical studies using cysteine-scanning mutagenesis to determine the extent of movement by S4 have demonstrated that S4 side chain accessibility to sulfhydryl reagents changes markedly during depolarization. Further, the substitution of histidine for a mid-S4 residue enabled investigators to detect a pH-sensitive current during depolarizing voltage-clamp pulses (47). These different sets of experiments indicate that a depolarizing step results in a sufficiently large outward (i.e., toward the extracellular surface) movement of the midpoint of S4 to translocate H⫹ across the membrane. How this outward movement of S4 leads to a conformational change that opens the pore region, permitting K⫹ permeation, is unclear at this point. As stated, no structural data presently exist on the S1–S4 voltage-sensing domain. However, very recent studies agree that the first two transmembrane segments (S1 and S2) have extensive lipid-exposed surfaces and are therefore probably located at the periphery or outer region of the Kv tetrameric complex (47a–47d). Whether the S3 segment is largely lipid exposed or more involved in tertiary interactions among segments is presently unclear (47a–47d). However, these recent studies do support the general notion of a network of charge

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interactions among S2, S3, and S4, i.e., interactions between negative residues that act as countercharges for the numerous basic S4 residues. Such charge interactions had been previously hypothesized to be important in the overall behavior of the voltage-sensing domain. Inactivation of Kv channels can result from two distinct processes referred to as N-type or C-type inactivation (48–52). N- and C-type inactivation are not mutually exclusive mechanisms. In N-type inactivation, the inner mouth of the pore region is occluded by the positively charged stretch of 앑20 amino acids at the start of the N-terminal region (so-called ‘‘ball-andchain’’ model). Generally, N-type inactivation is rapid (48–53). Data supporting this mechanism of inactivation include the following: (i) N-type inactivation is lost after N-terminal deletion and restored by the addition of short peptides derived from the N terminus (49, 52). (ii) Drug (e.g., TEA⫹) binding to the extracellular mouth of the pore does not alter N-type inactivation, whereas drug binding to the intracellular mouth of the pore does (54, 55); similarly, N-type inactivation is unaffected by changes in extracellular [K⫹] (51, 56, 57). (iii) Although the N terminus contains basic residues, its binding domain is near the intracellular channel surface, and therefore the positive charges sense very little of the transmembrane electrical field; consequently, N-type inactivation shows very little to no intrinsic voltage sensitivity (57). (iv) N-type inactivation is insensitive to point mutations at the outer mouth of the channel pore and the outer region of S6, mutations that do affect C-type inactivation (vide infra) (51, 52, 58). In C-type inactivation, which is typically slower than N-type, residues in the C-terminal region of the S5–S6 linker and the N-terminal end of the pore and S6 regions have been shown to be important modulators of the rate of inactivation (48–51). Several lines of evidence suggest the view that a conformational change in the external pore region contributes to C-type inactivation and that inactivation is not due to the effect of a tethered ball in the N- or C-terminus. These data include (i) persistence of C-type inactivation after removal of the N terminus (52); (ii) mutations of residues near the cytoplasmic end of the P loop and nearby region of S6 alter C-type inactivation (37, 58); (iii) binding of extracellular tetraethylammonium (TEA) or K⫹ to the extracellular part of the pore slows or prevents C-type inactivation (54, 58); and (iv) In Shaker K⫹ channels, C-type inactivation changes the external solute accessibility of a limited number of residues near the external mouth of the pore (50). An apparently distinct form of slow inactivation has been identified and called P-type inactivation (53). P-type inactivation and C-type inactivation appear to

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result in two sequential rearrangements around the P region and S4 during the onset of inactivation. The first rearrangement shuts the channel (P type). The second rearrangement stabilizes the closed conformation (C type). The P region is believed to form the outer mouth of the pore as well as the narrowest part. Using voltage-clamp fluorimetry with a fluorophore attached to the N-terminal end of the P region or to that of S4, Loots and Isacoff (53) obtained data that are consistent with two sequential arrangements around the P region and S4 during inactivation. These investigators proposed that both forms of inactivation involve the closure of a gate in the external part of the molecule (53). They further proposed that this gate is first closed in P-type inactivation and that in C-type inactivation a further rearrangement in or around S4 occurs that stabilizes this inactive conformation. Further, this stabilization in part results from the stabilization of S4 in its activated extruded transmembrane position. Despite the differences between N- and C-type inactivation, both processes share certain features. First, the rate of C-type inactivation is also voltage insensitive at potentials for which activation is complete. This suggests that C-type inactivation, like N-type inactivation, is coupled or partially coupled to activation, i.e., that a conformational change resulting from activation is needed to enable inactivation to occur. Second, neutralization of an S4 positively charged residue shifts both N- and C-type inactivation in a parallel fashion, suggesting that both processes require a similar degree of activation to proceed (56). In contrast, the reverse process, recovery from C-type inactivation, is voltage sensitive (56, 59). It has been hypothesized that this voltage sensitivity arises from a putative backward movement of the S4 voltage sensor (51) in a manner similar to that suggested for the ball-and-chain-type model of inactivation (34). Finally, in some Kv channels there appears to be a close coupling between the development of and recovery from inactivation, as interventions that slow C-type inactivation acccelerate its recovery. This relationship implies that interventions that raise the energy barrier for the development of inactivation, and as a result slow inactivation, destabilize the inactive state and accelerate recovery (51).

IV. ION CHANNEL GENES A. K Channel 움 Subunits Most cardiac K⫹ channel 움 subunits are orthologs of the Drosophila family of K⫹ channels. Mammalian cardiac K⫹ channels were cloned using Drosophila K channel probes to screen mammalian cardiac cDNA libraries (60–62). There are many more Kv channel

TABLE I Nomenclature of Ion Channels and Corresponding Genes cDNA

Gene

Kv1

KCNA

Kv2

KCNB

Kv3

KCNC

Kv4

KCND

HERG

KCNH

KvLQT

KCNQ

cDNAs than identified K⫹ currents, which has complicated the task of linking K⫹ channels with native K⫹ currents. Initially, four related subfamilies were cloned: Shaker (Kv1, KCNA), Shab (Kv2, KCNB), Shaw (Kv3, KCNC), and Shal (Kv4, KCND) (63) (Tables I and II). Transcripts from Kv1–Kv4 subfamilies have been detected in murine, rat, ferret, and human heart (64–67). While subfamily groups were originally assigned based on sequence similarity, it has also been established that these groupings also dictate the specificity of association (68, 69). K⫹ channel monomers can assemble with identical members or with other subfamily members to form heteromultimeric channels. For example, in the Kv1 subfamily, Kv1.4 can coassemble with Kv1.1, Kv1.2, and Kv1.5 to form functional heteromultimers (68, 69), but not members of the Kv2, Kv3, or Kv4 families. Related families of cDNAs, Kvs5–9, have been cloned from mammals (66–70), but only Kv5, Kv6, and Kv9.3 are found in heart (70–74). These subunits are electrically silent when expressed alone, but Kv5, Kv6, and Kv9.3 in heart may co-assemble with Kv2 channels and alter their function and expression (70–74). Linkage studies of patients with familial long QT interval syndrome have advanced our understanding of

TABLE II Correspondence of Cardiac Ionic Currents with Channel Subunits and Genes Current

Channel subunit

Gene

Ito-epi

Kv4.2 움 subunit Kv4.3 움 subunit

KCND2 KCND3

Ito-endo

Kv1.4 움 subunit Kv웁1.2 웁 subunit

KCNA4 KCNAB1

IKr

HERG1 움 subunit HERG1B 움 subunit MiRP1

KCNH1 KCNH1 KCNE2

IKs

KvLQT1 움 subunit minK

KCNQ1 KCNE1

IKur

Kv1.5 움 subunit

KCNA5

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voltage-dependent K⫹ currents in the heart, by identifying a novel gene, KvLQT1, and calling attention to one additional distantly related member of the family of voltage-dependent K⫹ channels, HERG (human ether-a`-go-go related gene) (8, 9, 75–80). The phylogenetic relationship between representative members of the K⫹ channel family is indicated in the dendrogram shown in Fig. 4. Kv subfamilies 1 and 4 are related more closely than Kv subfamilies 2, 5, 6, and 3, whereas KvLQT1 and HERG are related more distantly. The high degree of sequence similarity among Kv5.1, Kv6.1, Kv9.3, and Kv2.1 may explain why these three Kv channels inhibit the expression of and modify the gating properties of Kv2.1 channels preferentially (87–90). If these effects are mediated by heteromeric assembly among these different 움 subunit members, then Kv2.1, Kv5.1, Kv6.1, and Kv9.3 channels should be considered part of the Kv2 subfamily (Table III).

B. K⫹ Channel Ancillary Subunits A class of cytoplasmic proteins (웁 subunits) assembles with 움 subunits of the Kv1 subfamily. At least four different 웁 subunit genes have been isolated, (Kv웁1–4). There are the three alternatively spliced forms of Kv웁1, Kv웁1.1–Kv웁1.3, whereas Kv웁2, Kv웁3, and Kv웁4 are apparently unique (81–86). The C-terminal 323 amino acids of the 웁 subunits are highly conserved; their major sequence differences lie in their short N-terminal regions (83). Interaction with Kv1움 subunits appears to occur between the highly conserved NAB domain in the N terminus of the 움 subunit and the C terminus of

FIGURE 4 Cardiac voltage-gated K channels shown as a phylogenetic tree. A simplified dendrogram of voltage-dependent K⫹ channel genes (six transmembrane-spanning segments) described in heart. Protein sequences for each of the human channels were aligned using the ClustalW algorithm [Nucl. Acids Res. 22, 4673 (1994) as implemented in ‘‘Multiple Sequence Alignment’’ (InforMax)]. The phylogenetic tree was then generated using the neighbor-joining method [Saitou and Nei, Mol. Biol. Evol. 4:406 (1987)], also using Multiple Sequence Alignment. Note that the members of the Kv1 family are related more closely to each other than to other family members. Kv1.x and Kv4.x families are related more closely to each other than to members of Kv2, Kv5, and Kv6 families. Members of the HERG and LVLQT1 families are the most distantly related.

the 웁 subunit (83). N termini of Kv웁1 subunits produce inactivation by blocking the inner mouth of the pore region of the Kv움 subunits and can modify both activation and inactivation properties substantially (62, 87, 89) (Table III). C termini can produce significant increases in 움 subunit expression, possibly through a chap-

TABLE III Basis of Heterogeneity of Gene Expression and Function Example (reference) Multiplicity of gene products with similar functional properties Heteromeric assembly of subunits Electrically competent 움 subunits that associate to form channels with different functional properties Association of electrically silent with electrically competent 움 subunits to form channels of different functional properties Association with electrically silent 움 subunits that inhibit expression Coassembly of 움 and ancillary subunits Change in protein expression Change in function

Kv4.2 and Kv4.3 (2,128) Kv1.4 ⫹ Kv1.1, Kv1.2 or Kv1.5 (69) HERG1 ⫹ HERG1B (137) Kv2 ⫹ Kv5, Kv6 or Kv9.3 (70–74) KvLQT1 ⫹ truncated KvLQT1 (147) HERG ⫹ MiRP1 (96) KvLQT1 ⫹ minK (77) Kv1.4 ⫹ Kv웁1.2 (82) Kv1.5 ⫹ Kv웁1.2 (130) HERG ⫹ MiRP (96) KvLQT1 ⫹ minK (77)

Differential transcriptional control of 움 subunit expression

Kv4.2, Kv4.3 (109) HERG (129)

Posttranscriptional control of 움 subunit expression

Kv1.4 (109)

Differential expression of cell-signaling molecules

NOS, SODa (95a)

a

NOS, nitric oxide synthase; SOD, superoxide dismutase.

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erone-like effect (90). While it is not known whether all K⫹ channels are associated with 웁 subunits, some K⫹ channels in the brain exist as complexes with a stoichiometry of 움4웁4 (four 움 and four 웁 subunits) (91). It is possible that 웁 subunits also form heteromultimers (92–94). Hence, the potential for formation of 움–움, 움–웁, and 웁–웁 heteromultimers (87) is likely to have an impact on the degree of functional diversity of K⫹ channel function in vivo. Kv웁2 subunits have been crystallized and their struc˚ (91). The resultant structure is ture resolved at 2.8 A tetrameric, and each subunit is an oxidoreductase containing NADP⫹. This result confirms an earlier observation that a strong sequence homolgy exists between Kv웁s and oxidoreductases (95) and suggests a possible connection between the redox state of the cell and K⫹ channel activity (95a). A different class of K⫹ channel ancillary subunits are integral membrane proteins with one membranespanning segment, with the N terminus outside the cell. The first identified member of this family was previously called minK or IsK. It is now recognized that minK is a member of a larger family, referred to as MinK-related peptides (MiRP) (96). Whereas earlier studies suggested that the 130 amino acid minK could form a functional channel on its own, it is now clear that it is an ancillary subunit that associates with KvLQT1 to produce IKs . Another member of this family, MiRP1, coassembles with HERG to form a channel whose phenotype closely resembles IKr. Mutations in the MiRP1 gene may be associated with inherited and acquired cardiac arrhythmias (96).

V. PHYSIOLOGY OF CARDIAC VOLTAGE-DEPENDENT K CURRENTS Cardiac transmembrane action potential recordings show substantial differences in different regions of the heart. Differences in the notch of the action potential and the underlying Ito between the epicardium and the endocardium of the left ventricle are particularly illustrative of this point (5, 97, 98). Another example is the existence of a subpopulation of cells (M cells) that has been identified in the deep subepicardial layers of the canine ventricle. These M cells display longer action potential duration (phase 2), a steeper rate dependence of action potential duration, and a pharmacological responsiveness that differs from adjacent epicardial and endocardial layers (5, 97, 98). To understand the basis of differences in action potential duration, this section reviews the properties of the individual voltagedependent K⫹ currents as well as the regional differences in their properties and expression in the heart.

A. Ito1 Ito1 has been studied extensively in myocytes isolated from the working atrial and ventricular myocardium of many different species, including humans (99–109). One notable exception appears to be guinea pig ventricular myocytes, where Ito1 is minimal or absent. Ito1 is a rapidly activating and inactivating K⫹ selective current with activation properties that display conventional voltage dependence. Although the term ‘‘cardiac Ito’’ is applied frequently to this current, compelling evidence now exists to indicate that at least two functionally distinct Ito1 phenotypes exist within left ventricular myocytes (see later). Ito1 recorded from ferret right ventricular (RV) myocytes, the most extensively analyzed Ito1 to date, shows a high degree of uniformity (15) (Fig. 5). Ito1 in the ferret right ventricle is a relatively K⫹ selective current, with a PNa /PK ⫽ 0.08. Activation is a sigmoidal process implying at least three or more closed states, with conventional voltage-dependent time constants on the order of milliseconds (Fig. 5B). In contrast, the kinetics of inactivation are essentially voltage independent after the channel has been fully activated (Fig. 5C). Based on these kinetic observations, as well as the close overlap of the steady-state activation and inactivation curves, it was hypothesized that inactivation is partially coupled to activation, i.e., activation resulted in a conformational change that enabled inactivation to proceed. In contrast, whereas inactivation appears to display little or no intrinsic voltage dependence, recovery from inactivation is a rapid voltage-dependent process with time constants on the order of tens of milliseconds (see Fig. 6D). To account for this apparent discrepancy between voltageindependent inactivation and voltage-dependent recovery, it was hypothesized that recovery is coupled to voltage-dependent conformational changes in the channel that occur during deactivation (‘‘push-off ’’ model) (110). At the single channel level, ensemble average analysis of cell-attached single channel recordings demonstrated that the macroscopic current could be accounted for by a single type of K⫹ channel with a relatively low conductance (4–7 pS, 5.4 mM [K⫹] in the patch pipette) (Fig. 5D). In addition, the native Ito1 channel displays an interesting pharmacological phenotype, closed-state reverse use-dependent block by 4-aminopyridine (4-AP), a property shared by Kv4.2 but not Kv1.4 channels, see later (132). Subsequent patch clamp studies in ferret left ventricular (LV) ventricular epicardial myocytes indicated that these cells also possess an Ito whose phenotype is very similar to that displayed by RV myocytes (Fig. 6A); in particular, Ito in both RV and LV epicardial myocytes displays rapid recovery from inactivation with little or

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FIGURE 5 Representative voltage clamp recordings, I/V relationships of macroscopic Ito , activation kinetics, inactivation kinetics, and single channel recordings from ferret right ventricular myocytes. (A) Currents during depolarizing steps ranging between 10 and 100 mV (inset) and the mean peak I/V relationship obtained from six myocytes studied at 22⬚C. (B) Sigmoid activation kinetics of Ito (cooled to 12⬚C to resolve activation kinetics better) elicited by pulses to 20, 40, and 70 mV. Data were fit with a Hodgkin–Huxley-like a3i formulation (activation variable, n ⫽ 3), indicating that the channel must pass through multiple closed states prior to opening. (C) The process of inactivation is monoexponential in ferret right ventricular myocytes. Time constants of inactivation are voltage insensitive for depolarizing pulses ranging between 10 and 50 mV. (D) Representative single channel recordings obtained in the cell-attached configuration (patch potential 50 mV, K⫹ ⫽ 5.4 mM) and ensemble average inactivation properties at different test potentials that closely resemble those seen with the macroscopic currents. Reproduced with permission from Campbell et al., J. Gen. Physiol. 101, 571 (1993).

no ‘‘cumulative inactivation’’ at physiological heart rates (Fig. 6). It was also demonstrated that Ito1 in LV epicardial myocytes could be blocked by the spider toxin Heteropoda toxin 2 (HPTX2) (109, 111). When patch clamp studies were performed on ferret LV endocardial myocytes, these cells were also found to possess an Ito ; however, the density of Ito is 5–6 times lower in LV endocardial than in epicardial cells from the ferret left ventricle (Fig. 6B, C1 and C2). Significant differences in basic gating characteristics were also observed, particularly regarding steady-state inactivation, kinetics of inactivation, and recovery. In particular, the recovery kinetics of LV endocardial Ito were on the order of seconds (i.e., approximately 60 times slower than those of LV epicardial Ito, Fig. 6E), and as a result, inactivation

showed marked cumulative inactivation at physiological rates. The very slow recovery kinetics of Ito1 in the endocardial cells measured at 21–22⬚C in these experiments implies that this current makes a minimal contribution to repolarization at normal heart rates. However, when similar measurements were performed at 35–37⬚C in human myocytes, values for the time constant of recovery from inactivation ranged between 490 and 840 msec, suggesting that this current system could make a meaningful contribution to repolarization in human endocardial cells at physiological temperatures in vivo (108). Finally, in contrast to Ito in ferret LV epicardial cells, Ito in ferret LV endocardial cells was not blocked by 150 nM HPTX2. In summary, this work on ferret ventricular myocytes

FIGURE 6 Representative examples of Ito phenotypes in ferret left ventricular epicardial (A) and endocardial (B) myocytes. Currents elicited by depolarizing voltage clamp steps from a holding potential of ⫺70 mV at a pulse frequency of 0.167 Hz in epicardial cells and 0.05 Hz in endocardial cells. (C1) A comparison of mean current density–voltage (I/V) relationships for both Ito phenotypes and the inwardly rectifying K⫹ current, IK1 , in ferret LV epicardial and endocardial myocytes. (C2) The I/V relationship obtained from endocardial cells on an expanded scale. At 20 mV, peak Ito in endocardial cells is 앑20% of peak Ito in epicardial cells Two representative recovery patterns are recorded from a left ventricular epicardial (D) and an endocardial (E) myocyte. Note the difference in time scales. In both D and E, individual recovery waveforms were fit with single exponentials and the time constants are indicated. Recovery is 50–60 times slower in endocardial myocytes than in epicardial myocytes. Representative recovery waveforms (holding potential ⫽ ⫺70 mV) of rat heart Kv4.3 (F) and ferret heart Kv1.4 (G) clones expressed in Xenopus oocytes. Note the differences in time scales. Although the kinetics of recovery of Kv1.4 are very similar to those of native LV endo Ito, the kinetics of recovery of Kv4.3, while much more rapid, were still about five times slower than native LVepi Ito. Reproduced with permission from Brahmajothi et al., J. Gen. Physiol. 113, 581 (1999).

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has demonstrated conclusively the presence of at least two functional and quite distinct native Ito phenotypes in different regions of the left ventricle: (i) a rapidly recovering Ito in RV and LV epicardial myocytes, which is HPTX2 sensitive and displays closed state reverse usedependent block by 4-AP; and (ii) a slowly recovering Ito in LV endocardial myocytes, which is HPTX2 insensitive. Patch clamp studies on isolated LV endocardial and epicardial myocytes from other species and humans, while not as extensive, have also revealed similar results to those observed in ferret ventricular myocytes (102– 106, 108). While the functional implications of such a heterogeneous Ito phenotypic expression across the LV wall are presently unclear, it is clear that a modification of the term ‘‘cardiac Ito’’ is required. As will be described later, this heterogeneous Ito distribution very likely reflects expression of different Ito-generating 움 subunits (Kv1.4, Kv4.2, and Kv4.3) within different regions of the left ventricle. Such differences in current density are not confined to the ventricle, but have also been reported between different cell types of the right atrium, i.e., Ito1 may not be present in all atrial myocytes (107, 112).

B. IKur The ultrarapid component of the delayed rectifier current, IKur, is a very rapidly activating voltage-sensitive component of the delayed rectifier K⫹ current (Fig. 2B). It is a K⫹-selective, outwardly rectifying current with a single channel conductance between 10 and 14 pS (6, 113). Diversity in specific properties between species has been noted; however, in human right atrial myocytes, IKur appears to inactivate slowly and is much more sensitive to 4-AP (IC50 ⫽ 5.8–49 애M) than other voltage-sensitive K⫹ channels (2, 3, 17). It is also relatively insensitive to two classical K⫹ channel blockers, Ba2⫹ and TEA. The current has been identified primarily in atrial myocytes and its biophysical properties closely resemble those of the heterologously expressed Kv1.5 channel. IKur appears to be the predominant delayed rectifier current responsible for human atrial repolarization, whereas the role of this current in ventricular repolarization is species dependent (3). Studies suggest that while IKur does make a contribution to repolarization in the murine ventricle, it does not in the human ventricle (2, 3, 19).

C. IKr The selective blockade of IKr by E-4031 and dofetilide allowed investigators to separate two additional components of the delayed rectifier current in cardiac mus-

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cle (2–4,22–24, 26, 114, 115). The more rapidly activating component, IKr , has a rapid inactivation process (Fig. 2C). Because of its unique gating properties, IKr makes a unique contribution to repolarization. The rapid inactivation process competes with the activation process at depolarized potentials. As a result, there is very little current during the action potential plateau; however, the rapid recovery from inactivation enables the current to increase markedly during phase 2 and phase 3 of the action potential. As was first recognized by Shibasaki (116), IKr displays marked inward rectification at depolarized voltages. In addition, the rapid inactivation process, which overlaps activation, initially complicated the analysis of activation. To address some inconsistencies in previous analyses of gating, Liu et al. (26) used more complex voltage-clamp protocols in ferret atrial myocytes. They demonstrated that the activation of ferret IKr was actually much slower than that of Ito1 (26) (Figs. 7A and 7B). Furthermore, while the on rates of activation were voltage sensitive between ⫺10 and 30 mV, they were voltage insensitive at potentials positive to 30 mV. The voltage-dependent saturation in the on rate of activation makes IKr activation unique among Kv channels. Inactivation of ferret atrial IKr also showed several interesting properties. First, the time-dependent inactivation process was rapid, but surprisingly was also weakly voltage dependent (Figs. 7C and 7D). In addition, this analysis (26) showed that the macroscopic open channel conductance was nearly ohmic, indicating that the rapid inactivation process completely accounted for inward rectification of IKr. Second, the rapid inactivation process was shown to recover quickly and to have its own intrinsic voltage sensitivity (26, 117, 118). In fact, inactivation in this channel displays many attributes that have led to its classification as C type. Further understanding of the mechanisms underlying rapid IKr inactivation has come from studies on the HERG channel. Inactivation of HERG is sensitive to extracellular TEA⫹, permeant ions, and mutations near the extracellular mouth of the pore (51, 119–121). However, inactivation of HERG shows several other properties that are distinct from classic C-type inactivation, e.g., the rate of recovery is decreased or slowed by increases in extracellular K⫹ (51, 57, 119–121) and inactivation time constants are voltage sensitive at potentials positive to the threshold for activation (51, 117–121). IKr is increased paradoxically by extracellular K⫹, which is surprising given the reduction in K⫹ electrochemical gradient. The mechanism for this effect is a positive shift in the voltage dependence of channel inactivation without a change in the voltage dependence of activation (121). Changes mediated by an elevation of extracellular K⫹ have been determined to be clinically

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FIGURE 7 Activation and inactivation time courses of IKr . (A and B) How the activation time course is estimated from tail current measurements obtained from ferret atrial myocytes. Representative tail currents (A) were elicited by depolarizing pulses (to 20 mV) of variable duration. Mean time course of ferret atrial IKr activation (B) over the potential range ⫺10 to 50 mV. Data were fit (B) by a modification of a Hodgkin–Huxley model wherein voltage-insensitive rate constants governed the first step in the activation process and voltagesensitive rate constants governed the second step in the activation process. Note that (i) a voltage-insensitive step became rate limiting in the activation pathway and (ii) activation of IKr is nearly two orders of magnitude slower than that of Ito . (C and D) How IKr inactivation kinetics are estimated from a triple pulse protocol applied to voltage-clamped atrial myocytes. (C) Currents obtained from the end of a preconditioning pulse (500 msec, 50 mV), a return to ⫺40 mV for 50 msec to allow for recovery from inactivation, followed by a P3 pulse to allow for the redevelopment of inactivation at different potentials. Only currents during the 50msec pulse to ⫺40 mV and subsequent depolarizing P3 pulses are illustrated. Note the rapid redevelopment of inactivation during P3 pulses. (D) Time constant data obtained from inactivation data (䊉) shown in C along with recovery data (䊊) obtained from other experiments. The smooth line represents a best fit of a model where inactivation was coupled to activation. The voltage dependence of inactivation is weaker than that of activation, suggesting that it does not arise from coupling to activation and that it has its own intrinsic voltage sensitivity. Reproduced with permission from Liu et al., Biophys. J. 70, 2704 (1996).

important in treating patients with long QT interval syndrome caused by a mutation in HERG (3, 122).

D. IKs The other component of the delayed rectifier is the E-4031 and dofetilide insensitive component (23, 24, 145). The slowly activating component of the delayed rectifier K current, IKs, activates and deactivates extremely slowly (Fig. 2D). The slow deactivation of IKs appears to be an important determinant of rate-

dependent shortening of atrial and ventricular action potentials. At faster rates, IKs channels have less time to deactivate during diastole. As a result, IKs increases (‘‘cumulative activation’’) and progressively contributes to the acceleration of repolarization (24, 27, 123). IKs is a relatively K⫹-selective current with a threshold for activation that is positive to ⫺30 mV and a linear openchannel I/V relationship (3, 22, 124). IKs activation is extremely slow and shows a weak voltage dependence (22). Single channel conductance of IKs is small, with estimates ranging between ⬍1 and 6 pS (3,125). IKs is

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increased substantially by 웁 adrenergic agonists. The effects of divalents are of interest, as increased intracellular Mg2⫹ decreases, whereas elevated intracellular Ca2⫹ increases the current (3, 126, 127). To conclude, while the attributes of these four different K⫹ currents within individual myocytes have been analyzed in detail, it should be reemphasized that their properties are not uniform throughout the heart. For example, significant differences in the current density of the rapid and slow components of the delayed rectifier K⫹ current have been noted between different regions of the heart (5). While IKr has been described in SA and AV nodal cells and atrial and ventricular myocytes, the current is not universally found in all myocytes from these regions. Variation in the magnitude of the IKr tail currents has also been reported in ventricular myocytes isolated from the human heart (27, 111, 114, 115). Similarly, the density of IKs has been reported to be lower in the midmyocardial region than in the endocardial and epicardial regions (98). In addition, the density of IKs is lower in atrial than in ventricular myocytes, and in some species, such as cat, there is little or no IKs as compared to IKr (27). In sum, an abundance of phenotypical data indicates that there are marked differences in genes encoding for the different K⫹ currents between different regions of the heart.

VI. Kv CHANNEL GENE EXPRESSION AND THEIR RELATIONSHIP TO Kv CURRENTS IN THE HEART Commonly, protein function is determined through characterization of a purified protein. While this approach has been utilized in the case of sarcoplasmic reticulum ion channels, this would be a very difficult task in the case of cardiac K⫹ channels, and therefore assignment is typically made based on circumstantial evidence. Initially, the best evidence is provided by the comparison between biophysical properties of the native and the heterologously expressed current. Other evidence supporting the relationship between a K⫹ channel clone and a native current has been obtained from interventions that affect both heterologously expressed channel cDNA and native current in the same way. Examples include inhibition by drug, isoform specific antibodies, toxins, antisense cRNA, dominantnegative subunits, or gene knockouts (130). Finally, a critical factor in the case of cardiac K⫹ channels is the requisite demonstration that both the channel molecule and the native current be expressed in the same cell type and the area of the heart studied. In no case have all these criteria been met. Such an assignment is extremely complex, as there are many K⫹ channel genes (upwards of 30) and their expression in different regions of the

heart is variable. Additionally, the presence of electrically silent ancillary subunits presents an intimidating number of possible protein compositions that may explain the properties of a native current. One important avenue for narrowing down the possibilities is to understand the distribution patterns of different gene products in heart. Brahmajothi et al. (65) used fluorescent in situ hybridization (FISH) and immunohistochemistry on ferret isolated myocytes and heart tissue sections to determine the (i) fraction of myocytes expressing a particular Kv transcript or protein, (ii) where K⫹ channel transcripts are expressed in heart, (iii) whether differences in voltage-gated K⫹ channel expression exist between major anatomical regions of the heart, and (iv) the extent of uniformity of gene expression between myocardial cells within the major anatomical regions of the heart. The basic result of this work was that the presence of specific Kv channel transcripts was observed in only a fraction of cardiac myocytes analyzed. This result therefore raised the question: were these transcripts only expressed in certain specific regions of the heart? Because analysis of mRNA transcript distribution is itself limited by the potential lack of correlation between mRNA and protein expression (109), both FISH and immunolocalization studies were performed (109, 129). Such studies describing the distribution of ERG, Kv1.4, Kv4.2, and Kv4.3 are discussed further in the following sections (109, 129).

VII. PHYSIOLOGY, MOLECULAR BIOLOGY, AND MUTATIONS OF K CHANNEL CLONES A. Ito1 Homotetramers of Kv움 1.4, 4.2, and 4.3 produce rapidly activating and inactivating currents and are realistic candidate genes for Ito1 (1, 102, 109, 131) (Figs. 6F and 6G). As discussed previously, differences in peak current density and electrophysiological, biophysical, and pharmacological properties of Ito1 in the subendocardial and subepicardial regions of the ventricle (Figs. 6A–6E) indicate that there are at two least different functional Ito1 phenotypes (103, 105, 106, 109). The gating kinetics of Kv4.2/4.3 and the pharmacologic properties (4-AP, HpTx2) of Kv4.2 discussed previously suggest that these genes produce native Ito1 in epicardial cells (109, 131, 132). The distribution pattern of Kv1.4 protein in the ventricle, in conjunction with its slow recovery kinetics and insensitivity to HpTx2, makes it likely that this gene is responsible for native Ito1 in subendocardial myocytes of the left ventricle of both the human and the ferret heart (1, 69, 103, 105, 109) (Fig. 8).

FIGURE 8 Kv1.4, Kv4.2 and Kv4.3 움 subunit mRNA transcript and protein distribution in ferret sagittal ventricular tissue sections and compared. Relative mRNA levels were detected by FISH and are indicated by white fluorescence. Relative protein levels were detected by indirect immunofluorescence (IF) and are indicated by white fluorescence. Results were obtained from adjacent sagittal sections. (A) A view of a sagittal cut through the ventricle and great vessels. AO, aorta; CA, coronary artery; RV, right ventricle, LV, left ventricle. (B and C) Sections that have been probed with antisense and sense probes to cardiac troponin I (TnIc) and serve as positive and negative controls for this transcript. (D) A negative control signal to Kv4.2/Kv4.3 using a secondary antibody [see Brahmajothi et al. (149)] in the absence of primary antibodies. (E,G, and I) FISH results and (F, H, and J) IF results. Smaller numbered panels correspond to enlarged sections obtained from the selected regions indicated by white boxes. Note the general correspondence between Kv4.2 and Kv4.3 mRNA and protein levels, whereas Kv1.4 mRNA and protein levels do not correlate. Relative fluorescence intensity profiles of Kv1.4, Kv4.2, and Kv4.3 antibodies were measured in transverse sections obtained from the indicated levels in the basal, midventricular, and apical regions of the ventricle. The marked variation in relative intensity profiles measured from the different transverse sections indicates that protein levels vary between different small domains within adjacent regions as well as varying between different regions of the heart (regional localization). Reproduced with permission from Brahmajothi et al., J. Gen. Physiol. 113, 581 (1999).

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Kv4.2 transcript and protein have been reported in mouse, rat, ferret, and dog (128). Kv4.3 transcript and protein have been reported in rat, ferret, dog, and human heart (133). While Kv4.2 transcript is more abundant in the epicardium than in the endocardium of the rat and canine left ventricle, this is not the case for Kv4.3 (128). As a result, the absence of a difference in the distribution of Kv4.3 protein between epicardial and endocardial regions of the heart appears to be inconsistent with the reported differences in Ito density seen between these two regions of the human left ventricle (104, 105). The regional heterogeneity of Kv4.2/4.3 in the ferret ventricle may shed some light on this issue (109) (Fig. 8). In particular, this work demonstrated that marked differences in regional distribution between Kv4.2 and Kv4.3 exist and called attention to the need for accurately identifying the sites from which samples are taken for determination of transcript or protein expression. For example, close examination of Figs. 8H and 8I demonstrate that differences in site selection for procuring tissue samples, within the longitudinal axis of the left ventricle, would yield different conclusions about the relative distribution of Kv4.3 between the epicardium and the endocardium. Interestingly, unlike the ferret ERG, and Kv4 channels, there is considerable discrepancy between the distribution of Kv1.4 transcript and protein (Figs. 8E and 8F). The transcript is detected easily in both rat and ferret myocytes; however, the protein is undetectable in adult rat heart (6, 134) and is expressed at a low level in the endocardium and midmyocardium (free wall and septum) of the ferret ventricle (109). The discrepancy between the distribution of Kv1.4 transcript and protein in the heart, as well as the nonuniform distribution of Kv1.4 protein within a given myocyte type, may be related to the presence of 웁 subunits (90). Kv웁2 associates with the Kv1.2움 subunit early in channel biosynthesis when expressed heterologously in COS cells. These data suggest that Kv웁2 might exert a chaperone-like effect on associated Kv1.2움 subunits, mediating the maturation and surface expression of this channel. As Kv웁1 and Kv웁2 have been demonstrated to associate with Kv1.4 as well as Kv1.2 subunits, it is plausible that coexpression of one of these ancillary 웁 subunits may be necessary for the expression of the Kv1.4 protein in cardiac myocytes. Regardless of the underlying mechanism, the absence of a direct correlation between Kv1.4 mRNA and protein expression shows the importance of determining protein expression within well-defined anatomical regions and myocyte types when attempting to match a functional current phenotype with a cloned subunit. Nonuniform distribution of Kv1.4 and Kv1.5 channels has also been reported within individual cells

(132, 135). In Kv1.4 channels, the last four amino acids (ETDV) in the C terminus of Kv1.4 represent a PDZbinding domain. Interaction with members of the PDZ family likely accounts for the clustering of these channels at the intercalated discs (135). The functional consequences of such channel clustering are presently unclear.

B. IKur A rapidly activating sustained outward current occurs during depolarization in atrial but apparently not in human ventricular myocytes. Its rapid activation and minimal inactivation enable this current to make an important contribution to repolarization in atrial myocytes (Fig. 9A). The heterologously expressed Kv1.5 clone produces currents that have gating and pharmacological properties that resemble IKur closely (e.g., Kv1.5 current is the most sensitive to 4-AP among the Kv channels with a KD for 4-AP comparable to that reported for native IKur) (Fig. 9A). The distribution of Kv1.5 protein was studied by Mays et al. (130), who used antibodies directed against two distinct channel epitopes. They showed that Kv1.5 is present in both human atrial and ventricular myocytes. While the presence of the protein in the ventricle might argue that it cannot be responsible for a current that is restricted to the atrium, there are several plausible explanations for these data. For example, ventricular Kv1.5 may be associated with another Kv1.x clone or be associated with an inhibitory subunit. Additional data confirming the linkage between Kv1.5 and IKur were provided by Feng et al. (150), who demonstrated that IKur was decreased selectively in atrial myocytes by Kv1.5-directed antisense oligonucleotides.

C. IKr Homomultimers of heterologously expressed ERG produce currents whose gating kinetics closely resemble those of native IKr (118) (Fig. 9B). However, there are some significant differences, such as slower deactivation kinetics and different pharmacologic effects of dofetilide and E-4031 (118, 136). Coexpression of ERG with a cDNA encoding a MinK-related peptide 1 (MiRP1) produced a current that resembles native IKr more closely with respect to kinetics and pharmacology, suggesting that MiRP1 or a closely related protein is a subunit of the native channel (96). A complicating factor in understanding the structure of native IKr is the presence of multiple splice variants of ERG. Three N-terminal isoforms of mouse ERG1 have been identified (137, 138); however, only two have been detected in humans, HERG1 and HERG1b (139). These variants differ in the length and amino acid com-

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FIGURE 9 Three representative delayed rectifier current recordings obtained from isolated cardiac myocytes and their respective cloned channels. (A) IKur in a human atrial myocyte (left) and cardiac human Kv1. 5 current in a HEK293 cell (right). Reprinted with permission from Fedida et al., Circ. Res. 73, 210 (1993). (B) IKr in a neonatal mouse ventricular myocyte (left) and HERG current in a Xenopus oocyte (right). Reproduced with permission from Wang et al., Am. J. Physiol. 270, H2088 (1996) and Sanguinetti et al., Cell 81, 299 (1995). IKr is elicited by depolarizing pulses to potentials ranging between ⫺20 and 40 mV followed by a return to ⫺40 mV. The HERG current is elicited by depolarizing pulses to potentials ranging between ⫺50 and 20 mV, followed by repolarization to ⫺70 mV. Note that the magnitude of IKr and IHERG decreases with depolarizations positive to 0 mV. Tail currents are larger than currents elicited by depolarizing pulses because the rapid inactivation that occurs during depolarization recovers faster than the rate at which the channel deactivates following repolarization. (C) IKs in a guinea pig ventricular myocyte at 35⬚C (left) and a KvLQT1/ minK current expressed in a Xenopus oocyte at 22⬚C (right). Reproduced with permission from Sanguinetti and Jurkiewicz, Am. J. Physiol. 259, H1881 (1990) and Splawski et al., Nat. Genet. 17, 338 (1997). Currents were elicited by 7.5-sec depolarizing pulses ranging between ⫺20 and 60 mV followed by repolarization to ⫺50 mV. Note the slow activation and deactivation of IKs and the KvLQT1/minK current.

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position in the N-terminal portion of the protein. Human (H) and mouse (M) ERG have very similar electrophysiological properties. MERG1b (the short variant) deactivates rapidly, and the heteromultimer formed by the coassembly of MERG1a and MERG1b has deactivation kinetics that are faster than those of MERG1a or HERG1. A C-terminal splice variant of HERG1 (HERG USO) has also been reported, which shows different electrophysiological properties than those of the HERG homotetramer when coexpressed with HERG1 (140). Thus, if multiple HERG gene products are expressed in the ventricle, then the differences in activation and inactivation gating properties between the different HERG splice variants predict that the electrophysiological properties of IKr may not be uniform in heart cells. Further, if expression of the splice variants or MiRPs show different regional distribution, it could suggest considerable heterogenity of the native IKr current. However, regional differences in gating properties of IKr have not been reported to date. Although evidence is lacking for the nonuniform distribution of ERG splice variants and ancillary subunits, nonuniform distribution has been shown for ERG in the ferret heart. Ferret ERG mRNA is expressed in about half of ventricular and atrial myocytes. Analysis of ferret heart cross sections showed that ERG mRNA and protein are expressed, but are localized regionally. Interestingly, in ferret there were also differences in ERG expression in the ferret right atrium, where it was most abundant in the medial right atrium, especially in trabeculae of the atrial appendage and in SA nodal cells. Its detection in SA nodal cells may help explain the bradycardias seen in some patients with the familial long QT interval syndrome (129). Mutations in ERG have been linked to the second most common form of hereditary LQTS in patients, LQT2. Mutations in its gene, KCNH1, are scattered through the open reading frame (8, 76, 77). Thus far, KCNH1 mutations have been shown to be dominant. There are two obvious mechanisms for this dominance. One, supported by expression studies, demonstrates that for many of the different mutations the mutant channels can associate with wild-type channels to form a complex that is inactive or has greatly reduced activity. Another class of mutations prevents protein from being expressed off the mutant allele. Individuals with this type of mutation presumably develop LQTS due to loss of half of their functional HERG (76, 141). One additional point worth mentioning is that these mutations in HERG1 were sufficient to prolong the QT interval in these patients. This observation indicates that the expression of this protein throughout the ventricle is sufficiently broad to lengthen repolarization throughout the ventricle. A third mechanism is that some mutations,

when coexpressed with wild-type channels, shifted the gating properties of the channel, indicating that the abnormal phenotype resulted from altered protein function (142–144).

D. IKs IKs is an important component of the delayed rectifier K⫹ current recorded at positive potentials in the ventricle (Figs. 2D and 9C) and makes an important contribution to the plateau phase of repolarization with an increase in either frequency or plateau duration (24, 27). Heterologous expression of KvLQT1 alone produces a current that activates far more quickly than IKs (77, 78, 145, 146). However, coexpression with the ancillary subunit minK produces currents with gating kinetics and a pharmacologic profile that are very similar to the endogenous current (24, 77, 78, 145). MinK may also increase the expression of KvLQT1, which may be due in part to the fact that the heterooligomeric channel has significantly larger single channel currents with a higher opening probability (145). In addition, minK eliminates or greatly slows inactivation of KvLQT1 (146). The interaction between MinK and KvLQT1 probably occurs at a different site than is the case for 웁 subunits and Kv1.x channels, as minK has a single putative membrane-spanning domain. Interestingly, mutations in the membrane-spanning region of minK have been shown to produce changes in gating and permeation, suggesting that minK associates with part of the pore region of KvLQT1 channels (3, 149). A splice variant of KvLQT1 having an N-terminal truncation has been identified (KvLQT2) (147, 148). The truncated cDNA isoform reduces IKs expression when it is co-injected with the full-length cDNA isoform into Xenopus oocytes (147). The question of whether such a mechanism is operative in the intact heart has not yet been resolved, as it has not been determined whether both KvLQT1 and KvLQT2 are expressed in the same myocyte, nor has it been established that equimolar amounts of the mRNAs will result in similar protein expression. Regional heterogeneity of KvLQT1 transcript expression also is likely, as the transcript is expressed in just over half of ferret cardiac ventricular myocytes (65). Surprisingly, minK mRNA is only expressed in 10–18% of ferret ventricular myocytes and in 21–29% of ferret atrial myocytes. MinK associates with both ERG and KvLQT1 and increases the magnitude of the current over that seen with expression of the 움 subunit alone. The relatively sparse distribution of minK mRNA may account for some of the variability in IKs levels between the different regions of the canine ventricular wall that have been reported by Antzelevitch and colleagues (5).

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Mutations in KCNQ1 cause the LQT1 variant of familial LQTS (9, 77, 79) (Table II). This is the most common form of the disease and, as is the case for LQT2, the reported mutations are scattered throughout the open reading frame. Also, dominant negative suppression of wild-type KvLQT1 has been demonstrated. Mutations in KCNE1 cause one of the rare forms of hereditary LQTS, LQT5 (96).

6.

7.

8.

VIII. SUMMARY Elucidation of the molecular basis of the different K⫹ currents in the heart is progressing rapidly and has significantly advanced our understanding of the electrophysiology and biophysics of native cardiac K⫹ currents, as well as the molecular basis of the cardiac action potential. An additional variable that must be addressed is the nonuniform distribution of channel proteins and their modulators (7, 95a, 109). The heterogeneity of channel protein is greater than was originally anticipated, and the distribution patterns, as well as their underlying mechanisms, are just beginning to be addressed (Table III). Immunolocalization data from the ferret heart show that the variability of channel expression appears to result from a combination of variability among cells as well as regional sublocalization. Determination of the patterns of localization of K⫹ channel proteins within the human heart will have important implications regarding our understanding of the molecular basis of normal and abnormal function as well as for antiarrhythmic therapy.

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15 Inwardly-Rectifying K⫹ Channels in the Heart MASAYUKI TANEMOTO, AKIKAZU FUJITA, and YOSHIHISA KURACHI Department of Pharmacology II Graduate School of Medicine, Osaka University Osaka 565-0871, Japan

I. INTRODUCTION

the time period between action potentials. Therefore, K⫹ channels play clear roles in the formation of different phases of the cardiac action potential. K⫹ channels in cardiac myocytes can be classified into two major categories: inwardly rectifying K⫹ channels (Kir) and voltage-gated K⫹ channels (Kv). Cardiac Kir channels include classical Kir channels (IK1) (Kurachi, 1985; Sakmann and Trube, 1984b), G-protein-activated muscarinic K⫹ channels (KACh) (Kurachi et al., 1986a; Sakmann et al., 1983), Na⫹-activated K⫹ channels (KNa) (Kameyama et al., 1984), and ATP-sensitive K⫹ channels (KATP) (Noma, 1983). Kv channels that have been identified electrophysiologically in the heart are A-type transient outward K⫹ channels (Ito) (Coraboeuf and Calmeliet, 1982; Kenyon and Gibbons, 1979) and delayed rectifier K⫹ channels (IK), including IKur , IKr , and IKs (Coraboeuf and Calmeliet, 1982; Kenyon and Gibbons, 1979). IKur , IKr , and IKs are ultra rapidly, rapidly, and slowly activating components of IK , respectively. Kv channels are activated by depolarization during the action potential and initiate its repolarization, whereas Kir channels play diverse functional roles, such as setting of the resting membrane potential near the K⫹ equilibrium potential (IK1 channel), acetylcholine (Ach)-induced deceleration of the heart beat (KACh channel), and metabolic impairment-induced shortening of the action potential (KATP channel and possibly KNa channel). Different from Kv channels, Kir channels are not regulated by the membrane potential but by intracellular substances such as polyamines and Mg2⫹ ions (Fakler et al., 1994; Ficker et al., 1994; Isomoto and Kurachi, 1997; Lopatin et al., 1994; Matsuda et al., 1987;

The heart is a complex organ in which different regions have been specialized for the performance of relatively specific functions. In nodal cells, electrical activity is autogenic, whereas in Purkinje fibers, electrical activity is dedicated to the electrical signaling between atria and ventricule. In atrial and ventricular myocytes, electrical activity serves two functions: (1) to transmit the electrical signal to its neighboring myocytes with which it is connected via gap junction complexes and (2) to convert the electrical signal into mechanical activity. The latter is also known as electromechanical coupling. Therefore, in different regions of the heart the electrical activity of individual myocyte will differ. However, except for autogenic nodal cells, the myocyte action potential shares certain common characteristics. The action potential in cardiac myocytes has five phases (Fig. 1A). The rapid depolarizing phase (phase 0) is mediated by an abrupt influx of Na⫹ ions through voltage-gated Na⫹ channels. A subsequent small repolarization phase (phase 1) is achieved via K⫹ efflux flowing through a transiently activated K⫹ channel. This early outward current sets the initial plateau potential, thus influencing the behavior of subsequently activated ion channels and the duration of action potential. The plateau phase (phase 2) is characterized by minimal net ion flow due to the balance of an inactivating Ca2⫹ inward current and a K⫹ efflux through slowly activating voltage-gated K⫹ channels. In phase 3, the K⫹ permeability increases time dependently and results in complete repolarization. In phase 4, the K⫹ permeability sets the deep resting membrane potential and regulates

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FIGURE 1 Contribution of Kir channels to the cardiac action potential. (A) A schematic time course of action potential from cardiac myocytes. Numbers are assigned to different phases of the action potential. Kir channels determine the slope of phase 3 and the membrane potential of phase 4. (B) Schematic representations of action potential change evoked by inhibition (top) and activation (bottom) of Kir channels. By inhibiting Kir channels the membrane potential of phase 4 became shallower and the frequency of action potentials increases. However, by activating Kir channels the membrane potential of phase 4 goes down to the equilibrium membrane potential of potassium ions (EK) and action potentials disappear.

Yamada and Kurachi, 1995). These substances block the outward-going K⫹ currents through Kir channels above the K⫹ equilibrium potential, resulting in the inwardly rectifying property. Another feature common to Kir channels is that the channel conductance increases as the concentration of extracellular K⫹ increases. Also each Kir channel is regulated by intracellular specific ligands, G-protein (KACh), ATP (KATP), and Na⫹ (KNa). These features are essential for their different functional roles. In 1993, a K⫹ transporter Kir channel, Kir1.1/ROMK (Ho et al., 1993), and a classical Kir channel, Kir2.1/

IRK1 (Kubo et al., 1993a), were cloned by the expression cloning technique from the outer medulla of rat kidney and a mouse macrophage cell line, respectively. According to the analogy of Kv channels, it was assumed and subsequently proved that functional Kir channels require the association of four of these individual proteins or channel subunits. The cDNA encoding one of the pore-forming subunits of KATP channels (Kir6.1/ uKATP -1), as well as that of KACh channels (Kir3.1/ GIRK1), was cloned subsequently (Dascal et al., 1993; Inagaki et al., 1995c; Kubo et al., 1993b). All of these channel subunits exhibit the same primary structure. So far, more than 15 cDNAs encoding Kir channel subunits have been isolated in mammals. These cloned Kir channel subunits can be classified into four principle groups (Fig. 2): (1) Kir2.0/IRK subfamily, IRK channels (Kubo et al., 1993a; Morishige et al., 1993; Morishige et al., 1994; Takahashi et al., 1994); (2) Kir3.0/GIRK subfamily, G-protein-activated K⫹ channels (Dascal et al., 1993; Isomoto et al., 1996a; Krapinvinsky et al., 1995; Kubo et al., 1993b; Lesage et al., 1994); (3) Kir1.0 and Kir4.0/KAB subfamily, K⫹ transporter K⫹ channels (Bond et al., 1994; Ho et al., 1993; Takumi et al., 1995); and (4) Kir6.0/KATP subfamily, ATP-sensitive K⫹ channels (Inagaki et al., 1995a,c; Sakura et al., 1995). BIR9, which is referred to as Kir5.1, does not produce functional Kir channels when expressed by itself in Xenopus oocytes and may be able to associate specifically with Kir4.1 to form heteromultimeric Kir channels (Bond et al., 1994; Pessia et al., 1996). However, Kir5.1 does not possess a Walker type-A ATP-binding motif, which is present in the K⫹ transporter subfamily, and has only 36% sequence identity with both Kir1.1 and Kir1.2/ Kir4.1 and ⬍42% identity to other Kir channel subunits. Thus, Kir5.1 may belong to another subfamily of Kir channels. Kir2.4 (Toepert et al., 1998) and Kir7.1 (Doering et al., 1998; Krapinvinsky et al., 1998) are isolated members of the Kir family. Kir2.4 was reported to be expressed in brain, heart, and kidney. In the brain, it was expressed in the visceral motor cell column, the hypoglossal nucleus, and the choroid plexus. Its precise distribution in heart or kidney, however, has not been clarified yet. Kir7.1 was also reported to be expressed in brain choroid plexus, but not in heart. The chromosomal localization of several Kir channels has been determined in human and mouse. Their localization on mouse chromosomes is summarized in Fig. 3 (Isomoto et al., 1997; Mjaatvedt et al., 1995; Morishige et al., 1993, 1997; Mouri et al., 1998; Tada et al., 1997; Takumi et al., 1996; Wickman et al., 1997). The localization of other members of the Kir family is available on human chromosomes (Bock et al., 1997; Budarf et al., 1995; Chutkow et al., 1996; Derst et al., 1998; Erginel-Unaltuna et al., 1998; Gosset et al., 1997; Hugnot

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FIGURE 2 The amino acid homology tree of the two membrane-spanning domain inwardly rectifying K⫹ channel families. The relationships of the amino acid sequences of each subunit were calculated by the unweighted pair group method with arithmetic mean.

et al., 1997; Inagaki et al., 1995b; Lesage et al., 1995; Namba et al., 1997; Stoffel et al., 1994; Tsaur et al., 1995; Tucker et al., 1995; Verkarre et al., 1998). Kir subunits have common molecular motifs in their primary structure, i.e., two putative transmembrane domains (M1 and M2) and one potential pore-forming

loop (H5) (Fig. 4). Thus, the primary structure of these Kir channel subunits resembles that of S5, H5, and S6 segments of Kv channels (Jan and Jan, 1992, 1994; Pongs, 1992). In Kv channels, the S4 region, which possesses repeated positively charged amino acid residues, is presumed to be the voltage-sensing region of the

FIGURE 3 Schematic representation of chromosomal localization of Kir channels in mouse. Chromosomal numbers are shown on the top of each scheme. The size and the position of the box in the schemes represent the relative size and position of each gene. The name of each gene is shown on the right with the protein name in parentheses. The equivalent positions on human chromosomes are indicated on the left. The localization of other members of Kir subunits on human chromosomes has been determined: Kir1.1a-f/11q24, Kir3.1/2q24.1, Kir3.3/ 1q21-23, Kir4.2/21q22.2, Kir6.2/11p15, Kir7.1/2q37, and Sur1/11p15.

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FIGURE 4 Schematic representation of potassium channel structure. (Left) The predicted twodimensional conformation of each channel subunit. (Right) An approximation of the threedimensional conformation of channel forming units in the lipid bilayer. 6 TM, six transmembrane segment type potassium channel; 2 TM, two transmembrane segment type potassium channel; 4 TM, four transmembrane segment type potassium channel.

channels. Kir channel subunits do not possess a segment corresponding to the S4 region in Kv channels. Kir channel subunits are thought to be assembled to form homoor heterotetrameric functional channels (Corey et al., 1998; Inanobe et al., 1995; Jan and Jan, 1994; MacKinnon, 1991; Yang et al., 1995b). Progress in the molecular biology of Kir channels enables us to more clearly understand the structure– function relationship of channel biophysics, physiological regulation, and the pharmacology of these channels at the molecular level. This chapter summarizes current

understanding of the molecular properties and functional roles of Kir channels in myocardium.

II. CLASSICAL INWARDLY RECTIFYING K⫹ CHANNELS IN THE HEART (IK1) A. Physiological Roles of IK1 Channels The IK1 channel is a strong inwardly rectifying K⫹ channel that is constitutively active at all physiological

15. Kir in Heart

membrane potentials and formed the classical background Kir current. This channel is modulated by external K⫹ and blocked by external Cs⫹ and Ba2⫹. Its singlechannel conductance is approximately proportional to the root value of [K⫹]o . The IK1 channel is distinct from other Kir channels in its regulation and degree of rectification. It is not activated by transmitters such as acetylcholine and is not affected by the change of membrane potential. The inwardly rectifying property of IK1 is now believed to arise from the blockades of outward current flow through the channel caused by intracellular Mg2⫹ and polyamines (such as spermine and spermidine). IK1 is the dominant component of the resting conductance in cardiac myocytes, including Purkinje fibers as well as ventricular and atrial myocytes (Beeler and Reuter, 1970; Kurachi, 1985; McAllister and Noble, 1966; Rougier et al., 1968) but not in nodal cells (Noma et al., 1984). The single-channel conductance and mean open time at 앑 ⫺80 mV of IK1 in ventricular myocytes are 앑40 pS and 앑100 msec, respectively (Kurachi, 1985). Contributions of IK1 to the action potential are illustrated in the current–voltage relationship in Fig. 5. The large inward conductance at membrane potentials below the K⫹ equilibrium potential (EK) and the relatively large outward conductance at those just above EK set the resting membrane potential close to EK . The lack of outward conductance at plateau-level potentials caused by the strong inward rectification of IK1 prevents massive K⫹ efflux during the action potential plateau phase, resulting in the maintenance of depolarization by the small inward current through Ca2⫹ channels. When repolarization is initiated by activating the delayed Kv channels, a relatively large outward current passes through IK1 in the negative slope region of its current– voltage relation, which assists the rapid repolarization. Thus, IK1 plays important roles in (1) setting the resting potential, (2) maintaining the plateau phase, and (3) causing rapid repolarization.

B. Molecular Aspects of IK1 Channels Features of the IRK subfamily (Kir2.1–2.3/IRK1–3) closely resemble those of the cardiac background IK1 channel. In our laboratory, three Kir2.0/IRK subfamily members have been cloned from mouse brain cDNA libraries (Morishige et al., 1993, 1994; Takahashi et al., 1994). The amino acid sequence of mouse brain Kir2.1 shares 70 and 61% identity with Kir2.2 and Kir2.3, respectively. The amino acid sequence is well conserved in M1, M2, and H5, especially in H5 where they show differences from each other at only one amino acid residue. Xenopus oocytes injected with cRNAs derived from Kir2.0s expressed strong inwardly rectifying K⫹ currents (Figs. 6A and 6B). Hyperpolarizing voltage steps elicit

285

FIGURE 5 Current–voltage relationship and action potential in the ventricular myocyte. The current–voltage relationship for the instantaneous current under voltage clamp conditions is indicated by the solid line and that for the steady-state current recorded after 1 sec at each voltage by the broken line. The action potential is drawn sideways to correlate it to the current–voltage relationship above. Shaded area shows IK1 currents.

rapidly activated large inward currents. These currents are blocked by external Ba2⫹ and Cs⫹ in a concentrationand voltage-dependent manner. Single-channel conductances of Kir2.1, Kir2.2, and Kir2.3 are 앑22, 앑34, and 앑13 pS with 150 mM [K⫹]o , respectively. The steady-state Po of the Kir2.2 channel decreases with hyperpolarization, whereas those of Kir2.1 and Kir2.3 remain constant. The detailed gating kinetics analysis strongly indicates that the increase of long closed gaps (⬎200 msec) between clusters causes the prominent reduction of steady-state Po at hyperpolarized potentials in the case of Kir2.2. The dominant cardiac IK1 is reported to have a single-channel conductance of 30–40 pS with 150 mM [K⫹]o (Figs. 6C and 6D) and exhibits hyperpolarization-induced inactivation in the absence of blocking cations (Kurachi, 1985; McAllister and Noble, 1966). These properties are quite similar to those of Kir2.2 (Takahashi et al., 1994). Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of mRNAs obtained from isolated single ventricular and atrial myocytes showed that these myocytes express mRNA of only Kir2.2 among Kir2.0 (Matsumoto et al.,

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II. Cellular Electrophysiology

tively charged amino acid for a neutral one in stronginward rectifiers reduces the degree of inward rectification by decreasing the apparent affinity for polyamines and Mg2⫹ ions (Stanfield et al., 1994; Wible et al., 1994).

C. Modulation of IK1 Channels Collins et al. (1996) reported that Kir2.3 is inhibited by the physiological concentration of intracellular ATP (ATPi) (KD of 1.47 mM). This effect is antagonized by ADP in the physiological range, which implies that this channel is sensitive to the intracellular [ATP]/[ADP] ratio. Kir2.3 does not, however, correspond to cardiac KATP channels because native KATP channels exhibit weaker rectification and much higher sensitivity to ATPi . The inhibition of Kir2.3 currents by ATPi does not require Mg2⫹ and is mimicked by nonhydrolyzable ATP analogs, indicating that hydrolysis of ATP is not required. These effects, not observed in Kir2.1, may be specific to Kir2.3. However, Fakler et al. (1994b) showed that Kir2.1-mediated currents, after rundown upon excision of the patch, can be partly restored by application FIGURE 6 Single-channel recordings from cell-attached membrane patches of Xenopus oocytes expressing Kir2.0 channels (A and B) and of mouse ventricular myocyte (C and D). (A and C) Examples of the recording of patch current traces recorded at membrane potentials indicated in millivolt with each trace. The concentration of K⫹ was 150 mM in the pipette solution. Each of these patches appeared to contain only one channel. (B and D) Current–voltage relationships of the channel records shown in A (B) and C (D). Arrows indicate the membrane patch current level recorded when channels were closed. Reproduced from Matsumoto et al. (2000), with permission.

1999). Because Kir2.0 subunits seem to assemble as homotetramers (Yang et al., 1995b), at present, the IK1 channel is thought to be composed of a homotetrameric assembly of Kir2.2 subunits. Experiments with excised membrane patches suggest that the rectification of IK1 channels can result from block by intracellular Mg2⫹ and polyamines. It has been clarified that the differences in inward rectification between different Kir channels are attributable to the efficacy of these substances to block the outward-going K⫹ currents at two amino acid residues (receptor sites 1 and 2) located in the M2 domain (R1) and carboxylterminal regions (R2) of cloned Kir channels (Yang et al., 1995a). For cloned strong-inward rectifiers, which are believed to encode IK1 channels (Kir2.0/IRK subfamily) and KACh channels (Kir3.0/GIRK subfamily), one or two amino acid residues at these sites are negatively charged (Fig. 7). For weak-inward rectifiers such as the KATP channel, the corresponding sites contain only neutral residues. Indeed, substitution of a nega-

FIGURE 7 Mg2⫹ and polyamine interaction sites in Kir subunits. R1 and R2 indicate the approximate position of the negatively charged amino acid residues that are responsible for the sensitivity of the channel to intracellular Mg2⫹ and polyamine, which in Kir2.1 are aspartate at position 172 and glutamate at position 224, respectively. The table shows the amino acid residues at the analogous R1 and R2 sites in different Kir subunits. N, asparagine; G, glycine; D, aspartate; E, glutamate; and S, serine.

15. Kir in Heart

287

of 1 mM ATP with 1 mM free Mg2⫹ but not by nonhydrolyzable ATP analogs, suggesting that Kir2.1 is regulated by ATP hydrolysis [probably by phosphatidylinositol bisphosphate (PIP2)]. So far there has been no report on the specific regulation of Kir2.2.

III. G-PROTEIN-ACTIVATED MUSCARINIC K⫹ CHANNELS IN THE HEART (KACh) A. Physiological Roles of KACh Channels Acetylcholine released from axonal termini of vagal nerves decelerates the heart beat. The mechanism underlying ACh-induced bradycardia was clarified from the results of several physiological experiments (DelCastillo and Katz, 1955; Hutter and Trautwein, 1955; Noma and Trautwein, 1978; Osterrieder et al., 1981; Sakmann et al., 1983; Trautwein and Dudel, 1958). ACh activates a K⫹ current by binding to muscarinic ACh receptors. This K⫹ current flows through a specific population of K⫹ channels, named muscarinic K⫹ (KACh) channels. The KACh channel is present in cardiac atrial and nodal cells and is coupled to m2-muscarinic and A1-adenosine receptors through pertussis toxin (PT) or islet-activating protein (IAP)-sensitive G-proteins (GK) (Fig. 8) (Breitwieser and Szaro, 1985; Kurachi et al., 1986b; Pfaffinger et al., 1985). The concept that GK directly activates KACh channels was further strengthened by findings that this channel could be activated in cell-free inside-out patches of the atrial cell membrane by intracellular GTP (with agonist) (Kurachi et al., 1986a,b,c), its nonhydrolyzable analogs (Kurachi et al., 1986b,c), and by purified or recombinant G-protein 웁웂 subunits (in the absence of agonists) (Fig. 9) (Kurachi, 1989; Logothesis et al., 1987). These results also clearly showed the ‘‘membrane-delimited’’ nature of this system. To define the physiological interaction between GK and the KACh channel, the concentration-dependent effect of intracellular GTP (GTPi) on the KACh channel was examined. It was found that GTPi activates the KACh channel in a highly positive cooperative manner (Ito et al., 1991; Kurachi et al., 1990; Nakajima et al., 1992). Figure 10 shows the relationship between intracellular GTP and KACh channel openings in the presence of various concentrations of ACh. Even in the absence of agonist, some activation of the channel was caused by GTP with the internal solution containing 130 mM Cl⫺. This agonist-independent activation of the KACh channel was due to the basal turn-on reaction of GK , which requires the coupling of muscarinic ACh receptors or adenosine receptors with GK (Ito et al., 1991). By coupling of receptors and GK , the intrinsic GTPase

FIGURE 8 Effects of acetylcholine and adenosine on potassium currents in atrial myocytes. The whole cell current was recorded from atrial cells, and the solutions were applied for the periods indicated by the black lines above the trace. (A) The application of 1.1 애M of acetylcholine (ACh) increased the whole cell current (a). Theophylline showed no significant effects on the ACh-induced current, which was completely blocked by atropine. (b) The expanded cell current traces are indicated by numbers. (B) By applying 1 애M of adenosine (Ado), the current increased (a). Atropine showed no significant effect, but theophylline completely blocked the Ado-induced current. The blocking effect of theophylline on the Ado-induced current was washed out more rapidly than that of atropine on ACh-induced current. (b) The expanded cell current traces are indicated by numbers. (C) Schematic representation of proposed mechanisms of Ach and Ado activation of K⫹ channels in atrial cell membrane. Stimulation of their receptors by Ach or Ado functionally activate pertussis toxinsensitive G-proteins (Gi). Activated Gis will activate K⫹ channels. This scheme does not represent a quantitative relationship between the components and does not take into account possible intermediate steps.

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activity of GK was supposed to be inhibited by intracellular Cl⫺ (Gilman, 1987; Nakajima et al., 1992). As the concentration of ACh in the pipette was raised, it was observed that (1) the threshold concentration of GTP needed to open the channel decreased, (2) the GTP concentration for half-maximal activation of the channel decreased, and (3) the maximal relative channel open probability increased; however, (4) the Hill conefficient was constant at 앑3 and was independent of the ACh concentration (Fig. 10). These results indicate that ACh binding to the receptor increased both the maximal response and the apparent affinity of the KACh channel for GTP, which may be due to the facilitation of functional dissociation of GK (Kurose et al., 1986). The positive cooperative effects of GTP on the channel opening may be attributable to an interaction between GK and KACh channel (Ito et al., 1991; Kurachi et al., 1990). The simplest explanation is that the KACh channel has multiple (more than three) binding sites for the GK protein. Precise and reliable analysis of the single-channel kinetics of the KACh channel is difficult because multiple KACh channels are usually included in a single membrane patch from atrial myocytes (Fig. 10). In these cases, the spectral analysis of the channel currents is one of the most reliable and powerful ways to assess the channel kinetics (Fig. 11). The power spectrum con-

structed from inside-out patch recordings of KACh channel currents could always be well fitted with the sum of two Lorenzian curves irrespective of the GTPi concentration (Hosoya et al., 1996). This observation indicates that the KACh channel possesses three distinct open/ closed states. Because the channel possesses a single dominant open state (Sakmann et al., 1983), the equilibrium of the states can be described as C2-C1-O, where C2 and C1 represent closed states and O represents the open state. It is likely that the transition among these three states is responsible for the first open–close transitions of KACh channel currents observed at the singlechannel level (Fig. 11A). The corner frequencies of the two Lorenzian components were constant irrespective of GTPi concentration (Fig. 11B). The ratio of the powers of the two Lorenzian components at 0 HZ was also unaffected by the GTPi concentration. These results indicate that the kinetics of the fast open–close transition of the channel is not a function of GK activity. In other words, GK activates the KACh channel without altering the fast open–close kinetics of the channel. As the GTPi concentration was raised, the power of both Lorenzian components at 0 HZ became progressively larger (Fig. 11B), implying that GK increases KACh channel activity through a process too slow to be detected by spectral analysis. We assume the presence of another transition with slow kinetics between two

FIGURE 9 Muscarinic K⫹ channel activation by G웁웂 subunits but not by G움 subunits in the presence of acetylcholine (ACh). Arrowheads indicate zero current level. (A) An example of an inside-out membrane patch experiment obtained from a guinea pig atrial myocyte. Channel currents were recorded at ⫺80 mV with symmetrical 145 mM K⫹ solutions. The concentration of ACh in the pipette was 0.5 or 1 애M. Bars above each trace indicate the protocol for application of GTP, different G-protein subunits, and GTP웂S to the internal side of the patch membrane. The Gil움-GTP웂S and Gi2움-GTP웂S did not increase the current, whereas 10 nM of G웁웂 subunits increase the current as effectively as 10 애M of GTP웂S. (B) An example of an inside-out patch experiment showing the GDP form of G-protein 움 subunit decreased the muscarinic K⫹ channel, which was activated by GTP웂S. The experimental conditions were the same as in A.

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15. Kir in Heart

FIGURE 10 Concentration-dependent effect of intracellular GTP on the muscarinic K⫹ channel in the absence and presence of acetylcholine (ACh). (A) Examples of inside-out patch experiments obtained from guinea pig atrial myocytes. Channel currents were recorded at ⫺80 mV with symmetrical 145 mM K⫹ solutions. The concentration of ACh in the pipette was 0 or 1 애M as indicated. Bars above each trace indicate the protocol for application of different concentrations of GTP or 10 mM GTP웂S to the internal side of the patch membrane. A 3- to 10-fold increase in GTP concentration resulted in a dramatic increase of open probability (N · Po) of muscarinic K⫹ channels. (B) The relation between the concentration of GTP and N · Po of muscarinic K⫹ channels. N · Po is normalized to the maximal N · Po induced by 10 애M GTP웂S in each recording. Symbols and bars are mean ⫾ SD (n ⫽ 3). Continuous lines indicate the fit with the Hille equation for the relationship between GTP and channel activity in the presence of each concentration of ACh as indicated with each line. Reproduced from Ito et al. (1991), with permission of The Rockefeller University Press.

channel states U-A, where U represents ‘‘unavailable’’ and A represents ‘‘available’’ states of the channel. Within this framework, the U-A transition is independent of the fast transition C2-C1-O, and the A, but not the U, state allows the channel to be conducting when the channel passes into the O state. It is suggested that GK causes a shift of the equilibrium toward A to increase channel activity. This shift can be well explained by the Monod–Wyman–Changeux allosteric model, assuming

that the channel is composed of at least three functionally identical subunits (Fig. 11C).

B. Molecular Aspects of KACh Channels Kir3.1 from rat atrium was the first subunit of KACh channels to be isolated (Dascal et al., 1993; Kubo et al., 1993b). Two additional Kir3.1 homologues were isolated from brain and one from heart and designated

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FIGURE 11 Spectral analysis of muscarinic K⫹ channel currents in an inside-out patch. (A) Muscarinic K⫹ channel currents in the inside-out patch membrane from a guinea pig atrial myocyte. Channel currents were recorded at ⫺60mV with symmetrical 150 mM K⫹ solutions. The patch pipette also contained 0.5 애M acetylcholine. Different concentrations of GTP were applied to the internal side of the patch membrane as indicated above each current trace. (B) Power density spectra calculated from data shown in A. Each spectrum could be fitted with the sum of two Lorenzian functions. F1 and F2 indicated by arrows indicate the corner frequencies of the slow and the fast Lorenzian components, respectively. (C) (a) The fraction of the ‘‘available’’ state of the channels (A/(A⫹U)) was calculated from A. Symbols indicate the relationship between GTP concentration and the calculated A/(A⫹U). Lines indicate the fit of data with the Monad– Wyman–Changeux allosteric model with different ‘‘n’’ values. (b) Schematic representation of the Monad– Wyman–Changeux allosteric model. In this scheme, each muscarinic K⫹ channel is assumed to be a tetramer. Each subunit is in either the tense (T) or the relaxed (R) state, which is also represented by squares and circles, respectively. Independently of each other, each subunit in the T or R state binds with one activated G protein (solid circles) with the dissociation constant of KT or KR , respectively. In this model, all subunits in the same oligomer must change their conformations simultaneously. Therefore, the channel can be in either T4 or R4 states. T4 or R4 states are in equilibrium through an allosteric constant L. Reproduced from Hosoya et al. (1996), with permission of The Rockefeller University Press.

Kir3.2, Kir3.3, and Kir3.4, respectively. One more isoform was isolated from Xenopus oocytes and was designated Kir3.5 (XIR) (Hedin et al., 1996). The amino acid sequence of Kir3.5 is most homologous to that of Kir3.4. At least four different splice variants of Kir3.2 are transcribed from a single gene and are designated Kir3.2a, Kir3.2b, Kir3.2c, and Kir3.2d (Inanobe et al., 1999; Isomoto et al., 1996a; Stoffel et al., 1995). Kir3.2c is the longest form of the splice variants. Kir 3.2a is 11 amino acids shorter at its C terminus than Kir3.2c. Kir3.2d is 8 amino acids shorter at its N terminus than Kir3.2c. Kir3.2b is 87 amino acids shorter at its C terminus than Kir3.2a (Isomoto et al., 1996a). We also found at least one splice variant of Kir3.1. The mRNA expression of each subunit of Kir3.0s was examined in various tissues (Dascal et al., 1993; Iizuka et al., 1995; Krapinvinsky et al., 1995; Kubo et al.,

1993b; Lesage et al., 1994) (Table I). In the heart, only the expression of Kir3.1 and Kir3.4 mRNAs is detected. Immunohistochemical analysis using a specific antibody against Kir3.1 showed that Kir3.1 is localized homogeneously on the plasma membrane of atrial but not ventricular myocytes (Fig. 12A). Krapivinsky et al. (1995) revealed that Kir3.1 is immunoprecipitated with Kir3.4 from atrial myocytes. Furthermore, coexpression of Kir3.1 and Kir3.4 in Xenopus oocytes yields greatly enhanced KACh channel currents compared with expression of either of the subunits alone (Fig. 12B). Now it is generally believed that the Kir3.1 subunit alone fails to give rise to KACh currents in mammalian cell lines, including CHO, COS, and HEK cells (Krapinvinsky et al., 1995; Philipson et al., 1995; Spauschus et al., 1996; Wischmeyer et al., 1997). In Xenopus oocyte, Kir3.5 is expressed endogenously in various amounts dependent

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TABLE I mRNA Expression of Kir3.0s in Various Tissuea

Brain Heart Atrium Ventricle Lung Liver Spleen Pancreas Kidney Skeletal muscle Testis

Kir3.1

Kir3.2

Kir3.3

2⫹ b,c

2⫹ d,e

2⫹ d

⫹f

2⫹ b,c ⫹/⫺ b,c ⫹c ⫹/⫺ b

⫺ d,e ⫺ d,e ⫺ d,e ⫺ d,e ⫺e ⫹e ⫺ d,e ⫺ d,e ⫹e

⫺d ⫺d ⫺d ⫺d ⫺f ⫹/⫺ f ⫺d ⫹/⫺ d

2⫹ f,g ⫹/⫺ g or 2⫹ f

⫹/⫺ b,c

Kir3.4

⫺f ⫹f

a

Expression was examined by Northern blot analysis. 2⫹, strong expression; ⫹, distinct expression; ⫹/⫺, marginal expression; ⫺, no expression; no indication, not examined. b From Kubo et al. (1993). c From Dascal et al. (1993). d From Lesage et al. (1994). e From Stoffel et al. (1995). f From Iizuka et al. (1995). g From Krapivinsky et al. (1995).

on the individual frog, which may explain the inconsistent expression of G-protein-gated K⫹ channel currents in oocytes injected with Kir3.1 cRNA alone. These data indicate that the cardiac KACh channel is a heteromultimer composed of Kir3.1 and Kir3.4. The electrophysiological characters of the reconstituted channels, which contain Kir3.1 and Kir3.4, are also consistent with the electrophysiological studies from native cardiac myocytes. Kir3.0 clones contain various functional motifs, which may be important for the physiological function of the channels. Kir3.1 possesses an amino acid sequence homologous to the G웁웂-activating domain of adenylate cyclase 2 (N-X-X-E-R) in its C terminus. This is therefore the candidate for the G웁웂-activating site on the KACh channel (Reuveny et al., 1994). As with all other Kir channel subunits, Kir3.0s possess conserved cationic residues adjacent to the C-terminal end of the M2 domain, which were reported to be critically involved in the phosphatidylinositol 4,5-bisphosphate (PIP2)induced activation of Kir channels (Huang et al., 1998). Furthermore, all of the Kir3.0 subunits have an arginine–glycine–aspartate (RGD) motif (integrin-binding motif) in their linker region between M1 and H5 (Hynes, 1992). Integrins also recognize this RGD motif in KACh channels, and this motif is indispensable for the plasma membrane localization of Kir3.1 and Kir3.4. In fact, mutation of the RGD motif to RGE in these channel subunits decreased the G-protein-activated K⫹ current flowing through the channels reconstituted from these mutational channel subunits (McPee et al., 1998).

It was reported that either Gi- or Gs-coupled receptors, when expressed together with reconstituted KACh channels in Xenopus oocytes, can activate the channels (Lim et al., 1995). This may indicate that under the conditions where compartmentalization does not exist, as in Xenopus oocytes, the 웁웂 subunits released from Gs may be able to activate KACh channels. Because 웁-adrenergic agonists do not activate these channels in vivo, there must be some mechanism to guarantee the specificity of the native GK –KACh channel system in atrial cardiac myocytes. Further studies are needed to elucidate the mechanism for this.

C. Modulation of KACh Channels When the reconstituted KACh channel is expressed in Xenopus oocytes, the time course of the activation and deactivation is much slower than in native atrial myocytes (Krapinvinsky et al., 1995). Newly identified proteins known as regulators of G-protein signaling (RGS) serve to increase the activation rate of reconstituted KACh channels expressed in vitro (Doupnik et al., 1997; Saitoh et al., 1997). RGS proteins are members of a multigene family that enhance the intrinsic GTPase activity of certain G-proteins (mainly Gi /Go classes), probably by preferentially binding to and stabilizing G-protein 움 subunits in their transition state for the hydrolysis reaction (Koelle, 1997). Sixteen RGS homologues (RGS1–16) have been identified in mammals so far. Among them, RGS1, RGS3, RGS4, and RGS8 have been shown to shorten the time to peak of receptor-mediated activation of reconstituted KACh channels (Doupnik et al., 1997; Saitoh et al., 1997). Enhancement of GTPase activity by RGS proteins leads to an increase in the off rate of the G-protein-mediated reaction (Koelle, 1997). This effect, at least in theory, could abbreviate the time to peak even when the on rate of the reaction is not altered by the protein (Doupnik et al., 1997; Saitoh et al., 1997). In this case, the steady-state KACh channel activity should be decreased in the presence of a given concentration of an agonist. However, RGS proteins enhance the activation rate without changing the amplitude of the steady-state response in the reconstituted KACh channel system (Doupnik et al., 1997; Saitoh et al., 1997). One possible explanation of this phenomenon is that RGS proteins may also enhance the GDP/GTP exchange rate of GK . However, this could not be confirmed, at least in an in vitro system that lacked reconstituted receptor proteins (Saitoh et al., 1997). It is still possible that RGS proteins might increase the on rate of the GK-mediated reaction only in the presence of receptors or, alternatively, accelerate the subunit dissociation of GK . Further studies are necessary to identify

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FIGURE 12 Muscarinic K⫹ channel in rat heart. (A) Immunohistochemical analysis of Kir 3.1 proteins in the rat atrium (a) and ventricle (b). Homogeneous immunoreactivity was found on the plasma membrane of atrial, but not on ventricle myocytes. (B) Acetylcholine (ACh)-activated K⫹ currents observed in Xenopus oocytes expressing M2-muscarinic receptors (m2R) plus mouse Kir3.1 (m-GIRK1) and/or human Kir3.4 (h-GIRK4). ACh (1 애M)induced K⫹ currents at different membrane potentials were measured in the presence of 96 mM external K⫹ and are shown under the table. The combination of Kir3.1 and Kir3.4 increased Ach-induced current. The voltage-clamp protocol is depicted in the lower left corner.

the mechanism by which the RGS proteins accelerate the agonist-mediated KACh channel activation without affecting the steady-state response.

IV. ATP-SENSITIVE K⫹ CHANNELS IN THE HEART (KATP) A. Physiological Roles of KATP Channels The ATP-sensitive K⫹ (KATP) channel is a weakly inwardly rectifying K⫹ channel inhibited by intracellular ATP (ATPi) and activated by intracellular nucleoside diphosphates (NDPi). Thus, it provides a link between cellular metabolism and excitability (Terzic et al., 1994, 1995). The cardiac KATP channel may be involved in the increase of K⫹ efflux and shortening of the action

potential duration in the ischemic heart (Faivre and Findlay, 1989; Findlay et al., 1989; Nichols and Lederer, 1991). Both are major factors contributing to the electrophysiological abnormalities that predispose the heart to the development of reentrant arrhythmias (Gasser and Vaughan-Jones, 1992; Wilde, 1993; Wilde and Janse, 1994). However, opening of the cardiac KATP channel has also been implicated as a cardioprotective mechanism underlying ischemia-related preconditioning (Cole, 1993; Downey, 1992; Gross and Auchampach, 1992; Grover et al., 1992; Parratt and Kane, 1994; Yao et al., 1993). In cell-attached and excised patch recordings from human and guinea pig atrial myocytes, KATP channels are readily distinguished from IK1 , KACh , and KNa channels by their biophysical properties. The degree of inward rectification of KATP channels is much weaker than

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15. Kir in Heart

those of IK1 and KACh channels. The weak inward rectification of KATP channels is also mediated by Mg2⫹ (Findlay, 1987a,b; Terzic et al., 1995). With symmetrical 앑150 mM K⫹ ions, the conductance of the unitary inward current through KATP channels in cardiac myocytes is 앑70 to 90 pS (Ashcroft and Ashcroft, 1990; Heidbuchel et al., 1990; Horie et al., 1987; Kakei and Noma, 1984; Noma, 1983; Trube and Heschler, 1984), which is larger than those of IK1 and KACh channels (앑30 to 45 pS) (Kurachi, 1985; Kurachi et al., 1986a; Sakmann et al., 1983; Sakmann and Trube, 1984a) but smaller than that of the KNa channel (앑180 to 230 pS) (Kameyama et al., 1984; Wang et al., 1991). The KATP current (IKATP) is active at every potential and is essentially time and voltage independent (Nichols and Lederer, 1991; Takano and Noma, 1993; Terzic et al., 1995). KATP channels are known to be regulated by various intracellular factors such as ATPi (Fig. 13) and NDPi . ATPi is the main regulator of KATP channels and has two functions: (1) to close the channels and (2) to maintain channel activity in the presence of Mg2⫹ (Findlay and Dunne, 1986; Ohno-Shosaku et al., 1987; Takano et al., 1990; Trube and Heschler, 1984). The first action of ATPi is referred to as the ‘‘ligand action’’ because it is assumed to require the binding of ATPi to the KATP channel and persists as long as ATPi is bound to the channel. Typically, KATP channels have a very low open probability at physiological concentrations of ATPi . Half-maximum inhibition of the KATP channel in cardiac muscle cells is achieved by 20–30 애M of ATPi , which is independent of whether ATPi is in the free acid form (ATP4⫺) or is bound to Mg2⫹ (MgATP) (Ashcroft and Ashcroft, 1990; Findlay, 1988; Terzic et al., 1995). In pancreatic 웁 cells, however, it has been shown that Mg2⫹ increases half-maximum inhibition of the channel from 4 to 26 애M, suggesting that ATP4- is a more potent inhibitor of the pancreatic KATP channel than MgATP (Ashcroft and Kakei, 1989). Although information about the ATPi inhibition of KATP channels in smooth muscle is limited, MgATP is less effective than ATP4(Kajioka et al., 1991; Nelson and Quayle, 1995). Halfmaximum inhibition of the KATP channel in the rat portal vein is achieved by 29 애M of ATP4-, whereas MgATP is ineffective (Zhang and Bolton, 1996). The second action of ATPi is referred to as ‘‘hydrolysis dependent’’ because it apparently requires the hydrolysis of ATP in the presence of Mg2⫹ and can last for several hundreds of seconds after the removal of ATPi . The effect of ATPi on KATP channels depends on the state of the channel protein. When the channels are operative, ATPi inhibits channel opening. When the channels are not operative, treatment with MgATP restores channel opening. It has been shown that PIP2 added to the intra-

cellular side of the membrane could restore KATP channel activity after rundown (Shyng and Nichols, 1998) and that the restoration of KATP channel activity by MgATP can be blocked by wortmannin, a phosphoatidylinositol kinase inhibitor (Xie et al., 1999). NDPi , such as ADP, GDP, and UDP, also regulates KATP channel activity. NDPi has two distinct actions: (1) attenuation of ATP-induced channel inhibition by apparent competition to ATPi and (2) permission of KATP channel opening even after rundown (Beech et al., 1993a; Dunne and Petersen, 1991; Faivre and Findlay, 1989; Terzic et al., 1994a; Tung and Kurachi, 1991). The regulation of channel activity by nucleotides is modulated by several factors exogenous to the channel protein. One of these factors is hormones, including galanine and somatostatin, which are known to inhibit insulin secretion through the activation of G-proteins (Wille et al., 1988, 1989). It was reported that while the KACh channel is activated by G웁웂, the KATP channel is activated by a GTP-bound form of Gi움 , at least in the heart (Ito et al., 1992; Terzic et al., 1994b). KATP channels exhibit characteristic pharmacological properties: (1) they are selectively inhibited by antidiabetic sulfonylurea derivatives (SUs), such as glibenclamide and tolbutamide, and (2) they are activated by a certain class of vasorelaxant agents, such as pinacidil, nicorandil, and levcromakalim, which are collectively termed K⫹ channel openers (KCOs) (Table II). The properties of KATP channels vary among tissues. KATP channels in cardiac myocytes are activated by pinacidil but not by diazoxide. KATP channels in pancreatic 웁 cells are activated by diazoxide and only weakly by pinacidil. KATP channels in smooth muscle cells are activated effectively by both of these compounds (Ashcroft and Ashcraft, 1990; Nelson and Quayle, 1995; Terzic et al., 1995).

B. Molecular Aspects of KATP Channels Two Kir subunits belonging to the KATP subfamily, Kir6.1/uKATP-1 and Kir6.2/BIR, have been isolated (Inagaki et al., 1995a,c; Sakura et al., 1995). The predicted amino acid sequences show that Kir6.1 and Kir6.2 are around 70% homologous with each other and 40– 50% homologous with other members of the Kir channel family. The highly conserved motif in the H5 region, Gly–Tyr–Gly, found in all other members of the Kir channel family is Gly–Phe–Gly in both Kir6.1 and Kir6.2. Inagaki et al. (1995b) examined the distribution of their mRNAs by Northern blot analysis and showed that Kir6.1 is expressed ubiquitously in various tissues and that Kir6.2 is expressed in pancreas, heart, skeletal muscle, and brain. While the members of the Kir2.0/ IRK, Kir3.0/GIRK, or Kir1.0/KAB subfamily can func-

294

II. Cellular Electrophysiology

FIGURE 13 Inhibition of KATP channels by ATPi . (A) In the cell-attached configuration of the patch clamp technique (holding potential ⫺75 mV), no activity of the KATP channel can be recorded due to millimolar levels of ATP inside the cardiac cell. After patch excision to the inside-out configuration in an ATP-free solution, several KATP channels immediately appear. Application of micromolar concentrations of ATPi to the cytosolic side of the insideout patch membrane inhibits KATP channel openings. Reproduced from Terzic et al. (1995), with permission. (B) Examples of patch current traces recorded at the indicated membrane potentials. This membrane patch appeared to contain only one channel. The concentration of K⫹ was 140 mM in both sides of the membrane. Current–voltage relationship of this channel records is shown on the left.

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15. Kir in Heart

TABLE II Biophysical and Pharmacological Properties of Native and Cloned KATP Channels Single channel conductance a Native KATP channels Pancreatic 웁 cells

앑50–90 pS

Cardiac myocytes

앑70–90 pS

Skeletal muscle cells

앑55–75 pS

Smooth muscle cells

앑30 pS 앑100–250 pS h

SUR/Kir channels SUR1/Kir6.2

e

앑75 pS

SUR2A/Kir6.2

앑80 pS

SUR2B/Kir6.2

앑80 pS

SUR2B/Kir6.1

앑33 pS

Effect of ATPi

(IC50)b

Hill coefficient b

Glibenclamide (IC50 )

Tolbutamide (IC50 )

Pinacidil (EC50 )

Diazoxide (EC50 )

References l

Mg2⫹ (⫺)c: 앑5 애M Mg2⫹ (⫹)d: 앑15–45 애M Mg2⫹ (⫺) and (⫹): 앑20–100 애M

앑1 앑1 앑2–3

앑5–30 nM

앑5–20 애M

⬎500 애M

앑20–100 애M

1

앑5 nM

앑400 애M

앑10–30 애M

1–4

Mg2⫹ (⫺): 앑10–20 애M Mg2⫹ (⫹): 앑200 애M Mg2⫹ (⫺): 앑30–200 애M f Mg2⫹ (⫹): stimulatory at mM or inhibitory at 앑1 mM f

앑1.5–2 앑1.5–2

앑10–200 nM

앑50 애M

앑100 애M

No effect or inhibitory at 500 애M No effect

5–10

앑25 nM

앑350 애M

앑0.5 애M

앑40 애M

11–15

앑2–10 nM

앑5–30 애M

앑150 nM

앑120 애M

⬎70% inhibition at 1 애M ⬎90% inhibition at 3 애M

⬎70% inhibition at 500 애M

Mg2⫹ (⫺): 앑10 애M Mg2⫹ (⫹): 앑10–300 애M Mg2⫹ (⫺): 앑150 애M Mg2⫹ (⫹): 앑100–150 애M Mg2⫹ (⫺): 앑70 애M Mg2⫹ (⫹): 앑300 애M Mg2⫹ (⫺): no effect i Mg2⫹ (⫹): stimulatory at 1 mM, inhibitory at 10 mM j

앑1 앑2 앑2 앑2 앑1.5

g

g

No effect at 1 mM 앑10 애M 앑1 애M Stimulatory at ⱖ1 애M k

g

앑50 애M

16–19

No effect at 200 애M Stimulatory at 200 애M Stimulatory at 200 애M k

20, 21 22 23, 24

a

Values under symmetrical 150 mM K⫹ or similar conditions. Only applicable when ATP inhibits the channels. In the absence of intracellular Mg2⫹. d In the presence of intracellular Mg2⫹, whose concentration differs among studies but usually several hundred 애M to several mM in total. e The small conductance ATP-sensitive K⫹ channel or the nucleoside diphosphate-dependent K⫹ channel. f There is a controversy on the effect of ATP. Some groups reported only stimulatory effect, whereas others showed both stimulatory and inhibitory effects. g Data were obtained from smooth muscle cells of the mesenteric artery in which the nucleoside diphosphate-dependent K⫹ channels are known to exist. Because the experiments were done in whole cell configuration (Quayle et al., 1995), however, the single-channel conductance of the channels responding to these agents was unknown. h So-called large conductance of ATP-sensitive K⫹ channels in smooth muscle cells, which are so heterogeneous that no representative values are given to these channels. i This means no stimulatory effect. Inhibitory effect could not be examined because this channel does not possess spontaneous opening and because neither nucleoside diphosphates nor K⫹ channel openers activated the channel in the absence of Mg2⫹. j Effect of ATP on its own in the absence of spontaneous activity, nucleotide diphosphates, or K⫹ channel opener. k These effects of K⫹ channel openers are completely dependent on intracellular Mg2⫹ and nucleoside di- or triphosphates. l 1, Ashcroft and Ashcroft (1990); 2, Hamada et al. (1990); 3, Findlay (1992); 4, Faivre and Findlay (1989); 5, Weik and Neumcke (1989 and 1990); 6, Vivaudou et al. (1991); 7, Benton and Haylett (1992); 8, Allard and Lazdunski (1993); 9, McKillen et al. (1994); 10, Allard et al. (1995); 11, Kaijioka et al. (1991); 12, Beech et al. (1993a,b); 13, Kamouchi and Kitamura (1994); 14, Zhang and Bolton (1995 and 1996); 15, Quayle et al. (1995 and 1997); 16, Inagaki et al. (1995a); 17, Sakura et al. (1995); 18, Nichols et al. (1996); 19, Gribble et al. (1997a); 20, Inagaki et al. (1996); 21, Okuyama et al. (1998); 22, Isomoto et al. (1996); 23, Yamada et al. (1997); 24, Satoh et al. (1998). b c

tion as Kir channels for themselves (Ho et al., 1993; Jan and Jan, 1992, 1994; Krapinvinsky et al., 1995; Kubo et al., 1993b; Pongs, 1992), both Kir6.1 and Kir6.2 appear to require coupling with sulfonylurea receptors (SUR) to form functional KATP channels (Inagaki et al., 1995a, 1996; Isomoto et al., 1996b; Yamada et al., 1997). Inagaki et al. (1995c, 1996) demonstrated that the pancreatic 웁 cell and cardiac KATP channels are a complex composed of Kir6.2 and the sulfonylurea receptors, SUR1 and SUR2A, respectively (Fig. 14). SUR proteins are assumed to possess 17 putative transmembrane regions (Tusnady et al., 1997), two nucleotide-binding folds with Walker A and B consensus motifs, two N-linked glycosylation sites, and several protein kinase A- and C-dependent phosphorylation sites. The unitary conductance of the channel, which is composed of SUR1 and Kir6.2, is 76 pS in symmetric 140 mM K⫹ solution. ATPi inhibits the channel activity of this channel with a half-maximal value of 10 애M. These properties are similar to those of the pancreatic

웁-cell KATP channel (Cook and Hales, 1984; Dunne and Petersen, 1991; Findlay et al., 1985; Garrino et al., 1989). Northern blot analysis has revealed that SUR1 mRNA is expressed at a high level in pancreatic islets and at low levels in heart and skeletal muscle (Aguilar-Brown et al., 1995; Inagaki et al., 1995a). Thus, SUR1 seems to form the pancreatic 웁-cell KATP channel with Kir6.2. Mutations in the SUR1 protein have been shown to cause the KATP channel to become nonfunctional, which results in persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disease associated with unregulated insulin secretion (Kane et al., 1996; Thomas et al., 1995). The second sulfonylurea receptor, SUR2, has two isoforms designated SUR2A and SUR2B, respectively (Inagaki et al., 1996; Isomoto et al., 1996b). Sequence analysis indicates that they have the same animo acid sequence except for 42 amino acid residues in the carboxyl-terminal ends, suggesting that these two sulfonylurea receptors are formed by alternative splicing from a single gene. The amino acid sequence of

296

II. Cellular Electrophysiology

FIGURE 14 Molecular structure of ATP-sensitive K⫹ channels. Pancreatic, cardiac, and skeletal muscle ATP-sensitive K⫹ (KATP) channels are considered to be composed of two distinct subunits: a sulfonylurea receptor (SURs) and a Kir channel subunit, Kir6.2 (Inagaki et al., 1995a,c; Sakura et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996a,b; Aguilar-Bryan et al., 1997, 1998). With SUR2B, Kir6.1 composes the ‘‘small conductance KATP’’ or ‘‘nucleoside-diphosphate-dependent K⫹ (KNDP)’’ channel in vascular smooth muscle (Yamada et al., 1997).

SUR1 has 66 and 67% identity with those of SUR2A and SUR2B, respectively. One more splice variant of mouse SUR2A, which has a deletion of 35 amino acid residues in the intracellular loop between the 11th and the 12th membrane-spanning domains, was also cloned, but its function has not been examined (Chutkow et al., 1996). It is now designated SUR2C (Ashcroft and Gribble, 1998). Coexpression of Kir6.2 and either SUR2A or SUR2B also elicits KATP channel current with a single channel conductance of 앑80 pS in symmetric 앑150 mM K⫹ solution. The SUR2A/Kir6.2 channel activity is inhibited by ATPi in a concentration-dependent manner with a half-maximal value of 100 애M and is only partially inhibited by glibenclamide at 1 애M (Inagaki et al., 1996). In contrast to the SUR1/Kir6.2 channel, the SUR2A/Kir6.2 channel is activated by pinacidil but not by diazoxide without enough concentration of intracellular ADP (D’hahan et al., 1999). These are characteristics of cardiac and skeletal muscle KATP channels (Ashcroft and Ashcroft, 1990; Faivre and Findlay, 1990; Findlay, 1992; Fosset et al., 1988; Nichols and Lederer, 1991; Nichols et al., 1991; Terzic et al., 1994, 1995). The SUR2B/Kir6.2 channel is activated by both pinacidil and diazoxide, a characteristic that closely resembles the responses of the smooth muscle KATP channel to KCOs (Beech et al., 1993b; Kajioka et al., 1991; Lorenz et al., 1992; Nelson and Quayle, 1995; Standen et al., 1989). Thus, the pharmacological properties of KATP channels may be determined by the individual sulfonyl-

urea receptors. Without enough concentration of intracellular ADP, diazoxide activates KATP channels containing either SUR1 or SUR2B, but not SUR2A, the alternative splicing region between SUR2A and SUR2B may be a functional domain important for the diazoxide activation of KATP channels (Yamada et al., 1997). Interestingly, the sequence of the last 42 amino acids of SUR2B exhibits 74 and 33% identity with those of the corresponding region of SUR1 and SUR2A, respectively. Because the KATP channel containing SUR2A becomes diazoxide sensitive in the presence of 100 애M intracellular ADP, the intracellular ADP may change the conformation and an diazoxide accessibility of SUR2A. However, the functional domain for pinacidil may not involve the carboxyl-terminal end because pinacidil activates KATP channels reconstituted from SUR2A or SUR2B but not from SUR1 (Isomoto et al., 1996b). The RT-PCR analysis showed that SUR2A mRNA is expressed in heart, skeletal muscle, cerebellum, eye, and urinary bladder, whereas SUR2B mRNA ubiquitously distributes not only in these tissues but also in forebrain, lung, liver, pancreas, kidney, spleen, stomach, small intestine, colon, uterus, ovary, and fat tissue (Isomoto et al., 1996b). Using a probe that is common to SUR2 isoforms, in situ hybridization showed that SUR2 was expressed in the parenchyma of the heart and skeletal muscle and in the vascular structures of various tissues (Chutkow et al., 1996). Because SUR2A is believed to be the cardiac and skeletal muscle-type sulfonylurea

15. Kir in Heart

receptor, other isoforms of SUR2 would be expressed in the vascular structure. This supports the idea that SUR2B represents a part of the vascular smooth muscletype KATP channels. However, to clarify the distribution of each isoform of the sulfonylurea receptor, further studies using the specific probes are needed. The stoichiometry of SURs and Kir subunits in KATP channels was investigated with SUR1/Kir6.2 fusion constructs (Clement et al., 1997; Inagaki et al., 1997; Shyng and Nichols, 1998). These studies indicated that the KATP channels are hetero-octamers, consisting of four SURs interacting with four Kir6.2 subunits. Although Ammala et al. (1996) demonstrated that Kir6.1 also couples to SUR1 and acquires sulfonylurea sensitivity, other features of KATP channels, such as regulation by ATPi and NDPi and activation by K⫹ channel openers, have not been studied extensively on the channel reconstituted from Kir6.1 and SUR1. It has been found that Kir6.1 can form a smooth muscle NDPi-sensitive K⫹ channel with SUR2B (Satoh et al., 1998; Yamada et al., 1997).

C. Modulation of KATP Channels One of the hallmarks of native KATP channels is the inhibition of channel activity by micromolar concentrations of ATPi (Ashcroft, 1988; Noma, 1983; Terzic et al., 1995). Tucker et al. (1997) found that Kir6.2, which has 26 amino acids deleted from the carboxyl terminus (Kir6.2⌬C26), can be functionally expressed in the absence of SURs. A charge-neutralization mutation on lysine 185 of Kir6.2⌬C26 reduces the ATPi sensitivity of the Kir6.2⌬C26 channel by 앑40 times. The ATPi sensitivity of the Kir6.2⌬C26 channel was increased 5–8 times by coexpression of SUR1. Thus, it seems likely that the primary inhibitory ATPi-binding site resides in Kir6.2, whereas SUR1 increases the ATPi sensitivity of Kir6.2. Koster et al. (1998) showed that the complex of SUR1 and Kir6.2 where 30 amino acids were deleted from the N terminus (Kir6.2⌬N30) exhibited 앑10 times lower ATPi sensitivity than the complete SUR1/Kir6.2 channel. Interestingly, the complete SUR1/Kir6.2⌬N30 channel was also less sensitive to intracellular ADP (ADPi) and tolbutamide than the SUR1/Kir6.2 channel. Therefore, Kir6.2 might interact with SUR1 through its N terminus, and the low ATPi sensitivity of the SUR1/ Kir6.2⌬N30 channel might be due to impaired coupling between SUR1 and Kir6.2. Because ATPi inhibits SUR2A/Kir6.2 and Kir6.2⌬C26 channels with similar potencies (Ki of 앑100 애M) (Okuyama et al., 1998; Tucker et al., 1997), SUR2A may not substantially enhance the ATPi sensitivity of Kir6.2. The native cardiac KATP channel is 앑3 to 10 times more sensitive to ATPi than the SUR2A/Kir6.2 channel (Table II). Thus, some unidentified factors in cardiac myocytes might sensitize

297

the SUR2A/Kir6.2 channel to ATPi in vivo. In fact, various factors, including intracellular polyvalent cations and actin polymerization, have been suggested to affect the ATPi sensitivity of the native cardiac KATP channel (Deutsch et al., 1994; Hiraoka et al., 1996; Terzic and Kurachi, 1996). The SUR2A/Kir6.2 channel was equally sensitive to Mg2⫹-free and Mg2⫹-bound ATPi , whereas the SUR2B/Kir6.2 channel was more sensitive to Mg2⫹-free than Mg2⫹-bound ATPi . This difference could be ascribed to the difference in the amino acid sequence of the C terminus between SUR2A and SUR2B (Isomoto et al., 1996b). NDPi such as UDP exhibit distinct effects on the cardiac KATP channel before and after rundown (Terzic et al., 1995). UDP antagonizes the inhibitory effect of ATPi before rundown. After rundown, UDP restores the channel activity without attenuating the ATPi sensitivity of the channels (Tung and Kurachi, 1991). The SUR2A/Kir6.2 channel mimics such a dualistic response of the cardiac KATP channel to NDPi (Okuyama et al., 1998). Thus, SUR2A/Kir6.2 and native cardiac KATP channels respond to NDPi in a very similar way. Nichols et al. (1996) found that a human mutation (G1479R) in the second NBF of SUR1 abolished the antagonizing effect of ADPi on the ATPi-induced inhibition of the SUR1/Kir6.2 channel. It was also reported that Mg2⫹-ADPi inhibited the Kir6.2⌬C26 channel but activated the SUR1/Kir6.2⌬C26 channel (Tucker et al., 1997) and that either the K719A or the K1384M mutation of rat SUR1 abolished the activating effects of ADPi on the partial rundown of the SUR1/Kir6.2 channel both in the presence and in the absence of ATPi (Gribble et al., 1997). A hydrolysis-resistant 웁-methylene ADP failed to activate the SUR1/Kir6.2 channel. Thus, it is likely that hydrolysis of Mg2⫹-ADPi (and probably also the other NDPi) at the NBFs of SUR1 may be critically involved in the activating effect of the nucleotides. Although no corresponding studies have been done in SUR2s, a similar mechanism would probably underlie the activating effect of NDPi on the SUR2/ Kir6.2 channel. Kir6.2⌬C26 channels also exhibit rundown and can be reactivated with MgATPi (Tucker et al., 1997), indicating that the rundown/refreshment is primarily associated with a certain functional alteration of Kir6.2. PIP2 added to the intracellular side of the membrane could restore KATP channel activity after rundown (Hilgeman and Ball, 1996). Furthermore, the ATPi-mediated restoration of activity was inhibited by antibodies against PIP2 . Thus, PIP2 and its generation by ATP-dependent lipid kinases appear to be critically involved in spontaneous KATP channel activity. More recently, PIP2 was reported to shift the dose–response curves of KATP chan-

298

II. Cellular Electrophysiology

nel inhibition by ATPi toward higher concentrations. This result suggests that PIP2 metabolism may regulate the ATP sensitivity of KATP channels (Baukrowitz et al., 1988; Shyng and Nichols, 1998). The response of KATP channels to NDPi may have to be reexamined from this aspect.

V. Na⫹-ACTIVATED K⫹ CHANNELS (KNa) IN THE HEART A. Physiological Roles and Electrophysiological Properties of KNa Channels The cardiac KNa channel was first described by Kameyama et al., (1984). This channel is activated by Nai⫹ ⬎20 mM and shows inwardly rectification and has the highest conductance (⬎200 pS) among potassium channels in the heart. In inside-out membrane patches, relatively high concentrations of Na⫹ ions are required to activate KNa channels. The KD for activation was 66 mM with a Hill coefficient of 2.8 (Kameyama et al., 1984). The threshold for activation of the channel is around 30 mM. This low sensitivity of the channel for sodium ions raised the question about the eventual role of the channel, as the physiological intracellular Na⫹ concentration is estimated to be around 10 mM or less (Guarnieri, 1987; Pike et al., 1990). However, higher concentrations of intracellular Na⫹ may occur in pathological conditions, such as ischemia (Guarnieri, 1987; Pike et al., 1990), in digitalis intoxication (Eisner et al., 1984), and during perfusion with Ca2⫹ ,Mg2⫹-free medium (Rodrigo and Chapman, 1990). The sensitivity of KNa channels to Na⫹ is greater in cell-attached than in excised membrane patches. Rodrigo et al. (1993) suggested that the sensitivity of the channel in inside-out conditions is probably underestimated due to the loss of some intracellular factors. In neurons, however, the current has been claimed to play a physiological role as a repolarizing current during the action potential, where the KNa channel is believed to be activated by the flush-

ing of Na⫹ into cytoplasm during the upstroke of the action potential (Bader et al., 1985; Dryer, 1991).

B. Pharmacological Regulation of KNa Channels Many drugs have been tested but none can be considered selective for the KNa channel. The first drug described to block the KNa channel was the experimental drug R56578, known for its inhibition of the electrophysiological effects of digitalis intoxication (Vollmer et al., 1987). It inhibits other currents with even higher potency, such as the transient inward current (Leyssens and Carmeliet, 1991) and the slowly inactivating Na⫹ current (Carmeliet and Tygat, 1991). Block of the KNa channel has also been reported with other drugs (Luk and Carmeliet, 1990). The most efficient are flunarizine, aminodarone, nicardipine, verapamil, and tedisamil. KATP channel blockers such as glibenclamide or class I drugs (lidocaine) are not effective at therapeutic concentrations. Although many groups are trying to isolate the cDNA for KNa channels, none have been isolated yet.

VI. SUMMARY This chapter examined the most recent molecular biological information concerning inwardly rectifying K⫹ channels in myocardium. The combination of molecular biological and electrophysiological studies has developed the way toward understanding the properties of these channels: (1) their ultrastructure, (2) their mechanism of regulation, and (3) their distribution in various tissues. These results enable us to understand the different and specific characteristic properties of each channel subunit and the mechanism of action of drugs and regulators at the molecular level. Further studies to clarify the roles of the Kir channels in the heart are indispensable for understanding cardiac physiology and pathophysiology. Understanding cardiac physiology at the molecular level will allow the further development of treatment, strategies, and pharmacological agents to combat cardiac diseases.

Appendix: Two-Pore K⫹ Channels I. INTRODUCTION Since the cloning of the Shaker K⫹ channel (Tempel et al., 1987), many types of K⫹ channels have been identified by expression and homology cloning and by search-

ing in the GenBank for conserved K⫹ channel pore domain sequences. K⫹ channels can now be structurally classified into three major groups defined by whether they contain two, four, or six putative transmembrane (TM) regions. K⫹ channels with six TM regions include

299

15. Kir in Heart

FIGURE 15 The phylogenetic tree of 2P/4TM K⫹ channels. The phylogenetic tree indicates four subfamilies of 2P/4TM K⫹ channels defined by TWIK-1, TASK-1, TASK-2, and TREK-1 (associated with TRAAK). mTWIK-1 is mouse TWIK-1 (GenBank No. U33632). mTREK-1 is mouse TREK-1 (GenBank No. U73488). mTASK-1 (mc TBAK-1) is mouse TASK-1 (cTBAK-1) (GenBank Nos. AF006823 and AB013345). mTRAAK is mouse TRAAK (GenBank No. AF056492). hTASK-2 is human TASK-2 (GenBank No. AF084830).

voltage-dependent Kv channels, such as Shaker and HERG, and large and small conductance Ca2⫹-activated K⫹ channels, whereas those with two TM regions are the inwardly rectifying Kir channels of which there are seven subfamilies (Kir 1.0–Kir 7.1) (Do¨ring et al., 1998; Jan and Jan, 1997; Krapivinsky et al., 1998; Nichols and Lopatin, 1997). K⫹ channels with four TM regions have two poreforming domains (2P/4TM K⫹ channel) (Lesage and Lazdunski, 1999). Data produced by the Caenorhabditis elegans genome sequencing project have revealed an unexpectedly large extended gene family of K⫹ channels in this simple animal of less than 1000 cells (Wei et al., 1996). About 80 genes have been identified that encode K⫹ channel subunits of which an unusually large number (앑50) encode the novel class of 2P/4TM K⫹ channels. In mammal, five 2P/4TM K⫹ channel subunits—TWIK-1 (Lesage et al., 1996a), TREK-1 (Fink et al., 1996), TRAAK (Fink et al., 1998), TASK (cTBAK)-1 (Duprat et al., 1997; Kim et al., 1998), and TASK-2 (Reyes et al., 1998)—have been identified so far (Fig. 15). This appendix focuses on the electrophysiological, pharmacological, and regulatory properties of 2P/4TM K⫹ channels and discusses their physiological roles in the heart.

II. STRUCTURE OF 2P/4TM K⫹ CHANNELS In primary structure, 2P/4TM K⫹ channel proteins do not display any significant sequence similarity with other cloned K⫹ channel subunits except in the two regions that are highly homologous to the pore-like domain (P-domain) signature sequence. Even then the sequence conservation among 2P/4TM K⫹ channels is low (around 25% of amino acid identity) (Lesage and Lazdunski, 1999). Hydropathy analysis of 2P/4TM K⫹ channels predicts the presence of four TM regions (M1 to M4), with M1 and M2 flanking the first P domain

(P1) and M3 and M4 flanking the second P domain (P2) (see Fig. 4). Another important feature is the presence of an extended domain between M1 and P1 that is not found in Kv or Kir proteins. Finally, they do not contain a S4-like domain, which is responsible for the voltage dependence of Kv channels. Kv and Kir channel subunits, each of which contains a single P domain, have been shown to assemble functional ion channels by the association of four individual subunit proteins (see Fig. 4) (Jan and Jan, 1994; MacKinnon, 1991; Yang et al., 1995b). A tetrameric arrangement of K⫹ channel subunits therefore suggests that 2P/4TM K⫹ channels would assemble as dimers, as each subunit protein already contains two P domains (Lesage et al., 1996b). Actually, Western blot analysis of TWIK-1 (for Tandem of P domains in a Weak Inward rectifier K⫹ channel) reveals the presence of an immunoreactive complex of 65–80 kDa in the absence of reducing agents. This complex is dissociated by the reducing agent 웁-mercaptoethanol to give a band of 40 kDa. This suggests that TWIK-1 can form homodimeric complexes containing an interchain disulfide bond between two subunits. A mutant of TWIK-1 (C69S), in which the cysteine residue 69 located in the extracellular M1P1 loop is replaced by a serine, cannot form the 68- to 80kDa complex and also fails to produce active channels (Lesage et al., 1996b). All 2P/4TM K⫹ channels except TASK (cTBAK)-1 (for TWIK-related Acid Sensitive K⫹ channel and cardiac Two pore Background K⫹ channel) have a cysteine residue in the M1P1 loop. This indicates that disulfide bridge dimerization is not the rule to form functional 2P/4TM K⫹ channels. Another important feature is that TASK (cTBAK)-1 possesses a sequence, S-S-V, in the C-terminal end that corresponds to the anchoring protein, PSD-95, family binding motif (T/S-X-V), and both of these serine residues can be phosphorylated by protein kinase A (PKA) (Kim et al., 1998). Kir2.3, which possesses this motif, binds to PSD-95 through its C-terminal end, which is inhibited by its phosphorylation by PKA. It is therefore possible that the subcellular localization and/or the function of TASK-1 could be regulated dramatically by the interaction of PSD-95 family anchoring proteins and PKA-mediated phosphorylation.

III. ELECTROPHYSIOLOGICAL PROPERTIES A. Inwardly Rectifying TWIK-1 Currents In Xenopus oocytes, TWIK-1 expression results in time-independent currents that are instantaneous and noninactivating and selective for K⫹ (Lesage et al., 1996a). The current–voltage (I-V) relationship is nearly

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II. Cellular Electrophysiology

linear and saturates only for depolarizations positive to 0 mV. This weak inward rectification is also obtained with TWIK-1 currents recorded in the inside-out membrane patch in the presence of Mg2⫹ on the cytoplasmic side. This rectification disappears in the absence of Mg2⫹. The unitary conductance of individual TWIK-1 channel currents is 34 pS at ⫺80 mV in symmetrical 150 mMK⫹. The kinetic behavior of TWIK-1 channels consists of very rapid open–close transitions. TWIK-1 activity is associated with a strong hyperpolarization of the resting membrane potential (Em), with Em reaching a value close to EK in oocytes that express TWIK-1.

B. Outwardly Rectifying TREK-1 Currents Like TWIK-1, TREK-1 (for TWIK-Related K⫹ channel) expression results in instantaneous and noninactivating currents that are selective for K⫹ (Fink et al., 1996). Unlike TWIK-1, TREK-1 is an outward rectifier. When [K⫹]o is increased, an inward current is revealed that it saturates upon hyperpolarization, the I-V curve is shifted rightward, and the reversal potential of the currents closely followed EK . In inside-out patches, TREK-1 channel current kinetics are very rapid with a single-channel conductance of 48 pS ([K⫹]o ⫽ 150 mM).

sion of the patch, the channel activity is insensitive to acidification of the internal solution obtained either by modifying the Na2HPO4 /NaH2PO4 buffer ratio or by bubbling CO2 . The effect of pH on TWIK-1 channel activity is probably indirect through an unknown intracellular mechanism. TASK-1 (Duprat et al., 1997) and TASK-2 (Reyes et al., 1998) currents are not affected by modifications of internal pH but are very sensitive to variations of extracellular pH in a narrow physiological range (Fig. 16). As much as 90% of the maximum current is recorded at pH 8.4 and only 10% at pH 6.7. The external pH dependence of TASK-1 and TASK-2, recorded at 50 mV, indicates a pK value of 7.3 and a Hill coefficient of 1.6. The Hill coefficient of 앑1.6 found for the H⫹ concentration dependence of the TASK-1 and TASK-2

C. ‘‘GHK’’ Rectifying TASK and TRAAK Currents TASK-1 (Duprat et al., 1997; Kim et al., 1998), TASK-2 (Reyes et al., 1998), and TRAAK (Fink et al., 1998; for TWIK-Related Arachidonic Acid-stimulated K⫹ channel) currents are neither inwardly rectifying like TWIK-1 nor outwardly rectifying like TREK-1, but exhibit a novel type of behavior. Their currents show an outward rectification when external [K⫹] is low, but this rectification is not observed for high [K⫹]o . The rectification can be approximated by the Goldman–Hodgkin– Katz (GHK) current equation that predicts a curvature of the I-V plot under asymmetric ionic conditions. This indicates that they lack intrinsic voltage sensitivity. This behavior has not been observed in the case of K⫹ channels with one pore domain. In inside-out patches, TASK-1, TASK-2, and TRAAK currents have rapid kinetic behavior with a single-channel conductances of 16, 60, and 38 pS, respectively ([K⫹]o ⫽ 150 mM).

IV. REGULATION AND PHARMACOLOGY A. Modulation of TWIK-1, TASK-1, and TASK-2 by pH The activity of TWIK-1 is down regulated by internal acidification (Lesage et al., 1996a). However, after exci-

FIGURE 16 TASK (cTBAK)-1 is modulated by external pH changes in the physiological range. (A) Current–voltage relationships recorded from a TASK-1 (cTBAK-1)-expressing HEK293T cell with a voltage ramp ranging from ⫺100 to 50 mV, 500 msec in duration, from the holding potential of ⫺30 mV, in 40 mM K⫹ solution at pH 5.5 to 8.4. (B) pH dependence of TASK-1 (cTBAK-1) activity recorded in HEK293T cells at ⫺70 (䊊), ⫺50 (䊉), and 30 (䊐) mV (mean ⫾ SEM, n ⫽ 5). For current recorded at ⫺70, ⫺50, and 30 mV, pK values were 7.30, 7.34, and 7.33, respectively, showing that the inhibitory effect of external protons is not voltage dependent.

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currents is consistent with the idea that the channels are formed by the assembly of two subunits, as suggested by the biochemical characterization of TWIK-1 (Lesage et al., 1996b; see Section II).

B. Modulation of TWIK-1 and TREK-1 by Activation of Protein Kinases C and A TWIK-1 activity is upregulated by activation of protein kinase C (PKC) with phorbol-12-myristate-acetate (PMA) (Lesage et al., 1996a). However, PKC probably does not act by direct phosphorylation of the channel protein as mutation of the threonine (T161) in a PKC phosphorylation consensus motif does not abolish the upregulation by PMA. TREK-1 activity can be inhibited by activation of PKA and PKC in oocytes (Fink et al., 1996). Perfusion of a mixture of 3-isobutyl-1-methylxanthine and forskoline to increase intracellular cAMP levels results in inhibition of TREK-1 currents, whereas perfusion of PMA to activate PKC also results in inhibition, with the two types of inhibition being additive. These results suggest that TREK-1 activity is regulated via phosphorylation and dephosphorylation mechanisms implicating PKC as well as PKA. However, because applications of purified PKA and PKC to the internal side of channels in excised inside-out membrane patches have no effect, both mechanisms are probably indirect.

C. Activation of TRAAK and TREK-1 by Arachidonic Acid and Polyunsaturated Fatty Acids Arachidonic acid (AA) can increase TRAAK (Fink et al., 1998) and TREK-1 (Fink et al., 1998) currents. The AA-induced activation is not prevented by a mixture of inhibitors of the AA metabolism pathway, including nordihydroguaiaretic acid for lipoxygenase, indomethacin for cyclooxygenase, clotrimazole for epoxygenase, and 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of all three pathways. This result suggests that AA itself can directly stimulate TRAAK and TREK-1 and that stimulation does not require the production of another active eicosanoid (Fink et al., 1998). TRAAK and TREK-1 channels are also stimulated by other unsaturated fatty acids (FAs) but not by saturated FAs (Fink et al., 1998). The reversible effect of AA on TRAAK and TREK-1 is observed in inside-out patches. These results indicate that the effects of polyunsaturated FAs are probably direct, via binding either to the protein itself or to its immediate lipid environment, and that additional intracellular components are not needed.

D. Mechanosensitivity of TREK-1 and TRAAK TREK-1 channel activity, which is low in the cellattached configuration, can be enhanced by membrane stretch (Patel et al., 1998). TREK-1 is opened in a graded manner by membrane stretch, with a half-maximal activation at ⫺23 mm Hg pressure in inside-out patches. Negative pressure is significantly more efficient than positive pressure for TREK-1 activation (I⫺50 mm Hg / I⫹50 mm Hg : 3.3 ⫾ 0.3). In inside-out patches, an internal application of cytoskeleton-disrupting agents colchicine and cytochalasin D does not alter TREK-1 stretch activation (Patel et al., 1998). Amphipathic molecules are known to cause erythrocyte membranes to either crenate or form cups (Sheetz and Singer, 1974). These changes in cell shape are thought to be due to the preferential insertion of crenators into the external leaflet of the bilayer (Sheetz and Singer, 1974). The anionic crenator trinitrophenol (TNP) and the neutral crenator lysolecithin are able to mimic the activatory effects of stretch on TREK-1. In contrast, cationic amphipathic cup formers such as chlorpromazine and tetracaine inhibit TREK-1 channel activity. These results suggest that the expansion of the lipid bilayer may be responsible for TREK-1 opening, further implying that mechanical force may be transmitted directly to the channel via the lipid bilayer. TRAAK is also mechanosensitive (Fink et al., 1998). In cell-attached patch configuration, the TRAAK channel opens when a negative pressure is applied to the patch pipette. In contrast to TREK-1, colchicine strongly enhances the number of active TRAAK channels and the sensitivity to mechanical stretch. Cytochalasin D also enhances channel activity with, however, a weaker effect. Excision of the patch into the insideout configuration, which is known to disrupt cytoskeletal elements, produces a 10-fold increase in channel activation and significantly lowered the threshold for mechanical activation. Internal application of the membrane crenator TNP enhanced the opening of TRAAK by pressure. These data suggest that TRAAK channels may be sensitive to mechanical forces transmitted via the membrane, which is tonically repressed by the cytoskeleton. The mechanism of channel regulation through interaction with cytoskeletal components has not been elucidated.

E. Pharmacology of 2P/4TM K⫹ Channels Classical blockers of K⫹ channels are not very efficient on these channels. TWIK-1 (Lesage et al., 1996a), TREK-1 (Fink et al., 1996), TASK-1 (Duprat et al., 1997; Kim et al., 1998), TASK-2 (Reyes et al., 1998), and TRAAK (Fink et al., 1998) currents are insensitive to 4-aminopyridine (4-AP) and tetraethylammonium

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(TEA), except TWIK-1, which is slightly blocked by TEA (30% inhibition with 10 mM). TWIK-1 and TASK-2 are inhibited by quinine (TWIK-1; 50% of inhibition at 50 애M, TASK-2; IC50 ⫽ 20 애M), whereas TREK-1, TASK-1, and TRAAK are not. BaCl2 blocks 50% of TWIK-1 and TREK-1 currents at 0.1 mM. TASK-1 and TASK-2 are only sensitive to a high concentration of BaCl2 (TASK-1; 80% of inhibition with 3 mM, TASK-2; 55% of inhibition with 1 mM BaCl2). Finally, these 2P/4TM K⫹ channels are insensitive to toxins that block Kv or Kca channels (charybdotoxin, apamin, and dendrotoxin) and to the KATP channel blocker glibenclamide or to the KATP opener pinacidil.

F. Activation of TREK-1 and TASK-1 by Volatile Anesthetics TREK-1 and TASK-1 are activated by volatile general anesthetics (Patel et al., 1999). Chloroform, diethylether, halothane, and isoflurane activate TREK-1. Halothane and isoflurane activate TASK-1. TWIK-1 and TRAAK are not affected by these anesthetics (Patel et al., 1999). TASK-2 has not been examined. Residues in TREK-1 and TASK-1 proteins that are involved in activation by chloroform and halothane were identified using deletions and chimeras (Patel et al., 1999). Thus anesthetic-mediated TREK-1 opening depends on the C-terminal 48 amino acids of the channel, and the region of TASK-1 located between residues 242 and 248 (-VLRFMT-) confers its sensitivity to halothane. Fusions of the last 48 and 78 residues of TREK-1 to TASK-1 and TRAAK, respectively, did not confer sensitivity to chloroform, although the chimeras did have a prominent basal activity. The C-terminal region may not be the only structural element that confers chloroform sensitivity to TREK-1.

V. TISSUE DISTRIBUTION AND PHYSIOLOGICAL ROLES OF 2P/4TM K⫹ CHANNELS The reverse transcriptase–PCR analysis of mouse tissue shows that all 2P/4TM K⫹ channels are widely expressed and are found in excitable as well as nonexcitable tissues (Fink et al., 1998; Lesage and Lazdunski, 1999; Reyes et al., 1998). It is also clear that despite a quasi-ubiquitous distribution, each of them has a unique pattern of expression in terms of their relative levels in the examined tissues. For example, TWIK-1 was expressed at similar levels in the nervous system (brain, cerebellum, brain stem, and spinal cord) and the digestive tract (stomach, intestine, and colon), which is not the case for TASK-1, TASK-2, and TREK-1. However TWIK-1 expression in heart is very low in comparison to TREK-1 and TASK-1. TRAAK is expressed exclusively in brain, spinal cord, and retina (Fink et al., 1998). It is worthwhile to note that TWIK-1, TREK-1, TASK-1, and TASK-2, but not TRAAK, are expressed in embryos. In the heart, TREK-1 and TASK-1 are expressed (Table III). Both channels produce currents that are instantaneous and voltage independent (Duprat et al., 1997; Fink et al., 1996). They are open at all membrane potentials and are able to drive the resting membrane potential close to EK . This property indicates that they are probably the long-sought after background or leak K⫹ channels that play a major role in setting the resting membrane potential in the heart. Native K⫹ currents displaying these biophysical properties have been recorded as IKp in rat ventricular myocytes (Backx and Marban, 1993; Yue and Marban, 1988). This channel in ventricular myocytes is instantaneous and noninactivating. It is also voltage independent and exhibits an out-

TABLE III Tissue Distribution of mRNA for Two-Pore K⫹ Channels in the Mouse a Brain Cerebellum TWIK-1 TREK-1 TASK-1 (cTBAK-1) TASK-2 TRAAK

TWIK-1 TREK-1 TASK-1 (cTBAK-1) TASK-2 TRAAK

⫹⫹ ⫹⫹ ⫹ ⫾ ⫹⫹

⫹⫹ ⫹⫹ ⫹⫹ ⫾ ⫹⫹

Brain stem ⫹⫹ ⫹⫹ ⫹⫹ ⫾ ⫹⫹

Spinal cord Heart ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹

⫺ ⫹⫹ ⫹⫹ ⫾ ⫺

Ventricle Atria ⫺ ⫹⫹ ⫹⫹ ⫾ ⫺

Skeletal muscle Eye

⫺ ⫾ ⫹⫹ ⫾ ⫺

⫹ ⫹⫹ ⫾ ⫹ ⫺

⫹⫹ ⫹ ⫹ ⫹ ⫹

Kidney Liver Lung ⫹⫹ ⫹ ⫹ ⫹⫹ ⫺

⫹ ⫺ ⫺ ⫹⫹ ⫺

⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫺

Stomach

Intestine

Colon

Bladder

Pancreas

Thymus gland

Salivary

Uterus

Ovary

Testis

Embryo E16–18

Method

Reference

⫹⫹ ⫾ ⫹ ⫹ ⫺

⫹⫹ ⫾ ⫾ ⫹ ⫺

⫹⫹ ⫹ ⫾ ⫹ ⫺

⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫺

⫹ ⫺ ⫺ ⫺ ⫺

⫺ ⫹ ⫺ ⫺ ⫺

⫹⫹ ⫹ ⫹ ⫹ ⫺

⫹⫹ ⫹⫹ ⫹ ⫹ ⫺

⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫺

⫹⫹ ⫹⫹ ⫾ ⫾ ⫺

⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫺

RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR

Lesage and Lazdunski (1999) Lesage and Lazdunski (1999) Lesage and Lazdunski (1999) Reyes et al. (1998) Fink et al. (1998)

a Transcript fragments were amplified by PCR using specific primers and analyzed by Southern blot using internal ⫺, undetectable; ⫾, expressed moderately; ⫹, expressed; ⫹⫹, expressed strongly.

32

P-labeled ologonucleotides.

15. Kir in Heart

ward rectification. Its single-channel behavior shows rapid open–close kinetics. TREK-1 and TASK-1 channels share these properties and are candidates for underlying IKp , although the properties of the expressed cDNAs and the current in rat ventricular myocytes require further studies.

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16 Voltage and Calcium-Activated K⫹ Channels of Coronary Smooth Muscle JURE MARIJIC* and LIGIA TORO*,† Departments of *Anesthesiology and †Molecular & Medical Pharmacology University of California, Los Angeles School of Medicine Los Angeles, California 90095

I. INTRODUCTION

myocardial infarction (MI), unstable angina, and sudden death (3, 4). Many cases have been described of acute myocardial infarction in the absence of a significant decrease of coronary artery diameter due to arteriosclerosis, presumably because of segmental coronary artery spasm (5). Coronary vasospasm occurs when coronary smooth muscle (Fig. 1) is exposed to circulating or to in situ-produced vasoconstrictors or in the absence, reduction, or impairment of vasodilatory mechanisms. Coronary artery spasm is a major complication and cause of early morbidity and mortality and is a limiting factor in the performance of coronary artery bypass grafting (6, 7) and coronary artery balloon angioplasty (8), the ever more used method of coronary revascularization. Coronary artery spasm also plays an important role in the progression of arteriosclerosis as shown in clinical angiographic studies (9). In addition, an isolated coronary artery spasm may cause cardiac arrest even in the absence of angina, structural heart disease, or coronary artery narrowing. In one study, this type of patient presented an increased response to the vasoconstrictor methylergonovine, which suggests an altered function of the coronary arteries. Interestingly, all these patients were smokers with an average age of 44 years, suggesting a deleterious effect of smoking (10). It would be interesting to determine if smoking affects the function of KCa channels.

The purpose of this chapter is to review the biophysical and molecular characteristics of the coronary artery large conductance voltage and calcium-activated potassium (KCa ) channel, a key regulator of arterial tone. We will analyze its relationship to coronary blood flow, cardiac function, and drug effects. KCa channels are also frequently referred to as MaxiK, BK, or slo channels. Coronary blood flow is tightly coupled to myocardial oxygen consumption and cardiac performance (1). Unlike other vascular beds and tissues, there is very little reserve for increases in oxygen extraction in coronary circulation. This is because the myocardium extracts near maximal amounts of oxygen from the blood, even at times when the myocardial oxygen consumption is at its minimum (2). It is due to this high resting O2 extraction that any increase in myocardial oxygen consumption not accompanied with a proportional increase in coronary blood flow will lead to anaerobic metabolism and myocardial ischemia. This condition may be worsened by the establishment of a vicious cycle of myocardial ischemia, diminished cardiac function, and decreased blood pressure. Coronary and cerebral vascular beds are the only vascular beds in which even a very short period of inadequate blood supply may lead to permanent damage and cell death of the delicate cardiac and brain systems. Thus, coronary artery disease (CAD) will inevitably lead to heart malfunction. Although CAD has been related primarily to arteriosclerosis, it is now accepted that spasm of the coronary arteries is also a key player in the pathogenesis of acute coronary syndrome such as

Heart Physiology and Pathophysiology, Fourth Edition

II. KEY ROLE OF KCa CHANNELS IN THE REGULATION OF CORONARY ARTERY TONE Because the regulation of coronary blood flow is important for maintaining normal cardiac function, it is

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FIGURE 1 Rat coronary artery smooth muscle cells dissociated using collagenase.

necessary to understand the mechanisms involved in the regulation of coronary artery tone, as well as the targets of vasoactive substances affecting this regulation. Pioneering experiments suggesting the role of KCa channels in arterial tone by Brayden and Nelson (11) demonstrated that these channels are activated at increased intraluminal pressure and prevent excessive vasoconstriction. The concerted efforts by several research groups and the development of new potent and selective inhibitors and openers of ion channels have clearly shown that KCa channels are key regulators of vascular tone. In fact, the blockade of KCa channels by Iberiotoxin (IBTx), a selective blocker, induces a pronounced vasoconstriction of coronary arteries (Fig. 2). Several characteristics of KCa channels in coronary smooth muscle (CSM) make them critical in the regulation of coronary artery tone and therefore coronary blood flow. These characteristics include very high density (⬎10,000/cell), large conductance, responsiveness to changes in intracellular calcium concentration ([Ca2⫹]i), and in membrane potential, and their sensitiv-

FIGURE 2 Blockade of KCa channels with Iberiotoxin (IBTx) induces contraction of rat coronary artery rings. Coronary arteries were isolated, divided into 3- to 4-mm-long rings, and stretched to an optimum diameter (maximal increase in tone after exposure to high extracellular potassium). Addition of 100 nM IBTx produced a dramatic increase in the vessel tone, suggesting that IBTx-sensitive channels play an important role in the regulation of resting coronary artery tone. IBTx also induced oscillation in tension in some of the vessels. The tone of the control group (bottom line) remained unchanged during the same time period (Control). Each tracing is an average of three vessel rings.

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ity to both endogenous and exogenous vasoconstrictors and vasorelaxants (Table I). Furthermore, because of the extremely high input resistance of coronary artery smooth muscle cells (5–15 G⍀) (12), opening of only a few large conductance K⫹ channels may produce significant hyperpolarization and relaxation (13). Iberiotoxin has been an invaluable tool for the pharmacological ‘‘dissection’’ of the role of KCa channels in response to vasoactive substances. For example, pretreatment with blockers of KCa channels prevents the active response of vasodilators such as atrial natriuretic peptide (ANP) (14), nitric oxide (NO) (15, 16), and vasoactive intestinal peptide (VIP) (17). In summary, the responsiveness of KCa channels to multiple extracellular and intracellular messengers (18) puts these channels in a unique position to integrate multiple influences, local and global, dilatory and con-

stricting, and to regulate coronary flow and therefore cardiac function.

III. DISTRIBUTION OF KCa CHANNELS KCa channels are widely distributed among different excitable cells and tissues, including neurons, endocrine cells, epithelial cells, uterus, and skeletal muscle, and are especially abundant in smooth muscle (19–21). Interestingly, they are absent in myocardium. In the gastrointestinal tract, KCa channels are involved in the regulation of gastrointestinal motility (22) and secretion (23). In uterus these channels modulate uterine contractions (24), whereas in endocrine cells they regulate the secretion of hormones (25). In the central and peripheral nervous systems, KCa channels

TABLE I Pharmacology of KCa Channels Agent

Effect

Tissue

Mechanism of action

Reference

A23187 Acethylcholine Adenosine ADH Adrenergic agents Angiotensin II ANP Arachidonic acid ATP cGMP, GMP Cromakalim DHS-I Endothelin Estrogens GDP, GTP Glibenclamide H2O2 Hydrogen ions Niflumic acid Nitroglycerin NO NS 1619 Okadaic acid Pinacidil prostaglandin E2 Quinidine Relaxin Secretin Somatostatin Tedisamil Thromboxane A2 Vasopressin VIP Volatile anesthetics

앖 앖 앖 앖 앖 앗 앖 앖 앖 앖 앖 앖 앖, 앗 앖 앖 앗, 0 앖 앗 앖 앖 앖 앗 앖 앖 앖 앗 앖 앖 앖 앗 앗 앖 앖 앗

CSM CSM VSM Kidney CSM, GISM CSM VSM, Pituitary CSM VSM CSM, VSM VSM CSM CSM CSM VSM VSM CSM SM CSM CSM CSM, VSM VSM VSM CSM Kidney GISM Uterus Pancreas Pituitary VSM CSM CSM CSM CSM

[Ca2⫹ ]i , NO [Ca2⫹ ]i , NO GMP mediated Phosphorylation (P) P and G-protein-AC-PKA Ligand modulation cGMP mediated Ligand modulation P Ligand modulation Ligand modulation 웁-subunit binding [Ca2⫹]i cGMP dependent Ligand modulation Reversal of Pinacidil앖 Arachidonic acid pathway Ligand modulation 앖affinity for Ca2⫹ NO, cGMP, ligand modulation cGMP, ligand modulation Ligand modulation Inhibition of phosphatase Ligand modulation [Ca2⫹]i Ligand modulation Protein kinase A (PKA) P Protein de-Phosphorylation Ligand modulation Ligand modulation [Ca2⫹]i G-protein Ligand modulation

138 67 139 140 141, 62 142 139, 143 144 145 139, 146 107 30, 119 147 148 139 107 149 150, 151 112 106, 152 71 113 56 108 153 154 155 156 157 158 159 160 65 121

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regulate cell excitability as demonstrated in Drosophila where mutated KCa channels resulted in uncoordinated motor functions. Because of this phenotype, the gene encoding the KCa channel in Drosophila was named slowpoke (slo) (26). KCa channels are present in all the vascular beds examined, where they play a role in the regulation of regional blood flow and blood pressure by altering peripheral vascular resistance and intravascular filling pressure (preload)(15). As mentioned previously, the elevation of intraluminal pressure causes smooth muscle vasoconstriction (and depolarization) (27) that is opposed by the activation of KCa channels that function as a negative feedback mechanism by hyperpolarizing smooth muscle cells favoring relaxation (28). If this feedback mechanism fails, a vicious cycle of widespread vasoconstriction and hypertension may occur. Opposite to this situation, a generalized vasodilatation may occur in sepsis where endotoxin and mediators of inflammation release excessive amounts of NO, which activates KCa channels, producing generalized vasodilatation and vascular hyporeactivity to vasoconstrictors (29).

Consistent with the important role KCa channels in human coronary physiology, KCa channels are the predominant K⫹ channels both in number (⬎10,000 channels per cell) (30) and in conductance (150–300 pS with physiologic ion concentrations) in human coronary arteries. Gollasch et al. (31) demonstrated that KCa channels, unlike other K⫹ channels, are present in 100% of human coronary artery cells.

IV. FUNCTIONAL ANATOMY OF KCa CHANNELS: ROLE OF 움 AND 웁 SUBUNITS KCa channels are formed by at least two subunits, the 움 subunit that forms the channel pore and associated regulatory 웁 subunits (웁1–웁4) (32, 33) that regulate Ca2⫹ voltage sensitivity of the channel, gating, and pharmacology. The 움 subunit was first cloned in 1991 by Atkinson using the Drosophila slowpoke (Slo) mutant (26). Four 움 subunits associate to form the K⫹ selective pore. The structure of the K⫹ selective pore is highly conserved among species and other K⫹ selective channels (34). Similar to the recently crystallized K⫹ channel

FIGURE 3 Functional anatomy of 움 and 웁 subunits of KCa channels. (A) 움 and 웁 subunits

of KCa channels. The 움 subunit has seven transmembrane domains. The S0 domain and part of the exoplasmic NH2 terminus play an important role in the functional coupling between 움 and 웁 subunits. The intracellular COOH terminus of the KCa channel is long and has four hydrophobic regions. The 웁 subunit of KCa channel has two transmembrane domains and a long extracellular loop. The NH2 terminus and the COOH terminus are both intracellular. The ⌿ represents the consensus sequence for N-linked glycosylation. (B) the top view of a possible arrangement of four 움 and four 웁 subunits forming the KCa channel. The assembly of four 움 subunits forms a K⫹ selective pore.

16. Voltage and Calcium-Activated K⫹ Channels

from Streptomyces lividans, the four subunits may form an inverted cone cradling the selectivity filter in its outer ˚ ) and conend (35). The selectivity filter is short (12 A ⫹ ˚ tains two K ions approximately 7.5 A apart. The repulsive forces between these two ions increases the throughput rate while its geometry ensures high selectivity (35).

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sensing of the changes in intracellular calcium and membrane potential (32, 42). For example, at a given Ca2⫹ concentration, the voltage necessary to half activate the channels is decreased by about 100 mV (Figs. 4A and 4B) (42). Moreover, it appears that calcium serves as a ‘‘switch’’ that tightly couples 움 and 웁 subunits at concen-

A. Pore-Forming 움 Subunit Major differences between the pore-forming 움 subunit of KCa channels and other voltage-dependent K⫹ (Kv) channels are at the carboxyl and amino termini (Fig. 3). At the amino-terminal end, KCa channels contain an additional (seventh) transmembrane domain (called S0) that leads to an extracellular N terminus (36, 37). At the carboxyl terminus, the KCa channel protein is very long and contains several hydrophobic domains (S7–S10), which appear to be intracellular (37). 움-subunit domains perform various functions, including voltage sensing (S4), K⫹ conduction (loop between S5 and S6), and calcium sensing (region between hydrophobic segments S9 and S10). This last region is frequently referred to as a ‘‘calcium bowl’’ (38). The ‘‘calcium bowl’’ is highly conserved, in fact identical in all cloned Slo channels, consistent with its importance in regulating the function of KCa channels. The 움 subunit of KCa channels seems to be derived from a single gene and its diversity resides in alternative splicing of exons that generate isoforms with different kinetics, voltage sensitivity, and conductance (39). The 움 subunit of KCa channels has two functional modes: a Ca2⫹-independent mode (at [Ca2⫹]i ⱕ 100nM) and a Ca2⫹-modulated mode (at [Ca2⫹]i ⬎ 100 nM). Note that at ‘‘resting’’ [Ca2⫹]i, the MaxiK channel 움 subunit only senses voltage changes.

B. Regulatory 웁 Subunits Regulatory 웁 subunits (웁1–웁4) have a common membrane topology, but are encoded by different genes. They have two transmembrane domains linked with a long extracellular loop that is glycosylated (Fig. 3A). The region in the 움 subunit that is necessary for 웁subunit modulation includes part of the extracellular N terminus and the transmembrane S0 domain (34). The 웁1 subunit is especially abundant in smooth muscles (40), and its interaction with the pore-forming 움 subunit follows a one-to-one stoichiometry (41). The 웁2 subunit is highly expressed in fetal kidney (32), whereas the 웁4 (33) subunit seems to be brain specific. 1. Changes in Ca2ⴙ /Voltage Sensitivities In general, coupling with regulatory 웁 subunits (웁1 and 웁2) allows the 움 subunit a much more effective

FIGURE 4 Voltage (A and B) and calcium (C) sensitivity of KCa channels. Ionic currents were elicited in inside-out patches of Xenopus laevis oocytes expressing human 움 or 움 ⫹ 웁 subunits. The holding potential was 0 mV and intracellular Ca2⫹ concentration was 10 애M. Mean steady-state currents were measured at the end of 200-msec pulses and fractional open probability (FPo)-voltage (V) curves were constructed. In this example half activation potential, V1/2 was 18 mV for the 움 subunit alone (A) and was ⫺77 mV when the 웁 subunit was coexpressed (B), indicating increased voltage sensitivity by the presence of the 웁 subunit. (C) Functional coupling of 움 and 웁 subunits at intracellular Ca2⫹ concentrations greater than 100 nM. At intracellular Ca2⫹ concentration ⱕ100 nM KCa , channel activity becomes Ca2⫹ independent and purely voltage activated, and functional coupling between 움 and 웁 subunits is released. (Inset) The presence of the 웁 subunit alters activation kinetics. This effect is not calcium dependent and is present at low calcium concentrations. Reprinted from (33), copyright 1996, with permission from Elsevier Science.

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trations ⬎1 애M. In contrast, at low Ca2⫹ concentrations (앑100 nM) the functional coupling with the 웁 subunit appears to dissociate and the KCa channel becomes Ca2⫹ independent (Fig. 4C) (42). 2. Changes in Pharmacological Properties The 움 subunit of KCa channels seems to be less sensitive to the pore blocker IBTx when coexpressed with the 웁1 subunit (43) and less sensitive to CTX when coexpressed with the 웁2 subunit (44). For example, the apparent inhibitory dose for KCa channels formed by the 움 subunit alone is 앑1 nM, whereas it increases to 앑50 nM when the 웁1 subunit is present. Expression of the 웁 subunit is also needed for the activation of KCa channels by nanomolar concentrations of dehydrosoyasaponin I (DHS-I). It is interesting to note here that KCa channels are dramatically activated by DHS-I in human coronary artery cells, suggesting the presence of 움 ⫹ 웁 complexes in this arterial bed (30). 3. Changes in Kinetic Properties The 웁1 subunit alters activation and deactivation kinetics of the pore-forming 움 subunit (43, 45), making both processes slower. However, association with the 웁2 subunit induces fast-inactivating KCa currents. This effect is mediated by the N terminus of the 웁2 subunit that acts as a ‘‘ball’’ that binds to the open channel causing inactivation. The inactivating current is expressed preferentially in some neurons and adrenal glands, whereas in vascular smooth muscle, the noninactivating current is predominant (44).

V. MULTIPLE PATHWAYS INVOLVED IN THE REGULATION OF KCa CHANNEL ACTIVITY The binding of intracellular Ca2⫹ to the channel protein or changes in membrane potential are not the sole mechanisms that determine KCa channel activity, but other mechanisms, including hormones, second messengers, and endothelium-derived vasoactive substances, are also involved (Table I). These factors may work directly on the channel or indirectly via closely associated proteins. In most cases, this interaction results in altered voltage/Ca2⫹ sensitivity of the channel. An exception is the finding that 웁-estradiol can modulate the voltage sensitivity of the KCa channel exclusively in its Ca2⫹-independent conformation (46).

A. Membrane Potential In KCa channels, like in other voltage-gated channels, membrane depolarization promotes the displacement

of charged residues contained in the voltage sensor (S4 region) (Fig. 3), inducing gating currents and pore opening (47). KCa channels can be opened by membrane depolarization at virtually zero intracellular calcium concentration (42). As mentioned previously, at intracellular Ca2⫹ concentrations ([Ca2⫹]i) less than 100 nM the membrane potential is the main regulator of opening of these channels, as at these low [Ca2⫹]i they become calcium independent (Fig. 4C) (42).

B. Calcium Voltage and intracellular calcium concentration are the most important forces in the regulation of opening of KCa channels. Because the changes in membrane potential in vascular smooth muscle are relatively modest, the change in [Ca2⫹]i is the dominant force in the regulation of KCa channels. More than a decade ago Van Breemen and colleagues (48) suggested the existence of a subsarcolemmal compartment of elevated [Ca2⫹]i, which is separated from the bulk Ca2⫹ of the cytoplasm and the smooth muscle contractile machinery. This compartmentalization explains the discrepancy between KCa channel activity (indicating high local calcium concentration in the vicinity of KCa channels) and the low overall cytoplasmic calcium concentration measured with calcium-sensitive dyes such as Fura-2. It has been clearly demonstrated that KCa channel activity is controlled by alterations of subsarcolemmal Ca2⫹ levels, due to the nearby release of calcium from the sarcoplasmic reticulum (SR) (49). The local calcium release events (calcium sparks) are mediated through the opening of ryanodine receptors (Ca release channels) that induce the simultaneous opening of many KCa channels (spontaneous transient outward currents, STOCs) (28). As expected, STOCs are inhibited by ryanodine or the KCa channel blocker, Iberiotoxin (50). In addition to the release of Ca2⫹ from subsarcolemmal Ca2⫹ stores, colocalized Ca2⫹ channels may also provide local activating Ca2⫹. Guia et al. (51) demonstrated that slow voltage depolarization induces opening of KCa channels in rabbit coronary artery. This activation is dependent on L-type channel activity and extracellular calcium and occurs in the absence of increases in bulk cytosolic Ca2⫹. This suggests a functional local coupling between L-type calcium channels and adjacent KCa channels to restrict Ca2⫹ overload and cell death. An increased activity of KCa channels induced by Ca entry through Ca2⫹ channels would hyperpolarize the membrane to diminish Ca2⫹ channel activity. This regulatory feedback mechanism is also important in neurons, where KCa channels are also colocalized with Ca2⫹ channels (52).

16. Voltage and Calcium-Activated K⫹ Channels

C. Phosphorylation Coronary smooth muscle KCa channels are activated by cAMP (PKA) (53)- and cGMP(PKG)-dependent kinases (54), whereas phosphokinase C (PKC) inhibits channel activity (55). Numerous authors have provided evidence that the phosphorylation of KCa channels (or closely associated regulatory proteins) by PKG occurs after NO stimulation and cGMP production. Phosphorylation/dephosphorylation cycles are likely to occur, as Archer et al. (56) demonstrated that inhibition of phosphatase activity by okadaic acid in pulmonary artery caused a further increase of KCa current previously stimulated by cGMP. Using the cloned channel, Alioua et al. (57) demonstrated that KCa channels are direct substrates of PKGdependent phosphorylation. Moreover, the localization of residues relevant for functional activation has been detected for both PKG (58) and PKA (59); they are localized in the long intracellular C terminus (Fig. 3).

D. G-Protein KCa channels are modulated by the autonomic nervous system via adrenergic and cholinergic receptors

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coupled to G-proteins. It is well known that 웁2-adrenergic stimulation increases coronary blood flow. Some of this increase is due to the autoregulation of coronary blood flow; however, coronary artery vasodilatation is observed in vitro where influences of changes in myocardial oxygen consumption are removed (60). Cholinergic stimulation causes vasodilatation through stimulation of endothelial cells and NO release (Fig. 5). However, if the vessel is deendothelialized, acetylcholine (ACh) can act directly on smooth muscle cells, producing vasoconstriction. Adrenergic and cholinergic stimulation can modulate KCa channels directly by G-proteins or indirectly via the activation of kinases and Ca2⫹ release from intracellular stores. In coronary smooth muscle, 웁-adrenergic stimulation causes an increase in KCa channel activity by both direct action of Gs protein and phosphorylation via PKA (53). Direct G-protein modulation was demonstrated after reconstitution in lipid bilayers and stimulation with a 웁-agonist, which indicates that 웁 receptors, Gs proteins, and KCa channels have strong protein–protein interactions and may form tight complexes in the native membrane. Direct Gs-protein stimulation of KCa channels after 웁-adrenergic stimulation has also been demonstrated in airway myocytes and thus may be a com-

FIGURE 5 Interaction of endothelial cells (EC) and coronary artery smooth muscle cells (SMC). Endothelium releases multiple vasoactive substances, including nitric oxide (NO), prostaglandins (PGI2 , PGH2), thromboxane A2 (TXA2), and endothelium-derived hyperpolarizing factors (EDHF). Prostaglandins are synthesized from arachidonic acid (AA) by cyclooxygenase (COX). Binding of acetylcholine (ACh) to muscarinic receptors triggers the release of NO from the endothelium. Calcium and potassium channels, including KCa channels, are targets of some of these endothelial metabolites. These mediators and intracellular pathways play important roles in the regulation of the coronary artery smooth muscle. These pathways include activation of adenyl cyclase (AC), soluble guanylyl cyclase (sGC), and cGMP-dependent protein Kinase (PKG).

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mon mechanism of KCa channel regulation in smooth muscle (61). The response to isoproterenol is blocked by pretreatment with low doses of cholera toxin and the protein kinase A (PKA) inhibitor, suggesting that 웁2 agonists activate KCa channels via the G-protein– adenylate cyclase–PKA system (62). With respect to cholinergic stimulation, the action on KCa channels is less clear. For example, in visceral smooth muscles, both activation and inhibition of KCa channels have been reported. Activation seems to result from Ca2⫹ release from intracellular stores (63, 64), whereas inhibition is likely due to a membrane-delimited action of pertussis toxin-sensitive G-proteins (61, 65, 66). The identity of this G-protein is still an enigma; G움i-2 , G움i-3 , or G움o-1 were unable to mimic the muscarinic response of KCa channels in tracheal myocytes (61). However, in coronary smooth muscle, ACh has been reported to induce the activation of KCa channels via a release of Ca2⫹ from intracellular stores (67), which would predict a relaxation instead of a contraction in deendothelialized vessels. The physiological relevance of these two opposite responses (activation and inhibition of KCa channels) induced by ACh still needs to be addressed.

E. Mechanical Stretch KCa channels can also be activated by mechanical stretch. The level of activation of KCa channels is much greater in vessels at high intraluminal pressures than in vessels under low pressure, as the KCa channel blocker Charybdotoxin has a larger effect at higher pressures (11). This pressure-induced activation is independent of systemic factors such as intracellular calcium, as it is also observed in excised membrane patches (68).

F. Endothelium-Derived Vasoactive Substances Endothelium plays a central role in the regulation of vascular smooth muscle tone, including that of coronary artery. Although multiple pathways are involved in endothelium-induced relaxation, the major mechanism is via release of NO. L-NAME, an inhibitor of NO synthesis from L-arginine, has been a widely used tool to determine the role of NO. For example, exposure to L-NAME decreases hyperpolarization and relaxation of smooth muscle by acetylcholine demonstrating the participation of NO (69). The release of NO contributes to flow-related vasodilatation, reactive hyperemia, hypercapnic acidosis, the vasodilatation by adenosine, and the maintenance of flow in the presence of coronary artery stenosis (70).

1. Nitric Oxide Although there is evidence for direct action of NO on KCa channels (71), the majority of data supports that activation of guanylate cyclase (GC) (72), production of cGMP, and activation of PKG as the main pathway of NO and NO donor-induced smooth muscle dilatation. In native vessels, NO produced in the endothelium diffuses to adjacent coronary smooth muscle cells and triggers the GC–cGMP–PKG pathway and the phosphorylation of various target proteins (Ca2⫹ handling proteins), including KCa channels (Fig. 5). KCa channels are also modulated by constitutively active GC, i.e., in the absence of NO or NO donor vasodilators. (73). This property was demonstrated using 1H-[1,2,4]oxadiazolo[4,3,-움] quinoxalin-1-one (ODQ), a selective inhibitor of GC and thus cGMP production. In coronary artery cells, ODQ decreased the basal KCa channel open probability by 60% and, as expected, failed to inhibit the response to a nonhydrolyzable cGMP analogue, 8-bromo-cGMP. Interestingly, ODQ was more potent in reducing the vasodilation by NO donors than the KCa channel blocker Iberiotoxin, suggesting additional effectors besides KCa channels. 2. Arachidonic Acid and Fatty Acids Arachidonic and other fatty acids activate KCa channels. The activation by fatty acids seems to be independent of cyclooxygenase or lipoxygenase pathways, as fatty acids, which are not substrates to these enzymes, were also able to activate KCa channels (74). The effect is likely mediated by a direct action on the channel or a closely associated protein rather than via intracellular pathways (74). Ordway et al. (75) were the first to demonstrate that KCa channels from frog stomach could be activated directly by polyunsaturated fatty acids, such as arachidonic acid, and also by monounsaturated (linoelaidic) and saturated (myristic) free fatty acids. The primary requirement was the solubility of the free fatty acid. Later, a direct action of arachidonic acid (74) and its metabolite 11,12-epoxyeicosatrienoic acid (76) was demonstrated in vascular smooth muscle KCa channels and in smooth muscle-like mesangial cells (77) (important regulators of renal hemodynamics). Although arachidonic acid may increase guanylate cyclase activity (78), its effect on KCa channels seems to be direct and not through cGMP pathways (77). In addition to the direct effect of fatty acids on KCa channels, it is also possible that fatty acids may increase membrane fluidity, leading to changes in the Ca2⫹ sensor as proposed for 2-decanoic acid by Bregestovski et al. (79). It is also possible that fatty acids increase Ca2⫹ release from intra-

16. Voltage and Calcium-Activated K⫹ Channels

cellular stores (80) in close proximity to plasma membrane KCa channels, leading to their activation. It has been suggested that arachidonic acid is elevated in the serum and cytosol during cardiac ischemia (81). Thus, activation of KCa channels would increase coronary flow, alleviating ischemia. 3. Endothelium-Derived Hyperpolarizing Factors (EDHF) Endothelium-derived hyperpolarizing factors appear to play important roles, especially in the microvasculature (82). The chemical identity of EDHF has been related to several factors, such as epoxyeicosatrienoic acid (EET) (83), K⫹ (84), and anandamide (85). There is an emerging consensus that EDHF is unlikely to be a single factor and that considerable tissue and species differences exist for the nature and cellular targets of the hyperpolarizing factors. One of the potential targets is a KCa channel. Randall and Kendall (85) showed that anandamide, an endogenous cannabinoid derived from arachidonic acid, causes vasodilatation via opening of KCa channels. Hayabuchi et al. (86) demonstrated that EET also opens KCa channels via G-protein without changes in cAMP or cGMP. It is of interest that aging and hypercholesterolemia impair EDHF-mediated relaxation; however, the mechanisms of impairment are not clear (82).

G. Other Factors 1. Atrial Natriuretic Peptide (ANP) ANP dilates mesenteric arteries and increases KCa channel activity likely through the PKG pathway (14, 87).

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vasodilatation is mediated by opening potassium channels, other than KATP channels (KCa channels?), as their blockade with glibenclamide did not alter the response to testosterone (90). 웁-Estradiol is also a potent vasorelaxant of the coronary circulation and its effects appear to be at least in part through a direct activation of KCa channels (91, 92). 4. Oxygen and Carbon Monoxide (CO) Hypoxia suppresses KCa activity in pulmonary arteries (93, 94), depending on the intracellular redox state. KCa channels are inhibited by NADH, whereas NAD promotes their openings through a mechanism that is not completely understood (93). CO is a direct activator of KCa channels and a potent vasorelaxant whose action does not seem to be mediated by cGMP or G-proteins (95). In summary, KCa channels respond to a variety of stimuli by either increasing or decreasing their open probability and therefore hyperpolarizing or depolarizing the cell membrane and altering blood flow. These stimuli include intracellular calcium concentration, membrane potential, endothelial-derived vasoactive substances (including NO), prostacyclin, PGE2 , EDHF, and others. The activity of KCa channels is also modulated by several second messengers, including cGMP, GMP, GDP, GTP, and ATP, as well as hormones such as epinephrine, endothelin, vasopressin, ANP, antidiuretic hormone (ADH), secretin, somatostatin, TRH, and others. The ability of KCa channels to respond to a variety of intracellular local tissue factors and endotheliumderived factors, as well as hormones, makes these channels integrators of multiple stimuli.

VI. PHARMACOLOGY OF KCa CHANNELS 2. Nonadrenergic Noncholinergic Neurotransmitters: Substance P (SP) and Vasoactive Intestinal Peptide SP is a potent endothelium-dependent coronary artery vasodilator that appears to hyperpolarize coronary artery endothelial cells by opening KCa channels (88). It is not clear if SP can open KCa channels in vascular smooth muscle cells. VIP is also a potent vasodilator (89) whose action is partially inhibited by the KCa channel blocker Iberiotoxin (17). Thus, KCa channels are partly responsible for the vasorelaxing effects of VIP, possibly via a G-protein. 3. Sexual Hormones Testosterone and 웁-estradiol also have direct vasodilatory effects on smooth muscle. Testosterone-induced

A. TEA, CTX, IBTx, and Other Blockers of KCa Channels The KCa channel is distinguished from other K⫹ channels by its sensitivity to low concentrations of extracellularly applied tetraethylammonium (TEA) (96), Charybdotoxin (CTX) (97), and the selective blocker Iberiotoxin (98). External TEA is relatively effective in reducing the current amplitude of the KCa channel with a dissociation constant (Kd) in the range of 120–300 애M, whereas internal administration is much less effective (99, 100). Iberiotoxin is a peptide produced by the scorpion Buthus tamulus, which is a selective and potent blocker of KCa channels when applied to an external side of the smooth muscle cell membrane, with an apparent Kd of approximately 1 nM (98).

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B. KCa Channel Openers 1. NO Donors Nitric oxide containing vasodilators such as nitroglycerin (NTG) and sodium nitroprusside (SNP) mediate the majority of their vasodilatory effect by releasing NO either spontaneously (SNP) or during enzymatic degradation (NTG). PT 10, which neutralizes NO, inhibits the SNP response. In the 1970s, the effect of NO donors (SNP) was thought to be mediated by a decrease in Ca influx (101). However, the doses required to produce this effect were higher than those required for vasodilation, suggesting that a decrease in Ca2⫹ efflux was not crucial for SNP-induced vasodilation. Later, vasodilation by NTG and SNP was shown to be associated with hyperpolarization of the cell membrane, indicating the involvement of K⫹ channels (102). This involvement was also supported by the fact that the preconstriction of vascular smooth muscle with KCl (reduced K⫹ gradient) dramatically reduced vasodilation by SNP (103). The role of KCa channels has now been demonstrated by their blockade with CTX and IBTx, which leads to a drastic reduction of NTG (104) or SNP-induced vasodilatation (105). Moreover, an increase of KCa channel current by SNP has been demonstrated in human coronary arteries (106).

2. ‘‘Potassium Channel Openers’’ Although frequently thought of as selective KATP channel openers, pinacidil and cromakalim are potent openers of KCa channels. At concentrations less than 1 애M, these drugs doubled the open probability of the rabbit KCa channel at ⫺40 mV (close to the resting membrane potential). These effects were blocked by glibenclamide, although glibenclamide did not have an effect in the absence of KATP openers. Because of the large effect of pinacidil and cromakalim at clinically relevant concentrations, it is clear that this mechanism significantly contributes to the vasodilatation induced by these drugs (107). Bychkov et al. (108) also demonstrated that pinacidil is a nonselective activator of KATP and KCa channels in human coronary artery. These authors suggested that the conductance of KATP channels and their contribution to vasodilatation by pinacidil is small under physiological conditions and that the majority of the vasodilating effect of pinacidil is due to an increased activation of KCa channels (108).

has been proposed for aortic (109) and rat tail (110) arteries. However, in coronary arterial smooth muscle, KCa channels do not seem to play a role (111). 4. Fenamates and Structurally Related Benzimidazole Compounds Niflumic acid is a potent and readily reversible opener of KCa channels. The effect is dose dependent and involves an increased sensitivity of KCa channels to intracellular calcium and a leftward shift in the voltage– activation curve. The KCa channel-opening property does not appear to be a unique property of niflumic acid and is exhibited by other fenamates. In fact, flufenamic acid was as potent as niflumic acid, whereas mefenamic acid was three times less potent. Niflumic acid was more potent (more than four times) from the extracellular than intracellular space, suggesting an extracellular binding site. The fenamate receptor/binding site is different from TEA and CTX binding sites, as fenamates did not interfere with the KCa channel block by TEA and CTX. Fenamates are helpful tools in the study of functional properties of KCa channels and may be useful therapeutic tools as smooth muscle dilators (112). Several other related compounds have been shown to open KCa channels, including NS004 and its close derivative NS1619 (113–115). NS1619 also inhibited STOC and produced a pronounced dilatation of rat portal vein and aorta (113). Consistent with an activation of KCa channels, NS1619 causes vasodilatation. However, this effect is not prevented by blocking KCa with CTX or by reducing the K⫹ gradient (80 mEq KCl), suggesting that other or additional mechanisms of action are present. Indeed, in some reports, authors demonstrated that NS1619 is a potent blocker of Ca2⫹ channels, of voltage-dependent K⫹ channels, and of KATP channels (113, 116). 5. Nordihydroguaiaretic Acid (NDGA) NDGA, a lipoxygenase inhibitor and antioxidant, is also a potent opener of KCa channels in porcine coronary artery and is possibly a potential tool for designing more potent vasodilators and/or bronchodilators (117). Interestingly, NDGA was ineffective at low calcium concentrations, indicating that calcium is necessary, possibly as a way to couple 움 and 웁 subunits. 6. Dehydosoyasaponin I

3. Iloprost Iloprost, a prostacyclin analogue, is a potent vasodilator. The role of KCa channels in this vasodilator effect

DHS-I is a tripentene glycoside isolated from Desmodium adscedens and is a potent opener of KCa channels (118). In frog oocytes, DHS-I at nanomolar concentrations activated KCa channels only when 움 and

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웁 subunits were coexpressed (30). However, the presence of the 웁 subunit is not necessary if micromolar concentrations of DHS-I are used (44). DHS-I binds with 10- to 20-fold higher affinity to the open channel than to the closed. Binding of at least three or four DHS-I molecules is required for maximal activation of the channel (119). DHS-I reversibly activated macroscopic and single cannel activity of human coronary artery KCa channels, indicating that human coronary artery smooth muscle cell KCa channels are composed of 움 and 웁 subunits (30).

C. Volatile Anesthetics Volatile anesthetics, especially isoflurane, are potent dilators of coronary arteries. In fact, some authors have suggested that because of the potent vasodilating properties, isoflurane may cause coronary steal and ischemia. The role of KCa channels in coronary vasodilatation by isoflurane is not clear. Kokita et al. (120) suggested that a significant portion of the mesenteric artery smooth muscle hyperpolarization and vasodilatation by isoflurane in intact rats is mediated by opening KCa channels. This is contrary to the findings of Buljubasic et al. (121), who demonstrated that isoflurane actually decreased current though KCa channels in isolated coronary artery smooth muscle cells. The major difference between these two studies was that one investigated the effect in vivo (120) whereas the other studied direct effects in isolated cells (121), suggesting a possible role of endothelium, circulating factors, or the autonomic system in isoflurane-induced vasodilatation.

VII. PROTECTIVE EFFECTS ON CORONARY ARTERY MEDIATED TROUGH KCa CHANNELS A. Estrogens Estrogens protect against development and manifestation of coronary artery disease. Physiological levels of estrogen found in premenopausal women offer dramatic protection against coronary artery disease and its complications (122). In addition, maintenance of normal levels of estrogen by daily administration maintains a large part of this protection against coronary artery disease. The mechanisms of this protection are not clear; however, a growing body of evidence suggests the improvement of endothelial function. The protective effect of estrogens has been studied extensively in rats where protection is abolished by ovariectomy and the antiestrogen tamoxifen, and reestablished by the exogenous administration of estrogen. The presence of estrogen reduces myogenic tone dramatically (pressure-induced coronary artery contrac-

tion) in isolated coronary arteries. The difference was diminished by removal of the endothelium and by inhibition of NO synthase, although some reports suggest the involvement of smooth muscle cell iNOS (123–125). Isolated coronary arteries from female rats produced greater constriction in response to IBTx or KT-5823 (PKG inhibitor) than ovariectomized animals of the same age, suggesting an increased activity of KCa channels in normal animals (126). In addition to the effects of estrogen via the NO-PKG pathway, estrogens may directly open KCa channels; however, the physiological role of this effect is not clear as it is seen only at high estrogen concentrations (46, 54). Studies demonstrate that 웁-estradiol may directly activate KCa channels formed by 움 and 웁1 subunits (46).

B. Red Wine The protective effect of red wine, which appears to be much greater than the protection by other alcoholic beverages (127), may also be mediated through KCa channels. Red wine has been shown to increase cGMP levels and decrease the tone in human coronary arteries and rat aorta. This increase was endothelium dependent and likely involves the release of NO from endothelium (128). All wines did not produce this effect, but it was observed only after the administration of certain red wines produced ‘‘en barrique,’’ possibly due to their high content of phenolic substances (128).

VIII. ROLE OF POTASSIUM CHANNELS IN AGING AND DISEASE A. Aging The response to endogenous and exogenous (e.g., NTG) coronary vasodilators decreases with advanced age, but the response is not affected by endothelial dysfunction (129). Because NTG dilates coronary arteries via KCa channel activation (106), the decreased response to NTG in aging may be due to a decrease in the number of or in the openings of KCa channels in smooth muscle cells. An age-dependent decrease in the density of ion channels has also been observed in other tissues. For example, transient outward potassium current diminishes with age in myocardium (130). Apamin-sensitive potassium channels also decrease in rat skeletal muscle (131), and 4-AP-sensitive potassium channels are reduced in colonic smooth muscle (132). These facts underline the importance of determining the number, structure, gating, and role of all these channels in vascular smooth muscle in different age groups. Preliminary results (133) suggest that KCa channels in the membrane of rat and human coronary artery SMC

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may decrease with aging. In addition to the decreased response to vasodilators, increased age is associated with the increased contractile response of coronary artery to endothelin-1, serotonin, and angiotensins and likely to other vasoconstrictors. This would implicate increased susceptibility of older subjects to coronary artery spasm in response to these stimuli. Age-dependent changes in smooth muscle cells may be compounded by changes in endothelial function. Toda et al. (134) demonstrated an age-dependent decrease in endothelium-dependent dilation of coronary arteries exposed to ACh. However, the authors did not determine if the release of endothelium-derived factors is altered or if the aging process alters the response of the smooth muscle. In another study, Katano et al. (135) demonstrated an age-dependent increase in coronary vasoconstriction evoked by the injection of endothelin1 using isolated rat hearts. The alteration in endothelial function, as measured by release of EDRF and PGF1움 , was likely responsible for the increased vasoconstriction.

B. Hypertension Spontaneously hypertensive rats show an increased activity of TEA-sensitive KCa channels (136). The increase in KCa current may play an important protective role by limiting a raise in blood pressure through its vasodilator property. It seems that the increased activity of KCa channels in hypertension is due to an increase in the intracellular calcium concentration and not to a higher calcium sensitivity of KCa channels. It is interesting that the higher activity of KCa is not restricted to the vascular bed, but is also present in lymphocytes (137).

IX. SUMMARY Ca2⫹-activated potassium channels are widely distributed among different excitable cells and are especially abundant in smooth muscle, while absent in myocardium. KCa channels are the predominant K⫹ channels both in number (⬎10,000/cell) and in conductance (150–300 pS with physiologic ion concentrations) and are present in 100% of human coronary artery cells. KCa channels are formed by two subunits, the 움 subunit that forms the channel pore and associated regulatory 웁 subunits that regulate Ca2⫹ and voltage sensitivity of the channel, gating, and pharmacology. The responsiveness of KCa channels to multiple extracellular and intracellular messengers puts these channels into a unique position to integrate multiple signals, local and global, dilatory and constricting, and regulate coronary flow and therefore cardiac function. KCa channels also

play an important role in estrogen-induced protection against cardiovascular morbidity and mortality. Studies have demonstrated that 웁-estradiol may directly activate KCa channels by binding to its 웁 subunit. The protective effect of red wine may also be mediated through KCa channels. The role of KCa channels in vasodilatation by clinically used vasodilators, including nitroglycerin and sodium nitroprusside, has now been demonstrated, as well as the nitroglycerin-mediated increase of KCa channel current in human coronary arteries. In conclusion, KCa channels play an important role in the regulation of vascular smooth muscle tone in health and possibly in aging and in the development of disease processes. KCa channels are potential targets in the treatment of cardiovascular disorders.

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114. Olesen, S. P., Munch, E., Moldt, P., and Drejer, J. (1994). Selective activation of Ca2⫹-dependent K⫹ channels by novel benzimidazolone. Eur. J. Pharmacol. 251, 53–59. 115. Olesen, S. P., Munch, E., Weatjen, F., and Drejer, J. (1994). NS 004: An activator of Ca2⫹-dependent K⫹ channels in cerebellar granule cells. Neuroreport 5, 1001–1004. 116. Sargent, C. A., Grover, G. J., Antonaccio, M. J., and McCullough, J. R. (1993). The cardioprotective, vasorelaxant and electrophysiological profile of the large conductance calcium-activated potassium channel opener NS-004. J. Pharmacol. Exp. Ther. 266, 1422–1429. 117. Nagano, N., Imaizumi, Y., Hirano, M., and Watanabe, M. (1996). Opening of Ca2⫹-dependent K⫹ channels by nordihydroguaiaretic acid in porcine coronary arterial smooth muscle cells. Jpn. J. Pharmacol. 70, 281–284. 118. McManus, O. B., Harris, G. H., Giangiacomo, K. M., Feigenbaum, P., Reuben, J. P., Addy, M. E., Burka, J. F., Kaczorowski, G. J., and Garcia, M. L. (1993). An activator of calcium-dependent potassium channels isolated from a medicinal herb. Biochemistry 32, 6128–6133. 119. Giangiacomo, K. M., Kamassah, A., Harris, G., and McManus, O. B. (1998). Mechanism of maxi-K channel activation by dehydrosoyasaponin-I. J. Gen. Physiol. 112, 485–501. 120. Kokita, N., Stekiel, T. A., Yamazaki, M., Bosnjak, Z. J., Kampine, J. P., and Stekiel, W. J. (1999). Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane. Anesthesiology 90, 779–788. 121. Buljubasic, N., Rusch, N. J., Marijic, J., Kampine, J. P., and Bosnjak, Z. J. (1992). Effects of halothane and isoflurane on calcium and potassium channel currents in canine coronary arterial cells. Anesthesiology 76, 990–998. 122. Vyas, S., and Gangar, K. (1995). Postmenopausal oestrogens and arteries. Br. J. Obstet. Gynaecol. 102, 942–946. 123. Darkow, D. J., Lu, L., and White, R. E. (1997). Estrogen relaxation of coronary artery smooth muscle is mediated by nitric oxide and cGMP. Am. J. Physiol. 272, H2765–H2773. 124. Binko, J., and Majewski, H. (1998). 17 beta-Estradiol reduces vasoconstriction in endothelium-denuded rat aortas through inducible NOS. Am. J. Physiol. 274, H853–H859. 125. Knot, H. J., Lounsbury, K. M., Brayden, J. E., and Nelson, M. T. (1999). Gender differences in coronary artery diameter reflect changes in both endothelial Ca2⫹ and ecNOS activity. Am. J. Physiol. 276, H961–H969. 126. Wellman, G. C., Bonev, A. D., Nelson, M. T., and Brayden, J. E. (1996). Gender differences in coronary artery diameter involve estrogen, nitric oxide and Ca2⫹-dependent K⫹ channels. Circ. Res. 79, 1024–1030. 127. St. Leger, A. S., Cochrane, A. L., and Moore, F. (1979). Factors associated with cardiac mortality in developed countries with particular reference to the consumption of wine. Lancet 1, 1017– 1020. 128. Flesch, M., Schwarz, A., and Bo¨hm, M. (1998). Effects of red and white wine on endothelium-dependent vasorelaxation of rat aorta and human coronary arteries. Am. J. Physiol. 275, H1183– H1190. 129. Anderson, T. J., Meredith, I. T., Charbonneau, F., Yeung, A. C., Dyce, M., Selwyn, A. P., and Ganz, P. (1996). Nitroglycerininduced coronary vasodilation is not enhanced in patients with impaired endothelium-dependent dilation. J. Am. Coll. Cardiol. 28, 580–584. 130. Walker, K. E., Lakatta, E. G., and Houser, S. R. (1993). Age associated changes in membrane currents in rat ventricular myocytes. Cardiovasc. Res. 27, 1968–1977.

131. Vergara, C., and Ramirez, B. U. (1997). Age-dependent expression of the apamin-sensitive calcium-activated K⫹ channel in fast and slow rat skeletal muscle. Exp. Neurol. 146, 282–285. 132. Xiong, Z. L., Sperelakis, N., and Noffsinger, A. (1995). Ca2⫹ currents in human colonic smooth muscle cells. Am. J. Physiol. Gastrointest. Liver Physiol. 269, G378–G385. 133. Marijic, J., Li, Q., Song, M., and Toro, L. (1999). Anesth. Analg. 88(2S), S90–S90. [Abstract] 134. Toda, N., Bian, K., and Inoue, S. (1987). Age-related changes in the response to vasoconstrictor and dilator agents in isolated beagle coronary arteries. Naunyn Schmiedebergs Arch. Pharmacol. 336, 359–364. 135. Katano, Y., Ishihata, A., Morinobu, S., and Endoh, M. (1993). Modulation by aging of the coronary vascular response to endothelin-1 in the rat isolated perfused heart. Naunyn Schmiedebergs Arch. Pharmacol. 348, 82–787. 136. Rusch, N. J., De Lucena, R. G., Wooldridge, T. A., England, S. K., Cowley, A. W. Jr. (1992). A Ca2⫹-dependent K⫹ current is enhanced in arterial membranes of hypertensive rats. Hypertension 19, 301–307. 137. Furspan, P. B., and Bohr, D. F. (1990). Calcium sensitivity of Ca2⫹-activated K⫹ channels in spontaneously hypertensive stroke-prone rats. Hypertension 15 (Suppl.), 197–I101. 138. Bychkov, R., Gollasch, M., Ried, C., Luft, F. C., and Haller, H. (1997). Regulation of spontaneous transient outward potassium currents in human coronary arteries. Circulation 95, 503– 510. 139. Williams, D. L., Jr., Katz, G. M., Roy-Contancin, L., and Reuben, J. P. (1988). Guanosine 5⬘-monophosphate modulates gating of high-conductance Ca2⫹-activated K⫹ channels in vascular smooth muscle cells. Proc. Natl. Acad. Sci. USA 85, 9360–9364. 140. Guggino, S. E., Suarez-Isla, B. A., Guggino, W. B., and Sacktor, B. (1985). Forskolin and antidiuretic hormone stimulate a Ca2⫹activated K⫹ channel in cultured kidney cells. Am. J. Physiol. 249, F448–F455. 141. Scornik, F. S., Codina, J., Birnbaumer, L., and Toro, L. (1993). Modulation of coronary smooth muscle KCa channels by Gs alpha independent of phosphorylation by protein kinase A. Am. J. Physiol. 265, H1460–H1465. 142. Toro, L., Amador, M., and Stefani, E. (1990). ANG II inhibits calcium-activated potassium channels from coronary smooth muscle in lipid bilayers. Am. J. Physiol. 258, H912–H915. 143. White, R. E., Lee, A. B., Shcherbatko, A. D., Lincoln, T. M., Schonbrunn, A., and Armstrong, D. L. (1993). Potassium channel stimulation by natriuretic peptides through cGMP-dependent dephosphorylation. Nature 361, 263–266. 144. Scornik, F., and Toro, L. (1991). Modulation of Ca-activated K channels from coronary smooth muscle. In ‘‘Ion Channels of Vascular Smooth Muscle Cells and Endothelial Cells’’ (N. Sperelakis, and H. Kuriyama, eds.), pp. 111–124. Elsevier, New York. 145. Robertson, B. E., Corry, P. R., Nye, P. C., and Kozlowski, R. Z. (1992). Ca2⫹ and Mg-ATP activated potassium channels from rat pulmonary artery. Pflug. Arch. 421, 94–96. 146. Fujino, K., Nakaya, S., Wakatsuki, T., Miyoshi, Y., Nakaya, Y., Mori, H., and Inoue, I. (1991). Effects of nitroglycerin on ATPinduced Ca⫹⫹ mobilization, Ca⫹⫹-activated K channels and contraction of cultured smooth muscle cells of porcine coronary artery. J. Pharmacol. Exp. Ther. 256, 371–377. 147. Hu, S., Kim, H. S., and Jeng, A. Y. (1991). Dual action of endothelin-1 on the Ca2⫹-activated K⫹ channel in smooth muscle cells of porcine coronary artery. Eur. J. Pharmacol. 194, 31–36. 148. White, R. E., Darkow, D. J., and Lang, J. L. (1995). Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism. Circ. Res. 77, 936–942.

16. Voltage and Calcium-Activated K⫹ Channels 149. Barlow, R. S., and White, R. E. (1998). Hydrogen peroxide relaxes porcine coronary arteries by stimulating BKCa channel activity. Am. J. Physiol. 275, H1283–H1289. 150. Laurido, C., Candia, S., Wolff, D., and Latorre, R. (1991). Proton modulation of a Ca2⫹-activated K⫹ channel from rat skeletal muscle incorporated into planar bilayers. J. Gen. Physiol. 98, 1025–1043. 151. Kume, H., Takagi, K., Satake, T., Tokuno, H., and Tomita, T. (1990). Effects of intracellular pH on calcium-activated potassium channels in rabbit tracheal smooth muscle. J. Physiol. 424, 445–457. 152. Khan, S. A., Higdon, N. R., and Meisheri, K. D. (1998). Coronary vasorelaxation by nitroglycerin: involvement of plasmalemmal calcium-activated K⫹ channels and intracellular Ca⫹⫹ stores. J. Pharmacol. Exp. Ther. 284, 838–846. 153. Ling, B. N., Webster, C. L., and Eaton, D. C. (1992). Eicosanoids modulate apical Ca2⫹-dependent K⫹ channels in cultured rabbit principal cells. Am. J. Physiol. 263, F116–F126. 154. Wong, B. S. (1989). Quinidine blockade of calcium-activated potassium channels in dissociated gastric smooth muscle cells. Pflu¨g. Arch. 414, 416–422.

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155. Meera, P., Anwer, K., Monga, M., Oberti, C., Stefani, E., Toro, L., and Sanborn, B. M. (1995). Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A. Am. J. Physiol. 269, C312–C317. 156. Gray, M. A., Greenwell, J. R., Garton, A. J., and Argent, B. E. (1990). Regulation of maxi-K⫹ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation. J. Membr. Biol. 115, 203–215. 157. White, R. E., Schonbrunn, A., and Armstrong, D. L. (1991). Somatostatin stimulates Ca2⫹-activated K⫹ channels through protein dephosphorylation. Nature 351, 570–573. 158. Pfreunder, D., and Kreye, V. A. (1991). Tedisamil blocks single large-conductance Ca2⫹-activated K⫹ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein. Pflug. Arch 418, 308–312. 159. Scornik, F. S., and Toro, L. (1992). U46619, a thromboxane A2 agonist, inhibits KCa channel activity from pig coronary artery. Am. J. Physiol. 262, C708–C713. 160. Wakatsuki, T., Nakaya, Y., and Inoue, I. (1992). Vasopressin modulates K⫹-channel activities of cultured smooth muscle cells from porcine coronary artery. Am. J. Physiol. 263, H491– H496.

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17 Ion Channels in Vascular Smooth Muscle JUN YAMAZAKI and KENJI KITAMURA Department of Pharmacology Fukuoka Dental College Fukuoka 814-0193, Japan

tion. Several K⫹ channels contribute to the falling phase of the action potential, especially the voltage-dependent K⫹ and Ca2⫹-dependent K⫹ channels. The former is activated by membrane depolarization and the latter by an increase in [Ca2⫹]i as well as by membrane depolarization. In elastic vascular tissues, such as the aorta and pulmonary artery, action potentials are not generated following electrical stimulations; however, suppression of K⫹ channel activity reveals the ability these tissues to generate the action potentials, with the same pharmacological properties as those observed in the portal vein. As the physiological and pharmacological properties were the same whether spontaneous or evoked action potentials were examined, differences between portal vein smooth muscle cells and other vascular cells would seem to derive from the presence or absence of pacemaker activity. As described in the following sections, ion channels do not have a simple structure. Some ion channels gain their full functions when coexpressed with several regulatory proteins or pore-forming proteins. Some ion channels have a multimeric structure with different subtypes forming the channel pore. Furthermore, a number of nonfunctional channel-like proteins have also been identified, although their physiological roles are not fully understood. At present, there is a limited amount of molecular biological information available on the regulation of ion channels in vascular cells. Thus, in practice we are often limited to the pharmacological and physiological identification of the ion channels present in particular vascular cells.

I. INTRODUCTION The main biological function of vascular cells is the maintenance of blood pressure, which is essential for the transport of various substances to peripheral tissues. One of the main factors in the regulation of blood pressure is the smooth muscle tone of peripheral vascular tissues, especially arterial tissues, which is varied through the regulation of smooth muscle cell contraction. Although many factors contribute to vascular tone, they all ultimately affect the intracellular Ca2⫹ concentration ([Ca2⫹]i) within the vascular smooth muscle cell (Fig. 1). This chapter surveys the ion channels distributed in the membrane of vascular smooth muscle cells, as these channels comprise the most important machinery for the regulation of [Ca2⫹]i . Two types of vascular cells are recognized on the basis of their electrical activities. The portal vein is exceptional among vascular tissues in that it possesses spontaneous action potentials. Several types of Ca2⫹ and K⫹ channels contribute to these action potentials. In the guinea pig portal vein, two types of Ca2⫹ channels have been recorded, whereas in the rabbit portal vein only one type has been reported. In both tissues, action potentials are inhibited in Ca2⫹-free media or by application of organic or inorganic Ca2⫹ antagonists. However, tetrodotoxin (TTX), a potent Na⫹ channel blocker, does not suppress the action potentials seen in the portal vein, indicating that these action potentials are Ca2⫹ dependent. No other venous tissues and no arterial tissues produce spontaneous action potentials, but some do produce action potentials on neural or other stimula-

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FIGURE 1 Ion channels and their modulations by receptor activation. Activations of ion channels mobilize [Ca2⫹]i directly or indirectly via membrane depolarization or hyperpolarization.

II. Ca2⫹ CHANNELS Action potentials generated in the membrane of vascular smooth muscle cells are Ca2⫹ dependent, not Na⫹ dependent, as indicated by various pieces of evidence obtained using Ca2⫹ antagonists and tetrodotoxin. A major Ca2⫹ entry pathway is via the so-called L-type Ca2⫹ channels. However, as a reduction in the [Na⫹]o suppresses the action potential, Na⫹ channels do make some contribution to the generation of action potential in vascular smooth muscle cells. Two types of Ca2⫹ channels have been identified in various vascular smooth muscle cells: T- and L-type channels (Table I). Although in their essential nature these Ca2⫹ channels are practically the same as those in cardiac and neuronal cells, a variety of values for single channel conductance have been reported in different smooth muscle cells. For example, L-type channels found in aortic smooth muscle cells have half the value normally reported for single L-type channel conductance (12 pS) (Cafferey et al., 1986). This aortic channel is highly dihydropyridine (DHP) sensitive and has other L-type-like properties, except for the unusually small single channel conductance. Similar small DHP-sensitive Ca2⫹ channels have been recorded in guinea pig portal vein cells (Inoue et al., 1990b). In these portal vein cells, typical L-type

channels were also present. As no further report has been made since then, it is not clear whether these small L-type channels are restricted to the vascular smooth muscle cells in which they have been reported or whether they are ubiquitous in all vascular smooth muscle cells. T-type Ca2⫹ channels have been found in some arteries, such as mesenteric and basilar arteries (Benham et al., 1987; Yatani et al., 1987; Ganitokevich and Isenberg, 1990; Oike et al., 1990). This Ca2⫹ channel has the same properties as T-type channels found in cardiac and neuronal cells; i.e., this channel is activated and inactivated at low membrane potentials, shows rapid inactivation, is resistant to DHP derivatives and Cd, and has a small single channel conductance (8 or 12 pS). Different cloned T-type 움1 subunits were found to have slightly different single channel conductances (5 pS for 움1I, 8 pS for 움1G, and 11 pS for 움1H: Perez-Reyes et al., 1998; Cribbs et al., 1998; Lee et al., 1999). Although no subtype of the T-type Ca2⫹ channel has yet been identified in vascular smooth muscle cells, differences in single channel conductance among the T-type channels recorded in various vascular tissues may reflect differences in the actual subtypes present. T-type Ca2⫹ channels are considered to contribute to the triggering of the action potential and to pacemaker activity in both cardiac and

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neuronal cells. However, it is difficult to see what role Ttype Ca2⫹ channels might play in arterial smooth muscle cells, as many arterial cells have a resting membrane potential of ⫺50–70 mV, at which T-type Ca2⫹ channels are inactivated.

A. Molecular Structure of Ca2⫹ Channels in Vascular Smooth Muscle Cells All voltage-dependent Ca2⫹ channels, except T-type Ca2⫹ channels, are composed of multimeric protein molecules. These proteins are named 움1, 움2, 웁, 웃, and 웂 subunits. The channel pore is formed by the 움1 subunit alone; the other subunits act as modulators of its expression and function. The pharmacological properties of the various Ca2⫹ channels depend on the actual amino acid sequence of the 움1 subunit. At present, at least 10 types of 움1 subunits have been identified; four of them (움1S, 움1C, 움1D, and 움1F) are DHP sensitive and high voltage activated, three others (움1A, 움1B, and 움1E) are DHP insensitive and high voltage activated, and the remainder (움1G, 움1H, and 움1I) are low voltage activated (T type). It has been reported that the characteristics of the T-type Ca2⫹ channel, such as low voltage activation and inactivation and rapid inactivation, are

bestowed by 움1G, 움1H, or 움1I alone (Perez-Reyes et al., 1998; Cribbs et al., 1998; Lee et al., 1999) (Table II). All 움1 subunits possess 24 hydrophobic amino acidrich regions, each with a 17 to 25 amino acid chain, and the amino acid sequences of these regions are highly conserved. For example, 10 of these hydrophobic regions have been found to have identical amino acid sequences in their 움1A, 움1B, and 움1E subunits, and 7 regions were identical between 움1C and 움1D. However, DHP-sensitive and -insensitive 움1 subunits have no identical amino acid sequences. These regions are thought to be transmembrane regions, and each transmembrane region is linked to either a short or a long hydrophilic amino acid chain. Each set of 6 transmembrane regions (domain) is believed to form a quarter of the channel wall as a unit. It is well accepted that the No. 4 transmembrane region is a voltage-sensing region (as deduced from its regular sequence of polar amino acids), whereas either No. 5 or No. 6 transmembrane region or both form part of the channel pore (as deduced from their hydrophilic and hydrophobic compartments). Other transmembrane regions of each domain are thought to be channel stabilizers; however, the functional role of each of the transmembrane regions is still not entirely clear.

TABLE I Single Channel Conductances of Voltage-Dependent Ca Channels in Smooth Muscle Cells Category DHP sensitive

DHP insensitive

Big

Conductance (pS)

Tissue

Reference

30 29 28

Guinea pig taenia coli Human myometrium Guinea pig coronary artery

Yoshino et al. (1989) Inoue et al. (1990) Ganitkevich and Isenberg (1990)

Middle

25 25 25 25 23 22 21 20 19 18–24

Rabbit ileum Rabbit ear artery Rabbit mesenteric artery Guinea pig vena cava Rabbit basilar artery Guinea pig portal vein Rabbit coronary artery Rabbit ear artery Rat cerebral artery Dog saphenous vein

Inoue et al. (1989) Benham et al. (1987) Worley et al. (1986) Kawashima and Ochi (1987) Oike et al. (1990) Inoue et al. (1990) Matsuda et al. (1993) Aaronson et al. (1986) Quayle et al. (1993) Yatani et al. (1987)

Small

15–20 12 12 12 8

Rabbit mesenteric artery Guinea pig aorta Guinea pig portal vein Guinea pig taenia coli Rabbit mesenteric artery

Worley et al. (1986) Caffrey et al. (1986) Inoue et al. (1990) Yoshino et al. (1989) Worley et al. (1986)

Big

15 12 12

Guinea pig vena cava Rabbit basilar artery Human myometrium

Kawashima and Ochi (1987) Oike et al. (1990), Worley et al. (1986) Inoue et al. (1990)

Middle

8 8 8 7–9

Guinea pig coronary artery Guinea pig taenia coli Rabbit ear artery Dog saphenous vein

Ganitkevich and Isenberg (1990) Yoshino et al. (1989) Aaronson et al. (1986), Benham et al. (1987) Yatani et al. (1987)

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TABLE II Classification of Ca Channels Channel type T

L

N

P

Q

R

Threshold

Low

High

High

High

High

High

Activation voltage

Low

High

High

High

High

High

Inactivating rate

Rapid

Slow

Rapid

Slow

Intermediate

Intermediate

Locarization

Neurons Heart Smooth muscle

Ubiquitous

Neurons

Neurons

Neurons

Neurons

Function

Differnetiation Proliferation Pacemaker

Contraction Secretion

Neurosecretion

Neurosecretion

Neurosecretion

Neurosecretion

DHP derivatives Diltiazem Verapamil Flunarizine Mibefradil

Cilnidipine

Organic blockers

Flunarizine Mibefradil Inorganic blockers

Ni (ⱕ30 애M )

Peptide blockers 움1 subunit

움1G, 움1H, 움1I

Mibefradil

Daurizoline Mibefradil

Mibefradil

Mibefradil

Cd (ⱕ1 애M )

Cd (ⱕ1 애M )

Cd (ⱕ1 애M )

Cd (ⱕ1 애M )

Cd (ⱕ1 애M )

Calciseptine

웆Conotoxin GVIA

웆Agatoxin IVA (ⱖ3 nM )

웆Agatoxin IVA (ⱖ100 nM )

웆Agatoxin IIIA

움1S, 움1C, 움1D, 움1F

움1B

움1A

움1A

움1E

A complete sequence has been identified for the 움1 subunit from two smooth muscle tissues (rat aorta and rabbit lung), and these smooth muscle 움1 subunits show a more than 93% amino acid homology with the cardiac 움1C subunit, but only 65% homology with the skeletal muscle 움1S subunit (Biel et al., 1990; Koch et al., 1990). No other type of DHP-sensitive 움1 subunit has yet been identified. A search for the specific nucleotide sequence of the 움1C subunit has demonstrated that the 움1C subunit is also present in other smooth muscle tissues (uterus, intestine, stomach). There is at least one splice variant form of the 움1C subunit in brain as well as in smooth muscle cells; indeed, two types of 움1C subunits have been identified in the same tissue. One type (rbCI) is predominant in the aorta, and also in heart and brain, whereas the other (rbC-II) is predominant in colonic smooth muscle cells. The two types of 움1C subunit were in equal transcribed numbers in other smooth muscle cells (trachea, small intestine, and A7r5 cell line) (Rich et al., 1993) (Table III). Four types of 웁 subunit have been identified in various cells. Among these 웁 subunits, the 웁3 subunit is present in the small intestine, colon, and lung, whereas both 웁2 and 웁3 subunits are present in aorta and trachea (Castellano et al., 1993a; Collin et al., 1994; Hullin et al., 1992). From evidence such as this, it would appear that a combination of rbC-I type 움1C and both 웁2 and 웁3 subunits is predominant in aortic smooth muscle cells and that this combination might make up the predominant form of L-type Ca2⫹ channel in vascular cells. How-

ever, another combination (of rbC-II type 움1C and 웁3 subunits) makes up the predominant form of the intestinal L-type Ca2⫹ channel. As no investigation has yet been performed to establish whether particular channel characteristics are associated with a particular combination of subunits, no information is available to tell us whether these differences may have a significant influence over channel properties. However, experiments of this type may eventually provide us with molecular evidence that explains the heterogeneity of the L-type Ca2⫹ channels found in vascular and visceral smooth muscle cells. Generally, the transcription of 움1C alone has been thought to be sufficient for channels to be transported from the trans-Golgi apparatus to the membrane, but cotranscription of a 웁 subunit with an 움1C results in a much higher efficiency of channel expression in the membrane (Lory et al., 1993; Castellano et al., 1993a,b; Collin et al., 1994). Indeed, coexpression of a smooth muscle 움1C subunit with a skeletal muscle 웁1 subunit produced a larger Ca2⫹ current in chinese hamster ovary (CHO) cells. It is of interest that this combination resulted in a more efficient expression of Ca2⫹ channels than coexpression of a smooth muscle 움1C with a smooth muscle 웁3 subunit (Weling et al., 1995). Using a confocal microscope, Yamaguchi et al. (1998) directly proved that coinjection of an 움1C subunit with a 웁3 subunit increased the number of Ca2⫹ channels in the membrane. They therefore concluded that the 웁 subunit acts as a ‘‘chaperone’’ protein for the 움1 subunit, but

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it is not clear exactly how the 웁 subunit facilitates the translocation of the 움1C subunit to the membrane. Other possible factors leading to an increase in current density following the coexpression of 움1C and 웁 subunits have yet to be identified. Constantin et al. (1998) reported that co-expression of 움1C and 웁 subunits in Xenopus oocytes suppressed the switching between low and high open probability mode without significantly changing the mean open probability or the channel availability. They also noted that expression of an 움1C subunit alone produced normal channel activity, as recorded in native cardiac cells. As native cardiac Ca2⫹ channels consist of 움1C and 웁 subunits, as well as an 움2웃 subunit, there would seem to be differences in enviromental regulation between oocytes and cardiac cells. Bay K8644 did not increase the Ca2⫹ current in cells coexpressing 움1C with a 웁 subunit (Itagaki et al., 1992). As Bay K8644 increases the channel availability and open probability of the L-type Ca2⫹ channel, this result suggested that the 웁 subunit positively modulates the channel’s activity by interaction with the 움1C subunit. Another functional role of 웁 subunits is to shift the voltage-dependent activation curve in the negative direction and accelerate the inactivation of the L-type Ca2⫹ current. These effects are thought to be independent of the chaperoning role of the 웁 subunit (Castellano et al., 1993a,b; Lory et al., 1992, 1993; Yamaguchi et al., 1998). Castellano et al. (1993) and Yamaguchi et al. (1998) produced particularly good evidence for this idea.

Castellano et al. (1993) reported that a different order of the three 웁 subunits was required for maximal current amplitude than for voltage-dependent activation. Yamaguchi et al. (1998) elegantly demonstrated (1) that intracellular alkalinization by the suppression of proton ATPase selectively suppressed translocation of the 움1C subunit to the membrane without affecting the kinetics of the current and (2) that modulation of the channel kinetics by the 웁 subunit occurred at an early phase of the culture period, after which facilitation of channel translocation occurred. It has been found that, in the 움1C subunit, a 움 subunit-interacting domain (AID) exists at the linker between domain I and II and that an 웁-interacting domain (BID) exists at the middle of the 웁 subunit (Pragnell et al., 1994). Hence, interaction between 움1C and 웁 subunits may occur through these sites. A most plausible mechanism for the time- and voltage-dependent inactivation of the L-type current would be a 웁-type inactivation, i.e., a 웁 subunit protein being slowly translocated to the inner mouth of the channel and plugging the channel pore. However, when a 웁 subunit is not expressed, the Ca2⫹ current still showed weak time-dependent inactivation. This indicates that the inactivation gate is a feature of the 움1C subunit, with the 웁 subunit amplifying this function by acting as an auxiliary apparatus. As the 웁 subunit modulates the voltage-dependent channel kinetics, examining the interaction between the S4 transmembrane segment of the 움1C subunit and the 웁 subunit is also important

TABLE III Subtypes of Ca and Na Channels Identified in Smooth Muscle Cells Subunit Ca channel

Na channel

Subtype

Tissue

Reference

움1

움1C

Rat aorta Rat aorta Rabbit lung Canine colon Uterus Lung Stomach Small intestine Large intestine

Koch et al. (1989, 1990) Snutch et al. (1991) Biel et al. (1990) Rich et al. (1993) Koch et al. (1990) Koch et al. (1990) Koch et al. (1990) Koch et al. (1990) Koch et al. (1990)

웁

웁2

Trachea Aorta

Hullin et al. (1992) Hullin et al. (1992)

웁3

Trachea Aorta Human colon Human small intestine Human lung

Hullin et al. (1992) Hullin et al. (1992) Collin et al. (1994) Collin et al. (1994) Collin et al. (1994)

움2

움2c

Rat aorta

Williams et al. (1996)

움

Type III

Intestine

Gautron et al. (1992)

웁

웁1

Human uterus

Makita et al. (1994)

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II. Cellular Electrophysiology

if we are to discover the true role of this auxiliary protein (Cens et al., 1998). 움2 and 웃 subunits are synthesized by a single gene and are thought to be expressed as tightly bound proteins. There are at least three types of 움2 subunit (움2a, 움2b, and 움2c) (Williams et al., 1996). Using polymerase chain reaction analysis, 움2c has been found to be present in aortic cells. Although no data are available concerning the modulatory action of the smooth muscle 움2c subtype on the L-type Ca2⫹ current, Itagaki et al. (1992) reported that coexpression of a cardiac or aortic 움1C subunit with a skeletal muscle 움2 subunit (움2a) increased the current density without affecting its kinetics. They also demonstrated that this effect was weaker than that observed on coexpression of the 움1C subunit with a skeletal muscle 웁 subunit (웁1a), whereas coexpression of all three subunits produced a much larger Ca2⫹ current. There are considerable differences between the effects produced by different combinations of muscle type subunits and neuronal subunits. Thus, the muscle type 움2a웃 subunit did not alter the kinetics of the L-type Ca2⫹ current, whereas the neuronal type 움2b웃 subunit accelerated the inactivation quite markedly (Williams et al., 1992; Itagaki et al., 1992; Gurnett et al., 1996). Both the structure and properties of the pore-forming 움1A and 움1C subunits differ considerably, but it is uncertain whether such differences result from differences in the 움1 subunit itself or in the auxiliary subunits. Thus, experiments involving various combinations of smooth muscle type subunits are needed for a full understanding of the proper function of these subunits in the Ca2⫹ channels found in smooth muscle cells. The 웂 subunit has been found in skeletal muscle cells, but not in the L-type Ca2⫹ channels of cardiac and smooth muscle cells (Bosse et al., 1990; Jay et al., 1990; Powers et al., 1993). From its hydropathic profile, this subunit is thought to have four transmembrane regions with several putative N-glycosylation sites. Although experiments involving coexpression of cardiac 움1C and skeletal muscle 웂 subunits showed that the 웂 subunit may contribute to the stable expression of the 움1C subunit and that a cooperative effect with the 웁 subunit induced augmentation (Jay et al., 1990; Wei et al., 1991), the physiological relevance of this subunit in smooth muscle cells remains a subject for future investigation. Three types of low-voltage activated and rapidly inactivating channels have been identified. 움1G, 움1H, and 움1I subtypes. These subunits do not have the consensus sequence of BID in the linker between domain I and domain II, indicating that the T-type 움1 subunit does not interact with the 웁 subunit (Lambert et al., 1997; Cribbs et al., 1998). Although these subtypes alone produced typical T-type-like currents, the amplitudes of the single channel conductances were slightly different

(Perez-Reyes et al., 1998; Cribbs et al., 1998; Lee et al., 1999). In central nervous tissues, three types of T-type 움1 subunits are distributed abundantly (Telly et al., 1999), whereas 움1G and 움1H have been found in heart cells (Perez-Reyes et al., 1998; Cribbs et al., 1998; Lee et al., 1999). Unfortunately, the distribution of these subtypes in smooth muscle cells has not yet been reported.

B. Modulation of Ca2⫹ Channels by Receptor Activation There are several pathways by which Ca2⫹ channels can be modulated following receptor stimulation: by direct activation or inhibition by GTP-binding protein or by indirect activation or inhibition by protein phosphorylation through second messengers. Regulation of the resting membrane potential, by activation of the nonselective cation channels or K⫹ channels, and changes in [Ca2⫹]i also modify Ca2⫹ channel activity. Direct inhibition of neuronal 움1A, 움1B, or 움1E subunits by GTP-binding protein has been described (Dolphin, 1998; Furukawa et al., 1998a,b; Delmas et al., 1998; Stephens et al., 1998a,b; Page et al., 1998). In the case of these 움1 subunits, both G움 and G웁웂 subunits had inhibitory actions on Ca2⫹ channels. Furukawa et al. (1998a,b) and Delmas et al. (1998) demonstrated that G움 and G웁웂 subunits interact, respectively, with the C terminus and the linker region between domain I and domain II of the Ca2⫹ channel 움1 subunit. It is of interest that the consensus sequence of the G웁웂-binding site completely overlaps that of BID. Therefore, receptor activation should alter the 웁 subunit-mediated enhancement of channel activation. However, such G-proteinmediated channel inactivation was not seen in the cardiac 움1C subunit (Furukawa et al., 1998a,b). Dolphin (1998) and Delmas (1998) independently reached the conclusion that the consensus motif QXXER was essential for G웁웂 binding in neuronal 움1 subunits. In the same region, the DHP-sensitive 움1 subunit has a QXXEE motif instead of QXXER (Dolphin, 1998). Thus, evidence for G-protein-mediated inhibition of smooth muscle L-type Ca2⫹ channels is not convincing to judge from the existing molecular biological evidence. Several indirect pieces of evidence suggest that Gprotein, especially the G움 subunit, activates the 움1C Ca2⫹ channel. For example, in the rabbit saphenous artery receptor stimulation enhanced the L-type Ca2⫹ current after PKC activation by PDBu (Oike et al., 1992). Furthermore, Gs움, but not Gi움, has been reported to activate cardiac L-type Ca2⫹ channels when incorpolated in a planar membrane, even after sufficient pretreatment with PKA (Wang et al., 1993). Thus, potentiation of L-type currents by receptor activation is

17. Ion Channels in Vascular Smooth Muscle

not explained by stimulation of the intracellular second messenger system alone. Although a distinct interaction site for G움 has not been identified in the smooth muscle 움1C subunit, and no direct evidence has yet been provided for G-protein–움1C subunit interaction, direct activation of the G-protein may also contribute to the augmentation of the L-type Ca2⫹ channel current followings receptor stimulation. There are various phosphorylation sites in 움1 and 웁 subunits; however, we must be aware that not all sites are phosphorylated. Furthermore, it is uncertain whether phosphorylation always produces a functional change in Ca2⫹ channels even when these sites are phosphorylated. For example, in the skeletal 움1S subunit, serine residue 1854, which is located near the C terminus, has been reported to be the fastest and strongest PKA phosphorylation site; however, two other sites were only phosphorylated after denaturation or proteolytic cleavage (Rotman et al., 1992). In the 움1C subunit of rabbit airway smooth muscle, only two putative sites for potential PKA phosphorylation are present in the C terminus (R/KR/KXS/T; Biel et al., 1990), but this subunit also has three consensus sequences for PKA phosphorylation (RXS/T) in the C-terminal region and the linker between domain II and domain III. It is interesting that a 75 amino acid sequence, including a putative PKA phosphorylation site (RGT), is inserted in the linker between domain I and domain II of the 움1C subunit from the airway and that this region is located near AID. However, in the aortic 움1C subunit, five putative PKA phosphorylation sites are present in the C terminus (Koch et al., 1990). The latter authors noted, in the same paper, the presence of two amino acid sequences that differed from those in the cardiac 움1C subunit. However, the later demonstration of the full amino acid sequence for the rat cardiac 움1C subunit indicated that these differences were a species difference rather than a tissue difference (Snutch et al., 1991). The presence of three different lengths of mRNA message has been reported for the aortic 움1C subunit, and the deduced amino acid sequence of this subunit is believed to be transcribed from the medium size mRNA (Koch et al., 1990). This predicts the presence of a variant-spliced form of the aortic 움1C subunit, although no evidence has been presented to indicate whether the longer and shorter messages successfully transcribe functional 움1C subunits or whether the molecular mass and length of the protein synthesized by these messages are dependent on the message size. It has been reported that two sizes of neuronal 움1C subunit exist and that PKA phophorylates only the longer isoform (Hell et al., 1993a,b). These authors speculated that the shorter isoform might be produced by proteolysis of the longer one rather than by alternate splicing during transcription.

333

If this truncation of the 움1C subunit occurs after message translation in the vascular smooth muscle cell, the cAMPdependent phosphorylation site(s) of the vascular 움1C subunit could be eliminated (Zong et al., 1995). It has been well documented that cardiac L-type channels are positively modulated by cAMP-dependent metabolism. For example, 웁-adrenoceptor stimulation (웁 agonists), adenylate cyclase stimulation (forskolin), and application of cAMP or dibutylic cAMP all increased the open probability of the L-type channel as well as channel availability through PKA activation (Tsien et al., 1972; Ochi et al., 1984). However, cAMPdependent augmentation of the L-type current in smooth muscle cells is still controversial (Ohya et al., 1987; Ohya and Sperelakis, 1989a; Fukumitsu et al., 1990; Muraki et al., 1993). As shown in Fig. 2, in guinea pig coronary artery isoproterenol-induced augmentation of the L-type current was not mimicked by forskolin or cAMP. However, Muraki et al. (1993) reported that cAMP augmented the L-type current in trachea. If we assume that posttranslational proteolysis of the 움1C subunit occurs under both physiological and pathophysiological conditions, the smooth muscle 움1C subunit may lose its ability to undergo cAMP-dependent phosphorylation, as demonstrated for the cardiac 움1C subunit (Chang and Hosey, 1988; Schneider and Hofmann, 1988; Yoshida et al., 1990, 1992; Sculptoreanu et al., 1993). Several studies have been performed on the contribution of PKC-induced phosphorylation in the activation of smooth muscle L-type Ca2⫹ channels. Application of phorbol esters (PKC activators) has been demonstrated to augment the L-type Ca2⫹ current, whereas PKC inhibitors prevent this augmentation (Clapp et al., 1987; Loirand et al., 1990; Vivadou et al., 1991; Oike et al., 1992). However, Oike et al. (1992) reported that augmentation of the L-type Ca2⫹ current induced by H1-receptor stimulation was not inhibited by PKC inhibitors. However, evidence shows that PKC-mediated phosphorylation activates DHP-sensitive Ca2⫹ influx and that phosphorylation of the 움1S subunit, but not the 웁 subunit, is required for this activation (Gutierrez et al., 1994). This PKCmediated phosphorylation of the 움1S subunit occurred only when a 웁 subunit was coexpressed, suggesting that the location of the PKC phosphorylation sites was not the sole factor in this phosphorylation (Puri et al., 1997). There are several PKC phosphorylation sites in all 움1 and 웁 subunits, and in the case of the neuronal 움1C subunit, full and truncated 움1C subunits both preserved a PKC phosphorylation site (Stea et al. 1995). It has been reported that PKC-mediated phosphorylation of 움1C and 웁2a subunits occurs independently (Puri et al., 1997). However, electrophysiological experiments produced different results from the just-described biochemical experiments. Stea et al. (1995) demonstrated

334

II. Cellular Electrophysiology

that PMA, a phorbol ester, enhanced the peak Ca2⫹ current in cells coexpressing an 움1B or 움1E subunit with 웁1b, but did not enhance this current in cells coexpressing an 움1C or 움1A subunit with 웁1b. As the PKCmediated enhancement of the 움1B Ca2⫹ current was suppressed by replacement of the domain I-II linker with that from the 움1A subtype, they also concluded that PKC phosphorylation sites for functional modulation are located in domain I-II linker rather than in the C terminus. In this linker, 움1C, 움1D, and 움1S have no consensus motif for a PKC phosphorylation site (S/ TXR/K), whereas 움1A, 움1B, and 움1E all have this motif at a few sites. This indicates that phosphorylation by PKC of 움1C probably occurs in the C-terminal region and that phosphorylation of this site may augment the 움1C current. Phosphorylation of 웁1b by PKC does not modulate the activity of the 움1C Ca2⫹ channels; however, it is uncertain whether phosphorylation of the smooth muscle 웁2 or 웁3 subunit modulates that activity of the 움1C subunit.

C. Sites of Action of Ca2⫹ Channel Blockers It has been reported that at least two regions are important for the ability of DHP derivatives and 움1C subunits to produce current inhibition (S6 transmem-

brane region and linker between S5 and S6 in both domains III and IV; Grabner et al., 1996). Using photoaffinity labeling, Nakayama et al. (1991) found that DHP-binding sites in the skeletal muscle 움1S subunit were the regions between arginine (R988) and alanine (A1023) and between glutamate (E1349) and tryptophan (W1391) in the S5-S6 linker of domain III and the transmembrane S6 region of domain IV. Tang et al. (1993) confirmed the location of these DHP-binding sites using a chimeric 움1C/움1B subunit. Thus, results obtained from molecular and biochemical experiments are in good agreement. DHP molecules do not seem to have direct access to their binding sites from the aqueous phase, as DHP molecules have the ability to block Ca2⫹ channels from inside the patch membrane. At present, S6 transmembrane regions seem good candidates for primary interaction sites for DHP derivatives; however, it is uncertain how the drug molecule passes through to reach these sites in the 움1C subunit after intrusion into the membrane, as the S6 transmembrane segments are located in the core region of the channel pore (Fig. 3). In smooth muscle cells of rabbit intestine and portal vein, D600 was reported to inhibit Ca2⫹ channels from the extracellular side (Ohya et al., 1987; Leblanc and Hume, 1989), whereas in porcine coronary artery, neocortical neurons, and cardiac ventricular cell D890, a

FIGURE 2 Augmentation of the voltage-dependent Ca2⫹ current induced by isoproterenol, but not by forskolin in the guinea pig coronary artery. (A) Voltage-dependent Ca2⫹ currents in the presence and absence of 1 애M isoproterenol recorded at three different amplitudes of depolarizing pulses. (B) Relationships between Ica (relative amplitude) and concentrations of isoproterenol and noradrenaline. (C) Effects of isoproterenol, forskolin, and cAMP on the Ica (relative amplitude). (B) and (C) The amplitude of Ica before application of drugs is normalized as 1.0. (Z. Xiong and K. Kitamura, unpublished observations)

335

17. Ion Channels in Vascular Smooth Muscle

FIGURE 3 Schematic model of the Ca2⫹ channel 움1C subunit and putative binding sites for Ca2⫹ channel blockers and putative phosphorylation sites for PKA and PKC.

completely charged form of D600, inhibited the Ca2⫹ current from the inside of the membrane (Hescheler et al., 1982; Deisz and Prince, 1987; Klo¨ckner and Isenberg, 1991). Striessnig et al. (1990) concluded that D600 may bind to a region identical to one of the DHP-binding sites in the skeletal muscle 움1S subunit (between E1349 and W1391). However, this region, especially its intracellular part, is one of the most highly conserved regions among 움1 subunits, including DHP-insensitive types. In fact, only one residue differs between DHP-sensitive and -resistant 움1 subunits (W and S). However, the extracellular part through the SS2 region has diverse sequences in the various 움1 subunits. Therefore, it is hard to explain the electrophysiological discrepancies observed in these tissues simply by considering the structure of the phenylalkylamine-binding site; modulation by other regions of the 움1 subunit or by other subunits also needs to be considered. It is interesting that, in DHP-resistant 움1 subunits (움1A, 움1B, and 움1E), the serine residue (S1391) forms a PKA phosphorylation site (RXS), and thus the conformation or electric field of this area would be expected to be changed if this serine is phosphorylated. However, further precise experiments will be required to resolve this point.

D. Mode of Action of Ca2⫹ Channel Blockers Analysis of single channel opening showed that there were three opening modes for the cardiac L-type current, termed mode 0, 1, and 2 (Hess et al., 1984). It was reported that Ca2⫹ channel blockers, especially DHP derivatives, shift the mode from 1 (short opening) to 0 (no opening), whereas Ca2⫹ agonists and 웁 adrenoceptor stimulation shifted the mode from 1 to 2 (long opening) without a change in the mean open time (Hess et al., 1984; Kawashima and Ochi, 1988). Production of a similar modal shift by DHP derivatives was observed in smooth muscle L-type channels in the rabbit jejunum

(Inoue et al., 1990). Because the modal shift induced by 웁-adrenoceptor stimulation is mimicked by application of cAMP or PKA in cardiac cells, the PKA phosphorylation–dephosphorylation presumably regulates the channel state (Hess et al., 1984; Wang et al., 1993).

III. Na⫹ CHANNELS It is widely accepted that action potentials in smooth muscle cells are resistant to tetrodotoxin (TTX) and saxitoxin (STX), specific blockers of the fast Na⫹ channels in cardiac and skeletal muscle cells and neurons. Nevertheless, evidence has accumulated in favor of the presence of TTX-sensitive Na⫹ channels in smooth muscle cells (Okabe et al., 1988; Ohya and Sperelakis, 1989b; Okabe et al., 1990; Inoue et al., 1991; Muraki et al., 1991; Mironneau et al., 1992; Kao and Wang, 1994). Fast Na⫹ channels in smooth muscle are classified into two subtypes accordings to their TTX sensitivity, namely neuronal and cardiac types. The TTX-sensitive Na⫹ channel in the rabbit pulmonary artery has a very low IC50 value (ca. 10 nM), whereas those in rat portal vein, rat vena cava, rat and human colon, and guinea pig ureter have slightly higher IC50 values (Okabe et al., 1988, 1990; Muraki et al., 1991; Mironneau et al., 1992). However, in rat uterus and azygos vein, the presence of a TTX-resistant or TTX less sensitive Na⫹ current has been reported (Ame´de´e et al., 1986; Ohya and Sperelakis, 1989b; Yamamoto et al., 1993; Sturek and Hermsmeyer, 1986). A third type of Na⫹ channel (epitheliallike Na⫹ channel) has been identified in cultured A7r5 cells and in rat aortic and portal vein cells. This epithelial-like Na⫹ channel was voltage independent and insensitive to both TTX and amiloride, but sensitive to phenamil (Van Renterghem and Lazdunski, 1991). Although there is now considerable evidence for the existence of voltage-dependent Na⫹ channels in various

336

II. Cellular Electrophysiology

smooth muscle cells, their physiological roles are still uncertain. At the average resting membrane potential seen in smooth muscle cells (ca. -50 mV), only a small fraction of the Na⫹ channels present is utilized (Inoue et al., 1991; Sperelakis et al., 1992; Muraki et al., 1991). Inoue et al. (1991) showed Na⫹-dependent active responses with a tiny amplitude on membrane depolarization, suggesting that this small fraction of the Na⫹ channels might contribute to the initial membrane depolarization needed to trigger Ca2⫹-dependent action potentials in this tissue.

A. Molecular Basis of Na⫹ Channels

and as a single mutation in the S4 segment of domain II modified the slow inactivation of brain Na⫹ channels (Patton et al., 1992; West et al., 1992; Hartmann et al., 1994; Moran et al. 1994; Fleig et al. 1994), multiple sites may contribute to the channel’s inactivation. In contrast to the situation in the 움1C Ca2⫹ channel, phosphorylation by PKA and PKC has been reported to reduce the brain Na⫹ current (Numann et al., 1994; Muramatsu et al., 1994). PKC has also been reported to slow the inactivation of the cardiac Na⫹ current (Numann et al., 1994; Qu et al., 1996).

IV. K⫹ CHANNELS

⫹

Nine types of Na channels have been cloned: three in brain (rat I, rat II, rat III), two in ganglion cells (SNA, ANA2), and one each in skeletal muscle (SkM1 or 애1), cardiac and denervated skeletal muscles (H1), peripheral neurons (PN1), and glial cells (NaCh6) (Alexander and Peters, 1999). In addition to these functional Na⫹ channels, several nonfunctional Na⫹ channel-like proteins have been cloned in skeletal, cardiac, and uterine smooth muscle cells (Knittle et al., 1996). By blot hybridization analysis of rat I–III and glial cell-type Na⫹ channels, the rat III channel was only detected in intestinal smooth muscle (Suzuki et al., 1988; Gautron et al., 1992). Possibly, the rat III type Na⫹ channel might be expressed in vascular cells and the colon (Table III). However, gene types for TTX-resistant Na⫹ channels in the uterus and epithelial cell-like Na⫹ channels in A7r5 cells are not known. Like the voltage-dependent Ca2⫹ channels, functional Na⫹ channels are conformed as a multiheteromer of 움, 웁1, and 웁2 subunits with a stoichiometry of 1:1:1 in brain, whereas in skeletal muscle cells, the 웁2 subunit has not been identified (Fozzard and Hanck, 1996). The mRNA for the 웁1 subunit has also been identified in the human uterus (Makita et al., 1994). This message does not, however, necessarily indicate the presence of 웁1 protein, as the 웁1 RNA message does not translate to 웁1 protein in cardiac cells (Yang et al., 1993; Zeng et al., 1996; Cohen and Levitt, 1993). As an expression of the 움 subunit would be sufficient to produce channel function, including current inactivation (Noda et al., 1986; Goldin et al., 1986; Krafte et al., 1988, 1990), the role of the 웁1 subunit in cardiac and smooth muscle Na⫹ channels is not clear. The Na⫹ channel 웁 subunit is a single transmembrane protein associated with the 움 subunit. Although the 웁1 subunit accelerates the inactivation kinetics of brain and skeletal muscle Na⫹ channels (Catterall et al., 1991; Isom et al., 1992; Goldin, 1995), the 웁 subunit does not seem to form an inactivation gate. As partial deletion of the linker between domains III and IV abolished the fast inactivation of both brain and cardiac Na⫹ channels,

Seven types of K⫹ channels have been identified by their biophysical and pharmacological properties in smooth muscle cells: two Ca2⫹-dependent K⫹ channels, two delayed rectifier K⫹ channels, an inward rectifier K⫹ channel, an A-like channel, and an ATP-sensitive K⫹ channel.

A. Ca2⫹-Dependent K⫹ Channels Ca2⫹-dependent K⫹ channels are distributed abundantly in various smooth muscle cells and are classified into two types according to their single channel conductance and sensitivity to K⫹ channel blockers (maxiK or BK channel and SK channel). Iberiotoxin and apamin, respectively, are the selective blockers for these two channels. Charybdotoxin also effectively blocks the BK channel. The physiological role of the BK channel in smooth muscle cells is thought to lie in the cytoprotection against Ca2⫹ overload by the prevention of membrane excitation. It has also been reported that adenosine- and ACh-induced vasodilation or hyperpolarization is caused by the activation of BK channels through a cAMP-dependent pathway (as PKA phosphorylation activates the BK channel) (Carl et al., 1991; Minami et al., 1993b; Cabell et al., 1994). In addition to this PKA phosphorylation-dependent activation, it has been reported that a direct mechanism exists for BK activation by both cAMP and Gs움 protein in coronary arteries (Minami et al., 1993b; Scornik et al., 1993). However, in the case of cAMP-dependent direct activation, the possible presence of endogenous PKA was not taken into account, although the authors did employ insideout membrane patches (Minami et al., 1993b). Similarly, cGMP-dependent activation of BK channels through PKG has been reported in rabbit cerebral artery (Robertson et al., 1993). However, phorbol esters were shown to inhibit BK channels in whole cell and cell-attached membrane patches, indicating the presence of PKCmediated inhibition of BK channels (Kitamura et al., 1992; Minami et al., 1993a).

17. Ion Channels in Vascular Smooth Muscle

Two subunits, 움 and 웁, have been identified in the BK channels found in tracheal and aortic smooth muscle cells (Knaus et al., 1993). The 움 subunit has seven transmembrane regions and has a highly homologous sequence with mouse ‘‘mslo’’ and Drosophila ‘‘slopoke’’ proteins (Atkinson et al., 1991; Butler et al., 1993). Although a homotetramer of 움 subunits is sufficient to form the channel pore, the coexistence of the 웁 subunit is essential for the normal characteristics of Ca2⫹-dependent K⫹ channels, such as voltage- and Ca2⫹-dependent properties (McManus et al., 1995; Knaus et al., 1994; Garcia-Calvo et al., 1994). Both 움 and 웁 subunits have charybdotoxin-binding ability (Garcia-Calvo et al., 1994).

B. Delayed Rectifier K⫹ Channels Delayed rectifier K⫹ channels exist in all excitable cells, including smooth muscle cells. The physiological role of these channels is thought to provide a countercurrent against Na⫹ and Ca2⫹ channels. In smooth muscle, delayed rectifier K⫹ channels are classified according to their sensitivity to two K⫹ channel blockers: TEA and 4AP. Delayed rectifier K⫹ channels in rabbit intestine are sensitive to TEA and resistant to 4AP, whereas those in the pulmonary artery are sensitive to 4AP, but not to TEA (Ohya et al., 1986; Okabe et al., 1987). A lower TEA sensitivity was also reported in a cloned delayed rectifier K⫹ channel from dog colon (⬎10 mM for Kv1.2 and Kv1.5 channels; Overturf et al., 1994). 4AP-resistant delayed rectifier K⫹ channels have also been identified in other vascular and visceral smooth muscle cells (Noack, 1992; Imaizumi et al., 1989; Klo¨ckner and Isenberg, 1985; Ohya et al., 1986; McFadzean and England, 1992; Buljubasic et al., 1992; Gelband and Hume, 1992; Bonnet et al., 1991). Delayed rectifier K⫹ channels in vascular cells are activated by PKA and inhibited by PKA inhibitors (Edwards et al., 1993; Aiello et al., 1995). PKA may shift the activation curve of the delayed K⫹ current to the left, as reported in cardiac cells (Walsh and Kass, 1991). This suggests that delayed rectifier K⫹ channels will be activated at the resting membrane potential in smooth muscle cells, and so hyperpolarize the membrane. 4APsensitive K⫹ channels from dog colonic smooth muscle cells have been cloned, and these have a highly homologous sequence with Kv1.2 and Kv1.5 families (Hart et al., 1993; Overturf et al., 1994). These authors also showed that Kv1.5 was abundant in various tissues, including vascular, cardiac, and brain cells, whereas Kv1.2 was present in visceral smooth muscle and brain cells, but not in vascular or cardiac cells. These Kv1.2 and Kv1.5 channels have PKA phosphorylation sites in their N- and C-terminal regions, and phosphorylation in the N-terminal region is thought to be essential for channel

337

activation in cardiac Kv1.2 (Kennelly and Krebs, 1991; Huang et al., 1994). Voltage-dependent K⫹ channels are formed from a tetramer of 움 subunits, and Kv1.1, Kv1.2, Kv1.5, and Kv1.6 in the Kv1 family all show delayed rectifying properties (Chandy and Gutman, 1995). On the one hand, rapid inactivation of the current was observed in those channels coexpressing 웁1 snd 웁3 subunits (England et al., 1995; Heinemann et al., 1995; Sewing et al., 1996). On the other hand, a 웁2 subunit did not induce current inactivation of the Kv1.1 channel (Rettig et al., 1994). Thus, 웁 subunits regulate channel properties of delayed rectifier K⫹ channels and also act as chaperonelike proteins enhancing channel translocation (Shi et al., 1996). As a 웁 subunit has not yet been identified in smooth muscle cells, it is uncertain whether the 움 subunit alone or 움 and 웁 subunits together form the functional channel. Heteromeric formations involving different 움 subunits (Kv1.2 and Kv1.5) are also thought to exist in smooth muscle cells (Horowitz et al., 1995). The single channel conductance of delayed rectifier K⫹ channels found in rabbit portal vein and coronary artery was reported to be ca. 5–10 pS (estimation by noise analysis) (Beech and Bolton, 1989; Volk and Shibata, 1993), and these values are slightly smaller than those reported for porcine airway (13 pS) and canine colonic (19 pS) smooth muscle cells (Boyle et al., 1992; Carl et al., 1995). Single channel conductances in natural and cloned Kv1.2 and Kv1.5 channels from canine colon were reported to be 14 and 10 pS, respectively (Hart et al., 1993; Overturf et al., 1994; Carl et al., 1995). Because delayed rectifier K⫹ channels are formed as a heteromultimeric complex, as with multiple types of subunits, more precise studies with electrophysiological and molecular biological methods are needed to clarify their exact nature and properties (Table IV).

C. Transient K⫹ Channels A Ca2⫹-insensitive transient K⫹ current can be found in guinea pig ureter smooth muscle cells (Lang, 1989; Imaizumi et al., 1990), but not in vascular cells. This channel is sensitive to 4AP, but not to TEA (Lang, 1989), and both its pharmacological and biophysical properties were similar to those of the A-like current seen in neurons and the transient outward current seen in cardiac cells (Adams et al., 1980; Jophson et al., 1984). A single channel conductance of 14 pS was reported in ureter (Imaizumi et al., 1990). Thus, on the basis of 4AP sensitivity and single channel conductance, transient K⫹ channels and delayed rectifier K⫹ channels are not easy to distinguish. It was reported that a single gene spliced the various types of voltage-dependent K⫹ channels that do or do

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TABLE IV Single Channel Conductances of K Channels in Smooth Muscle Cells Category [Ca]i dependent

Conductance (pS) BK

200

SK ATP-sensitive [Ca]o dependent

[Ca] insensitive

Blocker

Tissue

Reference

Charybdotoxin Iberiotoxin TEA (low)

Ubiquitous

50

Apamin

Visceral tissue

20

Glibenclamide

Rat portal vein

Kajioka et al. (1990)

TEA

Rabbit portal vein

Inoue et al. (1986)

100

Benham et al. (1985) Inoue et al.(1985) Standen et al. (1989) Kajioka et al. (1991)

ATP-sensitive

30

Glibenclamide

Pig coronary artery

Inoue et al. (1989)

ATP-sensitive

258 135 50 35 24

Glibenclamide Glibenclamide Glibenclamide Glibenclamide Glibenclamide

Rabbit pregromerular arteriole Rabbit mesenteric artery Rabbit portal vein Pig coronary artery Rabbit portal vein

Lorenz et al. (1992) Standen et al. (1989) Kajoikka et al. (1991) Dart and Standen (1993) Beech et al. (1993) Kamouchi et al. (1997)

Delayed rectifier

19 13 7.3

Canine colon Porcine airway Rabbit coronary artery

Carl et al. (1995) Boyle et al. (1992) Volk and Shibata (1993)

KV1.2

14

Canine colon

Hart et al. (1993)

KV1.5

10

Canine colon

Overturf et al. (1994)

not have rapid inactivation properties (A-like Kv1.3 and Kv1.4 channels; delayed rectifier type Kv1.1, Kv1.2, and Kv1.5 channels) (Schwarz et al., 1988). Furthermore, coexpression of 웁1 or 웁3 subunits changed the channel’s time-dependent inactivation properties (Sewing et al., 1996). Therefore, two possibilities may explain the transient K⫹ channel found in smooth muscle cells; i.e., it may be formed by A-like channel type 움 subunits or by a complex of delayed rectifier type 움/웁 subunits.

D. Inward Rectifier K⫹ Channels Inward rectifier K⫹ channels have been identified in cerebral, mesenteric, and coronary arteries (Edwards et al., 1988; Quayle et al., 1993). In these arterial cells, stimulation with high K⫹ produced vascular relaxation instead of contraction (Edwards and Hirst, 1988; McCarron and Halpern, 1990). This peculiar property was well explained by the presence and activation of this inward rectifier K⫹ channel. In general, inward rectifier K⫹ channels are thought to stabilize the membrane potential at low levels in cardiac cells; however, in these arteries these channels are thought to stabilize the membrane potential at a high level by channel closure (Quayle et al., 1993; Kubo et al., 1993). The Kir2.0 family is thought to provide these inward rectifier K⫹ channels

in arterial cells on the basis of its strong rectification and large dependence on intracellular polyamines and Mg2⫹ (Matsuda et al., 1987; Quayle et al., 1997).

E. ATP-Sensitive K⫹ Channels KATP is thought to be opened under certain physiological conditions via receptor activation in smooth muscle cells. Adenosine, calcitonin gene-related peptide (CGRP), somatostatin, galanin, and 웁 adrenergic receptors have all been shown to activate the KATP in smooth muscle cells (Nelson et al., 1990, 1993; Quayle et al., 1994; Wellman et al., 1998; Kleppischt and Nelson, 1995; Nagao et al., 1996; Belloni et al., 1991; Daut et al., 1990; Sabouni, 1989; Nakashima and Vanhoutte, 1995). Because these receptors are coupled to adenylate cyclase via Gs protein, except the galanin receptor (Alexander and Peters, 1999), KATP is thought to be activated by PKA phosphorylation. However, ACh and vasopressin both inhibited the KATP by a mechanism involving coupling with phospholipase C (Martin et al., 1989; Wakatsuki et al., 1992). Zhang et al. (1994) reported that forskolin, cAMP, and PKA all opened KATP channels, whereas okadaic acid, a phosphatase inhibitor, prevented deactivation. In the case of adenosine receptors, Kleppischt and Nelson (1995) considered that adenosine

17. Ion Channels in Vascular Smooth Muscle

FIGURE 4 Regulation of KATP channel by receptor activations.

activated KATP channels through the A2 receptor, which is coupled with Gs protein. However, Dart and Standen (1993) reported that adenosine activated KATP chanels via the A1 receptor, which is coupled with the Gi protein. In the latter paper, direct introduction of the Gi움 subunit into the cytosol was shown to activate KATP channels through a membrane-delimited pathway. Galanininduced activation of KATP channels might also involve this mechanisms. To try to explain the inhibitory effects

339

induced by muscarinic receptor stimulation, Bonev and Nelson (1994) considered PKC phosphorylation mechanisms. Because vasopressin is also known to stimulate PLC and synthesize InsP3 and DG, vasopressin may possibly inhibit the KATP channel through PKC (Fig. 4). Although no drug-activating KATP channels has yet been used clinically, studies of KATP channels in smooth muscle were facilitated by the development of K⫹ channel openers (Quast and Cook, 1988; Wilson et al., 1988; Wilson, 1989; Buckingham et al., 1989; Edwards and Weston, 1994; Kitamura and Kuriyama, 1994). It is now well accepted that K⫹ channel openers directly open KATP channels in various cells, as cloned KATP channels can be activated by K⫹ channel openers (Ashford et al., 1994; Inagaki et al., 1995). As KATP channels open only in the presence of K⫹ channel openers (Kajioka et al., 1990, 1991), and as this effect is abolished under cellfree conditions, an endogenous (intracellular) activating factor(s) seems to be a prerequisite for KATP channel activation (Kajioka et al., 1991; Zhang and Bolton, 1995; Findlay, 1988; Dunne and Petersen, 1986; Lederer and Nichols, 1989; Tung et al., 1990; Shen et al., 1991; Kajioka et al., 1991; Beech et al., 1993; Kamouchi and Kitamura, 1994; Zhang and Bolton, 1995; Thuringer et al., 1995) (Fig. 5).

FIGURE 5 Whole cell and single KATP channel currents in the rabbit portal vein. (A) The KATP channel current is evoked by pinacidil and blocked by glibenclamide. (B) In a cell-attached patch-clamp condition, large unitary currents (BK channel currents) open spontaneously and small unitary current (KATP) are evoked only in the presence of pinacidil. Glibenclamide inhibits this pinacidil-induced KATP current. (C) Making an inside-out membrane patch, unitary KATP channel currents automatically close even in the presence of pinacidil. Application of GDP reactivates KATP channels. Unitary currents are blocked by either ATP or glibenclamide. (S. Kajioka, M. Kamouchi, and K. Kitamura, unpublished observations)

340

II. Cellular Electrophysiology

Mg2⫹-ATP and NDPs (nucleotides di phosphate) are thought to be endogenous regulators of KATP channel activation in cardiac and smooth muscle cells and in other cells (Kajioka et al., 1990, 1991; Zhang and Bolton, 1995; Findlay, 1988; Dunne and Petersen, 1986; Lederer and Nichols, 1989; Tung and Kurachi, 1991; Shen et al., 1991; Beech et al., 1993; Kamouchi and Kitamura, 1994; Zhang and Bolton, 1995; Thuringer et al., 1995). Mg2⫹ is an essential factor for ATPand NDP-induced activation, as ATP alone inhibits and NDPs alone weakly activate these channels (Shen et al., 1993; Beech et al., 1993; Kamouchi and Kitamura, 1994; Thuringer et al., 1995). Two possible roles have been considered for channel activation by these endogenous substances: nucleotide binding and phosphorylation. Although electrophysiological experiments did not distinguish between these possibilities (Kamouchi and Kitamura, 1994), the presence of nucleotide-binding domains and both PKA and PKC phosphorylation sites in KATP channel subunits seem to indicate that both mechanisms may operate during KATP channel activation. Therefore, Shen et al. (1991) and Kamouchi and Kitamura (1994) proposed the idea that KATP channels possess two channel states, operative and inoperative, depending on the activity of the endogenous modulators. They considered that K⫹ channel openers could open the channel only when it was

in the operative state. However, as in cardiac cells, spontaneously opened KATP channels were seen in the presence of NDPs, but not in the presence of a K⫹ channel opener under cell-free conditions (Tung and Kurachi, 1991; Beech et al., 1993; Zhang and Bolton; 1995). This indicates that KATP channels in smooth muscle cells can also be activated spontaneously under certain conditions, such as in the presence of both NDP binding and phosphorylation. This is consistent with KATP activation occurring through Gs-coupled receptor stimulation, such as by CGRP, isoprenaline, or adenosine. However, the presence of another factor(s) cannot be ruled out (Fig. 6). Modulatory actions of the cytoskeleton on KATP channels, especially on their inactivation mechanisms, were suggested by Furukawa et al. (1996). They speculated that channel stabilization in the membrane by Factin is essential for maintaining its activity. Although an interaction between KATP channels and F-actin has not been proved, this idea suggested the possible presence of additional endogenous mechanisms for channel reactivation. However, in the rabbit portal vein, cytocharacin D and phalloidin did not change the current inactivation in the presence of K⫹ channel openers (Kitamura and Kawakami, unpublished observations). Thus, it is not yet clear whether vascular KATP channels interact with the cytoskeleton.

FIGURE 6 Schematic state model of KATP channel. In this model, both K channel openers and endogenous channel modulators (Mg-ATP, GDP, UDP, etc.) are required to open the KATP channels (open state). In physiological conditions, PKA phosphorylation may be substituted for K channel openers. When neither K channel opener nor adenylate cyclase-activating receptor agonist is present, KATP channel is in the resting state and ready to open (operative state). However, the loss of endogenous modulators results in channel closure, even in the presence of K channel openers (inoperative state). This state represents the channel ‘‘run down,’’ which is always observed during the process, making cell-free membrane patches.

17. Ion Channels in Vascular Smooth Muscle

F. Molecular Basis of KATP Channels The pore-forming subunits of KATP channels are from the Kir6.0 family, with the subunits having weak inward rectifying properties; however, the additional presence of the sulfonyl urea receptor (SUR) is essential for producing the full function of this channel. Two subtypes of Kir (Kir6.1 and Kir6.2) and three types of SUR (SUR1, SUR2A, and SUR2B) have been cloned (Sakurai et al., 1995; Isomoto et al., 1996; Inagaki et al., 1995a,b, 1996; Chutkow et al.,1996). A combination of these subunits forms pancreatic, cardiac, and smooth muscle type KATP channels with a stoichiometry of 1:1 (Clement et al., 1997). Isomoto et al. (1996) showed that a combination of Kir6.1 and SUR2B could form the smooth muscle KATP channel on the basis of its pharmacological properties. Values obtained for the single channel conductance of these cloned KATP channels are within the range of 70–80 pS, and these values are not altered by changes in subunit combination (Kir6.2/SUR1: Sakura et al., 1995; Kir6.2/SUR2A and Kir6.2/SUR2B: Isomoto et al., 1995; Shindo et al., 1998; Kir6.2/SUR1 and Kir6.2/SUR2A: Inagaki et al., 1996; Kir6.2/SUR1: Inagaki et al., 1995a). Furthermore, Kir6.1 was shown to activate a current with a single channel conductance of 70 pS without coexpression of SUR (Inagaki et al., 1995b). As native KATP channels in various cells had values for single channel conductance similar to those seen in these cloned channels, combinations of these subunits may produce the functional KATP channels in each cell (cardiac cells, ca. 75–80 pS in symmetrical 140 mM K⫹ solutions: Noma, 1983; Han et al., 1993; Fan et al., 1990; Thuringer et al., 1995; mouse 웁 cell, 88 pS: Panten et al., 1990; RINm5F and HIT T15 cells, 50–55 pS: Ribalet et al., 1988; Niki et al., 1989). However, values for single channel conductance recorded in several vascular cells were less than 50 pS in symmetrical high K⫹ conditions, and some KATP channels have significantly smaller conductances than those recorded for cloned channels or cardiac cells (guinea pig urinary bladder, 10 pS: Bonev and Nelson, 1993; porcine coronary artery, 35 pS: Miyoshi and Nakaya, 1990; Dart and Standen, 1993; porcine urethra, 43 pS: Teramoto and Brading, 1996; rabbit portal vein, 24 and 50 pS: Kajioka et al., 1991; Beech et al., 1993; Kamouchi and Kitamura, 1994) (Table IV). Furthermore, in rabbit mesenteric artery, canine aorta, and rat ventromedial hypothalamic neurons, values obtained for single channel conductance were larger than 130 pS in symmetrical K⫹ (140 mM) or asymmetrical high K⫹ conditions (60/140 mM K⫹) (Standen et al., 1989; Kovac and Nelson, 1991; Ashford et al., 1990). As single channel conductance would be regulated by channel pore-forming subunits, the reason for this divergence of single channel conductance values needs to be

341

clarified; possible reasons include the existence of small auxiliary proteins to regulate the pore size, an energy barrier against potassium permeation, or the existence of a novel type of Kir6.0 family.

V. CHLORIDE CHANNELS IN VASCULAR SMOOTH MUSCLE Most mammalian cells are known to possess Cl⫺ channels in the plasma membrane, and these control cell homeostasis by regulating membrane excitability and cell volume. A number of papers indicate that vascular smooth muscle cells are no exception (Large and Wang, 1996; Strange et al., 1996). A unique feature of these Cl⫺ channels is their bidirectional behavior in changing the membrane potential during channel activation. The resting membrane potential in the majority of vascular smooth muscle cells is between ⫺75 and ⫺50 mV (Nelson et al., 1990), whereas the Cl⫺ equilibrium potential (ECl) lies between ⫺30 and ⫺20 mV (Large and Wang, 1996). Therefore, the direction of Cl⫺ flow can be readily reversed, with the actual direction being dependent on whether the membrane potential is more positive or more negative than ECl . Two functionally different types of Cl⫺ channels have been identified in vascular smooth muscle cells: Ca2⫹activated Cl⫺ channels and volume-regulated Cl⫺ channels. These channels are known to be regulated, respectively, by the intracellular Ca2⫹ concentration ([Ca2⫹]i) and by cell expansion or possibly membrane stretch. Irrespective of the type of Cl⫺ channel or its regulation, however, an important electrophysiological property of these channels is to depolarize the membrane at the resting membrane potential, and in turn to trigger the opening of voltage-dependent L-type Ca2⫹ channels, which is a prerequisite for smooth muscle contraction (Pacaud et al., 1991; Droogmans et al., 1991).

A. Ca2⫹-Activated Cl⫺ Current [ICl(Ca)] The first evidence for Ca2⫹-activated Cl⫺ channels in smooth muscle was obtained in rat anococcygeous muscle using the whole cell patch-clamp technique (Byrne and Large, 1987a). Since then, there have been a large number of reports showing Ca2⫹-activated Cl⫺ channels in a variety of vascular smooth muscle cells (for a review, see Large and Wang, 1996). Many pharmacologically active compounds have been found to evoke ICl(Ca) in vascular smooth muscle (rat pulmonary artery, Salter and Kozlowski, 1996; Wang et al., 1997; rat portal vein, Pacaud et al., 1991; rat aorta, Salter and Kozlowski, 1998; rabbit basilar artery, Kamouchi et al., 1997; rabbit portal vein, Wang and Large, 1991). The mechanism involved in the receptor agonist-evoked

342

II. Cellular Electrophysiology

ICl(Ca) is a rise in [Ca2⫹]i through the Gq protein– phospholipase C–inositol-1,4,5-triphosphate (IP3) pathway (Large and Wang, 1996). An alternate pathway may be activation of nonspecific cation conductance by the agonist, resulting in Na⫹ and/or Ca2⫹ influx and thereby membrane depolarization (Byrne and Large, 1988; Wang and Large 1991). As a consequence, Ca2⫹ enters through voltage-dependent L-type Ca2⫹ channels, which may accelerate the activation of ICl(Ca) . This idea is consistent with the finding that depolarizing pulses evoke an L-type Ca2⫹ current (ICa(L)) followed by activation of an inward tail ICl(Ca) in rabbit coronary artery (Lamb et al., 1994), rat pulmonary artery (Yuan, 1997), rabbit portal vein (Greenwood and Large, 1996), and rat portal vein (Pacaud et al., 1989). The primary source of Ca2⫹ is considered to be intracellular Ca2⫹ stores or sarcoplasmic reticulum (SR), as the effect of receptor agonists on ICl(Ca) is not attenuated immediately after the removal of external Ca2⫹ in various types of smooth muscle (Droogmans et al., 1991; Wang and Large, 1991). Moreover, simultaneous recording of the membrane current and [Ca2⫹]i has clearly demonstrated that Ca2⫹ release from the SR, but not an influx of extracellular Ca2⫹, is the main requirement for ICl(Ca) (Pacaud et al., 1992). These SR Ca2⫹ stores may be separated into at least two types in vascular smooth muscle cells: an IP3-induced Ca2⫹ release (IICR) store and a Ca2⫹-induced Ca2⫹ release (CICR) store (releasable by caffeine and ryanodine) (Iino, 1991; Tribe et al., 1994). Treatment with caffeine, ryanodine, heparin (an inhibitor of IICR), or thapsigargin (a Ca2⫹-ATPase inhibitor) alters Ca2⫹ release from or uptake into CICR or IICR stores. In rabbit coronary artery myocytes, the depolarization-evoked inward tail ICl(Ca) is inhibited by caffeine, suggesting the contribution of CICR stores to the production of ICl(Ca) (Lamb et al., 1994). Because an overlap between the two types of store is also found in certain types of smooth muscle, there could be species and tissue differences in SR stores involved in the activation of ICl(Ca) . Although the intracellular mobilization of Ca2⫹ stores is of primary importance, it should be noted that extracellular Ca2⫹ also plays a role by refilling Ca2⫹ into the SR stores [as the removal of external Ca2⫹ gradually, but not immediately, attenuates ICl(Ca)] (Ame´dee´ et al., 1990; Kamouchi et al., 1997). Much work has been done on spontaneous transient outward currents (STOC), which are thought to result from spontaneous openings of Ca2⫹-activated K⫹ channels (Benham and Bolton, 1986). Spontaneous inward currents following periodic Ca2⫹ release from SR stores at the resting membrane potential are called spontaneous transient inward currents (STIC). These currents have been recorded with the use of perforated patch techniques in various types of vascular smooth muscle (Wang et al., 1992; Hogg et al., 1993). In some cells,

STICs are often coincident with STOCs, although their kinetics appear to be different (ZhuGe et al., 1998). STICs are dependent on the Cl⫺ gradient across the cell membrane, and their biophysical characteristics are similar to those of the Cl⫺ currents evoked by agonists and to ICa(L) . The finding of spontaneous local [Ca2⫹] increases beneath the plasmalemma, termed ‘‘Ca2⫹ sparks,’’ seems to account for the Ca2⫹ source for STICs (ZhuGe et al., 1998; Kotlikoff and Wang, 1998). Concerning the biophysical properties of ICl(Ca) , the rank order of permeabilities for anions has been reported to be similar in different types of smooth muscles (I⫺ ⬎ Br⫺ ⬎ Cl⫺; for a review, see Large and Wang, 1996). ICl(Ca) is highly selective for Cl⫺ over cations. ICl(Ca) , which is triggered by depolarizing step pulses, has been found to exhibit a bell-shaped current–voltage (I-V) relationship similar to that observed for ICa(L) (Lamb et al., 1994; Yuan 1997). This phenomenon is likely to be due to the dependency of ICl(Ca) activity on the amount by which [Ca2⫹]i is increased by ICa(L) in close proximity to Ca2⫹-activated Cl⫺ channels. This interpretation is supported by the fact that the I-V relationship for ICa(L) activated by receptor agonists, or by Ca2⫹ photolytically released from a caged Ca2⫹ compound, has been shown to be mostly linear between ⫺50 and 50 mV (Wang and Large, 1991; Clapp et al., 1996; Wang et al., 1997). In cell-attached patches, the unitary conductance of the Ca2⫹-activated Cl⫺ channel was found to be low (1–3 pS), and its pharmacological properties were found to be similar to those of the macroscopic ICl(Ca) (Klo¨ckner, 1993). The properties of this single channel are comparable to those of channels with a low unitary conductance recorded by means of an inside-out excised patch technique or noise analysis in other tissues. In view of the substantial size of ICl(Ca) , the Ca2⫹-activated Cl⫺ channel would be expected to have a high membrane density despite its low unitary conductance. ICl(Ca) is strongly inhibited by a fenamate, niflumic acid (IC50 ⫽ 1–10 애M), in vascular smooth muscle cells, in good agreement with reports showing inhibition in other tissues (Lamb et al., 1994; Yuan 1997; Kamouchi et al., 1997; Salter and Kozlowski, 1998). A carboxylic acid derivative, antheracene-9-carboxylic acid (9-AC) (Hogg et al., 1993), and the stilbene compounds, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS) (Lamb et al., 1994, Hogg et al., 1994) and 4-acetamide-4⬘-isothiocyanostilbene-2,2⬘-disulfonic acid (SITS) (Hogg et al., 1994), have also been reported to inhibit ICl(Ca) with IC50 values ranging between 10 애M and 1 mM. To date, a definitive molecular identification of this channel has not been made. A 140-kDa Ca2⫹-activated Cl⫺ channel termed bCLCA1 has been cloned few years ago from bovine trachea (Cunningham et al., 1995). However, this cloned channel had a large unitary conductance (25–30 pS), distinct anion selectivity, and tol-

17. Ion Channels in Vascular Smooth Muscle

erance to niflumic acid and seems only to be present in the tracheal epithelium. This suggests that it may be a somewhat different type of Cl⫺ channel from those found in vascular and other nonvascular tissues. It has been reported that a mouse clone for bCLCA1 (mCLCA1) possesses similar pharmacological features to native Ca2⫹-activated Cl⫺ channels and has a ubiquitous distribution in heart, kidney, lung, spleen, and brain (Gandhi et al., 1998). Further studies will still be required to positively identify this Cl⫺ channel.

B. Volume-Regulated Cl⫺ Channel [ICl(vol)] A Cl⫺ current regulated by changes in cell volume [ICl(vol)] has been reported in various mammalian cells (for reviews, see Strange et al., 1996; Okada 1997). Exposing cells to hypotonic solution or cell inflation by applying a positive hydrostatic pressure via the patch pipette is known to activate this type of current. ICl(vol) is known to mediate the initial compensatory response to cell swelling (i.e., a decrease in [Cl⫺]i occurs, together with a reduction in [K⫹]i , and consequently water flows out of the cell, restoring cell volume to normal). This mechanism, termed regulatory volume decrease (RVD), is highly important for the homeostatic regulation of cell volume in mammalian cells (Strange et al., 1996). Although cells are unlikely to be exposed to excessive changes in osmolarity in their normal environment, RVD is thought to play a major role in regulating cell homeostasis in certain pathological states (e.g., edema, diabetes) (Garber and Kahalan, 1997). Another important function of volume-regulated Cl⫺ channels in excitable cells is the regulation of membrane excitability and contractility; activation of ICl(vol) depolarizes the cell membrane and results in the opening of L-type Ca2⫹ channels. This phenomenon could be of pathological relevance in ischemia-induced cell swelling and in electrophysiological abnormalities. In most mammalian cells, ICl(vol) has been shown to be outwardly rectifying in symmetrical Cl⫺ solutions, to exhibit the anion permeability sequence I⫺ ⬎ Br⫺ ⬎ Cl⫺ (Hagiwara et al., 1992; Kubo and Okada, 1992; Lewis et al., 1993), and to exhibit sensitivity to block by stilbene compounds (Hagiwara et al., 1992; Lewis et al., 1993; Yamazaki et al., 1998; Greenwood and Large, 1998), by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (Kubo and Okada, 1992; Lewis et al., 1993; Ehring et al., 1994; Jackson and Strange, 1993), by external ATP (Jackson and Strange, 1995), and by the antiestrogen tamoxifen (Ehring et al., 1994; Zhang et al., 1994; Vandenberg et al., 1994; Duan et al., 1997a). The unitary conductance for ICl(vol) is known to be intermediate, and it is estimated to be 20–50 pS (Solc and Wine, 1991; Duan et al., 1997a) (Fig. 7).

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There have been, however, only a few studies of ICl(vol) in vascular smooth muscle cells (Horowitz et al., 1999). A decrease in external osmolarity in canine pulmonary and renal arterial smooth muscle cells has been shown to induce cell swelling, accompanied by an outwardly rectifying Cl⫺ current with the anion permeability I⫺ ⬎ Br⫺ ⬎ Cl⫺ and a pharmacological profile similar to that observed in other mammalian cells (Yamazaki et al., 1998). A similar finding was also reported in rabbit portal vein smooth muscle cells (Greenwood and Large, 1998) and in cultured human aortic and coronary smooth muscle cells (Lamb et al., 1999). It is possible that this type of Cl⫺ channel plays a physiological role other than cell volume regulation in vascular smooth muscle. It has been hypothesized that the activation of Cl⫺ channels may be responsible for the intraluminal pressure-induced membrane depolarization and contraction seen in rat cerebral artery (myogenic tone), a phenomenon independent of endothelial and neuronal inputs (Nelson et al., 1997). In such a preparation, Cl⫺ channel blockers, DIDS, and indanyloxyacetic acid (IAA-94) each inhibit pressure-induced depolarization and contraction. Although Ca2⫹-activated Cl⫺ channels are expressed abundantly in smooth muscle (see the previous section), niflumic acid, a potent inhibitor of ICl(Ca) , did not alter the pressure-induced responses of regulation in rat cerebral artery (Nelson et al., 1997). This finding suggests that the Cl⫺ channel other than the Ca2⫹-activated Cl⫺ channel plays a role in the depolarization induced by membrane stretch or intraluminal pressure. This leads to speculation that the mechanism involved in myogenic tone may share the population of Cl⫺ channels that contributes to cell volume regulation. The molecular nature of these channels has not been established and several gene products have been proposed as molecular candidates for ICl(vol) (for reviews, see Okada 1997; Hume et al., 1999). These include multidrug resistance P-glycoprotein (P-Gp, MDR-1) (Valvarde et al., 1992), pIcln (Paulmichl et al., 1992), and ClC2 (Gru¨nder et al., 1992). Data suggest that P-Gp and pIcln are regulators of endogenous ICl(vol) , but not channels themselves. Among the ClC gene family (for a review, see Jentch 1996), ClC-2 is sensitive to cell volume and was thus at first proposed as a candidate, yet the product of this gene exhibits inward rectification with the anion permeability Cl⫺ ⱖ Br⫺ ⬎ I⫺ (Thiemann et al., 1992), each of which properties is strikingly different from the characteristics of ICl(vol) . Overexpression of ClC-3 in NIH/3T3 cells, which had been cloned from rat kidney (Kawasaki et al., 1994), was shown to increase outwardly rectifying Cl⫺ conductance drastically when the cell was exposed to a hypotonic solution (Duan et al., 1997b). ClC-3 has been found to possess biophysical and pharmacological profiles similar to those of ICl(vol) , indicating that ClC-3 may be responsible for the native ICl(vol) in

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FIGURE 7 Volume-regulated Cl⫺ currents in canine pulmonary artery. (A and B) Cells were exposed to the isotonic external solution (300 mOsm/kg) (a) and subsequently to the hypotonic external solution (230 mOsm/ kg) (b). Membrane currents evoked at potentials ranging from ⫺100 to 100 mV are shown. (C) Hypotonicityinduced depolarization is attenuated by DIDS (100 애M). A normal Tyrode solution and K⫹-rich pipette solution were used. (J. Yamazaki, unpublished observations)

TABLE V Expression of ClC Cl⫺ Channels in Smooth Muscle Cells Clone

Permeability

ClC2

Cl ⬎ Br ⬎ I

Voltage Cell volume [pH]o

Inward

Rat stomach,b intestineb Rabbit colonc Human stomachb Human aorta,b,d coronary arteryb,d

Thiemann et al. (1992) Furukawa et al. (1995) Sherry et al. (1997) Lamb et al. (1999)

ClC3

I ⬎ Br ⬎ Cl

Cell volume (stretch?)

Outward

Canine pulmonary artery,c renal arteryc Canine colonc Human aorta,b,d coronary arteryb,d Pulmonary arteryd,e

Yamazaki et al. (1998) Dick et al. (1998) Lamb et al. (1999) Lamb et al. (1999)

ClC5

I ⬎ Cl ⬎ Fa

[pH]o

Strong outward

Rat colonb Human aorta,b,d coronary arteryb,d

Sakamoto et al. (1996) Lamb et al. (1999)

ClC6

?

?

?

Human small intestine,b colonb

Brandt and Jentch (1995)

a

Regulation

Rectification

Tissue

The conductance sequence was reported to be Cl ⬎ Br ⬎ I at strongly positive potentials (Steinmeyer et al., 1995). b Northern blot analysis. c RT-PCR. d Cultured smooth muscle cells. e In situ hybridization.

Reference

17. Ion Channels in Vascular Smooth Muscle

mammalian cells. This gene product is of potential interest in vascular smooth muscle cells: as ClC-3 mRNA has been shown to be expressed in different types of vascular and visceral smooth muscle cells in a way corresponding well to the functional expression of ICl(vol) (Yamazaki et al., 1998; Dick et al., 1998) (Table V).

VI. NONSELECTIVE CATION CHANNELS Two types of nonselective cation channels have been reported to be present in smooth muscle cells; a hyperpolarization-activated nonselective cation channel and a receptor-operated nonselective cation channel. Hyperpolarization-activated channels have been recorded in cardiac muscle, neurons, and other excitable cells, and this channel is thought to contribute to pacemaker activity and to the quick recovery from the afterhyperpolarization (which permits the generation of a train of action potentials at a high frequency) (DiFrancesco, 1995; Pape, 1996). In smooth muscle cells, this current is also observed in cells that exhibit spontaneous electrical activity, such as those in the small intestine, uterus, bladder, and portal vein (Benham et al., 1987; Kamouchi et al., 1991; Green et al., 1996; Okabe et al., 1999). However, such channels are not present in vascular cells, except in the portal vein. The other type of nonselective cation channel is activated by receptor stimulation. This type of channel has been further classified into two types: ligand-gated and G-protein-coupled nonselective cation channels (Kuriyama et al., 1998). The P2X1 channel is an example of an ion channel activated by direct binding of the ATP molecule to the channel protein; this channel is present in vascular cells. This channel is activated by ATP and by several of its analogues, and the current is inactivated rapidly (Benham et al., 1987a; Benham and Tsien, 1988; Benham, 1989; Xiong et al., 1991). This channel permeates several cations, including Na⫹, K⫹, and Ca2⫹. The permeability ratio for Na⫹:Ca2⫹ has been reported to be 1:3–4 in various cells (Benham and Tsien, 1988; Honore´ et al., 1989). The P2X1 channel is thought to be a homotrimer of subunits with two transmembrane domains (Valera et al., 1994; Nicke et al., 1998). Although heteromultimer structures have been proposed for other P2X receptors, P2X1 mRNA signals have been predominantly observed in vascular cells, indicating the high likelihood of the presence of a homomultimer conformation (Soto et al., 1996; Garcia-Guzman et al., 1996; Le et al., 1997; Longhurst et al., 1996; Bo et al., 1998; Torres et al., 1998, 1999). The presence of a weak mRNA signal for a splice variant P2X1 subunit in rat cerebral and mesenteric arteries, as well as in cardiac and vas deferens smooth muscle cells, has been shown (Ohkubo

345

et al., 1999). Thus, a heteromultimer conformation involving wild and variant subtypes may be present in vascular cells. It has been reported that the 웂 phosphate of ATP interacts with the P-loop region located near the second transmembrane domain (Hansen et al., 1997). The P2X1 receptor shows a transient current with rapid inactivation, and for this channel property both transmembrane regions are essential; however, the P2X2 receptor channel, which is a noninactivating channel, acquires its inactivating property by C-terminal

FIGURE 8 PDBu/GTP-induced nonselective cation currents in rabbit portal vein. Three micromolar GTP웂S was added to the pipette solution (Cs⫹ aspartate), and 5 mM Ba2⫹-containing physiological salt solution was superfused in the bath. PDBu (0.3 애M) was applied to the bath. At (a) and (b), a ramp pulse (from ⫺100 to 50 mV) was applied from the holding potential of ⫺60 mV. Obtained currents by ramp pulses are demonstrated in B. (C) Relationships of the amplitude of PDBu/GTP-induced current and PDBu concentrations in the absence and presence of 3 애M GTP웂S. The PDBu/GTP-induced current was predominantly recorded in the presence of GTP웂S. (M. Oike and K. Kitamura, unpublished observations)

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truncation (Werner et al., 1996; Bra¨ndle et al., 1999). Therefore, the absence of a long C-terminal in the P2X1 subunit is also important for its inactivating properties, as is the presence of a P2X1-type transmembrane motif. The G-protein-coupled purinoceptor also activates a nonselective cation channel in the rabbit portal vein (Xiong et al., 1991). This current did not inactivate, and higher concentrations of ATP (submicromolar order) were required for channel activation, whereas 움웁-methylene ATP had only a very weak action. In contrast to the P2X1 receptor current, these authors also showed that the purinoceptor-mediated current was coupled to a pertussis toxin-insensitive G-protein and did not in practice permeate Ca2⫹ ions (Xiong et al., 1991). G-protein-coupled nonselective cation channels are also activated by stimulations of other receptors, which are generally coupled with Gq움 proteins (움 adrenoceptor in portal vein: Byrne and Large, 1988; Inoue and Kuriyama 1993; Yamada et al., 1996; endothelin in mesenteric artery: Chen and Wagoner, 1991; Enoki et al., 1995). Ionic properties similar to those of the pertussis toxin-insensitive current were also seen with GTP and PDBu when these were applied simultaneously (Oike et al., 1993). Therefore, it is likely that simultaneous modulation of the channel, through direct G-protein binding and PKC phosphorylation, is essential for its activation by receptor stimulation (Fig. 8).

‘‘window current’’ could be evoked. It is uncertain whether Ca2⫹ and Na⫹ influx through a window current is enough to elevate the [Ca2⫹]i and membrane depolarization, as reevaluation of the channel properties under the phsiological condition is necessary for elucidation of their physiological functions. Patch clamp experiments revealed various types of ion channels in vascular smooth muscle cells, and differences of electrical properties of vascular cells from visceral and cardiac cells might be caused by differences of distribution and density of each type of ion channel. The presence of nonselective cation channels activated by agonist binding to the receptors is especially important for direct and secondary activation of the voltagedependent ion channels, such as voltage-dependent Ca channels, and could specify the electrical activities of the vascular smooth muscle cells. However, it has been reported that first-order structures of the ion channels in vascular smooth muscle cells are not identical to the corresponding channels in other tissues. As there were very few reports showing that single or few points mutation of amino acid in the channel protein changed their pharmacological and physiological properties, speculation of channel properties from other known tissues was limited. Thus, further evidence with molecular biological experiments in vascular cells will be required for evaluating ion channels in vascular cells.

VII. SUMMARY

Acknowledgment

This chapter reviewed the several ion channels present in vascular smooth muscle cells. The physiological roles of some of them are uncertain, such as T-type Ca2⫹ and TTX-sensitive Na⫹ channels. Activation of these ion channels is speculated to contribute the initial membrane depolarization for generation of the action potentials in electrically active smooth muscle cells, and this might be correct in the case of small arterial cells having the lower resting membrane potential. In these vascular cells, a part of the ion channels is in the resting state at the resting membrane potentials, and initial membrane depolarization by receptor activation opens these channels to initiate the action potentials. However, no positive evidence has been proposed for their mechanisms in vascular smooth muscle cells. Furthermore, this idea is inapplicable to vascular cells having higher resting membrane potentials, such as aorta and pulmonary artery. Thus, other functions may need to be elucidated. Both T-type Ca2⫹ and TTX-sensitive Na⫹ currents close their inactivated gates rapidly by a sufficient depolarization; however, in a certain range of the membrane potential, channels are activated continuously with very low open probability. In such a condition, a so-called

We are grateful to Dr. R. J. Timms for correcting the English and for giving comments.

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18 Cardiac Pacemaker Currents D. DIFRANCESCO, A. MORONI, and M. BARUSCOTTI

ERIC A. ACCILI

University of Milan Department of Physiology and General Biochemistry 20133 Milan, Italy

School of Kinesiology Faculty of Applied Sciences Simon Fraser University Burnaby, British Columbia, Canada V5A 1S6

I. INTRODUCTION

presented that have finally shed light on the molecular structure of pacemaker channels and provided interesting clues as to how this structure is related to the unusual properties displayed by them.

The pacemaker activity of the heart originates in specialized myocytes located in the sinoatrial (SA) node. These specialized myocytes are known as ‘‘pacemaker’’ cells because, unlike cells from most other areas of the heart, they are able to beat spontaneously (e.g., West, 1955). Using voltage clamp techniques, the spontaneous activity of these cells has been found to arise from a phase known as the ‘‘slow diastolic’’ or simply ‘‘diastolic’’ depolarization because the rate is slow relative to the action potential upstroke and because it occurs during diastole. Depolarization occurs until threshold is reached for the subsequent action potential. The key current involved in the generation of this diastolic depolarization, and the modulation of this phase by neurotransmitters, is known as the cardiac ‘‘pacemaker’’ current. This current is activated by hyperpolarization and is carried by both Na⫹ and K⫹ ions. This current has also been called the hyperpolarizationactivated current because of its activation by hyperpolarization and the ‘‘funny’’ current (If) because of this and other biophysical characteristics that, at the time of discovery, were very unusual. For the purpose of this chapter we will refer to the native cardiac current as If . This chapter will provide highlights of the history regarding the discovery of this current and describe its unusual biophysical nature and modulation related to its important physiological role in pacemaking. The expression of the current in other regions of the heart under normal and pathophysiological conditions will be described. Finally, recent and exciting results will be

Heart Physiology and Pathophysiology, Fourth Edition

II. BACKGROUND ON THE PACEMAKER CURRENT Diastolic depolarization was originally thought to come about because of the decay of potassium conductance. Weidmann (1951) originally measured a decrease in membrane conductance during diastole in Purkinje fibers. The decay of potassium conductance seemed to be a reasonable way to explain the diastolic depolarization, and this was proposed by Vassalle (1966) based on voltage clamp measurements. Noble and Tsien (1968) and others described a current that was K selective and outward and decaying on hyperpolarization to the diastolic range of potentials. This current became known as IK2 in order to differentiate it from the classic delayed rectifier known as IK and from K-dependent inward rectification known as IK1 . An important property of this current, which lent support to the notion that it was important for pacemaker activity, was its modulation by catecholamines (Tsien et al., 1974). IK2 was reinterpreted as a current activated on hyperpolarization to potentials in the diastolic range, inward at these potentials, and carried by K⫹ and Na⫹. Two key observations led to the reinterpretation of IK2 . First, K⫹ depletion was found to alter the time course of currents during voltage clamp hyperpolarization in Pur-

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kinje fibers, thus producing a current reversal near the K⫹ equilibrium potential that was not real (DiFrancesco, 1981a,b). Second, a hyperpolarization-activated Na⫹ and K⫹ current was discovered in the SA node, which shared several properties with IK2 (Brown et al., 1979; DiFrancesco and Ojeda, 1980). This current became known as the ‘‘funny’’ current (If ) or the hyperpolarization-activated current (Ih ) due to its unusual properties. The notion that IK2 had been interpreted incorrectly as a K⫹ current, but was in fact identical to the ‘‘funny’’ current of the SA node, explained the disappearance of the IK2 current in Na⫹-free solutions (McAllister and Noble, 1966) and the fact that the current reversal was too negative for a K⫹ current (Cohen et al., 1976). The If current possesses properties that make it an ideal ‘‘pacemaker’’ current. The activation of this current by hyperpolarization and the fact that the current is inward in this range of potentials, due to its permeability to both sodium and potassium, make it ideal for the generation of pacemaker activity. A criticism of this view resulted from data suggesting that the range of pacemaker current activation was too negative relative to the range of pacemaker depolarization (Yanagihara and Irisawa, 1980). Factors that influence the variability of pacemaker current activation are discussed later. Other possible mechanisms for pacemaker generation have been suggested (Noble, 1984), including the activation of Ca2⫹ currents, the decay of the K⫹-delayed current (IK) in the presence of a ‘‘background’’ Na⫹ current, and a ‘‘sustained’’ inward current possibly carried by Ca2⫹ ions. These have been discussed in more detail elsewhere (Irisawa et al., 1993; DiFrancesco, 1993; Guo et al., 1995). However, it should be mentioned that the closing of K channels, i.e., the decay of outward current, cannot by itself generate depolarization. This is because I ⫽ ⫺C dV/dt holds in the SA node myocyte, where I is total ionic current, C is capacitive current, and dV/dt is the rate of change of membrane potential. I ionic must be negative, by definition inward, in order for dV/dt to be positive, i.e., in order to get depolarization. This is why the ‘‘IK decay’’ hypothesis requires the presence of an inward ‘‘background’’ current. Although this has often been assumed for computational purposes, the functional characterization of such a current is still scarce.

a cell has been voltage clamped at ⫺32 mV and hyperpolarized to potentials ranging from ⫺42 to ⫺72 mV. Hyperpolarization increases both the amount of inward current and the rate at which this current activates, and a sizeable fraction of If is already activated at ⫺42 mV. It has been reported by some authors that the range of activation of these currents is too negative to be involved in the generation of the diastolic depolarization. This range has proven to be quite variable. Pacemaker currents, measured in multicellular preparations, were originally reported to be activated on hyperpolarization from holding potentials of about ⫺35 mV (Brown et al., 1979; Brown and DiFrancesco, 1979). However, more negative voltages, near ⫺50 mV, have also been reported, suggesting a smaller role for this current in generation of the diastolic depolarization (Yanagihara and Irasawa, 1980). Measurement of the current in single cells has revealed a large variability in activation threshold ranging from ⫺35 mV (DiFrancesco et al., 1986) to ⫺65 mV (Denyer and Brown, 1990). Several factors may be responsible for this variability. Current rundown, in the form of a parallel negative shift in the current activation curve, has been shown to occur within a short

III. PROPERTIES OF CARDIAC If A. Kinetics If is activated by hyperpolarization and its threshold shows variablity. A distinguishing feature of these currents is their activation by hyperpolarization. In Fig. 1a,

FIGURE 1 (a) Current traces from a myocyte, isolated from the rabbit SA node, held at ⫺32 mV and pulsed to ⫺42, ⫺52, ⫺62, and ⫺72 mV. (b) A fully activated curve generated from a myocyte, isolated from the rabbit SA node, using the voltage protocol illustrated in the inset.

18. Cardiac Pacemaker Currents

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time frame (DiFrancesco et al., 1986), thus leading to a more negative threshold for activation. The washout of cAMP, which regulates the position of the activation curve (see later), is probably responsible for at least part of this ‘‘short-term’’ rundown. Because the position of the activation curve depends on cAMP, the natural variation in intracellular levels of this cyclic nucleotide will produce variability in the activation threshold of the pacemaker current between cells. Differences in the electrophysiological protocol for pacemaker current activation curves may also lead to variations in activation threshold. At depolarized voltages, longer hyperpolarizations are required for complete activation (DiFrancesco and Noble, 1989). Steps of 0.5 to 3 sec in duration (Denyer and Brown, 1990; Nakayama et al., 1984; van Ginneken and Giles, 1991) may lead to incomplete current activation at depolarized voltages and to a more negative estimate of the activation threshold. The kinetics of the pacemaker current allow for a significant contribution to the diastolic depolarization. The rate of activation of this current may seem slow relative to the the speed of action potential firing in the SA node. However, only a very small amount of net inward current is required to drive the diastolic depolarization. SA node cells have a mean capacity of about 30 pF (DiFrancesco, 1986; Denyer and Brown, 1990), and the rate of diastolic depolarization is normally around 0.1 mV/sec. Therefore, only 3 pA is required to depolarize the cell from the MDP to the action potential threshold. Experimental and theoretical analyses indicate that both the size and the kinetics of the pacemaker current are compatible with a contribution to the diastolic depolarization (Van Ginneken and Giles, 1991; DiFrancesco, 1991). More recently, it has been shown that If measured in cardiomyocytes from zebrafish mutants with slow heart rates, called slow mo, lacks a fast component that is seen in the wild-type zebrafish (Baker et al., 1998). This strongly supports the notion that If kinetics, as well as expression levels, are compatible with a major role for this current in the control of heart rate.

to a potential to fully activate the channel, which was ⫺112 mV in this example. The cell was then pulsed back to different test potentials. The current amplitude was measured at the instant it was pulsed to the test potentials indicated by the double-ended arrows. These amplitudes were plotted against the test potential to generate a linear fully activated curve. The reversal potential in this cell is approximately ⫺12 mV, indicating the mixed nature of the current. PNa /PK permeability ratios have been reported in the range of approximately 0.27–0.35, indicating that the channel has a preference for passing K (Frace et al., 1992; Ho et al., 1993). This property is an important one, physiologically speaking, as it determines that current flow is inward in the diastolic range of potentials and can therefore produce depolarization during diastole. An interesting feature of the fully activated conductance curve is the effect of increasing extracellular K⫹, which resulted in an increase in the inward portion of the fully activated current (Fig. 1c from DiFrancesco et al., 1986). This ‘‘activation-like’’ effect of extracellular K⫹ on If has also been observed in Purkinje fibers (DiFrancesco, 1982). Frace et al. (1992) have suggested that this effect is due to an interaction of Na⫹ and K⫹ binding to multiple sites within the pore, a result supported by other studies where external Na⫹ modified the PNa /PK permeability ratio of the If channel (Ho et al., 1993). Rundown of the If , in addition to a shift in the activation curve, may be also due to a decrease in conductance. This type of rundown differs from the one due to a negative shift of the activation curve. Rundown of the fully activated conductance is slower and may appear after approximately 20 min of recording (DiFrancesco et al., 1986). There is no change in the reversal potential of the current during this type of rundown, suggesting that a reduction in the number of channels or a decrease in the single channel conductance is responsible. This type of rundown is likely to be associated with a decrease in both intracellular cAMP and phosphorylation processes (see later).

B. Ionic Properties of Pacemaker Currents

C. If Single Channel Conductance

The fully activated relation is linear and reverses at potentials compatible with permability to both sodium and potassium. Detailed studies in Purkinje fibers determined that both sodium and potassium participate in carrying the pacemaker current (DiFrancesco, 1981). These results were later confirmed in the SA node (DiFrancesco et al., 1986). An example of the fully activated conductance curve from one cell from the SA node is shown in Fig. 1b (DiFrancesco et al., 1986). The inset indicates the protocol used to generate this relation. Briefly, the cell was held at ⫺32 mV and hyperpolarized

Channels underlying pacemaker currents have a very small single channel conductance. The mixed cationic nature of pacemaker currents led to the assumption that single channel conductance would be relatively large, much like other nonspecific cationic channels. Original attempts at measuring single channel currents in the SA node were not successful, leading to the suggestion that single channel currents were too small to resolve, possibly, because the current was carried by a mechanism more similar to transporters (Van Ginneken and Giles, 1985). However, using a two-pipette technique, single

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channel currents were measured in SAN myocytes (DiFrancesco, 1986). A linear current voltage relationship was generated and yielded a single channel conductance of approximately 1 pS.

IV. MODULATION OF PACEMAKER CHANNEL FUNCTION A. Classic Autonomic Modulation Heart rate is under control of the autonomic system. The control of the heart rate is primarily under the influence of the parasympathetic and sympathetic nervous system at the SA node. This region of the heart is densely innervated by these systems and contains receptors for the neurotransmitters and hormones released by these systems. Stimulation of the sympathetic nervous system leads to acceleration of heart rate through the 웁-adrenergic agonists norepinephrine and epinephrine, whereas stimulation of the parasympathetic nervous system leads to deceleration of heart rate through the muscarinic actions of ACh. Reviews have dealt with the actions of the autonomic nervous system on other current components (Hartzell, 1888; Irisawa et al., 1993; DiFrancesco, 1993). The focus here will be on how If is modulated by the autonomic nervous system. Sympathetic stimulation shifts the voltage dependence of f channel activation to more positive voltages, thus accelerating diastolic depolarization and heart rate. 웁-adrenergic stimulation has been known to increase IK2 in Purkinje fibers (Hauswirth et al., 1968; Tsien, 1974) and If in the SA node (Brown et al., 1979; Noma et al., 1980). Adrenaline increases the pacemaker current at diastolic potentials by shifting the activation curve to more positive voltages (DiFrancesco et al., 1986). An example of the action of 1 애M isoproterenol on the steady-state current, and the activation curve determined using a ramp protocol, is shown in Fig. 2. Here, a cell is held at ⫺35 mV and is subjected to a 60sec voltage ramp beginning at ⫺35 mV and ending at ⫺125 mV (Fig. 2a, note that time runs backwards). As can be seen from the steady-state current tracing in Fig. 2b, exposure to isoproterenol has no effect on the fully activated current (compare the maximal current at ⫺120 mV under control conditions and in the presence of isoproterenol). However, the activation curve shown in Fig. 2c is shifted to more positive potentials. The shift in channel activation provides a more inward current at diastolic potentials, thereby increasing the slope of this phase and accelerating heart rate. Increases in both pacemaker current and beating frequency can be recorded at fairly low isoprenaline doses (0.001–0.003 애M ) (Zaza et al., 1998). As can be seen in Fig. 3 (Di-

FIGURE 2 (a) Voltage ramp protocol, (b) steady-state current traces, and (c) activation curves generated from a myocyte isolated from the rabbit SA node. Adapted from Accili et al., 1997b, with permission.

Francesco, 1993), relatively low doses of isoproterenol increase beating frequency primarily by altering the diastolic depolarization with little effect on the shape of the action potential. This supports the idea that, at these low doses, the increase in beating frequency is due primarily to the modulation of If . Parasympathetic stimulation shifts the voltage dependence of If channel activation to more negative voltages, thus slowing diastolic depolarization and heart rate. Vagal stimulation had been suggested to cause slowing of beating frequency in the SA node by activation of a K⫹ current and the resulting hyperpolarization (Giles and Noble, 1976; Noma and Trautwein, 1978). More recently, it has been shown that ACh has another action, that of inhibiting the pacemaker current by a mechanism opposite to that of catecholamines. This involves a reduced cAMP production and a shift of the current activation curve to more negative voltages (DiFrancesco and Tromba, 1988a,b). Using a voltage ramp protocol, in Fig. 2 as just described, 1 애M ACh is seen to shift the If activation curve to more negative voltages without altering the fully activated current, i.e., an effect opposite to that produced by isoproterenol (Figs. 2b and 2c). The effect of ACh is sensitive to pertussis toxin,

18. Cardiac Pacemaker Currents

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FIGURE 3 Action potentials recorded from myocytes isolated from the rabbit SA node. Reprinted with permission from the Annual Review of Physiology, Vol. 55  1993 by Annual Reviews, www.AnnualReviews.org.

indicating the involvement of an inhibitory G-protein in these actions. Inhibition of the pacemaker current may be the primary mechanism of reducing heart rate in response to low concentrations of ACh (DiFrancesco et al., 1989). This is because the doses of ACh required for pacemaker current inhibition in the SA node are 20 times lower than those required for the activation of K⫹ channels; also, doses of ACh inhibiting If without activating IK,ACh are effective in reducing the beating frequency of isolated cells. These results are consistent with evidence showing that the reduction in heart rate in response to low ACh concentrations or moderate vagal stimulation occurs with only a reduction in the slope of the diastolic depolarization (Hirst et al., 1992). Also, it has been shown that the membrane conductance of pacemaker cells decreases in response to ACh or vagal stimulation, which is consistent with inhibition of If and not with activation of IK,ACh (Hirst et al., 1992). As can be seen in Fig. 3 (DiFrancesco, 1993), relatively low doses of ACh decrease beating frequency primarily by altering the diastolic depolarization with little effect on the shape of the action potential, which again agrees with the idea that, at these low doses, the decrease in beating frequency is due primarily to the modulation of If . ACh causes a negative shift in the activation curve of If in Purkinje fibers. However, this occurs only after 웁-adrenergic stimulation, probably reflecting low basal levels of cAMP (Chang et al., 1990). This difference in muscarinic actions between the SA node and Purkinje fiber is likely to be functionally important. When vagal tone is high, SA nodal firing frequency drops and may in fact stop completely. However, the lack of any marked effect on Purkinje fibers ensures that the ventricular rate of contraction and cardiac output is maintained. However, increases in the rate of Purkinje fiber contractions are limited under conditions of high sympathetic and parasympathetic activity. This is necessary to prevent extra ventricular beating under these conditions.

웁-adrenergic agonists increase, and muscarinic agonists decrease, cAMP levels suggesting that this messenger is crucial in producing the positive shift in the activation curve. Initially, cAMP was implicated as a second messenger important for the actions of adrenaline on IK2 in Purkinje fibers (Tsien et al., 1972). Cell-attached measurements further supported the role of a diffusible second messenger in the adrenaline-mediated modulation of If in the SA node (DiFrancesco, 1986). Subsequently, forskolin and IBMX, which increase intracellular cAMP levels by stimulating and inhibiting adenylate cyclase and phosphodiesterase, respectively, were found to shift the If activation curve to more positive voltages with no change in fully activated conductance (DiFrancesco and Tromba, 1988a). Although an action through protein kinase A was suspected by virtue of the fact that kinase inhibitors reduced the actions of 웁-adrenergic agonists on If in Purkinje fibers (Chang et al., 1991), it was then found that the direct application of cAMP to patches excised from nodal myocytes shifts the activation curve of the pacemaker current to more positive voltages due to a direct interaction of intracellular cAMP with channels according to a phosphorylationindependent mechanism (DiFrancesco and Tortora, 1991). The direct action of cAMP has no effect on the If fully activated conductance. In single channel measurements from excised patches, the direct action of cAMP decreases the latency to first channel opening, reflecting an increase in Po with no changes in single channel current, and the Po increase is due to a rightward shift of the voltage dependence of Po (DiFrancesco and Mangoni, 1994). The direct action of cAMP is the crucial mechanism by which neurotransmitters control the position of the activation curve, but some observations suggest that this may be controlled by other factors. In excised patches, the activation range of If moves to very negative potentials (DiFrancesco and Tortora, 1991). This very negative shift cannot be accounted for by the

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reduction or absence of cAMP and implies the existence of other regulatory factors. In Fig. 4, the intracellular components of autonomic modulation are presented. Activation of the 웁-adrenergic receptor leads to activation of Gs , stimulation of adenylyl cyclase, and an increase in cAMP; activation of the muscarinic receptor leads to the activation of Gi , inhibition of adenylyl cyclase, and a decrease in cAMP; finally, cAMP activates f channels by directly binding to them. This was the first description of an ion channel that could be modified both by voltage, as for voltage-gated channels, and by the binding of cAMP, as for ligand or cyclic nucleotide-gated channels. This modulation has been described where voltage and cAMP binding act as allosteric modulators of the channel according to hybrid Hodgkin–Huxley and Monod–Wyman– Changeux models (DiFrancesco, 1999). The reaction scheme is not linear but cyclic, and voltage and cAMP binding cooperate to modify the channel configuration. The similarities in actions of cAMP and voltage on channel kinetics have suggested that both may involve similar conformational effects on the channel and specifically on the voltage sensor (DiFrancesco and Tortora, 1991). The model predicts the shift in activation produced by cAMP and the sigmoidal dependence of this shift on cAMP concentration. A crucial hypothesis of this description is that the binding of cAMP is facilitated when the channel is in the open configuration. This readily explains the observation that cAMP produces larger effects on deactivation as compared to activation and suggests that this molecule is able to lock the channel in an open configuration. Phosphophorylation/dephosphorylation processes modify 웁-adrenergic modulation of If . In the SA node, the phosphatase inhibitor calyculin significantly increased the 웁-adrenergic-mediated shift in the pacemaker current activation curve (Accili et al., 1997a).

FIGURE 4 Modulation of the If channel through the cAMP pathway.

This finding is compatible with earlier studies showing that protein kinase inhibition reduces the actions of 웁adrenergic stimulation in Purkinje fibers (Chang et al., 1991). In the SA node, the phosphatase inhibitor had no effect on the position of the activation curve, implying that intracellular levels of cAMP remained unchanged. These observations suggest that the mechanism of action is likely upstream from the actions of cAMP. A similar mechanism has been suggested in rat pinealocytes, where calyculin A has been found to potentiate the accumulation of cAMP by isoproterenol but to have no effect on these levels in the absence of 웁adrenergic agonist (Ho and Chik, 1995). The importance of these observations is that phosphorylation processes can regulate input flowing through the 웁-adrenergic signaling pathway in the SA node myocyte. Phosphorylation processes have also been found to modify muscarinic regulation of If . Wang and Lipsius (1996) have found that removal of cAMP, after a 2-min exposure, leads to a rebound increase in If measured in pacemaker cells of the feline right atrium on cAMP readmission. Direct measurements of cAMP concentration in chick cardiomyocytes have demonstrated a rebound increase in cAMP levels in response to ACh withdrawl (Linden, 1987), and this reasoning has been invoked to explain the increase in If (Wang and Lipsius, 1996). This effect is eliminated by the kinase inhibitor H-89. Thus, this rebound enhancement of cAMP production and If may be related to the ability of phosphorylation to enhance 웁-adrenergic-mediated increases in If and cAMP accumulation mentioned earlier. Fast pathways, directly coupling the muscarinic receptor to the pacemaker current by G-proteins, have been proposed in the SA node. In the SA node, ACh also activates a muscarinic potassium conductance (IK,ACh ), but with an EC50 approximately 20 times larger than the EC50 for If inhibition (DiFrancesco et al., 1989). The activation of IK,ACh has been called a membranedelimited or fast muscarinic pathway because cytoplasmic components are not required and hence agonist binding produces a relatively rapid effect (Sakmann et al., 1983). This causes a hyperpolarization that contributes to the slowing of beating frequency. The binding of agonist to the muscarinic receptor activates a pertussis toxin-sensitive G-protein, which directly gates the channel (Pffafinger et al., 1985). Two pieces of evidence have emerged suggesting the possibility that such pathways participate in the modulation of the pacemaker current. First, the application of preactivated inhibitory G-proteins (Gi) to the cytoplasmic side of patches excised from isolated SA myocytes modulated the pacemaker current (Yatani et al., 1989). Second, carbachol inhibited whole cell If currents in SA node myocytes with time constants similar to those for the activation of IK,ACh by

18. Cardiac Pacemaker Currents

carbachol (Yatani and Brown, 1990). More recently, however, it has been determined that muscarinic inhibition of If by ACh is significantly slower than the activation of IK,ACh (Accili et al., 1998). Furthermore, the time constants for IK,ACh activation are proportional to ACh concentration. Support for two different pathways and for the lack of a fast pathway in the modulation of the pacemaker current come from experiments using outside-out patches where ACh was found to modulate only IK,ACh and to have no effects on If . That the cAMP pathway is the primary mechanism by which autonomic stimulation exerts its actions on If is also supported by previous studies showing that neurotransmitters do not affect the current when intracellular levels of cAMP are clamped with IBMX and forskolin (DiFrancesco and Tromba, 1988b) and evidence that If channels in cellattached patches can be modulated by externally perfused epinephrine (DiFrancesco, 1986). Differences in speed between the muscarinic pathways coupled to IK,ACh and If parallel data from knockout mice lacking GIRK4. Knockout mice have been produced that do not have GIRK4 (Wickman et al., 1998), which, together with GIRK1, is thought to underlie IK,ACh (Dascal et al., 1993; Kubo et al., 1993; Krapivinsky et al., 1995). Wickman et al. (1998) showed that, in response to vagal influences, heart rate changes occurred more gradually over 2–5 sec in knockout mice but changed in less than 2 sec in wild-type mice. The slower change in the knockout mouse correlates well with an action of ACh on If, whereas the fast change is compatible with an additional action on IK,ACh . These studies suggest separate roles for each current in vagally mediated changes in heart rate.

B. Modulation by Nonclassic Messengers Vasoactive intestinal peptide (VIP), a putative neurotransmitter in the SA node, shifts the pacemaker current activation curve to more positive potentials and increases heart rate. The SA node of the mammalian heart is rich in VIP innervation (Weihe et al., 1981; Crick et al., 1994; Steele and Choate, 1994; Accili et al., 1995). This peptide has been shown to cause positive chronotropic effects in vitro and in vivo (Christophe et al., 1984; Rigel, 1988), and its release in response to nerve stimulation has been demonstrated in parallel with concomittant increases in heart rate (Hill et al., 1993). The release of this peptide may partly explain excess tachycardia, which occurs after sustained vagal stimulation. As a result of its direct action on the SA node, VIP is able to accelerate the slope of the diastolic depolarization, increase the spontaneous rate by 14.8%, and shift the pacemaker current activation curve to more positive voltages without altering its fully activated conductance

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(Accili and DiFrancesco, 1996). Because of the importance of the pacemaker current in the generation of the diastolic depolarization in SA node myocytes, and hence heart rate, these data suggest that the shift in the activation curve of the pacemaker current is the mechanism by which VIP produces its positive chronotropic effect. The shift in the activation curve of the pacemaker current is similar to that produced by isoprenaline, suggesting similar mechanisms of action related to increases in cAMP. A similar effect of VIP has been demonstrated previously in canine Purkinje fibers (Chang and Cohen, 1994), which may contribute to its actions. Adenosine shifts the pacemaker current activation curve to more negative potentials and decreases heart rate. The release of adenosine is increased in the heart when oxygen availability decreases and acts on cells in a paracrine manner. This substance has ‘‘direct’’ negative inotropic effects, which were attributed primarily to its activation of an inwardly rectifying K channel (Belardinelli et al., 1988). However, more recent evidence has been obtained that demonstrates that adenosine shifts the pacemaker current activation curve to more negative voltages without altering the fully activated conductance (Zaza et al., 1996). The shift occurs with a Kd of approximately 0.079 애M, a concentration lower than those reported to activate the inwardly rectifying potassium current, which are in the 10–50 애M range (Belardinelli et al., 1988). These concentrations also significantly decrease beating frequency and the slope of the diastolic depolarization of isolated aggregates of SA node myocytes consistent with a mechanism acting primarily through the reduction of the pacemaker current. The mechanism is pertussis toxin sensitive and similar to that produced by muscarinic agonists and is compatible with the reduction of cAMP by the stimulation of A1 adenosine receptors.

C. Modulation of the Fully Activated Conductance of Pacemaker Current in the Heart Phosphorylation events have also been tied to increases in the fully activated conductance. The protein phosphatase inhibitor calyculin A increases the pacemaker current fully activated conductance in the SA node (Accili et al., 1997a). The increase had a time constant of 466 sec, which is much greater than for changes associated with direct gating by cAMP (time constant of about 5 sec; Accili et al., 1998). These times are similar to those required for increases in the fully activated conductance in neurons by cAMP/PKA-mediated processes (Tokimasa et al., 1990), suggesting that similar processes might mediate increases in fully acti-

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vated conductance in SA node myocytes. Experiments have suggested the involvement of tyrosine kinases, along with cAMP/PKA, in the regulation of fully activated conductance (Wu and Cohen, 1997). Inhibitors of these enzymes have been shown to reduce the maximal conductance of If in myocytes from the rabbit SA node. The reduction in maximal conductance produced by these inhibitors is augmented in the presence of CPTcAMP, a membrane-permeable analogue of cAMP, indicating a link between cAMP and tyrosine kinase pathways. Rundown of pacemaker currents measured in whole cell patch clamp experiments also links fully activated conductance, cAMP, and phosphorylation. As described earlier, rundown of the pacemaker current in the SA node occurs in two distinct ways (DiFrancesco et al., 1986). The first type of rundown is due to a negative shift in the activation curve and is related, at least partly, to a decrease in available cAMP. There is also a decrease in the fully activated relation. This occurs more slowly and becomes noticeable after about 20 min from the beginning of the experiment. This type of rundown does not occur in calyculin A-treated cells where increases in fully activated conductance, and the fully activated relation, were observed at times up to 30 min after the beginning of the experiment (Accili et al., 1997a). Both calyculin A-mediated increases and rundown-induced decreases in the fully activated conductance occur with similar time courses. Thus, there may be an association between this type of pacemaker current rundown and decreases in PKA-dependent phosphorylation. Neurohormonal stimulation has been shown to increase the fully activated conductance of the pacemaker current. In particular, the parathyroid hormone and the parathyroid hormone-related peptide have been shown to increase the fully activated conductance of If in the SA node, although a concomitant positive shift in the activation curve was not ruled out (Hara et al., 1998). These increases correlate with increases in beating frequency and in the slope of the diastolic depolarization compatible with an effect in If . Because PTH and PTHrelated peptide also increase cAMP production in cardiac myocytes (Rampe et al., 1991; Schluter et al., 1997), it is possible that the cAMP/PKA pathway is linked to the increase of the If fully activated conductance as mentioned previously. Nitric oxide may increase heart rate through an action on the If fully activated conductance. Sodium nitroprusside can increase the heart rate in patients who have undergone transplant, suggesting a direct action of this durg on the cardiac pacemaker (Levine et al., 1986). More recently, nitric oxide donors have been found to increase the beating frequency of SA node preparations from the guinea pig (Musialek et al., 1997). It was sug-

gested in these experiments that the increase is due to stimulation of the If fully activated conductance through a nitric oxide–cGMP-dependent pathway. The increase in both rate and If fully activated conductance occurred over a 3- to 5-min period, a time frame similar to the time required for phosphatase inhibitor-mediated increases in maximal conductance (Accili et al., 1997a). Interestingly, nitric oxide donors had no effect on the L-type calcium current in this and other studies of the SA node (Han et al., 1995).

V. If IN OTHER AREAS OF THE HEART If is found in other types of cardiac conduction tissue. Shortly after its description in the SA node and cardiac Purkinje fibers, If was found in the atrioventricular node of the rabbit (Kokubun et al., 1982). More recent studies have demonstrated that If is activated in the range of ⫺60 and ⫺90 mV in ovoid cells and at more negative potentials in rod-shaped cells of the AV node (Munk et al., 1996). The current density of pacemaker current also varies between these cell types, being approximately 25 times larger in ovoid cells than rod-shaped cells. This finding suggests a role for pacemaker current in spontaneous activity of these cells and also for the conduction through the AV node. Original observations of IK2 were made in Purkinje fibers, which are important for the conduction of the electrical impulse through to the ventricles. In this tissue, the activation range of If is more negative than in the SA node (Chang et al., 1990). Because of the importance of cAMP in determining the position of the If activation curve, the more negative activation range in the Purkinje fiber is probably related to a lower level of intracellular cAMP in the Purkinje fiber. More recently, If has been described in the ventricle. The first report describing a ventricular pacemaker current in the dog (Yu et al., 1995) has been followed by reports in ventricular myocytes of rat and human. In adult tissue, the role of the pacemaker current is not clear because of the apparently very negative activation voltage. However, the occurrence of pacemaker currents increases with aging and in spontaneously hypertensive rates and demonstrates a more positive activation threshold (Cerbai et al., 1994, 1996). The density of the pacemaker current in these animals is linearly related to the severity of cardiac hypertrophy and increases with 웁-adrenoceptor stimulation, suggesting that pacemaker currents may contribute to an increased propensity of the hypertrophied heart for arrhythmias. Interestingly, hypertrophied ventricular myocytes exhibit a diastolic depolarization. If is present in the human ventricle and atrium. Under

18. Cardiac Pacemaker Currents

normal conditions, and in cardiomyopathy, pacemaker currents have been found with midactivation voltages in the range of ⫺70 to ⫺110 mV (Cerbai et al., 1997; Porciatti et al., 1997; Hoppe et al., 1998a). The presence of If is bound to produce depolarization in myopathic cells, especially since it is known that plasma catecholamines are elevated, and the inward rectifer is reduced, in cardiomyopathy. Human atrial myocytes also express the pacemaker current, which is modulated by muscarinic, adenosinergic, and 웁-adrenergic stimulation as in the SA node and is inhibited by the antiarrhythmic agent propafenone (Porciatti et al., 1997; Hoppe et al., 1998b). These data suggest that therapeutic strategies can be envisaged to control atrial and ventricular tachycardias and other arrhythmias aimed at modulating pacemaker current function and expression.

VI. PHARMACOLOGICAL BLOCKADE OF PACEMAKER CURRENTS If is blocked by caesium ions. The blockade of If by Cs⫹ has been used to assess its contribution to the generation of the diastolic depolarization and spontaneous activity (Noma et al., 1983; Denyer and Brown, 1990). This ion slows, but does not stop, pacemaker activity in the SA node. However, the block brought about by this ion is voltage dependent, being most marked at more hyperpolarized potentials while having little effect on outward current (DiFrancesco, 1982; DiFrancesco et al., 1986). This is particularly interesting as even 5 mM blocks only a fraction of the fully activated current at diastolic potentials in the SA node. Thus, in spontaneously beating preparations, Cs⫹ is not ideal for dissecting the role of the pacemaker current in generation of the diastolic potential. Other molecules block If . More recently, substances known as specific bradycardic agents have been produced that are able to specifically inhibit pacemaker currents. Several substances have been developed that block If currents in a relatively specific manner. UL-FSrelated drugs are known to block pacemaker currents in a concentration, use, and frequency-dependent manner (Van Bogaert and Goethals, 1987). This substance behaves as an ‘‘open channel’’ blocker, as block occurs from the inside of the channel and is relieved on hyperpolarization (DiFrancesco, 1994). Another substance shown to decrease HR and to inhibit If with nanomolar affinity is ZD 7288 (BoSmith et al., 1993). This substance produces both a negative shift in the activation curve and a reduction in activation curve amplitude. However, the precise mechanism by which ZD 7288 produces the block has not been investigated in detail. The ability to inhibit pacemaker activity by targeting the pacemaker

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channel may be particularly important in clinical situations where a reduction in heart rate is desired but where other drugs, such as 웁 blockers, cannot be used. Also, the pharmacological blockade of If may be useful because of its possible involvement in arrhthmias as discussed earlier.

VII. DEVELOPMENTAL REGULATION OF PACEMAKER CURRENTS In the SA node of the newborn rabbit, If current density is higher than in the adult. The current density of If in the SA node has been found to decrease from 9 to 30 days of age (Accili et al., 1997b), corresponding with decreases in spontaneous beating over this approximate time period (Baruscotti et al., 1996). However, the position of the activation curve, and the modulation of the activation curve by muscarinic and 웁-adrenergic stimulation, does not change within this time period. The difference in If current density correlates with, and probably contributes to, greater intrinsic beating rates, rates of diastolic depolarization, and sensitivity to autonomic stimulation of the newborn rabbit heart (Toda, 1980; de Neef et al., 1983; Hewett and Rosen, 1985; Baruscotti et al., 1996), although other mechanisms, such as a neuronal-type I TTX-sensitive Na⫹ current, which is lost in the adult, may also contribute to spontaneous activity in the newborn (Baruscotti et al., 1996). How If current density is regulated during development may be related to phosphorylation events or other signal transduction pathways involved in the regulation of the fully activated conductance. The activation curve of If is shifted to more positive potentials in the newborn mammalian ventricle. In mammals, ventricular myocytes have the ability to beat spontaneously. In the adult rat ventricle, the threshold for If activation is negative relative to the SA node, as in other mammalian species (around ⫺113 mV; Robinson et al., 1998). Robinson et al. (1988) demonstrated that the threshold for activation in newborn ventricular cells is much more positive, in the range of ⫺70 mV. This could explain why myocytes isolated from the ventricle of younger animals are able to beat spontaneously. The activation curve of If in the ventricle can move back to more positive voltages after time in primary culture. In adult ventricular myocytes of the rat, the If threshold has been shown to move to more positive voltages after time in culture, from ⫺92 to ⫺63 mV measured at 4 and 12 days in culture, respectively (Fares et al., 1998). These authors also found that current density increased in this time period. Thus, a modeling process that results in dedifferentiation could move If into

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a range where it could more likely be activated and possibly contribute to ventricular arrhythmias. In the chick, a reduction of If current density occurs during development. If has been found in chick ventricular myocytes. The current is highly expressed in cardiomyocytes from a 3-day-old chick, diminished at 10 days, and is almost completely gone at 17 days. The reduction in current density parallels the decrease in spontaneous activity of these cells, implying an important role for this current in pacemaking (Satoh and Sperelakis, 1993).

VIII. SUBUNITS UNDERLYING THE FORMATION OF If CHANNELS A. If Channel Expression Has Been Detected in the Heart Although extensive cloning of voltage-gated channel superfamily members occurred in the 1980s, no hyperpolarization-activated channel gene was identified until 1997. An exciting discovery was made by Santoro et al. (1997), who reported the cloning from mouse brain of a putative member (mBCNG1) of a new family of channels. The cloning was accomplished by chance, using the yeast two-hybrid approach, while looking for proteins interacting with the SH3-binding domain of neural Src. Using the nomenclature suggested by Clapham (1998), BCNG-1 is now known as HCN1 and thus far its localization has been limited to the brain. Using complementary techniques of BLAST searches of an expressed sequence tag database, RT-PCR and screening of cDNA libraries, HCN2 (mouse and human), and HCN4 (human and rabbit) have now also been cloned (Ludwig et al., 1998, 1999; Santoro et al., 1998; Ishii et al., 1999; Vaccari et al., 1999). Northern blot and PCR analysis have shown that these isoforms are found in the heart. Human HCN2 and 4 were cloned from conduction tissue but are also found in contractile myocytes. HCN4 expression in the rabbit has been demonstrated specifically in the SA node. The functional expression of this channel results in currents with the hallmarks of If /Ih . The expressed currents are activated by hyperpolarization, permeable to K⫹ and Na⫹ and modulated by cAMP. HCN channels have the typical structure of voltagegated potassium channels. The primary structure indicates six transmembrane segments, a positively charged S4 segment acting as the voltage sensor, and the GYG pore sequence found in most known potassium selective channels. The proposed topology and functional domains of the channel are shown in Fig. 5, whereas a comparison of the primary structure of the human HCN2 with the mouse isoforms 1, 2, and 3 is shown in Fig. 6. At the amino acid level these HCN channels

FIGURE 5 Proposed topology and functional domains of the HCN2 channel subunit.

exhibit a high similarity to the ether-a-go-go (eag) family of channels, particularly in the transmembrance regions and to the cyclic nucleotide-gated family of channels in the cyclic nucleotide-binding region located in the C terminus. eag channels also contain a cyclic nucleotidebinding domain (CNBD) in their C terminus, but here the homology to HCN channels is lower. Channel subunits contain an unusual S4 segment comprising two regions (a ‘‘double-barreled’’ S4) of five positive amino acids. Clues as to the nature of HCN channel activation by hyperpolarization may reside in this unusual structure. The GYG pore sequence has been suggested to act as a potassium selectivity filter (Lipkind et al., 1995). If does show a preference for K⫹ ions over Na⫹ ions, but an important aspect of its physiology described earlier is the significant permeability to Na⫹, which is critical for the pacemaking role of the channel in the heart. Adding to this puzzle is that eag-related channels are very selective for K⫹ ions and yet they have a GFG pore sequence (Warmke et al., 1991). The question arises as to whether these channel subunits, alone or as heteromultimers, form the native If channel. In these studies, expression of the clones in mammalian cell lines results in currents that exhibit many similarities, and some differences, with the native If (Ludwig et al., 1998, 1999; Santoro et al., 1998; Ishii et al., 1999; Vaccari et al., 1999).

B. Kinetics of Cardiac HCN Channels The characteristics of current activation produced by expression of HCN2 and HCN4 are variable. The V1/2

18. Cardiac Pacemaker Currents

of the expressed current has shown great variability, ranging from ⫺87 to ⫺109 mV under basal conditions. This a range more negative than that found for If currents in native tissue: ⫺63 to ⫺97 mV in the rabbit SA node (e.g., van Ginneken and Giles, 1991; Denyer and Brown, 1990; Accili et al., 1997b); ⫺87 mV in the human atrium (Porciatti et al., 1997); and ⫺71 and ⫺111 mV in failing and normal human ventricle (Cerbai et al., 1997; Hoppe et al., 1998). An example of an activation curve of HCN2 expressed in HEK cells is shown in Fig. 7 (DiFrancesco, unpublished data). Figure 7a shows current traces from a cell hell at ⫺35 mV and pulsed to different test potentials as indicated. Each test potential was followed by a pulse to ⫺125 mV. The current amplitudes at ⫺125 mV were used to contruct a normalized activation curve shown in Fig. 7b. This curve shows that channel activation begins approximately in the range of ⫺65 mV. The variability in channel activation may re-

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flect differences in basal levels of cAMP but may also reflect other factors. For example, in patches excised from HEK cells expressing HNC2 and HCN4, the V1/2 of currents was shifted by 20–30 mV in the hyperpolarizing direction with respect to the position in whole cell conditions (Ludwig et al, 1998, 1999). A negative shift of the activation curve on excision also occurs in native If of the SA node (DiFrancesco and Tortora, 1991; DiFrancesco and Mangoni, 1994) as mentioned earlier.

C. Ionic Properties of HCN Channels The fully activated conductance is linear and reverses at potentials compatible with permeability to both sodium and potassium. Studies have shown that both Na⫹ and K⫹ are involved in carrying current produced by expressed HCN channels (Ludwig et al., 1998, 1999; Ishii

FIGURE 6 Multiple alignment of human HCN2 and mouse HCN1, HCN2, and HCN3 proteins. Residues identical to hHCN2 are indicated by a dash. A space indicates a gap. A stretch of 37 consecutive glutamines in mHAC2 is indicated as (Q)37 . Functional domains of the proteins are underlined (S1–S6: transmembrane domains, pore and CNBD: cyclic nucleotide-binding domain).

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FIGURE 7 Current traces recorded from an HEK cell transfected with human HCN2. (a) The myocyte was held at ⫺35 mV and pulsed first to ⫺55, ⫺85, ⫺98, ⫺100, and ⫺115 mV and was then followed by a pulse to ⫺125 mV. (b) An activation curve constructed from normalized amplitudes of current traces at ⫺125 mV in a.

et al., 1999). These studies have determined permeability ratios (PNa /PK) ranging from 0.19 to 0.31 for the different clones, indicating a preference for passing K⫹ as in the native channels. In addition, increases in external K⫹ have an ‘‘activation-like’’ effect as with the native channel (DiFrancesco, 1982), suggesting that there is an interaction between Na⫹ and K⫹ ions. However, no current was observed in rabbit HCN4 on the removal of external K⫹ as in the native If (Ishii et al., 1999; Frace et al., 1992). This last observation is of interest because an interaction between Na⫹ and K⫹ has also been postulated in the pore of cloned voltage-gated potassium channels (Korn and Ikeda, 1995). Like HCN channels, the cloned cardiac-delayed rectifier Kv1.5 displayed a negligible sodium conductance on the removal of potassium. However, in the absence of external K⫹, the neuronal delayed rectifier Kv2.1 displayed a large sodium conductance, which was inhibited by low concentrations of K⫹. Given the similarity in the pore sequences, espe-

cially in the GYG region, these characteristics may provide information on the structural determinants for selectivity in HCN channels. Currents produced by expressed HCN channels are blocked by Cs⫹ but not by Ba2⫹. Native If is blocked by Cs⫹, but this block is strongly voltage dependent (DiFrancesco et al., 1986). This voltage dependence is somewhat reflected in the currents produced by expression of the cloned ion channels. Mouse and human HCN2, and human HCN4, currents produced by hyperpolarizations to ⫺140 mV, which are in the fully activated range in these preparations, are almost completely blocked by 2 mM Cs (Ludwig et al., 1998, 1999). However, 3 mM Cs⫹ blocked approximately 71% of rabbit HCN4 currents produced by hyperpolarizations to ⫺90 mV, which is very close to the V1/2 (⫺87 mV) of the activation curve (Ishii et al., 1999). However, a more detailed study of the voltage dependence of Cs⫹ block, and of the differences between isoforms, is required.

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D. Modulation by cAMP The presence of a CNBD in the C terminus supports a direct effect of cAMP on the cloned channels as with the native channel. The presence of a CNBD strongly suggests that the actions of cAMP result from a direct interaction with HCN and If channels. On the amino acid level, this region bears a strong similarity to the CNBD of CNG channels. This binding site is related to those that bind cAMP in the catabolic gene activator protein of bacteria and the eukaryotic cyclic nucleotidedependent protein kinases (Shabb and Corbin, 1992). cAMP (0.3–1 mM intrapipette) is able to shift the activation curve by about 15–23 mV in the positive direction in cells expressing these subunits (Ludwig et al., 1998, 1999; Ishii et al., 1999). This is as compared to the maximal amount of 14.6 mV produced by the direct application of cAMP to excised patches from the SA node (DiFrancesco and Mangoni, 1994) and to a total shift of about 18 mV in whole cell conditions (Accili et al., 1997b). Values for K1/2 and Hill coefficient for cAMP binding in excised patches from SA node and HEK cells expressing HCN2 are also similar: 0.2 애M and 0.85 and 0.5 애M and 0.8, respectively. Finally, cAMP did not alter the fully activated conductance but increased the rate of activation of HCN2 channels (Ludwig et al., 1998).

IX. SUMMARY The If current has now been generally accepted to play an important role in the generation and modulation of pacemaking in the SA node and other conduction tissue. If is inward and activates on hyperpolarization at the termination of an action potential and is substantially activated when the membrane potential approaches the maximum diastolic potential in the range ⫺65/⫺70 mV. Its slow kinetics are compatible with the generation of a slow voltage depolarization of an approximately constant slope. Finally it is modulated in an appropriate way be neurotransmitters of the autonomic nervous system in a phosphorylation-independent manner. Modulation is achieved by the shifting of the activation curve along the voltage axis, a mechanism that can be controlled in a very precise manner by altering the intracellular levels of cAMP and hence controlling heart rate. Aspects of the role of If in other parts of the heart, in development and under pathological conditions, remain to be explored. If is found in the ventricle and atrium, but its function under normal conditions in the adult mammal seems limited. However, If may play a role in the excitability of these tissues in the developing

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animal that display a greater functional expression of the current. Knowledge of how If expression changes during development is necessary to understand not only development but also to provide a basis for treatment of heart disease in an age-specific way. Of special interest is the pronounced increase in functional expression that occurs in the ventricle under conditions of heart failure and hypertrophy. The increase in inward current under these conditions suggests that If may contribute to arrythmias in this tissue. Finally, the presence of If in the atrium and its modulation by antiarrhythmic drugs in this tissue suggest a role in physiology and pathophysiology of atrial excitability, which requires further investigation. VIP and adenosine-related substances may have important clinical applications related to their ability to modulate If . For example, glucagon, a peptide related to VIP, has been shown to increase cAMP levels in cardiac myocytes (Mery et al., 1997) and has been used to reverse symptomatic bradycardia in emergency situations where a 웁-adrenergic blockade exists (Love and Howell, 1996). Receptors for a glucagon-like peptide have also been shown to exist in the heart (Wei and Mojsov, 1995). Thus, both glucagon and GLP are probable modulators of the pacemaker current activation curve. The action of adenosine on If is very important given the use of this agent in clinical situations for improvement of conduction through the AV node. Its action may explain the pronounced bradycardia that sometimes occurs in response to adenosine administration under conditions of cardiac transplantation or weakened neural reflexes (e.g., Brodsky et al., 1995). The possible presence of other G-linked receptors in the SA node, and the presence of many paracrine, endocrine, and neurocrine substances in the blood and in different cells of the heart, suggests that additional modulators of the If activation curve remain to be be discovered. The knowledge of HCN primary structure will allow for the study of structural determinants of the channels’ ‘‘funny’’ properties as well as their expression. Interesting properties of these channels include activation by hyperpolarization, selectivity for both Na⫹ and K⫹, and modulation by cAMP. The structural basis of these characteristics can be tested for by using site-directed mutagenesis in combination with patch clamp studies. This information will be also important for understanding how naturally occurring mutations in the primary sequence of these channels affect the function of the channels and how they alter pacemaker and excitability properties of the heart. This approach has been very useful in understanding the relationship of several pathologies with channels, including mutations in the human ethera-go-go-related gene product, a potassium channel that

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has significant homology with the HCN channels, and an arrhythmia known as long QT syndrome (Sanguinetti et al., 1995). Structural knowledge will make it possible to generate drugs targeted to specific binding sites on the channels in order to modify channel function in a more specific way. It will also be possible to correlate factors altering functional expression with changes in localization, trafficking, posttranslational modifications, and quantification of subunits under normal and abnormal conditions. Knowledge of the primary structure allows for the possibility of devising drugs and strategies for targeting channel subunits directly and for altering their expression.

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and Cellular Oscillators’’ (J. W. Jacklet, ed.), pp. 59–85. Dekker, New York. Noma, A., and Irisawa, H. (1976). Membrane currents in the rabbit sinoatrial node cell as studied by the double microelectrode method. Pflu¨g. Arch. 364, 45–52. Noma, A., Morad, M., and Irisawa, H. (1983). Does the ‘‘pacemaker current’’ generate the diastolic depolarization in the rabbit SA node cells? Pflu¨g. Arch 397, 190–194. Noma, A., and Trautwein, W. (1978). Relaxation and noise analysis of the acetylcholine-induced potassium current in the sino-atrial node. J. Physiol. 284, 97P–98P. Ogino, K., Burkhoff, D., and Bilezikian, J. P. (1995). The hemodynamic basis for the cardiac effects of parathyroid hormone (PTH) and PTH-related protein. Endocrinology 136, 3024–3030. Pfaffinger, P. J., Martin, J. M., Hunter, D. D., Nathanson, N. M., and Hille, B. (1985). GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature 317, 536–538. Porciatti, F., Pelzmann, B., Cerbai, E., Schaffer, P., Pino, R., Bernhart, E., Koidl, B., and Mugelli, A. (1997). The pacemaker current I(f) in single human atrial myocytes and the effect of beta-adrenoceptor and A1-adenosine receptor stimulation. Br. J. Pharmacol. 122, 963–969. Rampe, D., Lacerda, A. E., Dage, R. C., and Brown, A. M. (1997). Parathyroid hormone: An endogenous modulator of cardiac calcium channels. Am. J. Physiol. 261, H1945–H1950. Ranjan, R., Chiamvimonvat, N., Thakor, N. V., Tomaselli, G. F., and Marban, E. (1998). Mechanism of anode break stimulation in the heart. Biophys. J. 74, 1850–1863. Rigel, D. F. (1988). Effects of neuropeptides on heart rate in dogs: Comparison of VIP, PHI, NPY, CGRP, and NT. Am. J. Physiol. 255(2 Pt 2), H311–H317. Robinson, R. B., Yu, H., Chang, F., and Cohen, I. S. (1997). Developmental change in the voltage-dependence of if, in rat ventricle cells. Pflu¨g. Arch. 433, 533–535. Roche, K. W., O’Brien, R. J., Mammen, A. L., Bernhardt, J., and Huganir, R. L. (1996). Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron 16, 1179– 1188. Rothermel, J. D., Stec, W. J., Baraniak, J., Jastorff, B., and Botelho, L. H. (1983). Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3⬘,5⬘-phosphorothioate. J. Biol. Chem. 258, 12125–12128. Sanguinetti, M. C., Jiang, C., Curran, M. E., and Keating, M. T. (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81, 299–307. Sakmann, B., Noma, A., and Trautwein, W. (1983). Acetylcholine activation of single muscarinic K⫹ channels in isolated pacemaker cells of the mammalian heart. Nature 303, 250–253. Santoro, B., Grant, S. G., Bartsch, D., and Kandel, E. R. (1997). Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels. Proc. Natl. Acad. Sci. USA 94, 14815– 14820.

Santoro, B., Liu, D. T., Yao, H., Bartsch, D., Kandel, E. R., Siegelbaum, S. A., and Tibbs, G. R. (1998). Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 29, 717–729. Satler, C. A., Vesely, M. R., Duggal, P., Ginsburg, G. S., and Beggs, A. H. (1996). Multiple different missense mutations in the pore region of HERG in patients with long QT syndrome. Hum. Genet. 102, 265–272. Satoh, H., and Sperelakis, N. (1993). Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: Developmental changes and modulation by isoproterenol and carbachol. Eur. J. Pharmacol. 240, 283–290. Shabb, J. B., and Corbin, J. D. (1992). Cyclic nucleotide-binding domains in proteins having diverse functions. J. Biol. Chem. 267, 5723–5726. Schluter, K. D., Weber, M., and Piper, H. M. (1997). Effects of PTHrP(107-111) and PTH-rP(7-34) on adult cardiomyocytes. Mol. Cell. Cardiol. 29, 3057–3065. Steele, P. A., and Choate, J. K. (1997). Innervation of the pacemaker in guinea-pig sinoatrial node. J. Auton. Nervous. Syst. 47, 177–187. Toda, N. (1980). Age-related changes in the transmembrane potential of isolated rabbit sino-atrial nodes and atria. Cardiovasc. Res. 14, 58–63. Tokimasa, T., and Akasu T. (1990). cAMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones. J. Physiol. 420, 409–429. Tsien, R. W. (1974). Effect of epinephrine on the pacemaker potassium current of cardiac Purkinje fibres. J. Gen. Physiol. 64, 293– 319. Tsien, R. W., Giles, W., and Greengard, P. (1972). cAMP mediates the effects of adrenaline on cardiac purkinje fibres. Nature 240, 181–183. van Ginneken, A., and Giles, W. (1985). If in isolated cells from the rabbit SA node. Biophys. J. 47, 496a. van Ginneken, A. C., and Giles, W. (1991). Voltage clamp measurements of the hyperpolarization-activated inward current I(f) in single cells from rabbit sino-atrial node. J. Physiol. 434, 57–83. Vassalle, M. (1966). Analysis of cardiac pacemaker potential using a voltage clamp technique. Am. J. Physiol. 210, 1335–1341. Warmke, J., Drysdale, R., and Ganetzky, B. (1991). A distinct potassium channel polypeptide encoded by the Drosophila eag locus. Science 252, 1560–1562. Wei, Y., and Mojsov, S. (1995). Tissue-specific expression of the human receptor for glucagon-like peptide-I: Brain, heart and pancreatic forms have the same deduced amino acid sequences. FEBS Lett. 358, 219–224. Wu, J. Y., and Cohen, I. S. (1997). Tyrosine kinase inhibition reduces i(f) in rabbit sinoatrial node myocytes. Pflu¨g. Arch. 434, 509–514. Yoo, S., Lee, S. H., Choi, B. H., Yeom, J. B., Ho, W. K., and Earm, Y. E. (1998). Dual effect of nitric oxide on the hyperpolarizationactivated inward current (I(f)) in sino-atrial node cells of the rabbit. J. Mol. Cell. Cardiol. 30, 2729–2738. Yu, H., Chang, F., and Cohen, I. S. (1995). Pacemaker current i(f) in adult canine cardiac ventricular myocytes. J. Physiol. 485, 469–483. Zhou, Z., Gong, Q., Epstein, M. L., and January, C. T. (1998). HERG channel dysfunction in human long QT syndrome: Intracellular transport and functional defects. J. Biol. Chem. 273, 21061–21066.

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19 Chloride Channels in Heart ROBERT D. HARVEY

JOSEPH R. HUME

Department of Physiology and Biophysics Case Western Reserve University Cleveland, Ohio 44106

Department of Physiology and Cell Biology University of Nevada School of Medicine Reno, Nevada 89557

nase C (ICl-PKC), cell volume (ICl-vol), cytoplasmic Ca2⫹ (ICl-Ca), and purinergic receptors (ICl-ATP), as well as a spontaneously active background Cl⫺ current. This list of cardiac chloride currents can now be simplified, as evidence shows that PKA and PKC, as well as purinergic receptor stimulation, may regulate the same anion channel and that the spontaneously active Cl⫺ current and ICl-vol are one in the same. The goal of this chapter is to provide a brief overview of the biophysical, pharmacological, and molecular properties of sarcolemmal Cl⫺ conductance pathways in the heart, their species and tissue distribution, and their known or predicted physiological roles. A more extensive discussion of various aspects of this subject can be found in several previously published reviews (Hume and Harvey, 1991; Ackerman and Clapham, 1993; Gadsby et al., 1995; Harvey, 1996; Hiraoka et al., 1998; Hume et al., 2000; Sorota, 1999; Gadsby and Nairn, 1999).

I. INTRODUCTION Chloride channels first emerged as the subject of electrophysiological studies in the heart in 1961, when Hutter and Noble and Carmeliet provided evidence suggesting that anion conductances contribute significantly to the electrical properties of cardiac muscle (for a review, see Hume and Harvey, 1991). Although not directly related to these initial observations, subsequent work led to the general agreement that a time- and voltage-dependent increase in chloride conductance was largely responsible for the initial rapid phase of repolarization of the action potential of cardiac Purkinje fibers. However, studies in the late 1970s raised serious doubts about the identity of this chloride conductance. Eventually, the development of methods for enzymatically isolating cardiac myocytes allowed for more careful characterization of cardiac conductance pathways using patch clamp techniques, but even then, the role of chloride channels in contributing to cardiac electrical activity remained obscure. It was not until 1989, when it was discovered that the 웁-adrenergic signaling pathway regulates a background or time-independent Cl⫺ conductance (Harvey and Hume, 1989; Bahinski et al., 1989), that interest in cardiac Cl⫺ channels reemerged. During the past decade, an increasing amount of effort has been devoted to the functional and molecular characterization of anion channels of cardiac cells and to efforts to reveal their physiological and possible pathophysiological role. At one point, at least six different Cl⫺ currents were functionally identified in cardiac cells. These included Cl⫺ currents regulated by 웁-adrenergic receptors/protein kinase A (ICl-PKA), protein ki-

Heart Physiology and Pathophysiology, Fourth Edition

II. CHLORIDE CHANNELS IN HEART A. PKA-Activated Chloride Channels 웁-adrenergic receptor stimulation regulates the activity of a number of different ion channels in the heart through a cAMP/PKA-dependent mechanism. This includes channels responsible for the L-type Ca2⫹ current (ICa-L) and the slow component of the delayed rectifier K⫹ current (IKs), in addition to ICl-PKA . Unlike 웁-adrenergically regulated cation currents, ICl-PKA does not appear to be active in the absence of agonists. Whole cell

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FIGURE 1 웁-adrenergic receptor-dependent activation of macroscopic ICl-PKA . This Cl⫺

current is not activated by methoxamine (500 애M), a selective 움1-adrenergic receptor agonist, but it is activated by isoproterenol (Iso; 30 nM), a selective 웁-adrenergic receptor agonist. (A) Time course of changes in background current measured at membrane potentials positive to ECl following exposure to selective adrenergic agonists. (B) Current traces recorded during voltage clamp steps to membrane potentials between 50 and ⫺120 mV. Each family of traces was recorded at the time point indicated in A. (C) Membrane potential (VM) dependence of difference currents (⌬I) obtained by digitally subtracting the indicated families of currents illustrated in B. Currents were recorded from guinea pig ventricular myocyte using the whole cell patch clamp technique ([Cl⫺]i ⫽ 20 mM; [Cl⫺]o ⫽ 150 mM). From Oleksa et al. (1996), with permission.

patch clamp experiments are perhaps the most common method used to study ICl-PKA in cardiac myocytes. Under conditions that block, inactivate, or otherwise eliminate cation currents, in the absence of a cAMP-generating agonist, voltage clamp steps over a wide range of membrane potentials typically elicit only a small leak current,

the reversal potential of which is not significantly altered by changes in the Cl⫺ equilibrium potential. However, subsequent exposure to cAMP-stimulating agents, such as the selective 웁-adrenergic receptor agonist isoproterenol, activates the time-independent Cl⫺ current, ICl-PKA (Fig. 1).

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1. Conductance Properties This 웁-adrenergically activated current is highly selective for Cl⫺ over cations. However, the reversal potential does deviate from the Cl⫺ equilibrium potential (ECl) when Cl⫺ is replaced with anions such as glutamate and gluconate, suggesting that it has a finite permeability to other anions. The anion selectivity sequence for this current is NO3⫺ ⬎ Br⫺ ⬎ Cl⫺ ⱖ I⫺ ⬎ F Ⰷ glutamate 앒 gluconate. Under physiological conditions, the concentration of Cl⫺ inside the cell is lower than that outside the cell. Typically, the intracellular Cl⫺ concentration of cardiac muscle is in the range of 10 to 40 mM. When ICl-PKA is recorded under such conditions, it exhibits outward rectification. When intracellular Cl⫺ is increased to equal the typical extracellular Cl⫺ concentration, the current–voltage relationship becomes linear. This behavior, which helps distinguish ICl-PKA from other cardiac Cl⫺ currents, is often attributed to a Goldman-type behavior, where rectification is a function of the gradient of permeant ions across the cell membrane. However, it has been clearly demonstrated that rectification of ICl-PKA is not dependent on the Cl⫺ gradient (Overholt et al., 1993). Instead, rectification is a function of the concentration and relative permeability of anions inside the cell. Movement of Cl⫺ from inside the cell to out (inward current) can be limited by a voltage-dependent block of the channel by relatively impermeant anions, such as glutamate or gluconate, which are commonly used as counteranions when experimentally altering Cl⫺ concentration. Unitary currents responsible for the macroscopic Cl⫺ current have been identified in guinea pig ventricular myocytes (Fig. 2) (Ehara and Ishihara, 1990). The single channel conductance rectifies when the intracellular Cl⫺ concentration is low and this rectification is relieved when the intracellular Cl⫺ concentration is increased,

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indicating that rectification of the macroscopic current reflects the conductance properties of the single channel. Rectification of single channel currents in cell-attached patches also indicates that rectification of this current occurs in vivo. Macroscopic ICl-PKA in heart is functionally indistinguishable from macroscopic ICl-PKA in epithelial cells, but the conductance of unitary currents initially attributed to the epithelial conductance was much larger (25–40 pS) than what was found in the heart (앑13 pS). However, defects in the epithelial Cl⫺ conductance are associated with the inherited disease cystic fibrosis (CF), and when the defective gene was identified, it became clear that the gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), was actually a Cl⫺ channel with unitary conductance properties (4–13 pS) similar to those of the Cl⫺ channel found in heart. Subsequent analysis of mRNA isolated from cardiac tissue clearly demonstrated that ICl-PKA in heart is associated with the expression of CFTR (Levesque et al., 1992; Nagel et al., 1992). 2. Regulation a. Protein Kinase A The primary physiological means of regulating the activity of ICl-PKA is through various G-protein-coupled receptors (GPCRs). The role of G-proteins in coupling 웁-adrenergic receptors and muscarinic receptors to the regulation of ICl-PKA in heart was established in early studies of the current (Hwang et al., 1992). Intracellular GTP was shown to be essential for the activation of ICl-PKA by 웁-adrenergic agonists, as well as for inhibition by muscarinic agonists. These effects of GTP can be attributed to the antagonistic actions of Gs and Gi on adenylate cyclase and the role that this plays in the same G-protein–adenylate cyclase–PKA pathway that

FIGURE 2 웁-adrenergic receptor activation of microscopic ICl-PKA . Channel activity was stimulated by exposure of a guinea pig ventricular myocyte to epinephrine (5 애M). Currents were recorded using the cellattached patch clamp technique ([Cl⫺]o ⫽ 150 mM). The membrane potential at which channel activity was recorded is reported relative to the cells resting membrane potential. The single channel conductance of the outward current was 13 pS. From Ehara and Ishihara (1990), with permission.

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regulates ICa-L and IKs (see Fig. 3). However, unlike some reports of direct G-protein coupling of receptors to Ca2⫹ and K⫹ channels, these Cl⫺ channels appear to be regulated solely through PKA-dependent phosphorylation. Because of this fact, ICl-PKA represents a model system for studies of receptor–G-protein–adenylate cyclase–PKA pathways in the heart. ICl-PKA has been used to study the intracellular signaling pathways involved in stimulatory responses to 웁-adrenergic and H2 histaminergic receptor activation, as well as the facilitation and/or antagonism of such responses by M2 muscarinic, 움1-adrenergic, and ETA-endothelin receptor stimulation. Regulation of ICl-PKA by GPCRs is the result of changes in cAMP production, which in turn affects the level of PKA-dependent phosphorylation of the channel protein. Activity of this Cl⫺ channel is directly proportional to the degree of phosphorylation. However, the channel protein has at least 10 putative PKA phosphorylation sites, most of which are found in the regulatory (R) domain (see Fig. 4), and not all appear to affect channel function equally. Evidence for at least two functionally distinct phosphorylation states has been identified on the basis of their differential sensitivity to dephosphorylation by okadaic acid-inhibitable phosphatase activity (Hwang et al., 1993).

FIGURE 4 Schematic diagram of the predicted secondary structure of the cystic fibrosis transmembrane conductance regulator. This Cl⫺ channel is predicted to have two transmembrane domains each with six membrane-spanning segments. Nucleotide binding domain 1 (NBD1), regulatory (R) domain, and nucleotide binding domain 2 (NBD2).

Although PKA-dependent phosphorylation is necessary for this Cl⫺ channel to open, phosphorylation alone is not directly responsible for gating of the channel. It is now well established that channel activity requires ATP interaction at two nucleotide-binding domains (NBDs) (see Fig. 4). Once phosphorylated, it is believed that ATP binding and hydrolysis at one NBD are responsible for gating the channel open, whereas ATP binding and hydrolysis at the other are responsible for gating the channel closed (Baukrowitz et al., 1994; Hwang et al., 1994). Although the cytoplasmic concentration of ATP in cardiac myocytes is typically much higher that that required to maintain maximal channel activity (⬍ 500 애M), it is feasible that alterations in the metabolic state of a myocyte could represent a pathological mechanism for regulating the activity of this channel. b. Protein Kinase C

FIGURE 3 Pathways regulating cardiac Cl⫺ channel activity. Norepinephrine (NE), acetylcholine (ACh), adenosine triphosphate (ATP), stimulatory G protein (Gs), inhibitory G protein (Gi), adenylate cyclase (AC), 웁-adrenergic receptor (웁), muscarinic receptor (M), purinergic receptor (P), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and protein kinase C (PKC). ICl-PKA is regulated by PKC and PKA; ICl-vol is activated by cell swelling (cell membrane depicted by broken line); ICl-Ca is activated by intracellular Ca2⫹ released from the sarcoplasmic reticulum. ATP activates a Cl⫺ current, possibly ICl-PKA .

Despite being regulated primarily by PKA-dependent phosphorylation, CFTR channels also contain several consensus phosphorylation sites for PKC. Studies involving epithelial CFTR have clearly demonstrated that the protein is phosphorylated by PKC, but the functional consequence of this action is complex. It has been shown that PKC phosphorylation can stimulate epithelial CFTR channel activity. However, the effects of PKC-dependent phosphorylation are variable, and the magnitude of PKC-dependent response is relatively small when compared to the effect of PKA. A more consistent finding is that PKC stimulation potentiates the rate and magnitude of subsequent PKAdependent responses. Similar results have been obtained in cardiac myocytes. The activation of endogenous PKC alone has variable effects on basal cardiac CFTR channel activity, and when it does appear to cause activation, the magnitude

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of the response is much smaller than that achieved through PKA-dependent stimulation alone. Furthermore, there is clear evidence that PKC-dependent stimulation can potentiate PKA-dependent responses (see Middleton and Harvey, 1998). In fact, phorbol ester activation of endogenous PKC is able to enhance the response to supramaximal PKA-dependent stimulation. Perhaps more importantly, however, inhibition of basal PKC activity in native cardiac myocytes limits subsequent PKA-dependent activation of the channel. This suggests that while PKC phosphorylation alone has little effect on channel activity, it is necessary in order for the channel to be able to respond to PKA stimulation. Current evidence suggests that macroscopic and/or unitary cardiac conductances attributed to the activation of PKC alone (ICl-PKC) can be explained by the PKCdependent regulation of CFTR channels. To date, there is very little evidence suggesting that PKC stimulation in heart leads to activation of a unique class of anion channels distinct from CFTR. The fact that PKC-dependent phosphorylation may be necessary to observe PKA-dependent activation of CFTR Cl⫺ currents has important implications. For example, it is possible that the lack of functional evidence for ICl-PKA in some cells may reflect differences in basal PKC activity. Conversely, the agonist-dependent activation of endogenous PKC may regulate cardiac CFTR function in the presence of elevated PKA activity. Although 움1-adrenergic receptor stimulation might be expected to regulate the activity of this cardiac Cl⫺ channel in a PKC-dependent manner, 움1-adrenergic receptor stimulation alone has little if any effect (see Fig. 1). Furthermore, 움1-receptor stimulation actually inhibits 웁-adrenergic regulation of channel activity via a PKC-independent mechanism (Oleksa et al., 1996). Nevertheless, PKC may still be involved in the regulation of cardiac CFTR possibly through purinergic and/or other signaling pathways. Extracellular ATP is known to have both positive and negative inotropic and chronotropic effects in the heart, which are species and purinergic receptor subtype dependent. Some of these effects may be mediated, at least in part, by the activity of a Cl⫺ current, ICl-ATP . ICl-ATP was first described in guinea pig atrial myocytes (Matsuura and Ehara, 1992) and has been subsequently studied in rat (Kaneda et al., 1994) and mouse (Levesque, 1995) ventricular myocytes, with the latter two studies suggesting involvement of a P2-purinergic receptor. To date, ICl-ATP represents the least studied cardiac Cl⫺ current. As such, there is uncertainty whether extracellular ATP activates a novel class of anion channels, or perhaps modulates other known types of anion channels. For example, ICl-ATP exhibits many of the same properties as ICl-PKA , including time independence and rectification affected by changes in intracellular but not

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extracellular Cl⫺ concentration. New evidence suggests that rather than activating a unique class of anion channels, P2-purinergic receptor stimulation in heart may be linked to the activation of CFTR Cl⫺ channels through a pathway involving both PKA and PKC (see Hume et al., 2000). 3. Pharmacology The sensitivity of ICl-PKA in heart to a variety of Cl⫺ channel antagonists is similar to epithelial CFTR channels. Although some discrepancies were initially reported, it is generally accepted that ICl-PKA is relatively insensitive to direct inhibition by stilbene derivatives such as 4-acetamido-4⬘-isothiocyanostilbene-2,2⬘-disulfonic acid (SITS), 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS), and 4,4⬘-dinitrostilbene-2,2⬘-disulfonic acid (DNDS). However, ICl-PKA is blocked by carboxylic acid derivatives such as 9-andthracene carboxylic acid (9-AC) and diphenylamine-2-carboxylic acid (DPC), arylaminobenzoates such as 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), clofibric acid analogues, and sulfonylureas such as glibenclamide. Clofibric acid and its analogues, p-chlorophenoxy propionic acid and gemfibrozil, may be the most potent inhibitors of ICl-PKA in guinea pig myocytes (Walsh and Wang, 1996). It should be noted that this pharmacologic profile is for compounds applied extracellularly to intact cells. While many of these compounds are lipid soluble, and may actually cross the cell membrane to reach their site of action, others are not membrane permeable and may have different effects when applied to the intracellular compartment. The inability of extracellularly applied stilbene derivatives to block this current is perhaps one of the most important features of its pharmacologic profile. Compounds that block ICl-PKA are not particularly selective and they will block other cardiac Cl⫺ currents as well. Stilbene derivatives are also notoriously promiscuous anion transport blockers and will block all other types of cardiac Cl⫺ currents. Therefore, the fact the CFTR is relatively insensitive to stilbenes is notable and makes the use of appropriate concentrations of these compounds a practical means of distinguishing ICl-PKA from other Cl⫺ currents. 4. Molecular Structure/Function CFTR is composed of 1480 amino acids, with a predicted secondary structure consisting of two transmembrane domains, each with six membrane-spanning segments, two NBDs, and an R domain (Fig. 4). The protein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which are structurally similar in terms

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of the organization of their transmembrane domains and NBDs. Over 100 members of this family have been identified, including P-glycoprotein (P-gp), which pumps hydrophobic compounds out of cells, and the sulfonylurea receptor, which combines with inward rectifier K⫹ channel subunits (Kir6.1, Kir6.2) to form functional ATP-sensitive K⫹ (KATP) channels. The two transmembrane domains of ABC proteins are believed to form the pathway for solute transport, whereas the two nucleotide-binding domains are believed to couple ATP hydrolysis to solute transport. CFTR is apparently unique among ABC transporters in that it forms an anion-selective channel. Site-directed mutagenesis has been employed in attempts to identify which regions of the channel protein may contribute to formation of the pore region of CFTR. Results have suggested that residues in the 1st, 5th, 6th, and 12th transmembrane-spanning segments of CFTR may form part of the ion conduction pathway. Despite extensive study, insight into the three-dimensional structure of the pore-forming region of CFTR is still limited. CFTR is also unique among ABC transport proteins in that it possesses a region referred to as the R domain. The R domain possesses most of the channel’s PKA consensus phosphorylation sties, as well as several of the PKC consensus phosphorylation sites. The current view of CFTR channel function suggests that the R domain may represent a blocking particle, which in its unphosphorylated form keeps the channel closed, but upon phosphorylation causes channel opening via a conformational change. Phosphorylation of the R domain alone, however, is insufficient to cause channel openings, as binding and hydrolysis of ATP to the NBDs are also required. Therefore, phosphorylation of the R domain may promote ATP binding to the NBDs, although the exact nature of the interactions between the R domain and the NBDs remains unclear. Functional studies indicate that the properties of the CFTR channel in nonhuman cardiac myocytes are largely indistinguishable from those of its human epithelial counterpart. This includes the single channel conductance, relative permeability to anions, pharmacologic profile, and regulation by PKA, PKC, and ATP binding and hydrolysis. However, molecular data suggest that there is at least one significant structural difference. Comparison of the amino acid sequence of human epithelial CFTR with the deduced sequence from rabbit heart suggests that the isoform of CFTR found in cardiac tissue is alternatively spliced. Exon 5, which encodes 30 amino acids in the cytoplasmic loop connecting the second two membrane-spanning segments, is absent from the rabbit cardiac isoform of the channel protein. Outside of the alternatively spliced region, the rabbit cardiac isoform displays greater than 91% identity to

human epithelial CFTR. Evidence for the presence of CFTR missing exon 5 (exon 5-) has been confirmed in rabbit, guinea pig, simian, and human heart using Southern analysis of reverse transcription polymerase chain reaction products (Warth et al., 1996). 5. Species/Tissue Distribution Electrophysiological studies indicate a significant species and tissue variability in the functional expression of ICl-PKA . This type of current has been reported in adult guinea pig, rabbit, cat, simian, and human cardiac myocytes, but it appears to be absent from adult canine, rat, and mouse cardiac myocytes. In species in which ICl-PKA has been identified, it is found most often in ventricular myocytes and less commonly or not at all in myocytes isolated from atrial or SA nodal tissue. Messenger RNA levels of CFTR in guinea pig atrium and ventricle strongly correlate with ICl-PKA densities measured electrophysiologically. Specifically, mRNA levels and ICl-PKA densities were highest in ventricular epicardial cells, lower in ventricular endocardial cells, and lowest (but not absent) in atrial cells (James et al., 1996). Evidence for the functional expression of ICl-PKA in human heart is controversial. The molecular evidence presently available strongly suggests that the CFTR message is expressed in both atrial and ventricular human myocardium (Levesque et al., 1992; Warth et al., 1996). Furthermore, in contrast to all other animal species yet examined, there is evidence for expression of both the exon 5⫹ as well as the exon 5⫺ isoforms in human and simian myocardium. Despite the presence of message, electrophysiological evidence for the expression of CFTR Cl⫺ channels in human heart is limited. A potential confounding factor is the fact that most (but not all) human studies have used atrial tissue. In future studies, it will be necessary to focus more on human ventricular myocytes, which may exhibit a higher density and more consistent expression of the CFTR gene product than atrial myocytes. It may also be necessary to consider other potentially confounding factors, such as the condition of the heart from which tissue is obtained, as well as the role that variations in basal PKC activity may play in determining functional responses. 6. Functional Role Activation of any Cl⫺ current will tend to move the membrane potential toward ECl , which is in the vicinity of ⫺40 to ⫺60 mV in cardiac cells. Because ICl-PKA is a time-independent background current, once activated it will tend to have a depolarizing influence on the resting membrane potential and a repolarizing influence during

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the action potential. The magnitude of its contribution will depend on the actual size of the Cl⫺ conductance relative to the total conductance of the membrane. In ventricular cells, this current has only a small depolarizing effect (just a few millivolts) on the resting membrane potential, but it contributes significantly to repolarization (Harvey et al., 1990). In the example illustrated in Fig. 5, the effect of ICl-PKA was determined by looking at the changes in action potential configuration before and after activation of this current by the 웁-adrenergic agonist isoproterenol. These experiments were conducted at room temperature and in the presence of nisoldipine. 웁-adrenergic stimulation has little effect on IKs at room temperature, and nisoldipine blocks ICa-L . These conditions limited the confounding effects that 웁-adrenergic stimulation would have had on the action potential due to enhancement of these cationic currents. The minor effect of ICl-PKA on the resting membrane potential can be explained by the fact that it contributes relatively little to the total resting membrane conductance, which in ventricular myocytes is dominated by the activity of inward rectifier K⫹ channels. However, these K⫹ channels deactivate during the upstroke of the action potential, at which time the contribution of ICl-PKA to the total membrane conductance becomes quite sig-

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nificant. The outward Cl⫺ current generated can then contribute significantly to repolarization. By acting to decrease action potential duration, ICl-PKA counters the prolonging effect that 웁-adrenergic stimulation has on the action potential due to stimulation of ICa-L . In this sense, the action of ICl-PKA could be viewed as beneficial in that it helps prevent abnormal prolongation of the action potential during sympathetic stimulation, when heart rate is increasing and cycle length is decreasing. 웁-adrenergic stimulation of IKs will also facilitate repolarization, although the relative importance of enhancing IKs vs activating ICl-PKA has not been determined.

B. Volume-Regulated Chloride Channels Volume-regulated anion channels are expressed ubiquitously in mammalian cells, and the activation of current conducted by such channels, ICl-vol , is believed to provide one of the initial triggers linking cell swelling to the subsequent loss of osmolytes and water, resulting in a regulatory volume decrease (RVD). Causing cells to swell by exposing them to hypotonic solutions is perhaps the most common technique used to activate ICl-vol . However, when using the patch clamp technique, cell swelling can be achieved by applying positive pressure to the inside of the cell via the patch pipette. ICl-vol also appears to activate spontaneously in some preparations, even in the absence of obvious pressure or osmotic gradients, but these spontaneously active currents can be suppressed by exposing cells to hypertonic extracellular solutions. Independent of how such currents are activated, they are often referred to as swelling-activated Cl⫺ currents (ICl-swell). However, the term ICl-vol may be more appropriate as these currents can also be regulated by cell shrinkage. 1. Conductance Properties

FIGURE 5 Contribution of ICl-PKA to the ventricular action potential. (A) Current clamp recording of action potentials from an isolated guinea pig ventricular myocyte under control (CTL) conditions and following exposure to isoproterenol (Iso) and Iso plus acetylcholine (ACh). (B) Voltage clamp recordings of membrane currents made just prior to recording the action potentials illustrated in A. The L-type Ca2⫹ current was blocked by nisoldipine, and 웁-adrenergic regulation of the delayed rectifier K⫹ current was limited by conducting the experiments at room temperature. ([Cl⫺]i ⫽ 20 mM; [Cl⫺]o ⫽ 150 mM). From Harvey et al. (1990), with permission of The Rockefeller University Press.

A volume-regulated Cl⫺ current has been described in virtually every type of cardiac myocyte in which it has been looked for. Irrespective of whether such currents activate spontaneously or are induced by exposure to hypotonic solutions or application of positive intracellular pressure, the macroscopic currents have similar properties. ICl-vol is largely time independent. However, at extremely positive membrane potentials (⬎50 mV) there is a characteristic time-dependent decline, which represents a voltage-dependent inactivation process. This is one feature that distinguishes ICl-vol from ICl-PKA . Another distinguishing property of ICl-vol is the fact that it exhibits outward rectification that is independent of the Cl⫺ concentration inside the cell (Vandenberg et al., 1994). Therefore, when the intracellular Cl⫺ concentration is high, ICl-vol will exhibit outward rectification,

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whereas ICl-PKA will exhibit a linear voltage dependence. As expected for a Cl⫺ current, the reversal potential of ICl-vol closely follows the Cl⫺ equilibrium potential (Fig. 6). Although the anion selectivity sequence has not been determined for the current identified in every preparation, in those preparations in which it has been studied, it is typically I⫺ ⱖ NO3⫺ ⬎ Br⫺ ⬎ Cl⫺ ⬎ F⫺

(Hagiwara et al., 1992; Vandenberg et al., 1994). In this case, the fact that I⫺ is significantly more permeant than Cl⫺ also distinguishes this current from ICl-PKA . Despite the apparent similarities in macroscopic currents, it remains to be determined whether spontaneously activated currents or currents activated by hypotonic- or pressure-induced swelling in various prepara-

FIGURE 6 Macroscopic ICl-vol activated by hypotonic-induced cell swelling. (A) Whole cell currents

recorded during voltage clamp steps to membrane potentials between 80 and ⫺100 mV before (a) and after (b–d) exposure of a rabbit atrial myocyte to a hypotonic extracellular solution containing various concentrations of Cl⫺. (B) Membrane potential (V) dependence of the swelling-induced current (I) recorded under conditions similar to those indicated in A. (C) Relationship between the extracellular Cl⫺ concentration ([Cl⫺]o) and the reversal potential (Erev) of the swelling-induced current. The solid line is the Nernst prediction for an ideal Cl⫺ conductance. From Duan et al. (1995), with permission.

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381

FIGURE 7 Microscopic Cl⫺ currents associated with hypotonic-induced cell swelling. Unitary currents recorded from a rabbit atrial myocyte using the cell-attached patch clamp technique ([Cl⫺]o ⫽ 108 mM). Channel activity was observed only after exposure of the cell to a hypotonic extracellular solution. The membrane potential at which channel activity was recorded is reported relative to the cell’s resting membrane potential (RP). Note that the current exhibits a pronounced outward rectification. From Duan et al. (1997), with permission.

tions are all conducted by the same type of anion channel. Some uncertainty exists with regard to the identification of unitary currents associated with ICl-vol in most mammalian cells, not just cardiac cells. Unitary currents that might be responsible for macroscopic ICl-vol in adult mammalian cardiac myocytes have been difficult to detect. In rabbit atrial myocytes, an outwardly rectifying Cl⫺-sensitive channel (ORCC) with an intermediate unitary conductance (앑60 pS) has been suggested to underlie the spontaneously active ICl-vol (Duan and Nattel, 1994). The relationship between spontaneously active channels and ICl-vol has also been examined directly by comparing the properties of unitary ORCCs in cell-attached membrane patches from myocytes exposed to isotonic and hypotonic solutions (Fig. 7). ORCCs with a unitary conductance of 앑28 pS were observed under both isotonic and hypotonic conditions (Duan et al., 1997a). However, active channels were more prevalent in patches from cells exposed to hypotonic solutions. In addition to exhibiting outward rectification and a similar unitary conductance, channels recorded under both conditions demonstrated a similar pharmacologic profile. Further studies will be necessary to determine whether a channel with the same properties is associated with ICl-vol in all cardiac preparations. 2. Regulation In cardiac cells, activation of ICl-vol does not appear to require phosphorylation by PKA and/or PKC, as ICl-vol can be activated in the presence of kinase inhibi-

tors. However, it has been reported that the inhibition of tyrosine kinase activity can block the activation of ICl-vol by exposure to hypotonic extracellular solutions. Furthermore, once activated by changes in cell volume, ICl-vol may be modulated by both PKC and PKA. PKC has perhaps the most potent effect on ICl-vol . In most, but not all, cases activation of PKC has been reported to inhibit both spontaneously active ICl-vol and ICl-vol activated by exposure to hypotonic solutions. Furthermore, this effect has been linked to the activation of 움1A-adrenergic receptors via a pertussis toxin-sensitive G-protein (Duan et al., 1995). The apparent effects of the cAMP/PKA-signaling pathway are much more inconsistent. cAMP- and/or PKA-dependent phosphorylation has been reported to produce both stimulatory and inhibitory effects, or no effect at all on ICl-vol in various preparations. A consistent feature of ICl-vol observed in native cardiac cells is a temporal lag between the onset of cell swelling and detectable activation of the current, suggesting that some metabolic or enzymatic intermediate may play a role in coupling changes in cell volume to channel activation. One possibility is that this reflects a tyrosine kinase-dependent phosphorylation mechanism. Another possibility is that activation of ICl-vol may actually involve a dephosphorylation process. This latter hypothesis has been proposed based, at least in part, on the fact that in many (although not all) preparations, ICl-vol can be inhibited by the stimulation of PKA and/ or PKC (Hall et al., 1995; Duan et al., 1999). In addition to dephosphorylation-dependent mechanisms, pro-

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cesses involving changes in cytoskeleton and membrane tension have been proposed. 3. Pharmacology As in many other types of cells, ICl-vol in cardiac cells is blocked by the stilbene derivatives, SITS and DIDS. The block by these compounds is usually voltage dependent, with outward currents inhibited more effectively than inward currents. ICl-vol is also blocked, although less potently, by carboxylic acid derivatives such as 9AC and DPC. The most extensive characterization of ICl-vol inhibitors has been conducted in canine atrial myocytes (Sorota, 1994). Niflumic acid (100 애M), NPPB (10–40 애M), and indanyloxyacetic acid 94 (IAA-94) (100 애M) produce complete block of ICl-vol , 9-AC (1 mM) and dideoxyforskolin (100 애M) produce partial block, and DIDS (100 애M) and DNDS (5 mM) block outward currents more effectively than inward currents. At these concentrations, other nonspecific effects of niflumic acid, IAA-94, and NPPB have been found, emphasizing that most of these compounds fall short of being considered selective antagonists of ICl-vol . One of the most potent inhibitors of ICl-vol is the antiestrogen compound tamoxifen, which at 10 애M has been shown to nearly completely block ICl-vol in various cardiac and noncardiac cells. The effect of this drug appears to be selective for ICl-vol over ICl-PKA at low concentrations (Vandenberg et al., 1994). the KATP channel inhibitor glibenclamide, which has been shown to inhibit both epithelial and cardiac CFTR Cl⫺ channels, also appears to significantly inhibit epithelial as well as cardiac ICl-vol in a voltage-dependent and reversible manner. There is also evidence that ICl-vol in cardiac myocytes can be inhibited with varying degrees of potency by extracellular cAMP and ATP. 4. Molecular Structure/Function P-glycoprotein (P-gp), a multidrug transporter, was initially suggested to underlie ICl-vol . This protein had promise because it belongs to the ABC superfamily of transporters, of which CFTR is also a member. Initial reports indicated that expression of P-gp correlated with expression of ICl-vol activity in several mammalian cell lines. However, it now appears likely that P-gp is not itself responsible for ICl-vol but may regulate endogenous ICl-vol in these cells. Another candidate proposed to underlie ICl-vol is pICln. This 235 amino acid protein, which is also expressed in the heart, has no obvious transmembrane-spanning regions. When expressed in Xenopus oocytes, pICln is associated with the expression of a Cl⫺ current with many of the properties of native ICl-vol . However, such studies are confounded by the presence

of an endogenous ICl-vol in oocytes, and it is now believed that pICln may regulate endogenous ICl-vol . The exact functional role of pICln remains elusive, and whether it forms an anion channel protein is currently being reevaluated. Another class of Cl⫺ channel proteins with promising candidates for ICl-vol comes from the ClC family of gene products. ClC genes encompass a large family of gene products that, when expressed, function as voltage-dependent anion channels. Expression of ClC-2 has been shown to yield volume-sensitive chloride channels, which are inwardly rectifying and have an anion selectivity of Cl⫺ ⱖ Br⫺ ⬎ I⫺. However, these characteristics are in contrast to the typical properties of ICl-vol found in most native mammalian cells, which exhibit outward rectification and an anion selectivity of I⫺ ⬎ NO3⫺ ⬎ Br⫺ ⬎ Cl⫺. Another member of the ClC family, ClC-3, is associated with the expression of a Cl⫺ conductance with the appropriate anion selectivity sequence. A fulllength ClC-3 cDNA has been cloned from guinea pig ventricle (gpClC-3) (Duan et al., 1997b), and transfection of gpClC-3 into NIH/3T3 cells yields a basally active chloride conductance that is modulated by changes in cell volume. Many properties of the current associated with the heterologous expression of gpClC-3 resemble those reported for native ICl-vol in heart and other tissues. These include (1) an outwardly rectifying unitary current with a slope conductance of 40 pS, (2) a greater selectivity of I⫺ than for Cl⫺, (3) time-dependent inactivation at positive membrane potentials, (4) inhibition of constitutively active current by hypertonic extracellular solutions, (5) further activation of the current by hypotonic extracellular solutions, and (6) inhibition by phorbol esters, stilbene derivatives, tamoxifen, and extracellular nucleotides. Information about the molecular structure of ClC Cl⫺ channel proteins is limited. ClC3 consists of 760 amino acids. As predicted for other members of the ClC Cl⫺ channel family, both carboxyterminal and amino-terminal ends of the protein are intracellular, and it is likely to possess 10 to 12 transmembrane segments.

5. Species/Tissue Distribution ICl-vol appears to be expressed ubiquitously in heart and has been observed in nearly every cardiac cell type examined, including canine atrial and ventricular myocytes, rabbit atrial and sinoatrial myocytes, cultured chick myocytes, guinea pig atrial and ventricular myocytes, feline ventricular myocytes, rat neonatal myocytes, and human atrial and ventricular myocytes. Where comparisons have been made, the density of ICl-vol appears to be significantly higher in atrial myo-

19. Chloride Channels

cytes than it is in ventricular myocytes (Vandenberg et al., 1994). 6. Functional Role A primary Physiological role of ICl-vol appears to be cell volume homeostasis. Activation of ICl-vol in cultured chick heart cells has been shown to partially mediate RVD (Zhang et al., 1993). In addition to contributing to RVD, ICl-vol also has the potential of significantly affecting the electrical activity of the heart. Because it is a largely time-independent current (at least over a physiologically relevant range of membrane potentials) and because it exhibits outward rectification, the contribution of this current to the electrical activity of ventricular myocytes is expected to be similar to that of ICl-PKA (see Fig. 5). However, directly demonstrating any effects of activating ICl-vol on the cardiac action potential is complicated by the fact that cell swelling has been reported to alter the activity of a variety of other electrogenic mechanisms in the heart. These include both slow and fast components of the delayed rectifier K⫹ current, KATP channels, the Na⫹ –K⫹ pump, the Na⫹ /Ca2⫹ exchanger, and nonselective cation channels. A number of reports have shown that cell swelling induces action potential shortening and membrane depolarization of cardiac cells. Swelling of guinea pig ventricular myocytes induced by exposure to hypotonic solutions has been shown to cause a small depolarization (4 to 5 mV) of the resting membrane potential and an initial brief lengthening of action potential duration followed by action potential shortening (Vandenberg et al., 1997). In most cells, the depolarization and action potential shortening could be partially prevented by DIDS, supporting the idea that ICl-vol contributes significantly to the swelling-induced changes in membrane potential. One potentially significant difference between the contribution of ICl-vol and ICl-PKA with respect to their contribution to cardiac electrical activity is the fact that ICl-vol is more prevalent in atrial cells. Activation of a background Cl⫺ conductance would be expected to have a much more pronounced effect on the resting membrane potential of an atrial cell, as the resting conductance of these cells is typically much lower than that of ventricular myocytes. In fact, swelling of dog atrial cells has been shown to cause a 15- to 20-mV depolarization of the resting potential, an effect that could be antagonized by niflumic acid and accentuated by making the equilibrium potential for Cl⫺ more positive (Du and Sorota, 1997). Activation of ICl-vol would also be expected to contribute significantly to the membrane conductance of nodal cells, although the exact effect that this would have on pacemaking activity is unclear. Because ECl is near the normal maximum diastolic poten-

383

tial of pacemaker cells, ICl-vol might actually be expected to stabilize diastolic membrane potential and slow spontaneous depolarization. In the absence of any countering effect, swellinginduced activation of ICl-vol alone might be expected to be arrhythmogenic. By causing an unopposed decrease in action potential duration, ICl-vol would effectively reduce the refractory period, making cells more vulnerable to premature excitation and the development of reentrant arrhythmias. Significant depolarization of the resting membrane potential, such a would be expected to occur in atrial cells, might also lead to the development of ectopic pacemaker activity. However, it is important to understand the context in which ICl-vol is activated. Under different conditions, activation of ICl-vol might be beneficial. For example, cardiac hypertrophy is typically associated with an increase in action potential duration. Under these conditions, ICl-vol-induced action potential shortening might be antiarrhythmic.

C. Calcium-Activated Chloride Channels The transient outward current (Ito), activated during membrane depolarization, has been studied extensively for many years and was initially referred to as the ‘‘early outward current,’’ ‘‘initial outward current,’’ or ‘‘positive dynamic current.’’ The transient nature of this current determines the role it plays in modulating cardiac electrical activity. Following Na⫹ and Ca2⫹ channel activation during the upstroke, rapid activation of Ito contributes to the initial repolarization phase of the cardiac action potential. The kinetics of Ito inactivation also influence the plateau and final repolarization phases of the action potential. Early evidence that Cl⫺ may be a charge carrier for Ito in the heart was complicated by poor voltage clamp control of multicellular preparations used for these studies. There were also difficulties effectively separating Cl⫺ sensitive components of current from overlapping K⫹ currents. These problems were due, at least in part, to the effect that some Cl⫺ substitutes had on free Ca2⫹ concentration, which indirectly affected Ca2⫹-activated currents. Furthermore, some Cl⫺ substitutes were also found to directly block K⫹ conductances. Using 4-aminopyridine (4-AP), a blocker of K⫹ current, subsequent studies concluded that Ito was composed of at least two components in most cardiac cells; a 4-AP-sensitive, Ca2⫹-insensitive current (Ito1) carried by K⫹ and a smaller 4-AP-insensitive, Ca2⫹-sensitive current (Ito2), initially assumed to be carried by K⫹. 1. Conductance Properties A reexamination of cardiac whole cell currents has clearly demonstrated that the Ca2⫹-activated current,

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Ito2 , is actually conducted by Cl⫺, not by K⫹ (Zygmunt and Gibbons, 1991; Zygmunt and Gibbons, 1992). This is illustrated by the fact that this Ca2⫹-dependent current is not abolished by replacing K⫹, but it is eliminated in low Cl⫺ solutions. Another difficulty associated with studying this Cl⫺ current has been separating it from ICa-L . The Cl⫺ current is activated by the release of Ca2⫹ from the sarcoplasmic reticulum (SR), and ICa-L is necessary to trigger SR Ca2⫹ release in the heart. As such, blocking ICa-L with nisoldipine or Cd2⫹ abolishes both currents. Conversely, increasing ICa-L with the 웁-adrenergic agonist isoproterenol augments both currents. It is also important to point out that Ca2⫹ released from the SR is responsible for the activation of ICl-Ca , not the Ca2⫹ that enters the cell via ICa-L . This is demonstrated by the fact that ICl-Ca can be abolished by treatment of cells with caffeine or ryanodine. These compounds eliminate SR Ca2⫹ release, but they do not block ICa-L . Studying this Cl⫺ current became easier once it was discovered that ICl-Ca , but not ICa-L , could be blocked by stilbene derivatives such as SITS and DIDS. This has made it possible to define ICl-Ca as a stilbene-sensitive current (Fig. 8). It has been clearly demonstrated that

FIGURE 9 Voltage dependence of ICl-Ca and ICa-L recorded from a rabbit atrial myocyte. (A) DIDS-sensitive current. Macroscopic currents were recorded during voltage clamp steps to membrane potentials between 80 and ⫺40 mV. Difference currents were obtained by subtracting current traces recorded in the presence of DIDS from those recorded in its absence (see Fig. 8). (B) Voltage dependence of peak ICl-Ca or DIDS-sensitive current (䊉) and peak ICa-L or DIDSinsensitive current (䊊). From Zygmunt and Gibbons (1992), with permission of The Rockefeller University Press.

FIGURE 8 Identification of ICa-Cl as a SITS-sensitive current. Macroscopic currents recorded from a dog ventricular myocyte during voltage clamp steps to 0, 20, and 40 mV from a holding potential of ⫺50 mV. (Top) Currents recorded in the absence of SITS consist of ICa-L and ICl-Ca . (Middle) Currents recorded in the presence of SITS primarily represent ICa-L . (Bottom) Digital subtraction of current traces recorded in the presence of SITS from those recorded in its absence yields the SITS-sensitive current, which primarily represents ICl-Ca . From Zygmunt (1994), with permission.

the reversal potential of the stilbene-sensitive current closely follows ECl , demonstrating that it is selective for Cl⫺ over cations. The selectivity sequence for anions is I⫺ ⬎ Br⫺ ⬎ Cl⫺ (Kawano et al., 1995). ICl-Ca , defined as a SITS- or DIDS-sensitive current, has a bell-shaped, current–voltage relationship (Fig. 9). Activation occurs at potentials positive to the threshold for the activation of ICa-L , peaks at potentials more positive than peak ICa , and declines at potentials approaching ECa . There is a dissociation in the time course of changes in bulk intracellular Ca2⫹ concentration and the time course of ICl-Ca ; the Cl⫺ current decays more quickly than the intracellular Ca2⫹ transient (Sipido et al., 1993). Such behavior could be explained if the channels responsible for this current exhibit voltage-dependent inactivation. Alternatively, it might reflect differences in the Ca2⫹ concentration in the bulk cytoplasm

19. Chloride Channels

385

and the Ca2⫹ concentration at the intracellular surface of the membrane. Evidence available at this time supports the idea that ICl-Ca behaves essentially as a ligandgated channel and its apparent time-dependence reflects changes in the concentration of intracellular Ca2⫹ near the channel. Because ICl-Ca channels do not appear to exhibit any voltage dependence, channel activity remains relatively constant in the presence of maintained Ca2⫹ levels (see Fig. 10). Information about the single channel behavior of ICl-Ca is limited, as only one study has described the unitary currents in cardiac myocytes. In inside-out membrane patches from canine ventricular myocytes, these channels exhibit a small single channel conductance (1.0–1.3 pS) with several properties similar to macroscopic ICl-Ca recorded from canine ventricular myocytes (Collier et al., 1996). This includes selectivity for Cl⫺, dependence on intracellular Ca2⫹ for activation, 4-AP resistance, and block by the anion transport blockers, niflumic acid and DIDS, and apparent lack of voltage dependence. These single channels are comparable to low-conductance (1–3 pS) Ca2⫹-activated Cl⫺ channels found in many other, noncardiac cell types. Despite the low single channel conductance, cardiac Ca2⫹-activated Cl⫺ channels have a rather high membrane density (앑3 애m⫺2). 2. Regulation Unitary Ca2⫹-activated Cl⫺ channel currents characterized in inside-out membrane patches from canine ventricular myocytes exhibit a much lower sensitivity to Ca2⫹i (Kd 앑 150 애M) than Ca2⫹-activated Cl⫺ channels described in some other types of cells. This may reflect the loss of a cytosolic component required for channel activation in detached membrane patches or it may reflect the presence of significant Ca2⫹ concentration gradients between the subsarcolemmal space and bulk cytoplasmic Ca2⫹, suggesting that these channels operate in a domain where Ca2⫹ levels reach quite high levels. It is now well established that subcellular gradients in intracellular Ca2⫹ concentration exist in cardiac cells. Calculations suggest that the subsarcolemmal Ca2⫹ concentration rises and falls more quickly and reaches a higher peak than the bulk Ca2⫹ concentration. In some noncardiac cell types, a rise in intracellular Ca2⫹ is believed to not only activate Ca2⫹-activaed Cl⫺ channels, but may also cause inactivation by stimulating the activity of a Ca2⫹-dependent protein kinase, such as PKC or Ca2⫹ /calmodulin-dependent protein kinase. Direct regulation of channels responsible for cardiac ICl-Ca by protein kinases, phosphatases, G-proteins, and other potential signaling pathways has not yet been examined in any detail.

FIGURE 10 Single Ca2⫹-activated Cl⫺ channels recorded in an inside-out patch of membrane excised from a dog ventricular myocyte. (A) When the patch was excised in a bath solution containing a high concentration of Ca2⫹, up to 16 channels were activated, as indicated by the size and distribution of the peaks in the amplitude histogram. (B) Exposing the patch to a nominally Ca2⫹-free solution resulted in a complete loss of channel activity. (C) Reexposure to a Ca2⫹-containing bath solution resulted in reactivation of channels in the patch, clearly demonstrating their Ca2⫹-dependent nature. Channel activity was recorded at a holding potential of ⫺130 mV ([Cl⫺]i ⫽ 150 mM; [Cl⫺]o ⫽ 5 mM). From Collier et al. (1996), with permission.

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3. Pharmacology As indicated previously, ICl-Ca is blocked by the stilbene derivatives, SITS and DIDS, at concentrations (100 애M–2 mM) that reportedly do not significantly inhibit Ca2⫹ currents (Zygmunt and Gibbons, 1991; Kawano et al., 1995). Both macroscopic ICl-Ca and unitary Ca2⫹-activated Cl⫺ channels are also blocked by niflumic acid (50 애M) (Collier et al., 1996). A variety of other anionic transport inhibitors, including carboxylic acid derivatives such as 9-AC and DPC, furosamide, NPPB, and IAA-94, at high concentrations have been reported to inhibit ICl-Ca in other types of cells, including smooth muscle. ICl-Ca in cardiac cells is also inhibited by the KATP channel inhibitor glibenclamide with an estimated EC50 앒 65 애M. 4. Molecular Structure/Function Definitive molecular identification of the protein responsible for small conductance Ca2⫹-activated Cl⫺ channels, which seem to be commonly expressed in various tissues and cell types, has yet to be made. A Ca2⫹activated Cl⫺ channel from bovine trachea (bCLCA1) has been cloned and functionally expressed in mammalian cells. This gene product is unrelated to other known Cl⫺ channel proteins. Its reported anion selectivity, large unitary conductance (25–30 pS), and insensitivity to niflumic acid make this an unlikely molecular candidate for the ubiquitous, small conductance Ca2⫹-activated Cl⫺ channels found in most mammalian cells. A more promising candidate is a protein with homology to bCLCA1, which has been cloned from a mouse lung cDNA library (mCLCA1) (Gandhi et al., 1998). mCLCA1 is a 902 amino acid protein, which, when expressed in HEK293 cells, gives rise to a Cl⫺ conductance activated by intracellular Ca2⫹, and inhibited by DIDS (300 애M) and niflumic acid (100 애M). Despite the fact that Northern analysis has demonstrated that mCLCA1 is expressed in cardiac tissue, any conclusion about whether this channel is responsible for ICl-Ca in cardiac myocytes requires further study. 5. Species/Tissue Distribution Although ICl-Ca has been studied mostly in rabbit atrial, ventricular, and Purkinje myocytes and canine ventricular myocytes, it has also been detected in sheep cardiac Purkinje fibers. Although this current appears to be absent in guinea pig ventricular myocytes, it is likely to underlie the Ca2⫹-sensitive component of Ito (Ito2) demonstrated in earlier studies involving calf Purkinje fibers, elephant seal atrial fibers, and feline ventricular myocytes. Ito has been measured in human atrial

and ventricular myocytes and is believed to play an important role in repolarization. Despite the fact that early studies suggested the existence of a Ca2⫹-sensitive component of Ito similar to Ito2 in human atrial tissue, the only study yet to look at the Cl⫺ dependence of Ito in human tissue failed to detect the presence of ICl-Ca in atrial myocytes. The 4-AP-resistant component of Ito detected was Ca2⫹ insensitive and was attributed to the voltage-dependent relief of 4-AP block of Ito1 (Li et al., 1995). 6. Functional Role Because activity of this channel correlates with changes in the concentration of intracellular Ca2⫹, ICl-Ca exhibits a pronounced time dependence. Like other types of Cl⫺ channels, ICl-Ca can generate inward or outward membrane current depending on where the membrane potential is relative to ECl . At positive membrane potentials, following SR Ca2⫹ release, ICl-Ca generates a transient outward current (Ito2), which along with Ito1 contributes to early repolarization in many cardiac cells. However, the actual magnitude of the contribution will depend on the amount of Ca2⫹ entering through Ltype Ca2⫹ channels as well as the numerous factors that control SR Ca2⫹ release. It has been suggested that elevation of ICl-Ca may serve as a negative feedback mechanism to limit Ca2⫹ entry through voltage-dependent Ca2⫹ channels by making the initial plateau level less positive. A transient inward current (ITI), originally characterized in cardiac Purkinje fibers exposed to toxic concentrations of digitalis, is believed to be responsible for the generation of oscillatory afterpotentials, resulting in a variety of cardiac arrhythmias. While Ca2⫹-activated nonselective cation channels and the Na⫹ /Ca2⫹ exchanger have been classically considered the charge carriers responsible for ITI , there is a significant amount of data supporting the idea that ICl-Ca may also play a significant role (Han and Ferrier, 1992; Zygmunt, 1994). The relative contributions of nonselective cation channels, the Na⫹ /Ca2⫹ exchanger, and ICl-Ca to the generation of ITI appear to be species and tissue dependent. In cells that do not exhibit Ito , or more specifically Ito2 , ICl-Ca would obviously not be expected to contribute to the generation of ITI and delayed after depolarizations.

D. Novel Chloride Channels The first member of the ClC family of voltage-gated Cl⫺ channels (ClC-0) was cloned from the Torpedo marmorata electric organ in 1990. At this time nine different ClC genes have been described in mammalian tissue, with differences in species and tissue distribution

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as well as function (Jentsch et al., 1999). The discovery of ClC-3 expression in heart and its proposed link to ICl-vol in native cardiac myocytes (Duan et al., 1997b) raise interesting questions regarding the possible expression and functional role of other mammalian ClC genes in heart. In addition to ClC-3, there presently is evidence for expression of at least five additional ClC genes in heart (for a review, see Hume et al., 2000). The functional significance of these other cardiac ClC genes is unknown at this time. Because the products of these genes represent potential candidates for existing or yet to be discovered chloride channels in sarcolemmal as well as intracellular membranes, they are certain to be the subject of future molecular biological and electrophysiological studies in mammalian heart.

III. SUMMARY It is becoming increasing clear that chloride conductance pathways can have an important influence on the electrical activity of the heart. As such, identification of the different types of chloride channels found in cardiac muscle is a very active area of basic research. Currently, there is evidence for at least three functionally distinct types of chloride channels in the heart. The first is an isoform of the cystic fibrosis transmembrane conductance regulator. This chloride-selective ion channel protein is regulated by protein kinase A-dependent phosphorylation, and the time-independent current that it generates plays an important role in sympathetic regulation of action potential duration, primarily in ventricular myocytes. The second type of cardiac chloride channel is one that is regulated by changes in cell volume. The molecular identity of the channel(s) underlying volumeregulated chloride conductances has been the subject of controversy. In at least some cardiac preparations it appears to be generated by ClC-3, a member of the ClC family of voltage-dependent chloride channel proteins. In addition to playing an essential role in cell volume homeostasis, this chloride conductance pathway can also contribute significantly to the electrical activity of atrial as well as ventricular myocytes. The third type of chloride channel found in cardiac muscle is calcium activated. This channel is responsible for the calcium-activated component of the transient outward current found in many types of cardiac cells. As such it contributes significantly to action potential repolarization. Furthermore, in some cardiac preparations, it may contribute to the transient inward currents associated with intracellular calcium overload and the generation of arrhythmogenic delayed after depolarizations. The molecular identity of the calcium-activated chloride channel in the heart has yet to be identified.

Bibliography Ackerman, M. J., and Clapham, D. E. (1993). Cardiac chloride channels. Trends Cardiovasc. Med 3, 23–28. Bahinski, A., Nairn, A. C., Greengard, P., and Gadsby, D. C. (1989). Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes. Nature 340, 718–721. Baukrowitz, T., Hwang, T.-C., Nairn, A. C., and Gadsby, D. C. (1994). Coupling of CFTR Cl⫺ channel gating to an ATP hydrolysis cycle. Neuron 12, 473–482. Collier, M. L., Levesque, P. C., Kenyon, J. L., and Hume, J. R. (1996). Unitary Cl⫺ channels activated by cytoplasmic Ca2⫹ in canine ventricular myocytes. Circ. Res. 78, 936–944. Du, X. Y., and Sorota, S. (1997). Cardiac swelling-induced chloride current depolarizes canine atrial myocytes. Am. J. Physiol. Heart Circul. Physiol. 272, H1904–H1916. Duan, D., Cowley, S., Horowitz, B., and Hume, J. R. (1999). A serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume. J. Gen. Physiol. 113, 57–70. Duan, D., Fermini, B., and Nattel, S. (1995). 움-Adrenergic control of volume-regulated Cl⫺ currents in rabbit atrial myocytes. Circ. Res. 77, 379–393. Duan, D., Hume, J. R., and Nattel, S. (1997a). Evidence that outwardly rectifying Cl⫺ channels underlie volume-regulated Cl⫺ currents in heart. Circ. Res. 80, 103–113. Duan, D., and Nattel, S. (1994). Properties of single outwardly rectifying Cl⫺ channels in heart. Circ. Res. 75, 789–795. Duan, D., Winter, C., Cowley, S., Hume, J. R., and Horowitz, B. (1997b). Molecular identification of a volume-regulated chloride channel. Nature 390, 417–421. Ehara, T., and Ishihara, K. (1990). Anion channels activated by adrenaline in cardiac myocytes. Nature 347, 284–286. Gadsby, D. C., Nagel, G., and Hwang, T.-C. (1995). The CFTR chloride channel of mammalian heart, Annu. Rev. Physiol. 57, 387–416. Gadsby, D. C., and Nairn, A. C. (1999). Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol. Rev. 79, S77–S107. Gandhi, R., Elble, R. C., Gruber, A. D., Schreur, K. D., Ji, H. L., Fuller, C. M., and Pauli, B. U. (1998). Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung. J. Biol. Chem. 273, 32096–32101. Hagiwara, N., Masuda, H., Shoda, M., and Irisawa, H. (1992). Stretchactivated anion currents of rabbit cardiac myocytes. J. Physiol. (Lond.) 456, 285–302. Hall, S. K., Zhang, J. P., and Lieberman, M. (1995). Cyclic AMP prevents activation of a swelling-induced chloride- sensitive conductance in chick heart cells. J. Physiol. (Lond.) 488, 359–369. Han, X., and Ferrier, G. R. (1992). Ionic mechanisms of transient inward current in the absence of Na⫹-Ca2⫹ exchange in rabbit cardiac Purkinje fibers. J. Physiol. (Lond.) 456, 19–38. Harvey, R. D. (1996). Cardiac chloride currents. NIPS 11, 175–181. Harvey, R. D., Clark, C. D., and Hume, J. R. (1990). Chloride current in mammalian cardiac myocytes: Novel mechanism for autonomic regulation of action potential duration and resting membrane potential. J. Gen. Physiol. 95, 1077–1102. Harvey, R. D., and Hume, J. R. (1989). Autonomic regulation of a chloride current in heart. Science 244, 983–985. Hiraoka, M., Kawano, S., Hirano, Y., and Furukawa, T. (1998). Role of cardiac chloride currents in changes in action potential characteristics and arrhythmias. Cardiovasc. Res. 40, 23–33. Hume, J. R., Duan, D., Yamazaki, J., Collier, M. L., and Horowitz, B. (2000). Anion transport in heart. Physiol. Rev.80, 31–81. Hume, J. R., and Harvey, R. D. (1991). Chloride conductance pathways in heart. Am. J. Physiol. 261, C399–C412.

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Hwang, T.-C., Horie, M., and Gadsby, D. C. (1993). Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl-conductance in mammalian heart. J. Gen. Physiol. 101, 629–650. Hwang, T.-C., Horie, M., Nairn, A. C., and Gadsby, D. C. (1992). Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance. J. Gen. Physiol. 99, 465–489. Hwang, T.-C., Nagel, G., Nairn, A. C., and Gadsby, D. C. (1994). Regulation of the gating of cystic fibrosis transmembrane conductance regulator Cl channels by phosphorylation and ATP hydrolysis. Proc. Natl. Acad. Sci. USA 91, 4698–4702. James, A. F., Tominaga, T., Okada, Y., and Tominaga, M., (1996). Distribution of cAMP-activated chloride current and CFTR mRNA in the guinea pig heart. Circ. Res. 79, 201–207. Jentsch, T. J., Friedrich, T., Schriever, A., and Yamada, H. (1999). The CLC chloride channel family. Pflu¨g. Arch. 437, 783–795. Kaneda, M., Fukui, K., and Doi, K. (1994). Activation of chloride current by P2-purinoceptors in rat ventricular myocytes. Br. J. Pharmacol. 111, 1355–1360. Kawano, S., Hirayama, Y., and Hiraoka, M. (1995). Activation mechanism of Ca2⫹-sensitive transient outward current in rabbit ventricular myocytes. J. Physiol. (Lond.) 486, 593–604. Levesque, P. C. (1995). ATPo but not cAMPi activates a Cl⫺ conductance in mouse ventricular myocytes. Cardiovasc. Res. 29, 336–343. Levesque, P. C., Hart, P. J., Hume, J. R., Kenyon, J. L., and Horowitz, B. (1992). Expression of cystic fibrosis transmembrane regulator Cl- channels in heart. Circ. Res. 71, 1002–1007. Li. G. R., Feng, J., Wang, Z., Fermini, B., and Nattel, S. (1995). Comparative mechanisms of 4-aminopyridine-resistant Ito in human and rabbit atrial myocytes. Am. J. Physiol 269, H463–H472. Matsuura, H., and Ehara, T. (1992). Activation of chloride current by purinergic stimulation in guinea pig heart cells. Circ. Res. 70, 851–855. Middleton, L. M., and Harvey, R. D. (1998). PKC regulation of cardiac CFTR Cl⫺ channel function in guinea pig ventricular myocytes. Am. J. Physiol. Cell Physiol. 275, C293–C302. Nagel G., Hwang, T.-C., Nastiuk, L., Nairn, A. C., and Gadsby, D. C. (1992). The protein kinase A-regulated cardiac Cl channel resembles the cystic fibrosis transmembrane regulator. Nature 360, 81–84.

Oleksa, L. M., Hool. L. C., and Harvey, R. D. (1996). 움1-Adrenergic inhibition of the 웁-adrenergically activated chloride current in guinea-pig ventricular myocytes. Circ. Res. 78, 1090–1099. Overholt, J. L., Hobert, M. E., and Harvey, R. D. (1993). On the mechanism of rectification of the isoproterenol-activated chloride current in guinea-pig ventricular myocytes. J. Gen. Physiol. 102, 871–895. Sipido, K. R., Callewaert, G., and Carmeliet, E. (1993). [Ca2⫹]i transients and [Ca2⫹]i-dependent chloride current in single Purkinje cells from rabbit heart, J. Physiol. Lond. 468, 641–667. Sorota, S. (1994). Pharmacologic properties of the swelling-induced chloride current of dog atrial myocytes J. Cardiovasc. Electrophysiol. 5, 1006–1016. Sorota, S. (1999). Insights into the structure, distribution and function of the cardiac chloride channels. Cardiovasc. Res. 42, 361–376. Vandenberg, J. I., Bett, G. C., and Powell, T. (1997). Contribution of a swelling-activated chloride current to changes in the cardiac action potential. Am. J. Physiol. Cell Physiol. 273, C541–C547. Vandenberg, J. I., Yoshida, A., Kirk, K., and Powell, T. (1994). Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J. Gen. Physiol. 104, 997–1017. Walsh, K. B., and Wang, C. (1996). Effect of chloride channel blockers on the cardiac CFTR chloride and L-type calcium currents. Cardiovasc. Res. 32, 391–399. Warth, J. D., Collier, M. L., Hart, P., Geary, Y., Gelband, C. H., Chapman, T., Horowitz, B., and Hume, J. R. (1996). CFTR chloride channels in human and simian heart. Cardiovasc. Res. 31, 615–624. Zhang, J., Rasmusson, R. L., Hall, S. K., and Lieberman, M. (1993). A chloride current associated with swelling of cultured chick heart cells. J. Physiol. Lond. 472, 801–820. Zygmunt, A. C. (1994). Intracellular calcium activates a chloride current in canine ventricular myocytes. Am. J. Physiol 267, H1984– H1995. Zygmunt, A. C., and Gibbons, W. R. (1991). Calcium-activated chloride current in rabbit ventricular myocytes. Circ. Res. 68, 424–437. Zygmunt, A. C., and Gibbons, W. R. (1992). Properties of the calciumactivated chloride current in heart. J. Gen. Physiol. 99, 391–414.

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20 Regulation of Cardiac Ion Channels by Phosphorylation, Ca2⫹, Cytoskeleton, and Stretch MASAYASU HIRAOKA, YUJI HIRANO, and SEIKO KAWANO

TETSUSHI FURUKAWA

Department of Cardiovascular Diseases Medical Research Institute Tokyo Medical and Dental University Tokyo 113-8510, Japan

Department of Physiology I Akita University School of Medicine Akita 010-8543, Japan

I. INTRODUCTION

many processes of these modulations are studied extensively in native cardiac myocytes, the clarification of these mechanisms at the molecular level is now in progress. This chapter describes how these factors, such as phosphorylation, intracellular Ca2⫹, cytoskeleton, and stretch, modulate the function of various ion channels.

The electrical activity of the heart and its modulation in response to physiological and pathophysiological stimuli are produced by the functional regulation of various ion channels individually or in concert. While most of these channels are operated primarily by voltage to generate action potentials under physiological condition, their functions are also influenced by the activity and quantitative level of intracellular second messengers. Among the actions of the second messengers, phosphorylation and dephosphorylation of the channel proteins or associated units and increased intracellular Ca2⫹ serve as important regulators of voltage-gated channels. A few channels are gated by ligands or mechanical stretch in cardiac membrane and contribute to the electrical activity of the heart as well. Ca2⫹ is one of the essential ligands to activate ligand-gated channels. There are at least two types of channels activated by an increase in the intracellular Ca2⫹ level: the Ca2⫹activated Cl⫺ channel and the nonselective cation channel. They contribute to changes in membrane potentials under physiological and pathological conditions. As cardiac membranes are exposed to mechanical stretch in a beat-by-beat basis and are prone to chamber dilatation causing deformation of the membrane and cell size under diseased states, stretch-activated channels may play a significant role in these conditions. There is accumulating evidence that the cytoskeleton can hold an important position as interconnecting signaling between the cell membrane and intracellular elements supplying second messengers and regulating the channel function. While

Heart Physiology and Pathophysiology, Fourth Edition

II. EFFECTS OF PHOSPHORYLATION A. Sodium Channels 1. Protein Kinase A (PKA)Dependent Phosphorylation The cardiac Na⫹ current (INa) is modulated by stimuli that increase cyclic adenosine 3⬘,5⬘-monophoshate (cAMP) and activate PKA-dependent phosphorylation. A reduction in INa and a shift in its availability are observed following application of 웁-adrenoceptor agonists that increase the activity of PKA-dependent phosphorylation in native cardiac myocytes (Gintant and Liu, 1992; Herzig and Kohlhardt, 1991; Ono et al., 1989; Schubert et al., 1990; Sunami et al., 1991). In contrast to these reports, an increase in the amplitude of INa without an apparent shift in its availability induced by 웁-adrenergic agonists was reported in rabbit cardiac preparations (Matsuda et al., 1992). Ono et al., (1993) showed that PKA activation shifts the voltage dependence of both activation and inactivation of INa in a hyperpolarizing direction. This explains why the increased activity of PKA-dependent phosphorylation can result in either an increase or a decrease in INa de-

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pending on the holding and test potentials used in different experiments. Recently, phosphorylation of the Na⫹ channels is suggested to cause changes in permeability accessible to Ca2⫹ (‘‘slip-mode conductance’’) (Santana, Gomez, and Lederer, 1998), but this interesting observation appears not to be supported by other groups and needs further clarification. In a cloned 움 subunit of the cardiac Na⫹ channel, there are eight candidate consensus PKA phosphorylation sites at the cytosolic loop interconnecting domains I and II (Catterall, 1997; Fozzard and Hanck, 1996; Marban et al., 1998), and only two of these serines are the targets for PKA-dependent phosphorylation (Murphy et al., 1996; Rossie et al., 1987). When cloned cardiac Na⫹ channels are phosphorylated by PKA, a significant increase in peak INa is observed without any effects on kinetic parameters (Schreibmayer et al., 1994; Frohnwieser et al., 1997; Murphy et al., 1996). To our knowledge, there are no data regarding the cloned cardiac Na⫹ channels that demonstrate downregulation of channel activity by PKA-dependent phosphorylation. In neural isoforms, there are five consensus sites for PKA-dependent phosphorylation in the intracellular linker between domains I and II (Catterall, 1997; Fozzard and Hanck, 1996; Marban et al., 1998). Phosphorylation of these sites of the brain channel reduces current amplitude without affecting gating (Smith and Goldin, 1997), which is in general agreement with observations in neurons (Li et al., 1992). The function of skeletal muscle isoforms of the Na⫹ channel is not affected by PKA (Catterall, 1997; Fozzard and Hanck, 1996; Marban et al., 1998). Therefore, subtype-specific modulation of PKA-dependent phosphorylation is present, possibly reflecting structural differences among different channel isoforms.

experimental conditions and the presence of different PKC isoforms, as well as tissue-specific Na⫹ channel isoforms from different species, my account for these discrepancies. Studies of cloned cardiac Na⫹ channels indicated that the consensus PKC site is most probably located at the interdomain III–IV linker. In the rat cardiac isoform (rH1), activation of PKC markedly reduced the amplitude of the Na⫹ channel current expressed in cells of a mammalian cell line with a negative shift in the steadystate inactivation curve. Phosphorylation of serine 1505 (S1505) at the interdomain III–IV linker was considered to mediate this effect (Qu et al., 1996). PKC activation in the human cardiac Na⫹ channel (hH1), however, produced voltage-independent reduction in the peak current without any changes in kinetics (Murray et al., 1997). The consensus PKC site in the III–IV interdomain linker similar to rH1 was responsible for the justdescribed changes. However, results of both studies indicate that the mutation of these sites reduced but did not eliminate the effects of PKC activation on the current, suggesting the presence of another site for phosphorylation. In rat brain Na⫹ channels, PKC activation resulted in a reduction in peak amplitude of the current and a retardation of macroscopic current decay. These effects were also elicited through the consensus PKC site in the III–IV interdomain linker (Numann et al., 1991). Therefore, tissue- and species-specific differences in response to PKC activation are also present. Because the significance and mechanism of Na⫹ channel modulation by protein phosphorylation have not been clarified, caution should be exercised when attempting to relate the effects in a heterologous expression system to physiological conditions.

B. Calcium Channels 2. Protein Kinase C (PKC)Dependent Phosphorylation Investigations on the effects of PKC activation on INa in native cardiac myocytes have yielded conflicting results. Most of these studies examined the effects of angiotensin II (ANG II) that activate PKC on INa in ventricular cells from different species. Results of two studies revealed an increased amplitude of INa in neonatal rat cardiac myocytes (Moorman et al., 1989; Benz et al., 1992). However, reports on the effects of PKC activators were not consistent: phorbol ester increased current amplitude in one study (Mooreman et al., 1989), whereas it reduced peak amplitude of the current in another (Benz et al., 1992). A separate study demonstrated that ANG II increased INa at low concentrations up to 1 애M but reduced it at high concentrations in guinea pig ventricular myocytes (Nilius et al., 1989). The reasons for these discrepancies are not clear. Different

Phosphorylation is a major physiological modulator of the L-type Ca2⫹ channel function, which is one of the main regulators of excitation and contraction of heart cells (see Chapter 13). 1. PKA-Dependent Phosphorylation A major target for 웁-adrenergic agonists through PKA-dependent phosphorylation is the L-type Ca2⫹ current (ICa. L ) (see Tsien et al., 1986; Hartzell, 1988; McDonald et al., 1994). An agonist binding to the 웁 receptor or nerve stimulation induces activation of the stimulatory G-protein (GS ), which in turn activates the catalytic site of adenylate cyclase and enhances the synthesis of cAMP in the cells. Then, increased cAMP activates PKA that phosphorylates the Ca2⫹ channel protein and/or an associated unit. Not only 웁-adrenergic agonists, but also other agonists, which include histamine

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(Hescheler et al., 1987), glucagon (Mery et al., 1990), and calcitonine gene-related peptide (Ono et al., 1989), that increase intracellular cAMP via stimulation of a different receptor can also produce a potentiation of the L-type Ca2⫹ channels. Likewise, drugs that inhibit phosphodiestrase (PDE) activity, causing increased intracellular cAMP, have a similar potentiating action on ICa. L . Application of 웁-adrenergic agonists is shown to increase the peak ICa. L in a dose-dependent manner. The voltage-dependent activation and inactivation may be slightly shifted to a hyperpolarizing direction (5–10 mV). The increase in peak current is achieved by an increase in the available number of Ca2⫹ channels that can be activated by depolarization and an increase in the probability of a channel opening without a change in the single channel conductance. Several mechanisms contributing to the increased probability of a channel opening are demonstrated: (1) an increase in the proportion of sweeps with channel activity (‘‘availability’’) (or a decrease in the number of null sweeps); (2) graded changes in gating behaviors, including prolongation of open times and shortening of closed times; and (3) potentiation of long openings or ‘‘mode 2’’ gating (Tsien et al., 1986; Hartzell, 1988; McDonald et al., 1994). The question as to whether these changes develop independently or through multiple processes occurring simultaneously is not clarified. While Yue et al. (1990b) proposed that graded alterations in gating time constants are the result of a new gating mode 2 during 웁-adrenergic stimulation, other studies (Hirano et al., 1994; Ono and Fozzard, 1993) argued that changes in open time constants independent from the potentiation of mode 2 gating behavior can occur at the same time, suggesting the involvement of multiple modulatory steps or different phosphorylation sites. The cardiac 움 subunit of the L-type Ca2⫹ channel (움1C ) has several candidate consensus sequences for PKA-dependent phosphorylation, and the cardiac 웁 subunit (웁2A ) also has such sequences (Catterall, 1997; Hofmann, Biel and Flockerzi, 1994). The issue as to whether specific consensus sequences, i.e., a single or multiple sites of 움1C and 웁2A subunits, are substrates for PKA-dependent phosphorylation for functional modulation has not yet been clarified. The main subunit, 움1C , of the channel purified from adult cardiac tissue appears to be the main substrate for PKA. Several groups reported a PKA-induced potentiation of currents through Ca2⫹ channels formed by 움1C alone in mammalian cells, although this increase was rather small or required pretreatment of the channel to PKA inhibitors in some cases. Others, however, could not reproduce these results. When expressed in Xenopus oocytes, currents through Ca2⫹ channels formed by 움1C were not potentiated by PKA or PKA-activating treatment, but were reduced by PKA inhibitors (Singer-Lahat et al., 1994).

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The latter findings could be explained by an abnormally high level of the basal phosphorylation in oocytes and dephosphorylation could reduce the current. Furthermore, the potential site of functional modulation by PKA appears to be at the C-terminal domain of 움1C , but the exact site or amino acid residue is still a matter of controversy. Some studies indicated that the phosphorylation of serine 1928 mediated the modulatory effect of PKA, whereas other studies suggested that serine 1627 and serine 1700 could be the phosphorylation sites (Perets et al., 1996; Leach et al., 1996; De Jongh et al., 1996). Involvement of PKA-dependent phosphorylation of Ca2⫹ channel 웁2A was also indicated (Haase et al., 1996). Most of these studies using cloned Ca2⫹ channels examined functional modulation at the macroscopic current level and observed increased peak current at varying degrees (Perez-Reyes et al., 1994; Zong et al., 1995; see Hofmann et al., 1994). Using recombinant cardiac 움1C and skeletal 웁 subunits expressed in baby hamster kidney (BHK) cells, we demonstrated a potentiation of the current by application of 8Br-cAMP at the single channel level with an increased number of channel openings and an increased duration of open times without changing the unitary current amplitude (Hirano et al., 1996), which were similar to the effects seen in native myocytes (Fig. 1). There are, however, still certain quantitative and qualitative differences in the responses of cloned channels compared to those of native myocytes. These may be due to differences in experimental conditions between native cells and expression systems, as well as unspecified proteins and subunit or tissue specificity of the channel. Gao et al. (1997) reported that channel phosphorylation and regulation are facilitated by submembrane targeting of PKA through association with an A-kinase anchoring protein called AKAP79. They proposed that the PKA-mediated regulation of the L-type Ca2⫹ channel is critically dependent on a function of AKAP79 and phosphorylation of 움1CA. One of the changes in gating behaviors leading to the upregulation of L-type Ca2⫹ channels by PKA-dependent phosphorylation is the induction of mode 2 gating (Tsien et al., 1986; McDonald et al., 1994). The mode 2 gating is also elicited by repetitive or strong membrane depolarization (Pietrobon and Hess, 1990) and, therefore, the prepulse-induced facilitation of the Ca2⫹ channel may be produced by phosphorylation. However, the involvement of PKA-dependent phosphorylation in the prepulse facilitation in the heart is not conclusive because of various conflicting results (Bourinet et al., 1994; Sculptoreanu et al., 1993; Hirano et al., 1999). 웁-adrenergic agonists accelereate pacemaker depolarization partly by increased ICa. L and partly by the activation of the hyperpolarization-activated inward

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FIGURE 1 Effects of 8Br-cAMP on a cloned cardiac L-type Ca2⫹ channel expressed in BHK cells. (A) Original current traces in the control (a) and during 8Br-cAMP application (b) obtained at the times indicated by arrows in B. (a and b, respectively). (B) Temporal profiles of channel open probability (NPo) for large (conventional) amplitude openings (top) and small amplitude openings (bottom). Each bar indicates average NPo values over five consecutive sweeps, including 0 for null sweeps. Period for the application of 8Br-cAMP (150 애M) is indicated by a thick line on the top. Records were obtained in the presence of Bay K8644. Reproduced from Hirano et al. (1996), with permission.

current (If ). If shows a positive shift in activation curve without changes in the fully activated current and an increase in the rate of the current activation. The probability of channel openings is increased by the presence of 웁-agonists at the single channel level (DiFrancesco, 1993). Intracellular modulation of this current, however, seems to be controlled directly by cAMP and not through phosphorylation in sinoatrial (SA) node cells (DiFrancesco and Mangoni, 1994; see Chapter 18). With the 웁-adrenergic receptor stimulation that increases cAMP/PKA-dependent phosphorylation, acetylcholine (ACh) always reduces ICa. L . The mechanism underlying this response can largely be explained by the muscarinic inhibition of adenylate cyclase activity through an inhibitory G-protein (Gi ), allowing cytosolic cAMP to return to basal levels (see Hartzell, 1988; McDonald et al., 1994). Adenosine also has similar antagonistic action on the stimulated ICa. L (Isenberg and Belrardinelli, 1984). In addition, ACh can produce inhibitory actions on ICa. L via a cAMP-independent pathway. Muscarinic stimulation increases cyclic guanosine 3⬘,5⬘monophosphate (cGMP) levels in cardiac myocytes, and this increased cGMP is postulated to be involved in the

inhibition of ICa. L by stimulation of PDE activity (type II) in frog ventricular myocytes (Hartzell, 1988). In mammalian ventricular myocytes, where the predominant PDE isoform is composed of type III, however, cGMP is found to facilitate cAMP-dependent activation of ICa. L (Ono and Trautwein, 1991). Another cGMPdependent pathway may be mediated by the activation of cGMP-dependent protein kinase (PKG) that inhibits ICa. L in both amphibian and mamalian cardiac myocytes (Mery et al., 1991). In relation to this hypothesis, increased guanylate cyclase activity induced by atrial natriuretic peptide (ANP) leading to cGMP accumulation has been shown to inhibit ICa. L (Le Grand et al., 1992; Tohse et al., 1995). Suppressive action by these agents on ICa. L appears to be mediated by increased cGMP and activation of PKG. 2. PKC-Dependent Phosphorylation In contrast to the marked potentiation of the channel function by PKA-dependent phosphorylation, PKCmediated responses mainly through 움-adrenergic stimulation in native cardiac myocytes were not conclusive

20. Regulation of Cardiac Ion Channels

because of inconsisting results depending on the species and experimental conditions used (Hartzell, 1988; Fedida, Braun and Giles, 1993; McDonald et al., 1994). Studies using the perforated patch-recording technique demonstrated a potentiation of the Ca2⫹ channel current by 움-adrenergic agonists, possibly through PKC activation (Liu and Kennedy, 1998; Zhang et al., 1998). Endothelin-1, another activator of PKC, was also shown to enhance ICa. L (Woo and Lee, 1999). The potentiation of the current is preceded by a transient suppression and is associated with transient suppression and a subsequent sustained increase in cell contraction. At a single channel level, potentiation is achieved by increasing the number of openings per sweep and by prolonging open times without effects on the unitary current amplitude (Zhang et al., 1998). Studies using cloned cardiac Ca2⫹ channels indicated functional modulation by PKC activation, but the results of these studies could not be reproduced in native myocytes (see Hofmann et al., 1994). In the Xenopus oocyte expression system, PKC activation produced an initial increase in the expressed current followed by a delayed decrease. The activation was dependent on the intracellular Ca2⫹ concentration (Bourinet et al., 1992). PKC-dependent phosphorylation and PKC-induced reduction in the current were shown to depend on an interaction between 움1CA and 웁2A subunits, whereas phosphorylation occurred independently in each subunit (Puri et al., 1997; Bouron et al., 1995).

C. Potassium Channels There are diverse types of K⫹ channels in the heart with different functions, modulating mechanisms and channel distributions in different cardiac regions and species (see Barry and Nerbonne, 1996; see Chapters 14 and 15). Some of these K⫹ channels are modulated by protein phosphorylation. 1. PKA-Dependent Phosphorylation The delayed rectifier K⫹ channel (Ik) is facilitated by 웁-adrenoceptor agonists with increased amplitude and a negative shift of the activation curve, without changing current kinetics (see Hartzell, 1988). The cardiac Ik is composed of two components, the rapidly activating component (Ikr) and the slowly activating one (Iks). The activating action on Ik by 웁-adrenergic agonists is limited to Iks (Sanguinetti and Jurkiewicz, 1990, 1992). The intracellular pathway for agonists following 웁-receptor stimulation is via the cAMP cascade, resulting in an increased activity of PKA-dependent phosphorylation (Hartzell, 1988; Walsh and Kass, 1988; Yazawa and Kameyama, 1990). Iks is composed of two

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molecular entities: a pore-forming subunit, KVLQT1, and a regulatory subunit, minK (Barhanin et al., 1996; Sanguinetti et al., 1996). It is known that the Iks current in Xenopus oocytes is the functional expression of the exogenous minK together with the endogenous KVLQT1 in the same species. The Iks current in the oocytes is upregulated by agents that increase cellular cAMP concentrations. In the presence of these agents, the current amplitude is increased and shows a faster activation rate without altering voltage dependence. These effects are mediated through PKA-dependent phosphorylation (Blumenthal and Kaczmarek, 1992; Kaczmarek and Blumenthal, 1997; Lo and Numann, 1998). There are, however, no consensus phosphorylation sites for PKA on the minK protein, and, therefore, the site for modulating the currents may be on the KVLQT1 protein. 2. PKC-Dependent Phosphorylation Effects of PKC activation on Iks in native cardiac myocytes were examined by application of 움-adrenergic agonists or PKC activators, yielding different results depending on the species used in experiments. Whereas PKC activation decreased Iks in rat cardiac preparations (Apkon and Nerbonne, 1998; Raven et al., 1989), it increased the current in guinea pig myocytes with a change in the slope of the activation curve (Tohse et al., 1987; Tohse et al., 1992; Walsh and Kass, 1988). Using the expression system of the cloned channels, the presence of PKC activators decreased the amplitude of currents expressed in oocytes from the rat or mouse minK gene (Busch et al., 1992; Honore et al., 1991; Kaczmarek and Blumenthal, 1997), whereas their presence enhanced expressed current from the guinea pig minK gene (Je Zhang et al., 1994; Varnum et al., 1993; Kaczmarek and Blumenthal, 1997). The Iks expressed from the human and cat minK was dually modulated by activation of PKC with time and by the concentrations of the activators: an initial increase followed by a later decrease in the peak current and an increase at low doses and a decrease at high doses of the activators (Lo and Numan, 1998). Different responses of the current to PKC activation in cloned channels from different species may partly depend on the serine residue at position 102 on the minK protein. In the same study, PKA activation only produced a sustained increase in Iks current. Premodulation by PKC prevented Iks modulation by PKA, and PKC had no effect on Iks after potentiation by PKA, suggesting the involvement of multiple interacting phosphorylation sites. Ca2⫹-insensitive and 4-AP-sensitive transient outward K⫹ currents (Ito.k or Ito1) are suppressed by 움adrenergic receptor agonists, which stimulate phospha-

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tidylinositol turnover and the subsequent activation of PKC (Apkon and Nerbonne, 1988; Raven et al., 1989; Fedida, Shimon, and Giles, 1990; see also Fedida et al., 1993). The responsible channel gene for Ito1 has not been definitely determined. This may be partly due to the fact that Ito.k recorded in various experiments has some subtle differences in time- and voltage-dependent properties, depending on tissues and species of the preparations used (see Barry and Nerbonne, 1996). Several molecular correlates are proposed and they include Kv1.4, Kv4.2, and Kv4.3. The expressed currents from each gene share partial similarity to the properties of native currents in myocytes, but a single gene does not reproduce all the current features. Therefore, Ito1 may be composed of heterogeneous channel genes depending on the tissues and species. It has been shown that the human Kv1.4 channel, which has multiple potential phosphorylation sites for PKC, is modulated by the PKC activator, with an initial increase followed by a significant reduction in the expressed current amplitude (Murray et al., 1994). 움-Adrenergic agonists, possibly through activation of PKC, have been shown to block the inward rectifier K⫹ current (IK1) in atrial and ventricular cells and to modulate acetylcholine-activated K⫹ current (IK. ACH) in atrial cells (Fedida et al., 1991, 1993; Ito et al., 1992). The ATP-sensitive K⫹ channel (KATP) in 웁 cells is upregulated by PKC activation, and this action is not well documented in the effects on the cardiac channel (Light et al., 1995). In contrast, 웁-adrenergic agonists activate KATP when [ATP]i is decreased substantially. This effect is achieved through a decrease in subsarcolemmal ATP concentration rather than a direct action on the channels by phosphorylation (Schakow and Ten Eick, 1994). 3. Other Kinase-Dependent Phosphorylations Activation of IK. ACH undergoes desensitization, causing inactivation of the channel activity (Pappano, 1990). The mechanism of desensitization has not been fully clarified, but phosphorylation of the muscarinic ACh receptor (mAChR) appears to be involved. The AChbound mAChR causes the G-protein to dissociate into 움 and 웁웂 subunits and, at the same time, 웁웂 subunits also activate muscarinic receptor kinase. In the presence of ACh, mAChR is quickly phosphorylated, presumably by the activated receptor kinase, and may cause uncoupling of the mAChR and G-protein (Kwatra et al., 1987; Haga et al., 1994). It has been suggested that G-proteincoupled, receptor kinase-dependent phosphorylation of mAChR is responsible for the slow phase of desensitization, and a soluble intracellular factor may be responsible for rapid desensitization (Shui et al., 1995).

D. Chloride Channels Various types of Cl⫺ channels are activated by agonists, stretching, or swelling and receptor stimulations in heart cells that may contribute to action potential changes and the genesis of arrhythmias (Hume and Harvey, 1991; Vandenberg et al., 1996; Hiraoka et al., 1998; see Chapter 18). 1. PKA-Dependent Phosphorylation Mammalian ventricular myocytes display a PKA-activated Cl⫺ current (ICl. PKA ) with a nearly linear whole cell current–voltage relationship in symmetrical 앑150 mM Cl⫺ solutions. The current was not activated under the basal condition, but was activated in the presence of agonists and receptor stimulation that increase cAMP in the cells. Single channel recordings confirmed that the characteristics are nearly identical to Cl⫺ channels of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). The unitary conductance is ohmic and 앑12 pS in symmetrical 앑150 mM Cl⫺ concentrations. The probability of opening is voltage independent, and the rates of opening and closing are very slow (Hume and Harvey, 1991; Gadsby and Nairn, 1999). Northern blot analyses and sequencing of the transcript revealed cardiac CFTR as a spliced variant of epithelial CFTR, and identical messengers were distributed in myocytes from the regions and species in which ICl. PKA was recorded (Horowitz et al., 1993; James et al., 1996). Thus, ICl. PKA is encoded by a cardiac variant of CFTR. The CFTR channel contains two nucleotide-binding domains (NBD), a regulatory domain, and multiple consensus sites for possible PKA-dependent phosphorylation. The channel activation appears to require PKAdependent phosphorylation as well as nucleotide binding and nucleotide hydrolysis. In a single open–close channel gating cycle, an individual channel hydrolyzes one ATP molecule at N-terminal NBD-I to open the channel and then binds and hydrolyzes a second ATP molecule at the C-terminal NBD-II to close the channel. This complex coordinated behavior of the two NBDs is orchestrated by multiple PKA-dependent phosphorylation events, some of which occur within the regulatory domain (see Gadsby and Nairn, 1999). A swelling-induced or stretch-activated chloride current (ICl. SWELL ) was found in heart cells of various species (see Vandenberg et al., 1996; Hiraoka et al., 1998). The signaling pathway between mechanical stretch and activation of the Cl⫺ channel is not known, but the involvement of protein phosphorylation and/or dephosphorylation is suggested. The current was not activated by the cAMP/PKA pathway, but was modulated following its activation by cell swelling or membrane stretch

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(Oz and Sorota, 1995). Protein tyrosine phosphorylation appeared to be required, at least partly, for its activation (Sorota, 1995). Forskolin and isoprenaline had variable effects on ICl. SWELL , depending on the tissue and species used. Brief exposure to both agonists was shown to stimulate the current in dog or human atrial myocytes (Oz and Sorota, 1995), whereas ICl. SWELL in cultured embryonic heart cells was inhibited by these agents (Zhang et al., 1994) and the current in rabbit supraventricular cells was not affected (Hagiwara et al., 1992). The current has been shown to be dually modulated by the cAMP/PKA pathway: an initial stimulation caused by the phosphorylation-independent process and a later inhibition mediated by PKA-dependent phosphorylation (Du and Sorota, 1997). 2. PKC-Dependent Phosphorylation Effects of PKC activation on ICl. PKA are quite complex and controversial. In guinea pig and feline ventricular myocytes, PKC stimulation can activate the Cl⫺ current, the properties of which, including single channel conductance, are quite similar to those of ICl. PKA (Collier and Hume, 1995; Walsh and Long, 1994; Zhang et al., 1994). It has not been confirmed whether PKC activation can elicit the same population of channels as ICl. PKA or different channels with similar properties. Zhang et al., (1994) described a PKA- and PKC-activated Cl⫺ current in feline ventricular myocytes and found that PKC was as effective as PKA in activating the current. PKA- and PKC-dependent responses were not additive. Middleton and Harvey (1998) demonstrated that endogenous PKC alone was not sufficient to activate ICl. PKA but could potentiate the PKA-activated current. The PKC-activated Cl⫺ current in guinea pig ventricular myocytes shared properties partly consistent with those of ICl. SWELL (Walsh and Long, 1994). In canine atrial cells lacking ICl. PKA , ICl. SWELL activated by positive-pressure inflation was stimulated by PKC activators but not by the 움-adrenoceptor agonist, phenylephrine (Du and Sorota, 1999). However, ICl. SWELL in rabbit atrial cells, which was activated under the basal condition, was inhibited by 움-adrenoceptor agonists via PKC stimulation (Duan et al., 1995). The channel protein mainly responsible for ICl. SWELL appeared to be C1C-3, and the expressed current in the cell line was inhibited by PKC activation (Duan et al., 1997). A subsequent study demonstrated that the C1C-3-expressed channel current, as well as the current in native myocytes from guinea pig heart, is opened by cell swelling and that the basally active current is partially inhibited by the activity of endogenous PKC. PKC activation closes both expressed and native channels, suggesting that phosphorylation/

dephosphorylation plays a crucial role in the regulation of ICl. Swell (Duan et al., 1999). It is not known, however, why different responses to PKC activation in ICl. SWELL are noted in different preparations of species.

III. EFFECTS OF Ca2⫹ Ca2⫹ has diverse and multiple effects on cardiac ion channels acting from both sides of the membrane, and this section mainly deals with functional modulations of cardiac ion channels by intracellular Ca2⫹.

A. L-Type Calcium Channel Intracellular Ca2⫹ concentration ([Ca2⫹]I) has dual effects on ICa. L : inactivation and potentiation. Ca2⫹-dependent inactivation is an important determinant of the time course of ICa. L during depolarization (Eckert and Chad, 1984: McDonald et al., 1994). This Ca2⫹dependent mechanism is observed with Ca2⫹ permeating through the channels, with an increased [Ca2⫹]I caused by Ca2⫹ release from the stores, and with steady-state effects of increased [Ca2⫹]I (Yue et al., 1990; Bates and Gurney, 1993; Hirano and Hiraoka, 1994). The inhibitory effect of Ca2⫹ on the channels results from a decrease in the probability of channel opening due to a reduction in subsequent reopenings and a shift of the gating mode toward prolonged closed states. This process appears to reflect a direct interaction between Ca2⫹ and the 움1-subunit of the channel protein (Imredy and Yue, 1994; De Leon et al., 1995). In addition to Ca2⫹dependent inactivation, various studies have demonstrated a potentiation of ICa. L by slight increases in [Ca2⫹]I (Gurney et al., 1989; Bates and Gurney, 1993; Hirano and Hiraoka, 1994; Romanin et al., 1992) (Fig. 2). Potentiation of ICa. L results from the increased probability of channel opening due to the increased number of nonblank sweeps (increased availability), as well as the increased number of openings during nonblank sweeps. It is also associated with reduction in the longer time constant of closed time and in the appearance of long openings. Ca2⫹-dependent potentiation has been attributed to the facilitation of ICa. L during repetitive depolarizations after a period of rest. Despite extensive biophysical analyses, the molecular basis of Ca2⫹dependent autoregulation remains unclear, although a putative Ca2⫹-binding EF-hand motif (De Leon et al., 1995) and a nearby consensus calmodulin-binding isoleucine–glutamine (‘‘IQ’’) motif (Rhoads and Friedberg, 1997; Soldatov et al., 1997; Zuhlke and Reuter, 1998) in the carboxy terminus of the 움1C subunit as a potential site have been suggested. Zuhlke et al. (1999) and Peterson et al. (1999) have demonstrated that cal-

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FIGURE 2 High [Ca2⫹]I-induced potentiation and subsequent inhibition in L-type Ca2⫹ channel activity recorded from a guinea pig ventricular myocyte. (A) Control and (B) approximately 10 min following switching of bath solution to Ca2⫹-containing solution. (Top) R340/380 ratio for intracellular Ca2⫹ concentration and (bottom) NPo of single channel activity during each depolarizing pulse of 0 mV delivered at 1 Hz. (C) Unitary current records during the period indicated by bars in A and B. With increasing [Ca2⫹]I channel activity, (NPo) was initially potentiated (b) from the control (a), but a further increase in [Ca2⫹]I inhibited channel activity (c) with some fluctuation of its level (d). Reproduced from Hirano and Hiraoka, (1994), with permission.

modulin is a critical Ca2⫹ sensor for both inactivation and facilitation of ICa. L and that the nature of the modulatory effects depends on residues within the IQ motif important for calmodulin binding.

B. Potassium Channels Intracellular Ca2⫹ modulates Ik, and the modulation is exclusively exerted on Iks (Sanguinetti and Jurkiewicz,

1992). The Ik modulation by internal Ca2⫹ is observed at a pCa level between 8 and 6 without changing the voltage-dependent properties (Tohse et al., 1987; Tohse, 1990). Ca2⫹-dependent activation is produced by the signaling pathway independent from PKC activation. The activation of Iks by intracellular Ca2⫹ appears to be caused by the Ca2⫹ –calmodulin complex, but is not mediated through phosphorylation by calmodulin kinase II in guinea pig ventricular myocytes (Nitta et al.,

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1994). The minK-expressed current is also upregulated by various treatments that increase cytoplasmic calcium levels (Busch et al., 1992; Honore et al., 1991; Kaczmarek and Blumenthal, 1997). It is intereting to note that calcium binding to calmodulin has been shown to be a key modulator of Ca2⫹ gating by small conductance, calcium-activated potassium channels (Xia et al., 1998). Intracellular Ca2⫹ produces a voltage-dependent block of IK1 ; the action is similar to that of Mg2⫹, indicating that it is one of the main mechanisms of inward rectification (Matsuda and Cruz, 1993). [Ca2⫹]I also produces inactivation of KATP and induces a quick inactivation and loss of channel activity (Findley, 1988; Furukawa et al., 1994).

C. Other Cation Channels Intracellular Ca2⫹ activates nonselective cation channels in cultured cardiac cells (Colquhoun et al., 1981). Similar types of channels are detected in adult ventricular myocytes. The channels are activated by [Ca2⫹]I at levels above 0.3 애M, and the channel open probability is increased to attain the maximum value of 0.93 at about 10 애M [Ca2⫹]I (Ehara et al., 1988). The increase in [Ca2⫹]I above certain levels or the Ca2⫹ overload in cells leads to the development of delayed afterdepolarizations (DADs) and triggered activity (January and Fozzard, 1988). DADs are produced by transient inward current (ITI), which is carried by certain combinations of electrogenic Na⫹ –Ca2⫹ exchange current, Ca2⫹-activated nonselective cation current, and Ca2⫹-

activated Cl⫺ current (Kass et al., 1978; January and Fozzard, 1988; Han and Ferrier, 1992; Zygmunt, 1994). The contribution of the nonselective cation current to the formation of ITI may not constitute a major part, as the reversal potential of ITI is not observed around 0 mV, a presumed reversal potential of the current carried by nonselective cation channels (Arlock and Katzung, 1985). An increase in [Ca2⫹]I from pCa ⫽ 10 to 7 results in an increased amplitude of hyperpolarization-activated inward current, If with a shift in the activation curve to positive potential without changes in kinetics (Hagiwara and Irisawa, 1989). This action of Ca2⫹ is different from that of 웁-adrenergic agonist-induced modulation of If and appears to be mediated by its direct action on the channels. The Ca2⫹-induced modulation of If may partly explain the observed accelerated pacemaker activity of the SA node with increased Ca2⫹.

D. Chloride Channels An increase in [Ca2⫹]I activates the Ca2⫹-activated Cl current (ICl. Ca) in atrial and ventricular myocytes and in Purkinje cells (Zygmund and Gibbons, 1992; Sipido et al., 1993; Kawano et al., 1995). ICl. Ca is activated by an increase in [Ca2⫹]I associated with a Ca2⫹ influx through the plasma membrane and subsequent Ca2⫹ release from the sarcoplasmic reticulum (Fig. 3). The routes of Ca2⫹ influx across the membrane depend mainly on ICa. L and partly on the reverse Na⫹ –Ca2⫹ exchange mechanism. The activated current, therefore, ⫺

FIGURE 3 Activation of ICl. Ca at various [Ca2⫹]I in a rabbit ventricular myocyte. (A) Traces of current sensitive to 4,4⬘-diisothiocyanatostilbene-2,2⬘-disulfonic acid (DIDS) at four different intracellular Ca2⫹ concentrations in pipette solution ([Ca2⫹]I). The pipette solution contained 1 nM Ca2⫹ (a), 10 nM Ca2⫹ (b), 0.1 애M Ca2⫹ (c), and 1 애M free Ca2⫹ (d). All experiments were conducted in the presence of 4 mM 4-aminopyridine (4-AP). Depolarizing pulses were applied from ⫺40 mV to test potentials indicated to the left of each trace. (B) Current–voltage relations of ICl. Ca. Data represent means ⫾ SEM. 䊉, 1 애M free Ca2⫹; 䊊, 0.1 애M Ca2⫹; 䉲, 10 nM Ca2⫹; and 䉮, 1 nM Ca2⫹. Currents were normalized by dividing by total cell capacity. Reproduced from Kawano et al. (1995), with permission.

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follows the time course of Ca2⫹ transient (Sipido et al., 1993; Zygmund and Gibbons, 1992; Kawano, et al., 1995; Kuruma et al., 1998). The channel responsible for carrying ICl. Ca appears to be a ligand-gated channel with [Ca2⫹]I serving as a ligand. A single channel study has demonstrated the existence of Cl⫺ channels that are activated in a [Ca2⫹]I-dependent manner. The channel activity is time independent when [Ca2⫹]I is held constant. The channel current is blocked by anion channel blockers similar to whole cell ICl. Ca (Collier et al., 1995). Because of these characteristics, the current mainly contributes to formation of the rapid repolarization phase (phase 1) and action potential shortening associated with the conditions of increased [Ca2⫹]I . The current may be partly involved in the formation of ITI and DADs (Han and Ferrier, 1992; Zygmunt, 1994).

IV. EFFECTS OF CYTOSKELETON AND ITS ASSEMBLY The cytoskeleton forms important elements that regulate cellular mechanics and it also serves as a pivotal component for cell signaling. Accumulating lines of evidence indicate that the cytoskeleton regulates ion channel function in various cell types (Janmey, 1998). In the heart, several different channels are affected by conditions of cytoskeletal assembly and disassembly. The voltage-dependent Na⫹ current in rat and rabbit ventricular myocytes is modulated by the application of cytochalasin D, which interferes with actin polymerization. Cytochalasin D reduced the whole cell peak INa by 20% and slowed current decay without affecting the voltage dependence of steady-state inactivation and recovery from inactivation. Single channel recordings revealed that cytochalasine treatment induced the bursting activity of Na⫹ channels by the induction of slower components that regulate the time for opening and closing of the channels (Undrovinas et al., 1995). The results suggest that the cytoskeleton regulates the gating of cardiac Na⫹ channels. In snail neurons, cytoskeletal disrupters and stabilizers affected the rundown of Ca2⫹ channels (Johnson and Byerly, 1993). Application of colchicine to dissociate microtubules into tublin facilitated the inactivation of L-type Ca2⫹ channels, whereas application of taxol to stabilize microtubules retarded inactivation with a prolonged mean open time in embryonic ventricular cells (Galli and DeFelice, 1994). They interpreted the results that microtubules could regulate the effective concentration of inactivating ions (local Ca2⫹) near the mouths of channels. The cytoskeleton also affects the function of cardiac K⫹ channels. In guinea pig ventricular myocytes, cytochalacine, a disrupter of actin filaments, accelerated the

appearance of the fourth substate in intracellular divalent cation-free conditions and also abolished Ca2⫹- but not Mg2⫹-induced rectification of IK1 (Mazzanti et al., 1996). Thus, cytoskeletal elements appear to control Ca2⫹-dependent substate expression and rectification in native IK1 . Cardiac KATP could be modulated by association with subsarcolemmal actin filament networks, and the channel opening was shown to be sensitive to the mechanical distortions of the membrane (Van Wagoner, 1993). In fact, application of actin filement disrupters, DNase I, to the internal side of patch membranes antagonized the ATP-induced inhibition of KATP (Terzic and Kurachi, 1996) and attenuated sulfonylurea-induced inhibition of the channel activity (Brady et al., 1996), suggesting the regulatory role of this channel by maintaining the integrity of the actin filament networks. KATP is inhibited by intracellular ATP, but in the absence of ATP the channel activity decreases with time, the process known as ‘‘rundown.’’ After the channel rundown, application of Mg-ATP, but not the free form of ATP, can restore channel activity. This restoration of IK. ATP by Mg-ATP is probably produced by utilizing ATPhydrolysis energy (Furukawa et al., 1994). Because actin polymerization also utilizes ATP-hydrolysis energy, a possible association of the rundown of KATP to actin polymerization and depolymerization was examined. Application of actin disrupters, cytochalasin, and DNase I accelerated channel rundown in excised patches of the membrane from guinea pig ventricular myocytes, whereas actin filament stabilizers, phalloidin, and PIP2 inhibited spontaneous and/or Ca2⫹-induced rundown (Fig. 4). After complete rundown, channel activity could not be restored by Mg-ATP alone, but application of F-actin with Mg-ATP could reinstitute channel activity (Furukawa et al., 1996). The results suggest that rundown and reactivation of IK. ATP are influenced by the assembly and disassembly of the actin cytoskeletal networks. Activation of a Cl⫺-selective conductance has been associated with volume regulatory processes in response to cardiac cell swelling. Little information is available regarding the signaling mechanisms responsible for the activation of these Cl⫺ channels. Several lines of evidence have implicated the cytoskeleton in cell volume regulation. Cell swelling is associated with changes in F-actin conformation in a variety of cell types (Mills et al., 1994). In cultured chick cardiac myocytes, swellinginduced changes in membrane conductance and F-actin architecture were monitored by whole cell patch clamp and fluorescence confocal microscopy. The results imply that the dynamic disassembly and reassembly of F-actin in response to cell swelling appeared to be a component of the volume transduction process regulating the activation of ICl. SWELL (Zhang et al., 1997).

20. Regulation of Cardiac Ion Channels

FIGURE 4 Effects of cytoskeletal stabilizors on KATP channel activity recorded from inside-out patch membranes from guinea pig ventricular myocytes. (A) Representative tracings depicting the effect of phalloidin on a fully activated channel (a), on a partially rundown channel (b), and on a channel almost completely rundown by exposure to 1 mM Ca2⫹ (c). (B) A tracing showing the effect of taxol on a fully activated channel. (C) Effect of phosphoinositolbisphosphate (PIP2) on spontaneous rundown of KATP channels. (a) A recording showing the effect of PIP2 on the fully activated channel, (b) on the partially rundown channel, and (c) on the completely rundown channel. (D) Effect of PIP2 on the Ca2⫹-induced rundown. (a) A representative experiment in which PIP2 was added to 0.1 mM Ca2⫹ and (b) in which PIP2 was added to 1 mM Ca2⫹. (E) Average channel activity (normalized to the control value) following rundown induced by 0.1 or 1.0 mM Ca2⫹ in the absence (open bars) or presence (hatched bars) of PIP2 . Numbers in parentheses beside the bars indicate the number of experiments. Reproduced from Furukawa et al. (1996), with permission.

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V. STRETCH AND OSMOTIC PRESSURE A variety of stretch-activated channels have been identified in myocardial cells. Mechanical deformation of membranes or cell swelling activates nonselective cation channels, K⫹ channels, and Cl⫺ channels (see Vandenberg et al., 1996). In addition, a number of voltage-dependent channels, including Ik and ICa. L , are mechanosensitive, although some controversial findings have been reported. Under the condition of mechanical stretch, action potential configurations and the level of resting potential are variously influenced. Stretch-activated channels may underlie the changes in electrical activity and form a basis for mechanoelectric feedback. (Lab, 1996). It was demonstrated that a stretch induces transient depolarization, leading to the development of abnormal automaticity, which may be caused by increased [Ca2⫹]i partly mediated by the stretch-activated channels permeable to Na⫹ and Ca2⫹ (Craelius et al., 1988; Ruknuden et al., 1993). The nonselective cation channel, which is sensitive to Gd3⫹ (Clemo and Baumgarten, 1997), is persistently activated in tachycardia-induced congestive heart failure, suggesting the involvement of this channel in the development of arrhythmias frequently encountered in this pathology (Clemo et al., 1998). A stretch can activate several K⫹ channels (Sigurdson et al., 1987; Kim, 1992; Van Wagoner, 1993). A stretch induced by hypoosmotic pressure also increases Iks, but not Ikr (Sasaki et al., 1994; Rees et al., 1995). It was also demonstrated that the Na⫹ –K⫹ pump current is stimulated by exposure to a hypoosmolar solution (Sasaki et al., 1994; Whalley et al., 1993). The mechanism of this pump stimulation has not been clarified, but phosphorylation and dephosphorylation of protein kinases associated with cell swelling might be involved (Sadoshima and Izumo, 1997). ICl. SWELL is activated by exposure to hypoosmotic superfusates or positive pressure inflation of the cells from various cardiac preparations (see Vandenberg et al., 1996; Hiraoka et al., 1998). The current may play a role in the volume regulation of cardiac cells (Clemo and Baumgarten, 1997). Alterations of cell volume during osmotic pertubation trigger multiple intracellular signaling events, including various second messenger cascades, phosphorylation or dephosphorylation of target proteins, and altered gene expression (Sadoshima and Izumo, 1997; Lang et al., 1998). Cell swelling has been shown to induce protein dephosphorylation, which may be due to decreased kinase activity and/or increased activities of serine/threonine protein phosphatase. However, cell shrinkage can cause protein phosphorylation. In fact, cell swelling and shrinkage have been shown to induce protein dephosphorylation and phos-

phorylation, respectively, in various cells, including cardiac myocytes (Hall et al., 1995). ICl. SWELL and its possible molecular counterpart, C1C-3, are inhibited by the activation of PKC. A serine residue (serine 51) within a consensus PKC phosphorylation site in the intracellular amino terminus of the C1C-3 channel protein is considered to be an important volume sensor of the channel (Duan et al., 1999). ICl. SWELL has been shown to be persistently activated in tachycardia-induced congestive heart failure (Clemo et al., 1999). Therefore, this current may play a role in abnormal electrical activity in this pathological condition.

VI. SUMMARY While it is widely accepted that phosphorylation through various kinases and an increase in [Ca2⫹]i play a crucial role in the functional modulation of various ion channels in native cardiac myocytes, their effects are sometimes diverse even in the same channel from different tissues and species. Different responses are not always reproducible in the recombinant system from the channel gene. This may be caused by subtype specificity of the molecular structure of the channel and/or intracellular signaling, depending on the preparations, or the exact molecular mechanism has not been elucidated. As to the actions of cytoskeleton and stretch for the regulation of channel function, their importance has increasingly been recognized. Clarification of these modulatory mechanisms will promote our understanding of physiology and pathophysiology of the heart.

Acknowledgment Secretarial assistance by N. Fujita is acknowledged.

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tion of currents by rat cardiac Na⫹ channels expressed in Xenopus laevis oocytes. Recept. Channels 2, 339–350. Schubert,B., Vandongen, A. M. J., Kirsch, G., and Brown, A. (1990). Inhibition of cardiac Na currents by isoproterenol. Am. J. Physiol. 258, H977–H982. Sculptoreanu, A., Rotman, E., Takahashi, M., Scheuer, T., and Catterall, W. A. (1993). Voltage-dependent potentiation of the activity of cardiac L-type calcium channel 움 1 subunits due to phosphorylation by cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 90, 10135–10139. Shui, Z., Boyett, M. R., Zang, W.-J., Haga, T., and Kameyama, K. (1995). Receptor kinase-dependent desensitization of the muscarinic K⫹ current in rat atrial cells. J. Physiol. (Lond). 487, 359–366. Sigurdson, W. J., Morris, C. L., Brezden, B. L., and Gardner, D. R. (1987). Stretch activation of a K⫹ channel in molluscan heart cells. J. Exp. Biol. 127, 191–209. Singer-Lahat, D., Lotan, I., Biel, M., Flockerzi, V., Hofmann, F., and Dascal, N. (1994). Cardiac calcium channels expressed in Xenopus oocytes are modulated by dephosphorylation but not by cAMPdependent phosphorylation. Recept. Channels 2, 215–226. Sipido, K. R., Callewaert, G., and Carmeliet, E. (1993). [Ca2⫹]I , transients and, [Ca2⫹]I-dependent chloride current in single Purkinje cells from rabbit heart. J. Physiol. (Lond). 468, 641–667. Smith, R. D., and Goldin, A. L. (1997). Phosphorylation at a single site in the rat brain sodium channel is necessary and sufficient for current reduction by protein kinase A. J. Neurosci. 17, 6068–6093. Soldatov, N. M., Zuhlke, R. D., Bouron, A., and Reuter, H. (1997). Molecular structures involved in L-type calcium channel inactivation: Role of the carboxyl-terminal region encode by exon 40-42 in 움1C subunit in the kinetics and Ca2⫹-dependent inactivation. J. Biol. Chem. 272, 3560–3566. Sorota, S. (1995). Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current. Pflu¨g. Arch. 431, 178–185. Sunami, A., Fan, Z., Nakamura, F., Naka, M., Tanaka, T., Sawanobori, T., and Hiraoka, M. (1991). The catalytic subunit of cyclic AMPdependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes. Pflu¨g. Arch. 419, 415–417. Terzic, A., and Kurachi, Y. (1996). Actin microfilament disrupters enhance KATP channel opening in patches from guinea pig cardiomyocytes. J. Physiol. (Lond). 492, 395–404. Tohse, N. (1990). Calcium-sensitive delayed rectifier potassium current in guinea pig ventricular cells. Am. J. Physiol. 258, H1200– H1207. Tohse, N., Kameyama, M., and Irisawa, H. (1987). Intracellular Ca2⫹ and protein kinase C modulate K⫹ current in guinea pig heart cells. Am. J. Physiol. 253, H1321–1324. Tohse, N., Nakaya, H., and Kanno, M. (1992). 움 1-Adrenoceptor stimulation enhances the delayed rectifier K⫹ current of guineapig ventricular cells through activation of protein kinase C. Circ. Res. 71, 1441–1446. Tohse, N., Nakaya, H., Takeda, Y., and Kanno, M. (1995). Cyclic GMP-mediated inhibition of L-type Ca2⫹ channel activity by human natriuretic peptide in rabbit heart cells. Br. J. Pharmacol. 114, 1076–1082. Tsien, R. W., Bean, B. P., Hess, P., Lansman, J. B., Nilius, B., and Norwicky, M. C. (1986). Mechanisms of calcium channel modulation by 웁 -adrenergic agents and dihydropyridine Ca agonists. J. Mol. Cell. Cardiol. 18, 691–710. Undrovinas, A. I., Shander, G. S., and Makielski, J. C. (1995). Cytoskeleton modulates gating of voltage-dependent sodium channel in heart. Am. J. Physiol. 268, H203–H214.

Vandenberg, J. L., Rees, S. A., Wright, A. R., and Powell, T. (1996). Cell swelling and ion transport pathways in cardiac myocytes. Cardiovasc. Res. 32, 85–97. Van Wagoner, D. R. (1993). Mechanosensitive gating of atrial ATPsensitive potassium channels. Circ. Res. 72, 973–983. Varnum, M. D., Busch, A. E., Bond, C. T., Maylie, J., and Adelman, J. P. (1993). The min K channel underlies the cardiac potassium current Iks and mediates species-specific responses to protein kinase C. Proc. Natl. Acad. Sci. USA 90, 11528–11532. Walsh, K. B., and Kass, R. S. (1988). Regulation of a heart potassium channel by protein kinase A and C. Science 242, 67–69. Walsh, K. B., and Long, K. J. (1994). Properties of a protein kinase C-activated chloride current in guinea pig ventricular myocytes. Circ. Res. 74, 121–129. Whalley, D. W., Hool, L. C., Ten Eick, R. W., and Rasmussen, H. H. (1993). Effect of osmotic swelling and shrinkage on Na⫹-K⫹ pump activity in mammalian cardiac myocytes. Am. J. Physiol. 265, C1201–C1210. Woo, S. H., and Lee, C. O. (1999). Effects of endothelin-1 on Ca2⫹ signaling in guinea-pig ventricular myocytes: Role of protein kinase C. J. Mol. Cell. Cardiol. 31, 631–643. Xia, X-M., Fakler, B., Rivard, A., Wayman, G., Johnson-Pais, T., Keen, J. E., Ishii, T., Hirschberg, B., Bond, C. T., Lutsenko, S., Maylie, J., and Adelman, J. P. (1998). Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395, 503–507. Yazawa, K., and Kameyama, M. (1990). Mechanisms of receptormediated modulation of the delayed outward potassium current in guinea-pig ventricular myocytes. J. Physiol. (Lond). 421, 135–150. Yue, D. T., Backx, P. H., and Imredy, J. P. (1990). Calcium-sensitive inactivation in the gating of single calcium channels. Science 250, 1735–1738. Yue, D. T., Herzig, S., and Marban, E. (1990b). 웁-Adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc. Natl. Acad. Sci. USA 87, 753–757. Zhang, J., Larsen, T. H., and Lieberman, M. (1997). F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes. Am. J. Physiol. C1215–C1224. Zhang, K., Barrington, P. L., Martin, R. L., and Ten Eick, R. E. (1994). Protein kinase-dependent C1⫺ currents in feline ventricular myocytes. Circ. Res. 75, 133–143. Zhang, S., Hiraoka, M., and Hirano, Y. (1998). Effects of 움 1-adrenergic stimulation on L-type Ca2⫹ current in rat ventricular myocytes. J. Mol. Cell. Cardiol. 30, 1955–1965. Zong, X., Schreieck, J., Mehrke, G., Welling, A., Schuster, A., Bosse, E., Flockerzi, V., and Hofmann, F. (1995). On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation. Pflu¨g. Arch. 430, 340–347. Zuhlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W., and Reuter, H. (1999). Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399, 159–162. Zuhlke, R. D., and Reuter, H. (1998). Ca2⫹-sensitive inactivation of L-type-Ca2⫹ channels depends on multiple cytoplasmic amino acid sequences of the 움 1c subunit. Proc. Natl. Acad. Sci. USA 95, 3287– 3294. Zygmunt, A. C. (1994). Intracellular calcium activates a chloride current in canine ventricular myocytes. Am. J. Physiol. 267, H1984– H1995. Zygmunt, A. C., and Gibbons, W. R. (1992). Properties of calciumactivated chloride current in the heart. J. Gen. Physiol. 99, 391–414.

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21 Cardiac Na⫹ /K⫹ Pump JOSEPH R. STIMERS Department of Pharmacology and Toxicology University of Arkansas for Medical Sciences Little Rock, Arkansas 72205

I. INTRODUCTION

transport has been shown to be constant over a wide range of conditions (Rakowski et al., 1989). This unequal exchange of ions during each transport cycle results in the Na⫹ /K⫹ pump generating a net outward current called Na⫹ pump current (Ip), i.e., the Na⫹ /K⫹ pump is electrogenic. This outward current will have an effect on membrane potential to cause it to become more negative (hyperpolarize). While Ip will tend to hyperpolarize the membrane potential, the extent of this effect on membrane potential will depend on the relative magnitude of other ionic currents and the conductance of the membrane. It has been shown that the Na⫹ /K⫹ pump will contribute about ⫺5 to ⫺10 mV to the membrane potential at rest. Although Ip should have a minimal effect during the upstroke of the action potential and repolarization, it is likely that Ip does contribute to the balance of currents during the plateau phase of the cardiac action potential influencing the action potential duration.

When considering cardiac function as a combination of electrical and mechanical activity, it is easy to understand the roles of Na⫹, Ca2⫹, and K⫹ channels as well as the varied Ca2⫹ regulatory mechanisms because of their direct role in action potential generation and contractility. What is often overlooked is the role of the Na⫹,K⫹-ATPase. While the Na⫹ current carried by Na⫹ channels is fast and large and obviously responsible for the upstroke of the cardiac action potential, the much slower Na⫹ efflux carried out by the Na⫹ /K⫹ pump averaged over time is larger than the Na⫹ influx mediated by Na⫹ channels. The Na⫹ /K⫹ pump-mediated Na⫹ efflux is larger because not only must it pump out all the Na⫹ brought in by the Na⫹ current but also the Na⫹ influx due to Na⫹ /Ca2⫹ exchange, Na⫹ /H⫹ exchange, Na⫹-K⫹2C1⫺ cotransport, Na⫹-coupled glucose and amino-acid transport, and other pathways for Na⫹ influx. The activity of the Na⫹ /K⫹ pump, directly or indirectly, sets up and maintains the ionic gradients necessary for the proper generation of action potentials and contraction.

B. Structure The Na⫹ /K⫹ pump exists in the cell membrane as a heterodimer of an 움 and a 웁 subunit. Evidence shows that these heterodimers come together as a tetramer (Hayashi et al., 1997), but the dimer is the functional unit for carrying out ion transport. As with other membrane proteins, there are multiple isoforms of each subunit of the Na⫹ /K⫹ pump with three 움 isoforms and two 웁 isoforms being common in mammalian cardiac myocytes. Much is now known about the molecular structure of the Na⫹ /K⫹ pump due to cloning of all the isoforms in

II. MECHANISM A. Stoichiometry of the Na⫹ /K⫹ Pump Although the Na⫹ /K⫹ pump exchanges Na⫹ for K⫹ during the transport cycle, the exchange is unequal. For every two K ions moved into the cell, three Na ions are moved out of the cell at the expense of one molecule of ATP (Fig. 1). This stoichiometric relationship for ion

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FIGURE 1 Stoichiometry of the Na⫹ /K⫹ pump in the cell surface

There also appears to be a 웂 subunit associated with the purified enzyme (Forbush et al., 1978). This subunit has been cloned and characterized from rat, dog, and pig tissues (Therien et al., 1997). It appears to be a transmembrane protein with an extracellular amino terminus. The 웂 subunit is not ubiquitous, but rather is only associated with the kidney Na⫹ /K⫹ pump and not in the heart or neurons (Therien et al., 1997). The 웂 subunit is not necessary for function of the Na⫹ /K⫹ pump, but may modify enzyme activity dependence on extracellular K⫹ (Be´guin et al., 1997).

membrane.

recent years, allowing the generation of various mutants and chimeras to identify functionally important regions of the molecule. The 움 subunit contains binding sites for both Na⫹ and K⫹, ATP (both high- and low-affinity sites), and cardiac glycosides. The 움 subunit also contains enzymatic activity for ATP, the phosphorylation site, and hormonal regulatory phosphorylation sites. Most models of the 움 subunit include 10 transmembrane domains with both amino- and carboxy-terminal ends being intracellular. Data suggest that cation-binding and transport sites lie within the transmembrane domains and that segments 4, 5, and 6 play a central role (Karlish, 1997). Experiments with affinity labels suggest that cation-binding sites have two negative charges where two K⫹ or two Na⫹ bind and a third neutral site for Na⫹ binding, which is within the membrane electric field. The major intracellular loop between segments 4 and 5 contains the ATP-binding domain and the catalytic activity of the ATPase. The amino-terminal contains two potential phosphorylation sites (serine 11 and 18; counting from mature form of rat 움1 isoform) for protein kinase C. Only one known protein kinase A (PKA)dependent phosphorylation site has been determined in the rat kidney 움1 isoform, located at serine 938, which is in the cytoplasmic linker between transmembrane segments 8 and 9 (Feschenko et al., 1997). While it remains to be demonstrated in the heart that these sites are phosphorylated, it is clear that both PKA and PKC activity does stimulate the Na⫹ /K⫹ pump in the heart. The 웁 subunit is composed of a single transmembrane domain with the amino-terminal end being intracellular. About 80% of the 웁 subunit is located extracellularly and is highly glycosylated. The 웁 subunit is important for proper folding of the 움 subunit and for its translocation to the cell surface membrane (Colonna et al., 1997). Some reports have suggested that the 웁 subunit might play a role in modifying the affinity of the 움 subunit for transported cations; however, in the human Na⫹ /K⫹ pump expressed in Xenopus oocytes, little effect of the 웁 subunit was found (Crambert et al., 2000).

C. Similarities/Differences between 움 Isoforms Because the Na⫹ /K⫹ pump exists as multiple isoforms, there has been much effort directed toward finding functional differences between them to try to explain the physiological role of each isoform. Most of this work has been done in animal models with the best detail obtained in rat. The most striking difference in rat is that the 움1 isoform has a low affinity for cardiac glycosides, about 100-fold lower than either 움2 or 움3 isoforms. However, this apparently is not the case in humans, as evidence obtained using cloned human 움 and 웁 isoforms has shown that the apparent affinity for ouabain is 4–20 nM for all three isoforms (Crambert et al., 2000). All isoforms of the Na⫹ /K⫹ pump have an affinity for extracellular K⫹ that is 1–2 mM without much variation between species either. The last factor commonly studied is the affinity for intracellular Na⫹. In rat, 움1 and 움2 isoforms have an affinity approximately 10 mM, but the 움3 isoform has a lower affinity of about 30 mM (Zahler et al., 1997). This appears to be also true for human isoforms, but to a lesser degree, with the apparent affinity of the 움1 and 움2 isoforms being about 10 mM, but the 움3 isoform is about 20 mM (Crambert et al., 2000). It still remains to be determined if these differences are physiologically significant.

D. Kinetic Mechanism Unlike ion channels that operate passively and gate in response to membrane potential or ligand binding, the Na⫹ /K⫹ pump is an active, energy-requiring mechanism. The Na⫹ /K⫹ pump utilizes the energy stored in ATP to drive the conformational changes necessary for ion translocation against their electrochemical gradients. Under resting physiological conditions, K⫹ is near its equilibrium and so its transport requires little energy. However, Na⫹ is far from its electrochemical equilibrium and so transporting it out of the cell should be expected to require the input of energy. Figure 2 shows a modified Post-Albers schematic diagram used to rep-

21. Cardiac Na⫹ /K⫹ Pump

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FIGURE 2 Modified Post-Albers scheme of the kinetic mechanism of the Na⫹ /K⫹ pump. E denotes the enzyme and E1 and E2 the two major conformational states. Na⫹, K⫹, ATP, and ouabain binding to the enzyme are indicated. E1*P indicates the high energy conformation. f and b indicate forward and backward rate constants for each reaction step.

resent the kinetic states of the Na⫹ /K⫹ pump. The enzyme (E) is believed to exist in two major conformational states: E1 and E2. The E1 conformation has high affinity for Na⫹, a low affinity for K⫹, and can be phosphorylated by ATP in the presence of Mg2⫹ as a cofactor. The E2 conformation has a lower affinity for Na⫹, but has a high affinity for K⫹. The binding and release of the ligands of the pump and the cycling back and forth between E1 and E2 conformations give rise to the cyclic model of Na⫹ /K⫹ pump action shown in Fig. 2. Beginning with the E1 conformation bound with ATP (upper left corner), the enzyme has high affinity for Na⫹ with the binding sites facing the intracellular compartment, which normally has a low concentration of Na⫹ of 5–10 mM. In most experiments the affinity for Na⫹ under physiological conditions is also about 5–10 mM so the pump binds intracellular Na⫹ rapidly: E1–ATP ⫹ 3 Nai 씮 Na3 –E1–ATP ⫹

(1)

⫹

While this scheme depicts all three Na binding simultaneously, evidence suggests that two Na⫹ bind first at the sites of the two fixed negative charges in the binding pocket, followed by the third Na⫹ to another site. Once Na⫹ has bound, the enzyme catalyzes the hydrolysis of ATP and transfers the high-energy 웂-phosphate to itself at aspartate-369 with a high energy bond: Na3 –E1–ATP 씮 Na3 –E1*P ⫹ ADP

(2)

This high-energy bond, like that formed in the active complex between myosin and actin, is an acyl phosphate,

where the phosphate is linked to the carboxyl group of aspartate. The phosphorylated states E1–P and E2–P have been shown to be energetically distinct by their ability to phosphorylate ADP to ATP. The E1–P conformation can readily phosphorylate ADP to ATP and is said to be ADP sensitive. However, the low-energy E2–P conformation is not ADP sensitive and does not phosphorylate ADP readily. The energy stored in this bond is used in the conformational change to E2 with the translocation of Na⫹ from inside to outside the cell. The energy in this highenergy bond is then used to drive a conformation change in the enzyme, resulting in the occlusion of Na⫹ so that it is no longer accessible to the intracellular compartment and the major conformational change to E2, translocating the Na⫹ extracellularly: Na3 –E1*P 씮 [Na3]–E1*P 씮 E2–P ⫹ 3 Nao⫹

(3)

Again the translocation of Na⫹ shown in Eq. (3) is represented as a single step; however, evidence suggests that first one ion is deoccluded and released, accounting for the majority of the voltage dependence of the translocation process, followed by the release of the other two Na⫹ (Rakowski et al., 1997). An interesting observation about the E2–P conformation is that it binds cardiac glycosides, such as digoxin or ouabain, with highest affinity. This has been determined by observations showing that Na⫹ and ATP promote ouabain binding while K⫹ inhibits it. This binding of cardiac glycosides to a particular kinetic state of the

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pump accounts for the ionic and activity dependence of apparent affinity of the glycosides. This also explains the well-known clinical observation that serum K⫹ levels are critical for digoxin therapy of heart failure. Next, the free enzyme in the E2–P conformation has low affinity for Na⫹ and high affinity for K⫹ and so picks up extracellular K⫹. Although it is still uncertain how the binding sites change affinity, it is likely that the molecular rearrangement involved in changing from the E1 to the E2 conformation alters the binding sites such that the ligand affinity is also altered. K⫹o binding also promotes dephosphorylation of the enzyme: E2–P ⫹ 2 Ko⫹ 씮 K2 –E2 ⫹ Pi

(4)

At this stage, ATP binds again and is followed by occlusion of K⫹: K2 –E2 ⫹ ATP 씮 K2 –E2–ATP 씮 [K2]–E2–ATP

(5)

The final step is the conformational change back to E1, with deocclusion and release of K⫹ intracellularly, restoring the enzyme to its original conformation: [K2]–E2–ATP 씮 E1–ATP ⫹ 2 Ki

(6)

III. PHYSIOLOGY Physiological activity in a cardiac myocyte depends on the coordinated action of a number of ion transport, ion channel, and ion-buffering mechanisms to control the contractile apparatus. Figure 3 shows a simplified diagram of a cardiac myocyte with some of these mechanisms illustrated. The activity of the Na⫹ /K⫹ pump, di-

rectly or indirectly, sets up and maintains the ionic gradients necessary for the proper generation of action potentials and contraction. The direct actions of the Na⫹ /K⫹ pump are to use the energy stored in ATP to transport Na⫹ out of the cell in exchange for transporting K⫹ into the cell. This sets up the normally large concentration gradients for Na⫹ and K⫹ so that when Na⫹ or K⫹ channels open, those ions will move across the membrane passively, down their electrochemical gradients, generating currents necessary for the cardiac action potential as well as the resting membrane potential. The indirect actions of the Na⫹ /K⫹ pump result from other transport mechanisms utilizing the energy stored in the Na⫹ gradient setup by the Na⫹ /K⫹ pump to drive their activity. Note that several of the passive transporters in Fig. 3 are coupled to the influx of Na⫹. By using the energy stored in the Na⫹ gradient, these transporters are able to move other ions, such as Ca2⫹ or H⫹, out of the cell. One of the most important mechanisms regarding cardiac function is the Na⫹ /Ca2⫹ exchange. Using the Na⫹ gradient and membrane potential to drive its activity, the Na⫹ /Ca2⫹ exchange transports Ca2⫹ out of the cell, helping to maintain its very low level in the cytosol. The low level of intracellular Ca2⫹ then allows the heart to relax and provides the driving force for the influx of Ca2⫹ through the Ca2⫹ channels when they open during the cardiac action potential. This Ca2⫹ influx, as shown in Fig. 3, activates Ca2⫹ release from the sarcoplasmic reticulum to activate myocardial contraction. The activity of the Na⫹ /K⫹ pump makes this complex cascade of events possible. Consequently, the modulation of Na⫹ /K⫹ pump activity can alter the excitability and contractility of cardiac myocytes, as discussed later.

A. Role in Ion Regulation: Housekeeping

FIGURE 3 Simplified cardiac myocyte showing some relevant ion transport, ion channel, and ion-buffering mechanisms. In the sarcolemma starting from the upper right corner and proceeding clockwise are Na⫹ /H⫹ exchange, Na⫹-K⫹-2C1⫺ cotransporter, C1⫺ /HCO3 exchange, Ca2⫹ -ATPase (or Ca2⫹ pump), Na⫹ /Ca2⫹ exchange, Na⫹ /K⫹ pump, K⫹ channel, Ca2⫹ channel, and Na⫹ channel. The structure within the cell is the sarcoplasmic reticulum (SR) with a Ca2⫹ release channel and a Ca2⫹ pump.

Clearly the most important function of the Na⫹ /K⫹ pump is its role in cellular homeostasis. The Na⫹ /K⫹ pump extrudes Na⫹ from the cell in exchange for K⫹ transport into the cell at the expense of ATP. This activity maintains the steep transmembrane gradients for Na⫹ and K⫹ normally found in excitable cells, such as muscle and nerve. In the heart, Na⫹ /K⫹ pump activity maintains intracellular Na⫹ below 10 mM, despite a high (about 145 mM) extracellular Na⫹ concentration. However, intracellular K⫹ is kept high (about 135 mM) while extracellular K⫹ is low (about 4 mM). This low level of internal Na⫹ acts as a sensitive regulator of Na⫹ /K⫹ pump activity because as intracellular Na⫹ rises, pump activity increases and as Na⫹ falls, pump activity decreases (Fig. 4, left). This is an example of negative feedback control because increasing Na⫹ increases pump activity, which decreases Na⫹. This feedback control is very sensitive because resting intracellular Na⫹

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FIGURE 4 Na⫹ /K⫹ pump current is regulated by intracellular Na⫹ (left), extracellular K⫹ (center), and membrane potential (right). These three graphs illustrate the typical relationship between Ip and these parameters assuming that other factors are optimal for activating the Na⫹ /K⫹ pump.

is near the level that half activates the pump, about 10 mM. Based on this feedback control of Na⫹, it is clear that the Na⫹ /K⫹ pump plays a role in regulating the level of intracellular Na⫹, much like a thermostat regulates the temperature in a room. It has also been suggested that the Na⫹ /K⫹ pump plays a critical role in regulating extracellular K⫹. This is not so obvious because normal serum K⫹ levels of 4–5 mM are close to saturating (⬎80%) the Na⫹ /K⫹ pump (Fig. 4, center). However, evidence in skeletal muscle shows that serum K⫹ is a sensitive regulator of Na⫹ /K⫹ pump activity and expression level (Clausen, 1998). Furthermore, because the transmembrane K⫹ gradient has a strong influence on resting membrane potential and membrane potential also regulates Na⫹ / K⫹ pump activity, these two factors can combine to alter both Na⫹ /K⫹ pump activity and excitability (Dobretsov and Stimers, 1996). Skeletal muscle, which accounts for a large fraction of total body water, acts as a buffer for serum K⫹, with up- or downregulation of Na⫹ /K⫹ pump site density or activity accounting for this regulatory ability. In hypokalemic patients, Na⫹ /K⫹ pump activity in skeletal muscle can be reduced markedly (50% or more) with a pronounced loss of cellular K⫹. Whereas the kidney is the final regulator of total body K⫹ in the long term, skeletal muscle buffers serum K⫹ over the intermediate term (minutes to hours). To accomplish this regulation of serum K⫹, the Na⫹ /K⫹ pump is sensitive to a number of hormone systems, such as insulin and catecholamines (Clausen, 1998). Insulin and catecholamines have been shown to increase the activity of the Na⫹ /K⫹ pump. In skeletal muscle, which has a predominantly 움2 isoform of the pump, the mechanism for increasing activity is by increasing the affinity of the Na⫹ /K⫹ pump for Na⫹ so that it operates faster at a given level of intracellular Na⫹. In the heart, which is a predominantly 움1 isoform, it has also been shown that catecholamines stimulate Na⫹ /K⫹ pump activity (Pecker et al., 1986; Ellingsen et al., 1987), but the mechanism

is by increasing the rate of K⫹ deocclusion and release intracellularly (Dobretsov et al., 1998). This may represent one of the functional differences between the isoforms that account for their physiological significance; however, further research is needed to clarify this contention.

B. Role in AP: Electrical Because the Na⫹ /K⫹ pump is electrogenic, exchanging three Na⫹ ions for two K⫹ ions, it will generate an outward current. This current will contribute to the resting membrane potential. Experiments have shown that under physiological conditions this contributes about ⫺5 mV to resting potential in ventricular myocytes. (Stimers et al., 1990). In partially depolarized muscle, as seen during ischemia, this hyperpolarizing effect of the Na⫹ /K⫹ pump can be much larger for several reasons. First, depolarization will itself increase pump current (Fig. 4, right). Second, depolarization will reduce membrane conductance due to the effect on the inward rectifier K⫹ channel. Finally, K⫹ accumulation in extracellular spaces will activate the pump. In normal or ischemic myocardium, during the plateau phase of the action potential, depolarization will enhance the Na⫹ /K⫹ pump current due to its voltage dependence. This enhanced current will allow the pump to remove the Na⫹ influx through the Na⫹ channels more effectively, but it may also influence the plateau phase of the AP and may hasten repolarization.

C. Role in Contractility: Mechanical Activity of the Na⫹ /K⫹ pump is important to myocardial contractility because of its indirect effect on the Ca2⫹ level of the cytosol. When Na⫹ /K⫹ pump activity is decreased by any factor (e.g., hyperpolarization of Vm , hypokalemia, cardiac glycosides), the level of intracellular Na⫹ increases because it is not being pumped

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out as efficiently (assuming that the Na⫹ influx is not also changed). This increased Na⫹ level reduces the transmembrane Na⫹ gradient, which thus reduces the energy available for other Na⫹-coupled transport mechanisms. Of particular interest in this case is the Na⫹ /Ca2⫹ exchanger. Reduced activity of the Na⫹ /Ca2⫹ exchanger results in an increased level of intracellular Ca2⫹, which increases, the contractile force of the myocytes. Regulation of contractility can also be affected by changing the efficiency with which the Na⫹ /K⫹ pump is able to regulate the K⫹ concentration in the restricted extracellular spaces between myocytes. If extracellular K⫹ rises due to decreased Na⫹ /K⫹ pump activity, then membrane depolarization will depress the cardiac action potential and shorten its duration. These factors can result in a decrease in contractility. Additionally, these changes may result in changes in impulse generation and propagation, potentially leading to arrhythmia.

IV. REGULATION OF THE Na⫹ /K⫹ PUMP The Na⫹ /K⫹ pump is now known to be regulated directly in both its activity and its site density by a number of chemical agents. Table I lists several of these agents and their effects on the Na⫹ /K⫹ pump. Some of the listed effects are still controversial and not all have been demonstrated in cardiac muscle. Kidney and skeletal muscle Na⫹ /K⫹ pump studies have produced evidence for some of these effects. In skeletal muscle, upregulation is induced by training, thyroid hormones, or glucocorticoids, whereas downregulation is seen in hypothyroidism, cardiac insufficiency, myotonic dystrophy, McArdle disease, K⫹ deficiency, and after muscle inactivity (Clausen, 1998). Evidence also shows that some of the effects listed in Table I may depend on the tissue or species in which the study was done. The effect of K⫹ deficiency to reduce Na⫹ /K⫹ pump site density has only been shown in skeletal muscle and only the 움2 isoform was decreased (Clausen, 1998). Also the effect of exercise training in Na⫹ /K⫹ pump site density was demonstrated in skeletal muscle. Perhaps the most controversial effect is adrenergic agonists on the Na⫹ /K⫹

pump, in part because of the multiple receptor classes and the varied signal transduction mechanisms activated by those receptors. A brief discussion is given next; however, many of these issues have yet to be resolved.

A. Protein Kinase A Activation 웁-adrenergic stimulation in the heart causes the Na⫹ / K pump to be phosphorylated via the G-protein– adenylate cyclase–protein kinase A pathway. 웁-adrenergic receptors are coupled to a heterotrimeric G-protein containing the G움s subunit. Activation of the receptor causes the G-protein to disassociate, releasing the G움s subunit to activate adenylate cyclase. Adenylate cyclase synthesizes cyclic AMP from ATP, which then activates cyclic AMP-dependent protein kinase (PK-A). PK-A then phosphorylates the Na⫹ /K⫹ pump, increasing its activity. Experiments in rat heart have shown that this increased activity is due to an enhanced transport, deocclusion, and release of K⫹ intracellularly (Dobretsov et al., 1998). In the guinea pig heart, the mechanism appears to be much more complicated, but the Na⫹ /K⫹ pump is still stimulated by 웁-adrenergic agonists (Gao et al., 1994) In the kidney it had been believed for many years that dopamine (activates PK-A) inhibits the Na⫹ / K⫹ pump, but evidence has shown that this was an artifact of experimental conditions that could be overcome by proper oxygenation of the tissue to reveal the expected stimulation of the pump, as dopamine promotes Na⫹ retention (Kiroytcheva et al., 1999). However, evidence from the kidney suggests that the increased activity is caused by the incorporation of more Na⫹ /K⫹ pump sites into the surface membrane (Carranza et al., 1998) ⫹

B. Protein Kinase C Activation The most controversial mechanism for activation of the Na⫹ /K⫹ pump is via receptors that activate PK-C (Ca-dependent protein kinase). In rat heart (Stimers and Dobretsov, 1995) and in guinea pig heart (Gao et al., 1999; Wang et al., 1998), PK-C has been shown to mediate the stimulation of the Na⫹ /K⫹ pump via the activation of 움-adrenergic receptors. Using rat 움1 ex-

TABLE I Regulators of the Na⫹/K⫹ Pump Increases Naⴙ /Kⴙ pump

Decreases Naⴙ /Kⴙ pump

Activity

Site density

Activity

Site density

Adrenergic agonists Insulin

Thyroid hormone Adrenal steroids Training

Cardiac glycosides Endogenous digitalis-like factors

K⫹-deficiency Diabetes Heart failure

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pressed in a kidney cell line, it was shown that PKC activation caused increased Na⫹ /K⫹ pump activity (Pedemonte et al., 1997). However, in other expression systems, opposite results have been obtained. Using HeLa cells with an endogenous human 움1 Na⫹ /K⫹ pump or transfected with rat 움1 , 움2 , 움3 , or 움1-M32 (truncated amino-terminal between 1 and 32 residues), it was found that activation of PK-C inhibited all these forms of the enzyme (Nestor et al., 1997). However, because 움1-M32 lacks putative PK-C phosphorylation sites, this result suggests that the inhibition of the Na⫹ /K⫹ pump by PKC is either at an as yet unidentified phosphorylation site or is by some indirect mechanism, which could then be cell specific, depending on the activity of this indirect pathway in each cell type. An interesting study looking at the effect of different PK-C isoforms on different Na⫹ /K⫹ pump 움 isoforms expressed in Xenopus oocytes showed that inhibition or stimulation of Na⫹ /K⫹ pump activity (not site density) could occur depending on the isoform combination being investigated (Vasilets, 1997). All of this suggests that regulation of the Na⫹ /K⫹ pump by PK-C may be cell and species specific.

C. Cardiac Glycosides and Endogenous Digitalis-like Factors Clinically, the most important regulator of the Na⫹ / K pump is the class of drugs known as cardiac or digitalis glycosides; common agents include digoxin and ouabain. These drugs have been in use clinically for over 200 years for the treatment of congestive heart failure. Cardiac glycosides bind directly to the Na⫹ /K⫹ pump, inhibiting its activity. Na⫹ /K⫹ pump inhibition leads to an increase in intracellular Na⫹, reducing the Na⫹ gradient. The reduced Na⫹ gradient reduces the energy available to drive the Na⫹ /Ca2⫹ exchange and so intracellular Ca2⫹ rises, increasing myocardial contractility. However, if Ca2⫹ rises too much, Ca2⫹ overload of the myocardium can result in spontaneous contractions and arrhythmias. As discussed earlier, cardiac glycosides bind to a specific conformation of the Na⫹ /K⫹ pump and compete with extracellular K⫹ for binding to that conformation. This competition leads to the well-known clinical interaction between serum K⫹ levels and digitalis toxicity. In heart failure patients, treatment with diuretics to decrease blood volume and afterload on the heart can cause a significant loss of K⫹, leading to hypokalemia. Concurrent treatment with digoxin to increase myocardial contractility can result in toxicity because the hypokalemia will increase the apparent affinity of digoxin for the Na⫹ /K⫹ pump, resulting in too much inhibition and resulting Ca2⫹ overload in the myocardium.

In addition to these exogenous cardiac glycosides, there is considerable evidence for the existence of endogenous digitalis-like factors. The existence and functional role of these factors have been reviewed (Jortani and Valdes, 1997). Although not yet proven, these factors may act like hormones regulating the biological activity of the Na⫹ /K⫹ pump. One of these factors is believed to be a 12-kDa protein that is elevated in patients with congestive heart failure, chronic renal failure, hyperaldosteronism, and essential hypertension (untreated) (Gonick et al., 1998). Other factors found in the brain have been shown to be steroid compounds similar to ouabain and have been implicated in the sympathoexcitatory and pressor responses in salt-sensitive hypertension and sympathetic hyperactivity in congestive heart failure (Budzikowski et al., 1998). It has also been postulated that arrhythmias seen following acute myocardial infarction may be due to an inhibition of Na⫹ /K⫹ pump activity. A clinical study found that on the first day following acute myocardial infarcation, there was a significant increase in the plasma digitalislike factor, which returned to normal levels by the next day (Bagrov et al., 1996). This result suggests that the digitalis-like factor may be involved in myocardial ischemia-induced arrhythmogenesis after acute myocardial infarcation.

⫹

V. PATHOPHYSIOLOGY A. Cardiac Hypertrophy and Heart Failure In patients with heart failure due to various causes, there is a significant reduction in Na⫹,K⫹-ATPase site density ranging from 20 to 50% reduction (Schmidt et al., 1997). This reduction occurs with ischemic heart disease, dilated cardiomyopathy, hypertrophic heart disease, and aortic valve disease. In fact, a linear correlation exists between the degree of heart failure, as measured by left ventricular ejection fraction, and Na⫹,K⫹-ATPase site density or enzyme activity (Ishino et al., 1999) In human heart failure, it has been shown that both Na⫹ /K⫹ pump site density (measured by 3[H]ouabain binding) and enzyme activity are reduced (Schwinger et al., 1999; Bundgaard and Kjeldsen, 1996). This reduction in activity was accompanied by a specific reduction in 움1 and 움3 isoforms to 60 and 70% of control levels, respectively, with no significant change in 움2 isoform or the Na⫹ /Ca2⫹ exchange (Schwinger et al., 1999). Interestingly, in this study, total site density was reduced to 61% of control whereas enzyme activity was reduced to 58% of control, showing good correlation between these two measures. These facts also point out the dominant

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contribution of the 움1 isoform to enzyme activity in the human heart. A further extension of this finding is the correlation found between the level of Na⫹,K⫹-ATPase activity and left ventricular ejection fraction in heart failure patients (Ishino et al., 1999). This suggests that loss of Na⫹ /K⫹ pump activity impairs the heart from performing normal contractile function. One of the major clinical symptoms of heart failure is the presence of fatigue in response to mild or moderate exercise. Lunde et al. (1998) concluded that fatigue in heart failure patients is likely associated with poor control of intracellular Ca2⫹. To the extent that intracellular Ca2⫹ levels are regulated by the Na⫹ /K⫹ pump as described earlier, the downregulation of the Na⫹ /K⫹ pump in both skeletal muscle and myocardium may contribute to this very important symptom. It has also been shown that normalizing K⫹ and Mg2⫹ levels in heart failure patients can have beneficial effects on Na⫹ /K⫹ pump site density and overall symptoms of fatigue, improving the quality of life for these patients (Dorup, 1996).

B. Arrhythmia A review by Levi et al. (1997) has described some of the fundamental mechanisms by which the Na⫹ /K⫹ pump is involved in the generation of myocardial arrhythmias. Through its well-known interactions with the Na⫹ /Ca2⫹ exchange mechanism, Na⫹ /K⫹ pump inhibition will result in a rise in intracellular Ca2⫹. This can lead to Ca2⫹ overload of the sarcoplasmic reticulum, which in turn results in the spontaneous release of Ca2⫹ into the cytosol, initiating a delayed after-depolarization. This is the most common arrhythmia resulting from digitalis intoxication in heart failure patients. What Levi et al. (1997) also described was the effect increased wall stress in a Ca2⫹-loaded heart can have on arrhythmogenesis. They suggested that the increase in contractile force caused by the rise in intracellular Ca2⫹ may be arrhythmogenic, as this will increase wall stress and energy demands in the ventricle. In addition, the rise of intracellular Na⫹ will modulate a number of ion channels and affect the regulation of intracellular pH, which may also cause arrhythmias. An additional factor to be considered in the arrhythmogenic action of the Na⫹ /K⫹ pump is its effects on regulating extracellular K⫹. It has been shown that the activity of the Na⫹ /K⫹ pump in skeletal muscle is critical in regulating serum K⫹ levels (Dorup, 1996; McDonough and Thompson, 1996). Whereas the kidney is clearly the ultimate buffer for serum K⫹ over the long term, in the relatively short term (minutes to hours), skeletal muscle serves as a K⫹ buffer by either increasing Na⫹ /K⫹ pump activity to take up excess K⫹ or reducing pump activity to release K⫹ into the serum when it is

low. Conditions that can cause acute changes in serum K⫹ include sudden changes in physical activity, a Krich/poor diet, and diuretics. In addition to these challenges, a number of pathological or pharmacological conditions can interfere with the ability of the skeletal muscle Na⫹ /K⫹ pump to accommodate changes in serum K⫹ levels. These can include heart failure, which will reduce Na⫹ /K⫹ pump activity, and its treatment with digitalis, which will further reduce activity. However, the hormonal responses (elevated catecholamines) to improve contractility will tend to enhance Na⫹ /K⫹ pump activity. Also, ischemic heart disease reduces pump activity, but the hormonal response (high catacholamines) will tend to enhance pump activity. These complex interactions make it difficult for Na⫹ /K⫹ pumps in the skeletal muscle to adequately regulate serum K⫹ levels in these patients. Because deviations of either high or low serum K⫹ can result in arrhythmia generation, it is likely that this will contribute to arrhythmogenesis in these patients. One of the best clinical indications for antiarrhythmic therapy is the use of 웁-adrenergic receptor blockers following acute myocardial infarction. Drugs such as atenolol have been shown to reduce the incidence of arrhythmias, as well as to increase the survival in these patients. While an obvious mechanism for this therapy is to counteract the effects of elevated catecholamines on myocardial Ca2⫹ fluxes to prevent spontaneous contractions and Ca2⫹ overload-induced arrhythmias, the Na⫹ /K⫹ pump may also play a role. Coronary occlusion has been shown to cause a drop in serum K⫹ levels (Nordrehaug et al., 1985), which may lead to an increased incidence of fibrillation. Thus the use of 웁 blockers could also suppress arrhythmias by inhibiting (or removing the stimulation of) the Na⫹ /K⫹ pump, allowing serum K⫹ to rise back to normal levels.

VI. SUMMARY The Na⫹ /K⫹ pump is a vital component in the surface membrane of all excitable cells. It is directly or indirectly responsible for setting up and maintaining the ion gradients necessary for excitation and contraction in the heart. Its structure and mechanism of action are complex, consisting of multiple isoforms and assuming multiple conformations during the transport cycle. Although involved in basic homeostatic mechanisms of the cell, it is highly regulated by a number of physiological, pathological, and pharmacological mechanisms. While the Na⫹ /K⫹ pump has been the target of study since the late 1950s, there is still much to learn about its role in cardiac physiology.

21. Cardiac Na⫹ /K⫹ Pump

Bibliography Bagrov, A. I., Kuznetsova, E. A., and Fedorova, O. V. (1996). Endogenous digoxin-like factor in myocardial infarction. Klin. Med. 74, 15–17. Be´guin, P., Wang, X. Y., Firsov, D., Puoti, A., Claeys, D., Horisberger, J. D., and Geering, K. (1997). The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function. EMBO J. 16, 4250–4260. Budzikowski, A. S., Huang, B. S., and Leenen, F. H. (1998). Brain ‘‘ouabain,’’ a neurosteroid, mediates sympathetic hyperactivity in salt-sensitive hypertension. Clin. Exp. Hyperten. 20, 119–140. Bundgaard, H., and Kjeldsen, K. (1996). Human myocardial Na,K-ATPase concentration in heart failure. Mol. Cell. Biochem. 163–164, 277–283. Carranza, M. L., Rousselot, M., Chibalin, A. V., Bertorello, A. M., Favre, H., and Fe´raille, E. (1998). Protein kinase A induces recruitment of active Na⫹,K⫹-ATPase units to the plasma membrane of rat proximal convoluted tubule cells. J. Physiol. 511, 235–243. Clausen, T. (1998). Clinical and therapeutic significance of the Na⫹,K⫹ pump. Clin. Sci. 95, 3–17. Colonna, T., Kostich, M., Hamrick, M., Hwang, B., Rawn, J. D., and Fambrough, D. M. (1997). Subunit interactions in the sodium pump. Ann. N. Y. Acad. Sci. 834, 498–513. Crambert, G., Hasler, U., Beggah, A. T., Yu, C., Modyanov, N. N., Horisberger, J. D., Lelievre, L., and Geering, K. (2000). Transport and pharmacological properties of nine different human Na,KATPase isozymes. J. Biol. Chem. 275, 1976–1986. Dobretsov, M., Hastings, S. L., and Stimers, J. R. (1998). Na⫹-K⫹ pump cycle during 웁-adrenergic stimulation of adult rat cardiac myocytes. J. Physiol. 507, 527–539. Dobretsov, M., and Stimers, J. R. (1996). Characterization of the Na/K pump current in N20.1 oligodendrocytes. Brain Res. 724, 103–111. Dorup, I. (1996). Effects of K⫹, Mg2⫹ deficiency and adrenal steroids on Na⫹,K⫹-pump concentration in skeletal muscle. Acta Physiol. Scand. 156, 305–311. Ellingsen, O., Sejersted, O. M., Leraand, S., and Ilebekk, A. (1987). Catecholamine-induced myocardial potassium uptake mediated by 웁1-adrenoceptors and adenylate cyclase activation in the pig. Circ. Res. 60, 540–550.[Abstract] Feschenko, M. S., Wetzel, R. K., and Sweadner, K. J. (1997). Phosphorylation of Na,K-ATPase by protein kinases: Sites, susceptibility, and consequences. Ann. N. Y. Acad. Sci. 834, 479–488. Forbush, B., Kaplan, J. H., and Hoffman, J. F. (1978). Characterization of a new photoaffinity derivative of ouabain: Labeling of the large polypeptide and of a proteolipid component of the Na,K-ATPase. Biochemistry 17, 3667–3676. Gao, J., Cohen, I. S., Mathias, R. T., and Baldo, G. J. (1994). Regulation of the 웁-stimulation of the Na⫹-K⫹ pump current in guineapig ventricular myocytes by a cAMP-dependent PKA pathway. J. Physiol. 477, 373–380. Gao, J., Mathias, R. T., Cohen, I. S., Wang, Y., Sun, X., and Baldo, G. J. (1999). Activation of PKC increases Na⫹-K⫹ pump current in ventricular myocytes from guinea pig heart. Pflu¨g. Arch. Eur. J. Physiol. 437, 643–651. Gonick, H. C., Ding, Y., Vaziri, N. D., Bagrov, A. Y., and Fedorova, O. V. (1998). Simultaneous measurement of marinobufagenin, ouabain, and hypertension-associated protein in various disease states. Clin. Exp. Hyperten. 20, 617–627. Hayashi, Y., Kameyama, K., Kobayashi, T., Hagiwara, E., Shinji, N., and Takagi, T. (1997). Oligomeric structure of solubilized Na⫹ / K⫹ -ATPase linked to E1/E2 conformation. Ann. N. Y. Acad. Sci. 834, 19–29.

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Ishino, K., Botker, H. E., Clausen, T., Hetzer, R., and Sehested, J. (1999). Myocardial adenine nucleotides, glycogen, and Na,KATPase in patients with idiopathic dilated cardiomyopathy requiring mechanical circulatory support. Am. J. Cardiol. 83, 396– 399. Jortani, S. A., and Valdes, R. J. (1997). Digoxin and its related endogenous factors. Crit. Rev. Clin. Lab. Sci. 34, 225–274. Karlish, S. J. (1997). Organization of the membrane domain of the Na/K-pump Ann. N. Y. Acad. Sci. 834, 30–44. Kiroytcheva, M., Cheval, L., Carranza, M. L., Martin, P. Y., Favre, H., Doucet, A. and Fe´raille, E. (1999). Effect of cAMP on the activity and the phosphorylation of Na⫹,K⫹-ATPase in rat thick ascending limb of Henle. Kidney Int. 55, 1819–1831. Levi, A. J., Dalton, G. R., Hancox, J. C., Mitcheson, J. S., Issberner, J., Bates, J. A., Evans, S. J., Howarth, F. C., Hobai, I. A., and Jones, J. V. (1997). Role of intracellular sodium overload in the genesis of cardiac arrhythmias. J. Cardiovasc. Electrophysiol. 8, 700–721. Lunde, P. K., Verburg, E., Vollestad, N. K., and Sejersted, O. M. (1998). Skeletal muscle fatigue in normal subjects and heart failure patients: Is there a common mechanism? Acta Physiol. Scand. 162, 215–228. McDonough, A. A., and Thompson, C. B. (1996). Role of skeletal muscle sodium pumps in the adaptation to potassium deprivation. Acta Physiol. Scand. 156, 295–304. Nestor, N. B., Lane, L. K., and Blostein, R. (1997). Effects of protein kinase modulators on the sodium pump activities of HeLa cells transfected with distinct alpha isoforms of Na,K-ATPase. Ann. N. Y. Acad. Sci. 834, 579–581. Nordrehaug, J. E., Johannessen, K., Von der lippe, G., and Myking, O. L. (1985). Malignant arrhythmia in relation to serum potassium in acute myocardial infarction. Am. Heart J. 110, 944– 948.[Abstract] Pecker, M. S., Im, W. B., Sonn, J. K., and Lee, C. O. (1986). Effect of norepinephrine and cyclic AMP on intracellular sodium ion activity and contractile force in canine cardiac Purkinje fibers. Circ. Res. 59, 390–397. Pedemonte, C. H., Pressley, T. A., Cinelli, A. R., and Lokhandwala, M. F. (1997). Stimulation of protein kinase C rapidly reduces intracellular Na⫹ concentration via activation of the Na⫹ pump in OK cells. Mol. Pharmacol. 52, 88–97. Rakowski, R. F., Bezanilla, F., De weer, P., Gadsby, D. C., Holmgren, M., and Wagg, J. (1997). Charge translocation by the Na/K pump. Ann. N. Y. Acad. Sci. 834, 231–243. Rakowski, R. F., Gadsby, D. C., and De weer, P. (1989). Stoichiometry and voltage dependence of the sodium pump in voltage-clamped, internally dialyzed squid giant axon. J. Gen. Physiol. 93, 903– 941. Schmidt, T. A., Bundgaard, H., and Kjeldsen, K. (1997). Regulation of myocardial Na,K-ATPase concentration in experimental and human heart disease. Ann. N. Y. Acad. Sci. 834, 676–679. Schwinger, R. H., Wang, J., Frank, K., Muller-Ehmsen, J., Brixius, K., McDonough, A. A., and Erdmann, E. (1999). Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na⫹,K⫹-ATPase activity but unchanged Na⫹-Ca2⫹ exchanger protein levels in human heart failure. Circulation 99, 2105–2112. Stimers, J. R., and Dobretsov, M. (1995). Effect of phorbol ester on Na/K pump current in cultured adult rat cardiac myocytes. FASEB J. 9, A66. Stimers, J. R., Shigeto, N., and Lieberman, M. (1990). Na/K pump current in aggregates of cultured chick cardiac myocytes. J. Gen. Physiol. 95, 61–76. Therien, A. G., Goldshleger, R, Karlish, S. J., and Blostein, R. (1997).

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Tissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase. J. Biol. Chem. 272, 32628–32634. Vasilets, L. A. (1997). Diversity of regulatory phosphorylation of the Na⫹ /K⫹-ATPase from mammalian kidneys and Xenopus oocytes by protein kinases: Characterization of the phosphorylation site for protein kinase C. Cell. Physiol. Biochem. 7, 1–18.

Wang, Y., Gao, J., Mathias, R. T., Cohen, I. S., Sun, X., and Baldo, G. J. (1998). 움-Adrenergic effects on Na⫹-K⫹ pump current in guinea-pig ventricular myocytes. J. Physiol. 509, 117–128. Zahler, R., Zhang, Z. T., Manor, M., and Boron, W. F. (1997). Sodium kinetics of Na,K-ATPase 움 isoforms in intact transfected HeLa cells. J. Gen. Physiol. 110, 201–213.

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22 Cardiac Na⫹ –Ca2⫹ Exchanger: Pathophysiology and Pharmacology JUNKO KIMURA Department of Pharmacology Fukushima Medical University School of Medicine Fukushima 960-1295, Japan

I. INTRODUCTION

et al., 1969). The first report in the heart was made by Reuter and Seits (1968), who observed external Na⫹dependent 45Ca2⫹ efflux in the guinea pig atria, where an intracellular Na⫹-dependent Ca2⫹ influx was also discovered (Glitsch et al., 1970). Detailed kinetics of the exchanger was studied by Wakabayashi and Goshima (1981a,b), who first used dissociated cultured heart cells from mouse and chick embryo. In the 1980s, mainly two groups, one led by John Reeves and another by Ken Philipson, published various papers on Na⫹ –Ca2⫹ exchange by the flux method using radioactive Na⫹ and Ca2⫹ in cardiac membrane vesicles and established basic properties of the Na⫹ –Ca2⫹ exchanger. These flux studies predicted that Na⫹ –Ca2⫹ exchange would generate a membrane current, as the exchange ratio was more than 2Na⫹ against each Ca2⫹, underlined by the theory of the electrogenic Na⫹ –Ca2⫹ exchanger by Mullins (1981). Indeed the membrane current generated by the exchanger was identified by the whole cell voltage clamp method (Kimura et al., 1986; Mechmann and Pott, 1986; Hume and Uehara, 1986). The amino acid sequence of the Na⫹ –Ca2⫹ exchanger was first cloned in dog heart (Nicoll et al., 1990). There are now three isoforms of the Na–Ca2⫹ exchanger: cardiac type (NCX1), skeletal muscle type (NCX2), and brain type (NCX3) (Linck et al., 1998). Various new methods have been developed to investigate Na⫹ –Ca2⫹ exchanger activity and function, including a giant patch method (Hilgemann, 1989), antisense oligonucleotide inhibition (Lipp et al., 1995), and overexpression (Adachi-Akahane et al., 1997). A computer program for simulation of the cardiac electrical activity incorporating the Na⫹ –Ca2⫹ exchange cur-

The Ca2⫹ ion is essential in cardiac muscle contraction. Ca2⫹ ions enter cardiac myocytes through Ca2⫹ channels on each heart beat. Once Ca2⫹ enter the cells, Ca2⫹ is released from the sarcoplasmic reticulum (SR) by opening ryanodine receptors or Ca2⫹ release channels. The released Ca2⫹ binds to troponin-C of the contractile protein and induces contraction. The cardiac muscle relaxes when Ca2⫹ returns to SR by the Ca2⫹ pump or Ca2⫹ ATPase. Although there is a species difference, a major fraction (70–90%) of Ca2⫹ required for contraction is released from the SR and the minor fraction (10–30%) enters the cells from extracellular milieu (Bassani et al., 1994). The amount of Ca2⫹ entered should be removed from the cell on each heart beat, as otherwise the cell is overloaded with Ca2⫹. A specific membrane transporter called the ‘‘Na⫹ –Ca2⫹ exchanger’’ is a main player of the Ca2⫹ extrusion in cardiac myocytes. Excellent and exhaustive reviews on the Na⫹ –Ca2⫹ exchanger from various aspects are available (Janvier and Boyett, 1996; Matsuda et al., 1997; Hryshko and Philipson, 1997; Reeves, 1998; Mochizuki and Jiang, 1998; Blaustein and Lederer, 1999). This chapter focuses on relatively less described aspects, i.e., the relation to various cardiac diseases and pharmacology of the cardiac Na⫹ –Ca2⫹ exchanger.

II. HISTORICAL BACKGROUND Na⫹ –Ca2⫹ exchange was first described in crab nerves (Baker and Blaustein, 1968) and in squid axons (Baker

Heart Physiology and Pathophysiology, Fourth Edition

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rent has been also available (DiFrancesco and Noble, 1985).

III. BASIC CHARACTERISTICS OF THE Na⫹ –Ca2⫹ EXCHANGER The following characteristics of the exchanger were found using the whole cell voltage clamp and giant patch clamp technique in cardiac ventricular cells from guinea pig. The coupling ratio of Na⫹ –Ca2⫹ exchange is 3Na⫹ : 1Ca2⫹ (Kimura et al., 1986; Yasui and Kimura, 1990; Ehara et al., 1989), although this value has been challenged by 4 : 1 (Fujioka et al., 2000). Ca2⫹ efflux or the ‘‘forward’’ mode of Na⫹ –Ca2⫹ exchange generates an inward membrane current. Ca2⫹ influx or the ‘‘reverse’’ mode of the exchange generates an outward exchange current. The Na⫹ –Ca2⫹ exchanger reverses its direction at a membrane potential (ENCX) predicted by Eq. (1), where n is stoichiometry and R, T, and F are gas constant, absolute temperature, and Faraday constant, respectively. (n ⫺ 2) ENCX ⫽ ⫺RT/F ln 兵(Cao /Cai )(Nai /Nao )n 其 (1) The reversal potential of the exchange depends on intraand extracellular concentrations of Na⫹ and Ca2⫹. The affinities of Na⫹ and Ca2⫹ on the exchanger were estimated from apparent half-maximal concentrations (Km) that activated the exchange current; Km values are 90, 20, 0.2–1.4, and 0.6–6 애M for external Na⫹, internal Na⫹, external Ca2⫹, and internal Ca2⫹, respectively (Miura and Kimura, 1989; Matsuoka and Hilgemann, 1992). Intracellular Ca2⫹ is not only transported but also activates the exchanger with the half maximum of 22–300 nM (Miura and Kimura, 1989; Hilgemann et al., 1992). In contrast, intracellular Na⫹ inactivates the exchanger (Hilgemann, 1990). The exchanger is accelerated at a higher temperature (Q10 ⫽ 2–4) (Kimura et al., 1987; Hilgemann et al., 1992). The exchange mode is consecutive but not simultaneous (Li and Kimura, 1991; Hilgemann et al., 1991; Khananshvili, 1990). Intracellular ATP depletion decreases the exchanger activity possibly by decreased PIP2 (Hilgemann and Ball, 1996). Na⫹ –Ca2⫹ extrudes Ca2⫹ from the cell (Beukelmann and Wier, 1989). Ca2⫹ influx via Na⫹ –Ca2⫹ exchange induces Ca2⫹ release from the SR, but the efficacy of triggering Ca2⫹ release is considerably less than Ca2⫹ influx from L-type Ca2⫹ channels (Sipido et al., 1997).

IV. STRUCTURE AND LOCALIZATION The canine cardiac Na⫹ –Ca2⫹ exchanger (NCX1) has 970 amino acids with a molecular mass of 110 kDa

(Nicoll et al., 1990). It has a large internal loop containing about 520 amino acids. From the hydropathy analysis, the exchanger molecule was initially interpreted as a monomeric protein with 11 transmembrane segments. However, the structure has been reinterpreted to have 9 transmembrane segments instead of 11 (Cook et al., 1998; Nicoll et al., 1999; Iwamoto et al., 1999). The large internal loop contains a Ca2⫹ regulatory site and an exchanger inhibitory peptide (XIP) region. Synthetic XIP specifically inhibits the exchanger as described later in the chapter (Li et al., 1991). The cardiac type of exchanger (NCX1) is distributed ubiquitously and is most abundant in the heart and the brain and at low levels in the retina, skeletal, and smooth muscle (Komuro et al., 1992). Na⫹ –Ca2⫹ exchanger proteins were found over the entire sarcolemma of the cardiac myocytes, but the density is higher in T tubules than in the external sarcolemma (Kieval et al., 1992; Frank et al., 1992; Chen et al., 1995).

V. DEVELOPMENT AND AGING There are 앑2.5 times the amount of exchanger protein in newborn sarcolemma compared with the adult preparation in rabbit (Artman et al., 1992). Levels of mRNA of the sarcolemmal Na⫹ –Ca2⫹ exchanger are maximal near the time of birth and then decline postnatally (Boerth et al., 1994). In chicken heart, Na⫹dependent Ca2⫹ uptake by Na⫹ –Ca2⫹ exchange showed a sixfold increase during the development of heart from day 5 to young adult (Prakash et al., 1996). Koban et al. (1998) reported that Na⫹ –Ca2⫹ exchanger mRNA levels increased gradually and reached maximum levels during the embryo and in newborn rat or day 1 and then decreased after 12 days, whereas SR-Ca2⫹ ATPase mRNA increased gradually and reached maximum levels in adult (Studer et al., 1997; Koban et al., 1998). The lowest mRNA level was found in 6- and 18-month-old-rat, whereas mRNA of a 24-month-old senescent rat was 50% above that seen at 6 and 18 months of age (Koban et al., 1998). In human heart, the levels of mRNA encoding the exchanger were significantly lower in fetal hearts than in adult heart (Komuro et al., 1992). In the aged rat heart (24 months old), cardiac exchanger mRNA and protein decreased more than that of young rat (3 months old) (Janapati et al., 1995). Klitzner et al. (1995) suggested that the role of the exchanger may be different during development. In the neonate, the exchanger may be involved more directly in excitation–contraction coupling. T tubules develop after birth. The Ca2⫹ channel antagonist diltiazem had little or no effect on tension generation in neonatal papillary muscle when the plateau potential was main-

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tained using the voltage clamp technique (Klitzner et al., 1995), suggesting that the Ca2⫹ current may not contribute Ca2⫹ directly for contraction in neonatal heart cells and that Ca2⫹ enters through the Ca2⫹ influx mode of the Na⫹ –Ca2⫹ exchange.

VI. SPECIES DIFFERENCE Mammalian species difference of the Na⫹ –Ca2⫹ exchanger is small in the amino acid sequence but large in functional aspects. The number of amino acids of guinea pig, dog, rat, and human cardiac Na⫹ –Ca2⫹ exchanger proteins are 970, 970, 971, and 973, respectively. The nucleic acid sequence of the guinea pig clone is 91, 93, and 89% identical to dog, human (Komuro et al., 1992), and rat cardiac exchangers. The deduced amino acid sequence is 98, 98, and 95% identical to dog, human, and rat heart exchangers, respectively (Tsuruya et al., 1994). The ratio of Na⫹ –Ca2⫹ exchange current densities was 4 : 2 : 1.5 : 1 for hamster ⬎ guinea pig Ⰷ human ⬎ rat myocytes (Sham et al., 1995). In the giant patch method, the Na⫹ –Ca2⫹ exchange current amplitude was also found to be two- to threefold higher in guinea pig than in rat (Keppl and Hartung, 1996).

Cross et al. (1998) reported that overexpression of the Na⫹ –Ca2⫹ exchanger reduced the postischemic recovery of cardiac contractile function measured by the left ventricular pressure and energy metabolites and concluded that the Na⫹ –Ca2⫹ exchanger may play a role in ischemia/reperfusion injury. On studies of females, however, it appears that this exacerbation of ischemia/ reperfusion injury by overexpression of the Na⫹ –Ca2⫹ exchanger can be overcome partially by female-specific hormones such as estrogen. Slodzinski and Blaustein (1998) investigated the turnover time of the exchanger in cultured neonatal rat cardiomyocytes. The half-life (t1/2) of the Na⫹ –Ca2⫹ exchanger protein was 33 hr. Knockdown of the Na⫹ –Ca2⫹ exchanger by antisense oligodeoxynucleotides for at least 4 days did not exhibit spontaneous Ca2⫹ transients or respond to Na⫹-free medium; however, cyclopiazonic acid, an inhibitor of Ca2⫹ uptake by SR, and caffein, which releases Ca2⫹ from the SR, induced prolonged elevation in [Ca2⫹]i . In contrast, control myocytes exhibited spontaneous Ca2⫹ transients, and the sustained cytosolic Ca2⫹ concentration rose only when external Na⫹ was removed (Slodizinski and Blaustein, 1998).

VIII. DISEASES AND Na⫹ –Ca2⫹ EXCHANGE

VII. OVEREXPRESSION AND KNOCKDOWN

A. Hypothyroidism

The cardiac Na⫹ –Ca2⫹ exchanger has been overexpressed in transgenic mice (TG) and in cultured cells. In monitoring intracellular Ca2⫹ signals, the decay of Ca2⫹ transient was significantly faster in overexpressed TG myocytes than control (Adatchi-Akahane et al., 1997; Yao et al., 1998). The basal force of contraction was not significantly different between control and cardiac myocytes of transgenic mice overexpressed with the Na⫹ –Ca2⫹ exchanger (Ba¨umer et al., 1998; Yao et al., 1998). In the presence of a Na⫹ channel agonist, however, the developed tension increased more significantly in the overexpressed atria than in the control, but the diastolic tension was unchanged between the two. An increase in the force of contraction by isoprenaline was not significantly changed between the control and the overexpressed atria (Ba¨umer et al., 1998). Therefore, an increase in intracellular Na⫹ concentration seems essential and is the most effective factor for Na⫹ –Ca2⫹ exchanger to modify the force of contraction. In CCL39 fibroblasts overexpressed with NCX1, the resting Ca2⫹ level or the Ca2⫹ content of the intracellular stores was not changed. However, the agonist-induced Ca2⫹ transient was smaller in transfected cells than that of control (Iwamoto et al., 1998).

Na⫹ –Ca2⫹ exchanger mRNA in hypothyroid myocardium is elevated more than twofold compared to euthyroid rats (Koban et al., 1998).

B. Myocardial Infarction (MI) Three weeks after MI, myocytes isolated from rat hearts had a decreased Na⫹ –Ca2⫹ exchange current (Zhang et al., 1998). In those MI rats, however, 6–8 weeks of high-intensity sprint training (HIST) ameliorated the maladaptation of post-MI myocytes seen in the sedentary rat heart. HIST increased reverse INCX significantly without affecting the amount of the exchanger protein detected by immunoblotting in MI myocytes. Eight weeks after myocardial infarction, rabbit ventricular cells were enlarged with a decreased Ca2⫹ current (31%) and an increased Na⫹ –Ca2⫹ exchange current density (32%) (Litwin and Bridge, 1997).

C. Hypoxia, Ischemia, and Reperfusion Injury Mochizuki and Jiang (1998) have written an excellent review on these topics. Anoxia induced by argon gas in

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a semiclosed, air-excluding patch clamp chamber with the perfusate bubbled with pure nitrogen gas decreased the Na⫹ –Ca2⫹ exchange current in guinea pig ventricular myocytes significantly (Shigematsu and Arita, 1999). This effect was due to intracellular acidification, as adjusting intracellular pH with a high concentration of HEPES buffer prevented the decrease of the exchange current (Shigematsu and Arita, 1999). Acid pH has been known to inhibit the Na⫹ –Ca2⫹ exchange current (Earm and Irisawa, 1986; Doering and Lederer, 1993). Ch’en et al. (1998) simulated myocardial ischemia and reperfusion using the Oxsoft Heart computer model.

D. Acute Pressure Overload An acute pressure overload induced a rapid increase of cardiac Na⫹ –Ca2⫹ exchanger mRNA expression in the cat ventricle (Kent et al., 1993). This enhanced exchanger expression appeared within 1 hr after the onset of right ventricular pressure overload and was sustained during cardiac overloading for at least 4 hr. Maintenance of this pressure overload for 48 hr evoked an increase in the production of exchanger protein. Veratridine but not insulin treatment also increased mRNA of the Na⫹ – Ca2⫹ exchanger, indicating that pressure overload and Na⫹ influx may produce a Na⫹ –Ca2⫹ exchanger via a common mechanism.

E. Hypertrophy Controversial results have been reported in iNCX activity during cardiac hypertrophy. Na⫹ –Ca2⫹ exchange activity decreased up to 50% in sarcolemmal vesicles from 30 to 70% hypertrophied rat heart by pressure overload (4 weeks after aortic stenosis) (Hanf et al., 1988). Na⫹ –Ca2⫹ exchanger mRNA levels were reduced and contraction was depressed in left ventricular hypertrophy induced by the banding of abdominal aorta for 16 weeks in rats (McCall et al., 1998). However, no change was found in moderately and severely hypertrophied rat myocytes (Momtaz et al., 1996; Delbridge et al., 1997). In contrast, an increase in iNCX density has been reported in mildly hypertrophied guinea pig myocytes (Ryder et al., 1993) and in cardiomyopathic Syrian hamster myocytes (Hatem et al., 1994). In human heart, mRNA, protein, and Na⫹-dependent 45Ca2⫹ uptake increased in hypertrophied/failing myocardium because of coronary artery disease and dilated cardiomyopathy (Studer et al., 1997). Studer et al. (1997) suggested that there is a common underlying mechanism in the control of Na⫹ –Ca2⫹ exchanger expression in the immature and hypertrophied myocardium.

The Syrian cardiomyopathic hamster develops a progressive cardiomyopathy characterized by cellular necrosis, hypertrophy, and eventually cardiac dilatation and congestive heart failure. Na⫹ –Ca2⫹ exchange was normal until 15 days and then increased by 400% at 30 days; at 360 days, Na⫹ –Ca2⫹ exchange was decreased by 50% (Wagner et al., 1989). Enhancement of the Na⫹ – Ca2⫹ exchange current was also described in this animal at 220–300 days old (Hatem et al., 1994).

F. Heart Failure An increased Na⫹ –Ca2⫹ exchanger may increase the arrythmogenic potential of the failing heart. The levels of mRNA of the exchanger were unchanged between control and the myocardium from a patient with endstage heart failure (Komuro et al., 1992).

G. Diabetes A diabetic cardiomyopathy is characterized by defects in both diastolic and systolic function. Cardiac sarcolemmal Na⫹ –Ca2⫹ exchanger activity decreases in streptozotocin- or alloxan-induced diabetic rat (Khatter and Agbanyo, 1990; Allo et al., 1991; Shaffer, 1991; Golfman et al., 1996, 1998; Chattou et al., 1999). The decreased activity was reversed by insulin treatment (Golfman et al., 1998). Exchanger mRNA was not changed between control and diabetic heart (Shaffer et al., 1997; Golfman et al., 1998).

IX. PHARMACOLOGY OF Na⫹ –Ca2⫹ EXCHANGE Unlike digitalis for the Na⫹ –K⫹ pump and tetrodotoxin for Na⫹ channels, no specific inhibitor has yet been found for the Na⫹ –Ca2⫹ exchanger. However, a variety of compounds have been demonstrated to inhibit the exchanger.

A. Inorganic Cations Inorganic ions that can substitute for Na⫹ or Ca2⫹ in the exchange are rare. Na⫹ cannot be substituted by any other monovalent ions such as Li⫹, Cs⫹, or K⫹. St2⫹, but not Ba2⫹, can substitute for Ca2⫹ (Kimura et al., 1987). Most of the divalent and trivalent cations block Na⫹ – Ca2⫹ exchange with different potencies. The potency order of inhibition of intracellular Na⫹-dependent Ca2⫹ uptake is Co2⫹ ⬎ Sr2⫹ ⬎ Mg2⫹ in cultured mouse myocytes (Wakabayashi and Goshima, 1981) and chick heart cells (Wakabayashi and Goshima, 1981). In ventricular sarcolemmal vesicles of the dog heart, intracellular Na⫹-induced Ca2⫹ uptake was also inhibited by La3⫹ ⬎

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Nd3⫹ ⬎ Tm3⫹ 앑 Y3⫹ ⬎ Cd2⫹ Ⰷ Sr2⫹ ⬎ Ba2⫹ 앑 Mn2⫹ Ⰷ Mg2⫹ (Trosper and Philipson, 1983). In the guinea pig heart, external Mg2⫹ is a weak blocker of an outward exchange current with 50% inhibition at 12.5 mM (Kimura, 1996). Mg2⫹ was competitive with external Ca2⫹, but Mg2⫹ did not block the inward exchange current (Ca2⫹ efflux) (Kimura, 1996). Ni2⫹ at 5 mM inhibits Na⫹ –Ca2⫹ exchange current completely and reversibly (Ehara et al., 1989, Kimura et al., 1987). Ehara et al. (1989) concluded that Ni2⫹ competes with external Ca2⫹ at the binding site in addition to some other allosteric effect. Iwamoto and Shigekawa (1998) found that Ni2⫹ inhibited NCX1 and NCX2 about 10-fold more potently than NCX3 in NCX-transfected fibroblasts. Those cations, however, are all nonspecific inhibitors of the Na⫹ – Ca2⫹ exchanger because they also affect other channels, such as Ca2⫹ channels.

B. Polypeptides The canine cardiac Na⫹ –Ca2⫹ exchanger (NCX1) has 970 amino acids with a molecular mass of 110 kDa (Nicoll et al., 1990). The topological structure has a large internal loop containing about 520 amino acids. A region of 20 amino acids in the internal loop has a sequence homologous to the calmodulin-binding site and its function is considered to be an autoinhibitory site. The synthetic polypeptide of this region blocked the exchanger activity completely (Li et al., 1991; Chin et al., 1993) and was called the ‘‘exchanger inhibitory peptide (XIP).’’ Basic and aromatic residues are most important for the inhibitory function of XIP (He et al., 1997). Another peptide reported to inhibit the exchanger is the positively charged cyclic hexapeptide Phe-ArgCys-Arg-Cys-Phe-CONH2 (FRCRCFa) (Khananshvili et al., 1995). This peptide inhibits both the forward and the reverse mode of Na–Ca2⫹ exchange in flux studies. It does not compete with Na⫹ or Ca2⫹ and therefore does not affect binding sites. It prevents the ion translocation through the exchanger at the cytosolic side of the membrane. Although this peptide did not go through the membrane, the myristyl form of the peptide became permeable through the membrane and blocked the exchanger when applied externally without affecting other currents (Khananshvili et al., 1997). An amino acid, taurine, had no effect on NCX (Katsube and Sperelakis, 1996).

C. Synthetic Compounds Amiloride derivatives represented by 3⬘,4⬘-dichlorobenzamil (DCB) block Na⫹ –Ca2⫹ exchanger (Kaczorowski et al., 1985). DCB inhibited the so-called tran-

sient inward current (iti), whose component has been largely attributed to the Na⫹ –Ca2⫹ exchange current, with a half-maximal concentration of DCB at 30 애M (Lip and Pott, 1988). DCB inhibited an inward Na⫹ – Ca2⫹ exchange current (Ca2⫹ efflux) with a half-maximal concentration at 17 애M (Watano et al., 1996). Interestingly, DCB did not inhibit an outward exchange current up to 30 애M (Watano et al., 1996). Another inhibitor developed more recently is KBR7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulfonate) (KBR) (Watano et al., 1996; Iwamoto et al., 1996). KBR inhibited the outward Na– Ca2⫹ exchange current (Ca2⫹ influx) with a half-maximal concentration at 3 애M, whereas the inward exchange current (Ca2⫹ efflux) was less sensitive to KBR (halfmaximal inhibition at 17 애M). The block by KBR is opposite to that of DCB. KBR competes with external Ca2⫹ (Watano et al., 1996; Watano and Kimura, 1998). Therefore, KBR might affect the external Ca2⫹-binding site. Among the three isoforms of the exchanger, KBR was found to inhibit NCX3 threefold more potently than NCX1 and NCX2 (Iwamoto and Shigekawa, 1998). The author reinvestigated the direction-dependent block of KBR under bidirectional ionic conditions where an inward exchange current flows in voltages positive to the reversal potential of the exchange current (ENCX) and an outward current in voltages negative to ENCX . ENCX was set in the middle of the voltage range examined. The drug blocked both inward and outward components of the bidirectional exchange current with equal potency. In other words, both directions of the current were blocked equally (Kimura et al., 1999). This discrepancy could be explained by a slow time course of dissociation of the drug from the exchanger molecule. The cardioprotective effect of KBR was demonstrated in the ischemia/reperfusion heart by monitoring intracellular Ca2⫹ (Haffner et al., 1999). This drug also inhibited ouabain-induced arrhythmia (Watano et al., 1999). Quinacrine (de la Pena et al., 1987) and calmodulin inhibitors such as W-7 and trifluoperazine (Kimura, 1993) inhibit the exchanger. The effect of W-7 disappears if trypsin is perfused intracellularly. More recently, it was found that 2,3-butanedione monoxime, a well-known inhibitor of muscle contraction, also inhibited the exchange current (Watanabe et al., 1998).

D. Effect of Digitalis Digitalis cardiac glycosides enhance the force of contraction of the heart. This drug has been used for heart failure for more than 2000 years, but the precise mechanism of the drug has not been fully described. Its action as a cardiotonic agent is caused indirectly by

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FIGURE 1 Computer simulation of Na⫹ –Ca2⫹ exchange current. Effects of various [Ca2⫹]i at 5 and 10 mM [Na⫹]o on ENCX and the current. ENCX shifts in the negative direction as [Na⫹]i increases and [Ca2⫹]i decreases.

blocking the Na⫹ –K⫹ pump. Inhibition of the Na⫹ –K⫹ pump induces intracellular Ca2⫹ rise, and this Ca2⫹ rise has been attributed as a direct cause of the cardiotonic effect. Why does Ca2⫹ rise by inhibiting the Na⫹ –K⫹ pump? It is estimated that blocking the Na⫹ –K⫹ pump accumulates intracellular Na⫹. Accumulated Na⫹ accelerates the reverse mode of exchange to increase Ca2⫹ entry and/or inhibit Ca2⫹ efflux by inhibiting the Ca2⫹ efflux mode of the exchange. Figure 1 shows the effect of Na⫹ accumulation on current–voltage relationships of the Na⫹ –Ca2⫹ exchange current with 3 : 1 stoichiometry by a computer simulation. The Na⫹ –Ca2⫹ exchange current reverses at the equilibrium potential, which shifts in the positive direction as the intracellular Ca2⫹ concentration rises. The inward current increases as internal Ca2⫹ rises, indicating that the Ca2⫹ efflux mode operates at more positive potentials. In contrast, at 10 mM Na⫹ inside, every reversal potential at each internal Ca2⫹ concentration shifts in the more negative direction compared to those at 5 mM [Na⫹]i . This negative shift indicates that Ca2⫹ entry is promoted and that the Ca2⫹ efflux is retarded by the intracellular Na⫹ increase. Interaction of the Na⫹ –K⫹ pump and the Na⫹ –Ca2⫹ exchanger in intracellular Na⫹ and Ca2⫹ concentrations was demonstrated by the whole cell clamp method (Fujioka et al., 1998). A chronic treatment of ouabain increased Na⫹ –Ca2⫹ exchanger mRNA and protein levels in cultured cardiac myocytes (Vemuri et al., 1989). Endogenous ouabain has been discovered, and its relation to various diseases, especially essential hypertension, may be via the Na⫹ – Ca2⫹ exchange (Blaustein, 1993).

X. SUMMARY Although a specific inhibitor has not been found, various inhibitors of the Na⫹ –Ca2⫹ exchanger have been developed, including polypeptides and isotheourea derivatives. These drugs will contribute in the research of the exchanger. The inhibitory effect of amiodarone on the exchanger indicates that more drugs currently in use may have such an effect in addition to various other effects. The effect on the exchanger may be contributing to the cardioprotective effect of the drug. The levels of mRNA, protein, and function of the Na⫹ –Ca2⫹ exchanger change dramatically during development, postnatal stages, and aging. Surprisingly, the changes are also prominent in various pathological conditions of the heart and the changes occur rapidly. However, mechanisms of the causes of those changes are unknown. Whether there is a genetic disease that involves the Na⫹ –Ca2⫹ exchanger is also a future question. More work is clearly needed.

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Iwamoto, T., Watano, T., and Shigekawa, M. (1996). A novel isothiourea derivative selectively inhibits the reverse mode of Na⫹ /Ca2⫹ exchange in cells expressing NCX1. J. Biol. Chem. 271, 22391–22397. Janapati, V., Wu, A., Davis, N., Derrico, C. A., Levengood, J., Schummers, J., and Colvin, R. A. (1995). Post-transcriptional regulation of the Na⫹-Ca2⫹ exchanger in aging rat heart. Mech. Ageing Dev. 84, 195–208. Janvier, N. C., and Boyett, M. R. (1996). The role of Na-Ca exchange current in the cardiac action potential. Cardiovas. Res. 32, 69–84. Kaczorowski, G. J., Barros, F., Dethmers, J. K., Trumble, M. J., and Cragoe, E.J., Jr. (1985). Inhibition of Na⫹ /Ca2⫹ exchange in pituitary plasma membrane vesicles by analogues of amiloride. Biochemistry 24, 1394–1403. Khananshvili, D., Mester, B., Saltoun, M., Wang, X., Shaulov, G., and Baazov, D. (1997). Inhibition of the cardiac sarcolemma Na⫹ /Ca2⫹ exchanger by conformationally constrained small cyclic eptides. Mol. Pharmacol. 51, 126–131. Katsube, Y., and Sperelakis, N. (1996). Na⫹ /Ca2⫹ exchange current: Lack of effect of taurine. Eur. J. Pharmacol. 316, 97–103. Kent, R. L., Rozich, J. D., McCollam, P. L., McDermott, D. F., Thacker, U. F., Menick, D. R., McDermott, P. J., and Cooper, G., IV (1993). Rapid expression of the Na⫹-Ca2⫹ exchanger in response to cardiac pressure overload. Am. J. Physiol. 265, H1024–H1029. Keppl, M., and Hartung, K. (1996). Rapid charge translocation by the cardiac Na-Ca exchanger after a Ca concentration jump. Biophys. J. 71, 2473–2485. Khananshvili, D. (1990). Distinction between the two basic mechanisms of cation transport in the cardiac Na⫹-Ca2⫹ exchange system. Biochemistry 29, 2437–2442. Khananshvili, D., Shaulov, G., Weil-Maslansky, E., and Baazov, D. (1995). Positively charged cyclic hexapeptides, novel blockers for the cardiac sarcolemma Na(⫹)-Ca2⫹ exchanger. J. Biol. Chem. 270, 16182–16188. Khatter, J. C., and Agbanyo, M. (1990). Mechanisms of increased digitalis tolerance in streptozotocin-induced diabetic rat myocardium. Biochem. Pharmacol. 40, 2707–2711. Kieval, R. S., Bloch, R. J., Lindenmayer, G. E., Amesi, A., and Lederer, W. J. (1992). immunofluorescence localization of the NaCa exchanger in heart cells. Am. J. Physiol. 263, C545–C550. Kimura, J. (1996). Effects of external Mg2⫹ on the Na-Ca exchange current in guinea pig cardic myocyes. Ann. N. Y. Acad. Sci. 779, 515–520. Kimura, J., Watano, T., Kawahara, M., Sakai, E., and Yatabe, J. (1999). Direction-independent block of bi-directional Na⫹ /Ca2⫹ exchange current by KB-R7943 in guinea pig cardiac myocytes. Br. J. Pharm. Kimura, J., Miyamae, S., and A. Noma. (1987). Identification of sodium-calcium exchange current in single ventricular cells of guinea pig. J. Physiol. 384, 199–222. Kimura, J., Noma, A., and Irisawa H. (1986). Na-Ca exchange current in mammalian heart cells. Nature 319, 596–597. Kimura, J. (1996). Effects of external Mg2⫹ on the Na-Ca exchange current in guinea pig cardic myocyes. Ann. N. Y. Acad. Sci. 779, 515–520. Kimura, J., Watano, T., Kawahara, M., Sakai, E., and Yatabe, J. (1999). Direction-independent block of bi-directional Na⫹ /Ca2⫹ exchange current by KB-R7943 in guinea-pig cardiac myocytes. Br. J. Pharm. Klitzner, T. S., Chen, F., Raven, R. R., Wetzel, G. T., and Friedman, W. F. (1995). Calcium current and tension generation in immature mammalian myocardium: Effects of diltiazem. J. Mol. Cell. Cardial. 23, 807–815.

Koban, M. U., Moorman, A. F. M., Holtz, J., Yacoub, M. H., and Boheler, K. R. (1998). Expressional analysis of the cardiac NaCa exchange in rat development and senescence. Cardiovas. Res. 37, 405–423. Komuro, I., Wenninger, K. E., Philipson, K. D., and Izumo, S. (1992). Molecular cloning and characterization of the human cardic Na⫹Ca2⫹ exchanger cDNA. Proc. Natl. Acad. Sci. USA 89, 4769–4773. Li, J., and Kimura, J. (1991). Translocation mechanism of cardiac NaCa exchange. Ann. N. Y. Acad. Sci. 639, 48–60. Li, Z., Nicoll, Collins A., Hilgemann, D. W., Filoteo, A., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991). Identification of a peptide inhibitor of the cardiac sarcolemmal Na⫹-Ca2⫹ exchanger. J. Biol. Chem. 266, 1014–1020. Linck, B., Qiu, Z., He, Z., Tong, Q., Hilgemann, D. W., and Philipson, K. D. (1998). Functional comparison of the three isoforms of the Na⫹ /Ca2⫹ exchanger (NCX1, NCX2, NCX3). Am. J. Physiol. 274, C415–C423. Lipp, P., and L. Pott. (1988). Voltage dependence of sodium-calcium exchange current in guinea-pig atrial myocytes determined by means of an inhibitor. J. Physiol. 403, 355–366. Lipp, P., Schwaller, B., and Niggli, E. (1995). Specific inhibition of Na-Ca exchange function by antisense ologodeoxynucleotides. FEBS Lett. 364, 198–202. Litwin, S. E., and Bridge, J. H. B. (1997). Enhanced Na⫹-Ca2⫹ exchange in the infarcted heart. Circ. Res. 81, 1083–1093. Matsuda, T., Takuma, K., and Baba, A. (1997). Na⫹-Ca2⫹ exchanger: Physiology and Pharmacology. Jpn. J. Pharmacol. 74, 1–20. Matsuoka, S., and Hilgemann, D. W. (1992). Ion and voltage dependencies of the transport cycle. J. Gen. Physiol. 100, 963–1001. McCall, E., Ginsburg, K. S., Bassani, R. A., Shannon, T. R., Qi, M., Samarel, A. M., and Bers, D. M. (1998). Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am. J. Physiol. 274, H1348–H1360. Mechmann, S., and L. Pott. (1986). Identification of Na-Ca exchange current in single cardiac myocytes. Nature 319, 597–599. Miura, Y., and J. Kimura. (1989). Sodium-calcium exchange current, dependence on internal Ca and Na and competitive binding of external Na and Ca. J. Gen. Physiol. 93, 1129–1145. Mochizuki, S., and Jiang, C. (1998). Na⫹ /Ca2⫹ exchanger and myocardial ischemia/reperfusion. Jpn. Heart J. 39, 707–714. Momatz, A., Coulombe, A., Richer, P., Mercadier, J.-J., and Coraboueuf, E. (1996). Action potential and plateau ionic currents in moderately and severely DOCA-salt hypertrophied rat hearts. J. Mol. Cell. Cardiol. 28, 2511–2522. Mullins, L. J. (1981). ‘‘Ion Transport in the Heart.’’ Raven Press, New York. Nicoll, D. A., Longoni, S., and Philipson, K. D. (1990). Molecular cloning and functional expression of the cardiac sarcolemmal Na-Ca exchanger. Science 250, 562–565. Nicoll, D. A., Ottolia, M., Lu, L., Lu, Y., and Philipson, K. D. (1999). A new topological model of the cardiac sarcolemmal Na-Ca exchanger. J. Biol. Chem. 274, 910–917. Prakash, P., Meera, P., and Tripathi, O. (1996). Developmental changes in Ca2⫹-uptake, Na⫹,Ca2⫹-exchange and Ca2⫹-ATPase in freshly isolated embryonic, newborn and adult chicken heart. Reprod. Fertil. Dev. 8, 71–78. Reeves, J. P. (1998). Na⫹ /Ca2⫹ exchange and cellular Ca2⫹ homeostasis. J. Bioenerg. Biomembr. 30, 151–160. Reuter, H., and Seits, N. (1968). The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J. Physiol. 195, 451–470. Ryder, K. O., Bryant, S. M., and Hart, G. (1993). Changes in cell length consequent on depolarization in single left ventricular myo-

22. Cardiac Na⫹ –Ca2⫹ Exchanger cytes from guinea-pigs with pressure-overload left ventricular hypertrophy. Proc. R. Soc. Lond. B 253, 35–42. Schaffer, S. W., Ballard-Croft, C., Boerth, S., and Allo, S. N. (1997). Mechanisms underlying depressed Na⫹ /Ca2⫹ exchanger activity in the diabetic heart. Cardiovasc. Res. 34, 129–136. Schaffer, S. W. (1991). Cardiomyopathy associated with noninsulindependent diabetes. Mol. Cell. Biochem. 107, 1–20. Sham, J. S. K., Hatem, S. N., and Morad, M. (1995). Species differences in the activity of the Na⫹-Ca2⫹ exchanger in mammalian cardiac myocytes. J. Physiol. (Lond.) 488, 623–631. Shigematsu, S., and Arita, M. (1999). Anoxia depresses sodiumcalcium exchange currents in guinea-pig ventricular myocytes. J. Mol. Cell Cardiol. 31, 895–906. Sipido, K. R., Maes, M., and Van de Werf, F. (1997). Low efficiency of Ca2⫹ entry through the Na⫹-Ca2⫹ exchanger as trigger for Ca2⫹ release from the sarcoplasmic reticulum. Circ Res. 81, 1034–1044. Slodzinski, M., and Blaustein, M. P. (1998). Na⫹ /Ca2⫹ exchange in neonatal rat heart cells: Antisense inhibition and protein half-life. Am. J. Physiol. 44, C459–C467. Studer, R., Reinecke, H., Vetter, R., Holtz, J., and Drexler, H. (1997). Expression and function of the cardiac Na⫹ /Ca2⫹ exchanger in postnatal development of the rat, in experimental induced cardiac hypertrophy and in the failing human heart. Basic Res. Cardiol. 92, Suppl. 1, 53–58. Trosper, T. L., and Philipson, K. D. (1983). Effects of divalent and trivalent cations on Na2⫹-Ca2⫹ exchange in cardiac sarcolemmal vesicles. Biochim. Biophys. Acta 731, 63–68. Tsuruya, Y., Bersohn, M. M., Li, Z., Nicoll, A. N., and Philipson, K. D. (1994). Modecular cloning and functional expression of the guinea pig cardiac Na⫹-Ca2⫹ exchanger. Biochim. Biophys. Acta 1196, 97–99. Vemuri, R., Longoni, S., and Philipson, K. D. (1989). Ouabain treatment of cardiac cells induces enhanced Na-Ca exchange activity. Am. J. Physiol. 256, C1273–C1276.

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23 Na⫹ /H⫹ Exchanger and pH Regulation M. PUCE´AT Research Center of Macromolecular Biochemistry CNRS UPR 1086 Cedex 5, France

equivalent H⫹ and HCO⫺3 ions into and out of the cell. A Na⫹ /H⫹ antiport and a Na⫹-dependent HCO⫺3 transporter regulate pH from acidosis, whereas a Na⫹-independent Cl⫺ /HCO⫺3 exchanger and the recently described Cl⫺ /OH⫺ exchanger (CHE) regulate pH from alkalosis (Puce´at, 1999) (Fig. 1). The Na⫹ /H⫹ antiport is the predominant alkalinizing ion transporter in cardiac cells and is the only one to allow the cell to recover fully from an acidic challenge (Puceat et al., 1993).

I. INTRODUCTION Intracellular pH (pHi) regulates many cellular processes, including cell metabolism, Ca2⫹ homeostasis, gene expression (Isfort et al., 1993), cell motility and contractility, cell–cell coupling (Orchard and Kentish, 1990), cell adhesion (Tominaga and Barber, 1998), and cell death (McConkey and Orrenius, 1996) (Gottlieb et al., 1996). Maintenance of steady-state pH is thus mandatory for a normal cell function. In heart, the excitation–contraction coupling of the cardiomyocyte, as well as cell–cell communication (i.e., cardiac conduction), is tightly dependent on pH. Most mammalian cells, including the cardiomyocyte, feature a steady-state cytosolic pH (pHi) of 7.1–7.2. Taking into account the metabolic activity, the thermodynamic and the resting membrane potential of a cell, and if protons were distributed passively across the plasma membrane according to the electrochemical gradient, pHi would be much more acidic (around one unit pH lower than extracellular pH, i.e., roughly 7.3). For example, assuming a resting membrane potential of ⫺80 mV for a cardiac cell, a calculation in accord to the Nernst equation gives an intracellular pH of 6.1 for a cell that would be bathed in an extracellular medium of pH 7.4. A pHi of 6.1 would be incompatible with cellular processes, such as metabolic pathways catalyzed by pH-sensitive enzymes, cardiac contractility mediated by pH-sensitive contractile proteins, or cardiac conduction mediated by a pHsensitive conductance of gap junction (Orchard and Kentish, 1990). To regulate and maintain a steady-state value of cytosolic pH, the cardiac cell possesses ion transporters at its plasma membrane that drive H⫹ or

Heart Physiology and Pathophysiology, Fourth Edition

II. EXPRESSION, MOLECULAR IDENTITY, AND ROLE OF Na⫹ /H⫹ EXCHANGE IN HEART PATHOPHYSIOLOGY A. Protein NHE1 Mediates Cardiac Na⫹ /H⫹ Exchange The Na⫹ /H⫹ antiport is an electroneutral ion exchanger that pumps H⫹ ions out of the cell when its activity is increased by an intracellular acidic load (Figs. 1 and 2). The H⫹ efflux is driven by the inwardly directed Na⫹ electrochemical gradient. First discovered in kidney and intestine, the antiport has now been found in most tissues of eukaryotic cells. The diuretic amiloride and its chemical derivatives (dimethy1- or ethylsisopropyl amiloride) are the most potent inhibitors of Na⫹ /H⫹ exchange and have thus been broadly used to investigate the role of the antiport in many cell functions. The differential amiloride sensitivity of Na⫹ /H⫹ exchange (Counillon et al., 1993b) in various tissues predicted the existence of several isoforms of the protein that mediates this ion exchange. Indeed Na⫹ /H⫹ exchange

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FIGURE 1 pH regulatory ionic transporters in cardiac cells. NBC, Na⫹-dependent HCO3⫺ cotransport; NHE, Na⫹ /H⫹ antiport; AE, anion exchanger; CHE, Cl⫺ /H⫹ exchanger.

al., 1993; Collins et al., 1993) have been cloned. These isoforms have a more restricted tissue distribution. NHE2 and NHE4 are poorly expressed in the heart. Similar to NHE1, NHE2, NHE3, NHE4, and NHE5 are likely to display 12 transmembrane domains. NHE6 is expressed in numerous tissues with the highest mRNA level in brain, skeletal muscle, and heart. Composed of 12 transmembrane domains, it contains a putative inner mitochondria membrane targeting signal at its N-terminal domain. Expressed in HeLa cells, NHE6 localized in perinuclear cluster, which is consistent with mitochondria localization. It may thus mediate the mitochondrial Na⫹ /H⫹ exchange in cardiac cells (Orlowski and Grinstein, 1997).

B. Intracellular Localization of NHE1 in Cardiac Cell is performed by different proteins belonging to the multigenic NHE family (Na-H exchanger). So far six members of the NHE family that share 30 to 60% homology in their sequence of amino acids have been cloned. The first NHE gene (NHE1) was cloned by Sardet et al. (1989) by gene complementation of an antiporterdevoid fibroblast cell line. NHE1, which displays the greatest sensitivity for amiloride (EC503 mM for amiloride, 80 nM for methylpropylamiloride) or for the most recent Na⫹ /H⫹ inhibitor HOE694 (3-methylsulfonyl - 4 - iperidinobenzoyl) guanidinemethanesulphonate; EC50:0.16 mM ) (Counillon et al., 1993b), is a housekeeping gene. A critical amino acid (Phe 167) was found to be responsible for the amiloride sensitivity of NHE1 (Counillon et al., 1993a). NHE1 is widely expressed in virtually all tissues and is the predominant NHE isoform in cardiac cells, as cloned by Fliegel et al. (1991) from a rabbit cardiac cDNA library. This is a N- and Oglycosylated protein (Counillon et al., 1994) of 815 amino acids migrating in SDS-PAGE with an apparent molecular mass of 110 kDa in most species, including the mouse (Fig. 3). The NHE1 protein expressed in the antiport-deficient cell line forms stable dimers. However, individual subunits of NHE1 function independently within the oligomeric state (Fafournoux et al., 1994). Glycosylation of the protein does not affect the rate of ion transport (Counillon et al., 1994; Haworth et al., 1993), but may play a role in antiport sorting. The antiport contains 10 or 12 transmembrane domains (Fig. 3). The glycosylation sites are located in a long external loop between transmembrane domains 1 and 2. The amino-terminal domain is sufficient to transport the ions in an amiloride-sensitive manner (Wakabayashi et al., 1992). In addition to NHE1, NHE2, NHE3, NHE4, and NHE5 (Tse et al., 1992; Orlowski et al., 1992; Wang et

Early work by Pierce and Philipson (1985) suggested that the Na/H antiport was localized in the sarcolemmal fraction of canine heart. More recently, Petrecca et al., (1999) used in situ immunofluorescence and found that NHE1 is localized specifically in intercalated disks and

FIGURE 2 Functional Na⫹ /H⫹ antiport in cardiomyocytes. Cardiac cells loaded with Snarf-1 are bathed in an extracellular HCO3⫺-containing medium. pHi is monitored on a single cell with a CCD camera. After an acidic challenge triggered by the removal of NH4Cl, pH recovery is first mediated by a HCO3⫺-dependent transporter in the presence of EIPA, an inhibitor of the NHE1. pH recovery is accelerated greatly and complete when the Na⫹ /H⫹ antiport is functional after washout of EIPA.

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FIGURE 3 Structure of NHE1. vol, volume-regulatory domain; CHP-R, calcineurin homologue protein-binding domain, Amino acid numbers are indicated by an arrow. (Inset on the right) Expression of mouse heart NHE1 protein. NHE1 was immunoprecipitated from a lysate of mouse ventricular cells and blotted with a specific anti-NHE1 antibody (provided by Dr. J. Pouyssegur).

in T tubes. NHE1 is, however, absent from the lateral sarcolemma (Fig. 4). No difference of localization was found in atrium or ventricles (Petrecca et al., 1999). The physiological relevance of such an intracellular localization of NHE1 remains unknown.

C. Regulation of NHE1 by Ca2⫹, Cell Volume, and Protons The C-Terminal region of NHE1 features an ATPbinding site, two calmodulin-binding sites, and a calcineurin homolog protein (CHP)-binding region that all confer a Ca2⫹ sensitivity to the antiport. The C-terminal tail of the antiport also contains many phosphorylation sites and is thus involved in neurohormonal regulation of the ion-exchange activity (Wakabayashi et al., 1994b). The Na⫹ /H⫹ antiport is regulated by cell volume (Demaurex and Grinstein, 1994). NHE1 is stimulated by hypertonicity and is inhibited in hypotonic media. A volume-sensitive domain has been mapped in NHE1 (Grinstein et al., 1992; Nath et al., 1996). A Volumeor osmolarity-sensitive site(s) exists between the NH2terminal domain and residue 566 of NHE1 (Fig. 3) (Bianchini et al., 1995). A phosphorylation-independent mechanism is likely to underlie the volume regulation of the antiport (Grinstein et al., 1992). This volumesensitive site is therefore different from the site(s) postulated to mediate the stimulatory effects of calcium and growth factors. So far, the physiological significance of this regulation is still unclear in cardiac cell function.

In isolated rat cardiomyocytes in vivo, a clear and steep pH dependence of Na⫹ /H⫹ exchange activity was found. The set point of the antiport (pH threshold to switch on the Na⫹ /H⫹ exchange) was found to be 7.2. As shown previously by Vaughan Jones’s group and others, we also observed that the antiport is rather inhibited by extracellular protons (Puceat et al., 1993). This inhibition follows a linear relationship (Wallert and Frohlich, 1989). The pH sensitivity of NHE1 is a complex mechanism. It is regulated by an autoinhibitory domain (AA 636656) that prevents protonation of the pH sensor (Wakabayashi et al., 1997a) and another domain that is more directly responsible for the maintenance of pH sensitivity (AA 567635)(Wakabayashi et al., 1994b). Amino acids 515–595 in the C-terminal domain are particularly critical for the pH sensitivity of the antiport (Wakabayashi et al., 1997b).

D. Regulation of Na⫹ /H⫹ Antiport Activity by Intracellular ATP A drop in intracellular ATP decreases the activity of the Na⫹ /H⫹ antiport dramatically in several cell types. This effect is observed independently of the NHE isoform expressed (NHE1, NHE2, or NHE3) (Kapus et al., 1994; Wakabayashi et al., 1997b), demonstrating that the ATP regulatory site is conserved among members of the NHE family. The ATP dependence of NHE1 cannot be only explained by the requirement of a phosphorylation. Indeed, mutation of all known phosphory-

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FIGURE 4 Expression and immunolocalization of NHE1 in ventricle (A) and atrium (C). (B and D) Higher magnifications of A and C, respectively [immunofluorescence images are from Petrecca et al. (1999), with permission].

lation sites (Goss et al., 1994) does not abolish the metabolic regulation of the antiport. The dependence of the antiport on ATP is thus rather complex; the cytoskeleton is likely to participate in such a mechanism. A nondiffusible effector that binds ATP is probably required to activate the antiport in the presence of ATP (Demaurex et al., 1997). This ATP-dependent regulation may be of importance in cardiac ischemia.

E. Regulation of Na⫹ /H⫹ Antiport Activity by Neurohormones and Growth Factors It has been recognized for many years that besides protons, the Na⫹ /H⫹ antiport is regulated by external agonists, including hormones and growth factors. Proton affinity or the maximal rate of ion exchange (Vmax) of NHEs and thus the rate of ionic transport are increased when the cell is stimulated by neurohormones or growth factors. Phosphorylation of NHE proteins was first proposed to be responsible for activation of the ion transport because okadaic acid, a serine threonine phospha-

tase inhibitor, alone or together with growth factors, induces activation of the antiport (Sardet et al., 1991). In isolated cardiac cells, purinergic agonists, 움1- and 웁-adrenergic agonists, endothelin, and angiotensin II regulate the activity of the Na⫹ /H⫹ exchanger (Puceat et al., 1993; Puceat and Vassort, 1995). Most of these agonists activate the ionic exchange activity by shifting the pH sensitivity of the antiport toward alkaline values. Protein kinase C (PKC) has been involved in NHE1 regulation. Human but not rabbit NHE1 (Fliegel et al., 1991) displays a PKC consensus site (Ser 648). However, mutation of this amino acid does not affect phorbol ester- or thrombin-induced activation of the ionic exchange (Wakabayashi et al., 1994b). Direct phosphorylation of NHE1 by PKC is probably not required for activation of the antiport. Furthermore, PKC stimulation has various effects on the rate of Na⫹ /H⫹ exchange. The phorbol ester PMA (phorbol 12-myristate 13-acetate) inhibits NHE3 activity but activates NHE1 and NHE2 expressed in PS120 NHE-deficient cells (Tse et al., 1993), although we failed to observe such an effect

23. Na⫹ /H⫹ Exchanger and pH Regulation

in isolated cardiomyocytes (Puceat et al., 1993). Growth factors and serum that also activate PKC stimulate the three NHE isoforms in PS120 cells (Levine et al., 1993). It should be noted that NHE1 is mainly activated following a rise in proton affinity (Paris and Pouyssegur, 1984; Puceat et al., 1993), whereas the Vmax of NHE2 and NHE3 is regulated by external stimuli (Levine et al., 1993; Tse et al., 1993). NHE1 does not feature any protein kinase A (PKA) phosphorylation sites. The effect of PKA on NHE activity is quite controversial and depends on the cell type and, in turn, on the NHE isoform expressed. NHE activity can thus be either activated or inhibited by agents that increase intracellular cAMP. The regulation of NHE activity by PKA is even less simplistic. NHE3 is activated by the treatment of cells with the 웁-adrenergic agonist isoproterenol, but is inhibited by forskolin stimulation that activates the adenylyl cyclase directly (Noel and Pouyssegur, 1995). Lefkowitz’s group elucidated this apparent discrepancy. They showed that the occupancy of the 웁2-adrenergic receptor with isoproterenol triggers the binding to the receptor of the NHE regulatory factor (NHERF), a factor that inhibits the Na⫹ /H⫹ antiport in a PKA-dependent manner. NHERF contains two distinct protein interaction PDZ domains: NHERFPDZ1 and NHERF-PDZ2. NHERF binds to NHE3 through one of these PDZ domains. Binding of NHERF to the 웁2-adrenergic receptor displaces NHERF from the antiport and thus relieves the inhibition of NHE3 (Hall et al., 1998). A human homologue of NHERF has been cloned. This homologue, the neurofibromatosis 2 tumor suppressor protein, binds to merlin, an ERM (Ezrin, Radixin, Moesin) family protein that makes the link with the cytoskeleton actin. The protein complex NHE–NF2 tumor suppressor ERM suggests a potential role of NHE3 in cytoskeleton rearrangement (Murthy et al., 1998). On addition to the role of NHERF, it is interesting to briefly point out that a novel Na⫹ /H⫹ exchange activity that requires Cl⫺ ions was found in crypt cells of the rat distal colon. This activity is fully inhibited by 0.5 mM DIDS, an inhibitor of Cl⫺-dependent transporters, and by 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl⫺-channel blocker. A polyclonal antibody to the cystic fibrosis transmembrane conductance regulator (CFTR) partially inhibits the Cl⫺-dependent proton gradient-driven Na⫹ uptake, suggesting that the Cl⫺-dependence of Na⫹ /H⫹ exchange involves a Cl⫺ channel that may be the CFTR and/or the outward rectifying Cl⫺ conductance (Rajendran et al., 1999). It has been reported that NHERF-PDZ1 binds to the CFTR C terminus. This protein–protein interaction further suggests a potential regulatory role of NHERF CFTR function (Wang et al., 1998). It is still unknown whether this regulation occurs in heart,

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a tissue in which CFTR is expressed. Because NHE1 cannot be phosphorylated by PKA nor features PDZ domains, how a 웁-adrenergic agonist modulates NHE1 activity in cardiac cells is still mysterious and requires further investigation. A mechanism involving an interactive protein regulated by PKA similar to the one that modulates NHE3 may be expected. NHE1 displays two Ca2⫹ –calmodulin-binding sites and a calcineurin homologous protein (CHP)-binding domain that also binds Ca2⫹. Binding of phosphorylated CHP inhibits the antiport (Lin and Barber, 1996). A 24-kDa protein, which has not been identified, coimmunoprecipitates with NHE1 (Goss et al., 1996). It is striking that 24 kDa is the size of CHP. This observation allows for speculation that it may indeed be CHP. ‘‘In vivo,’’ Ca2⫹ has been reported to have no effect (Puceat et al., 1993), to decrease (Levine et al., 1995), or to increase antiport activity (Gupta et al., 1994). Whether a Ca2⫹ –calmodulin-dependent kinase regulates the activity of cardiac NHE by direct phosphorylation has not yet been proven. A direct effect of Ca2⫹ or calmodulin on NHE may also explain the stimulatory effect of Ca2⫹ on NHE activity (Bertrand et al., 1994; Wakabayashi et al., 1994a). Pouyssegur’s group proposed as early as 1991 (Sardet et al., 1991) that a specific NHE1 kinase may phosphorylate NHE1 following cell stimulation with growth factors. The NHE1 kinase would be downstream of mitogenactivated protein kinase (MAPK) or extracellular regulated kinases (ERK) that integrate the signal from both PKC- and tyrosine kinase-dependent pathways. Using a dominant negative form of p44 MAPK and inhibitors of MAPK kinase, Bianchini et al. (1997) showed that p42/p44 MAPKs play a predominant role in NHE1 regulation by neurohormones and growth factors. Berk’s group identified p90rsk as a potential NHE kinase. Both inhibition of p42/p44 MAPK and Ca2⫹-chelating agents prevent p90rsk activation, demonstrating that this NHE kinase is downstream of a MAPK-signaling pathway (Fig. 5). p90rsk may mediate the angiotensin II-induced activation of Na⫹ /H⫹ antiport in vascular smooth cells in hypertension (Phan et al., 1997; Takahashi et al., 1997) and in cardiac cells. More recently, Takhashi et al. (1999) showed that p90rsk is a serum-stimulated kinase that phosphorylates serine 713 of NHE1; using a dominant negative mutant of p90rsk, transfected in 293 cells they also showed that the kinase is a mediator of NHE1 activation in vivo. Involvement of p42/p44 MAPK and the downstream p90rsk kinase in the phosphorylation of cardiac NHE1 was shown in in vitro assays using a recombinant carboxy-terminal domain of NHE1 and partially purified extracts of cardiac cells (Moor and Fliegel, 1999). The authors further found that inhibition of p42/p44 MAPKs by PD98059 in neonatal rat cardiac

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dependent regulation of NHE1 may be of importance in cardiac hypertrophy, a situation in which these Gproteins play a key role.

G. Regulation of NHE1 Expression in Cardiac Cells

FIGURE 5 Possible signaling pathways that regulate NHE1 activity. A, agonist; R, receptor; TK, tyrosine kinase; DAG, diacylglycerol.

cells prevents serum-and endothelin-induced activation of Na⫹ /H⫹ antiport activity. The MAPK cascade may also mediate purinergic and 움1-adrenergic effects on cardiac NHE, as this signaling pathway is strongly stimulated by these agonists. The phosphorylation state of NHE1 has never been investigated in cardiac cells after neurohumoral stimulation.

F. Regulation of NHE1 by G-Proteins The G-protein-mediated mode of regulation has opened a new field of investigation. In 1994, the small oncogenic G-protein ras was proposed as a long-term regulator of NHE1 activity (Kaplan and Boron, 1994). The same year, a constitutively activated form of G움13 , a member of the Gq family, expressed in HEK293 cells was reported to activate the Na⫹ /H⫹ exchange mediated by NHE1 (Voyno-Yasenetskaya et al., 1994). Later on, the same group found that G움13 activates the antiport through an independent stimulation of the Rho family proteins, Cdc42 and RhoA. Cdc42 effect is mediated by MEKK1 (MAPK kinase kinase) while RhoA is not (Hooley et al., 1996). Furthermore, activation of NHE by RhoA is required for the formation of actinic stress fibers, a cell phenotype typical of RhoA activation (Nobes and Hall, 1994). This clearly indicates that stimulation of the Na⫹ /H⫹ antiport, and thus changes in pHi, is an event that occurs downstream of G움13 and RhoA activation (Vexler et al., 1996). This signaling pathway, which involves p160 Rho kinase, regulates integrin-induced cell adhesion (Tominaga and Barber, 1998). However, expression of a constitutively activated form of G움12 in HEK293 and CCL19 fibroblasts inhibits NHE1 ion-exchange activity but stimulates NHE2 and NHE3 ion exchange (Lin et al., 1996). The role of these Gproteins in NHE1 regulation has to be investigated in cardiac cells. More specifically, the ras-, Rho-, or cdc42-

Expression of NHE1 is regulated in the course of cardiac development. NHE1 mRNA is more abundant in neonates than in adults. Similarly, the ion-exchange activity is higher in neonatal rat cardiomyocytes than in adult rat myocytes (Fliegel and Wang, 1997). Other stimuli affect NHE1 gene expression. Pressure overload on the ventricle that develops in rabbit after aortic constriction increases the level of NHE1 mRNA by twofold compared to sham-operated rabbits (Takewaki et al., 1995). In cardiac papillary muscle isolated from SHR rats in which hypertension is associated with hypertrophy, NHE activity is also higher than in muscles of normotensive rats (Perez et al., 1995). Nuclear factors that bind to the AP2 site present in the NHE1 gene promoter may be involved in the long-term increase in cardiac NHE1 expression. Three AP1 sites are also present in the promoter of the NHE1 gene. Sustained extracellular acidosis that increases NHE1 expression is associated with a rise in expression of c-fos and cjun, two components of AP1 (Fliegel and Wang, 1997; Wakabayashi et al., 1997b). We have found that 24 hr of aldosterone treatment of neonatal rat cardiomyocytes in culture enhances activity of the Na⫹ /H⫹ exchange (Korichneva et al., 1995). More insignt into the longterm regulation of exprssion NHE1 gene is required, more specifically in association with diseased myocardium.

H. NHE1 Is Involved in Cardiac Pathologies It has been shown that the cardiac Na⫹ /H⫹ antiport plays an important role in ischemic–reperfusion injury of the heart. During myocardial ischemia, intracellular acidosis develops quickly. This activates the exchanger, which extrudes H⫹ from the cell in exchange for an influx of Na⫹ ions into the myocyte. Because ischemia is accompanied with depletion in intracellular ATP, ATPdependent pumps, such as the Na⫹ /K⫹ ATPase, are not effective anymore and the cell is unable to handle the overload of Na⫹. This switches on the Na⫹ /Ca2⫹ exchanger and in turn triggers a Ca2⫹ overload, which can lead to deleterious cardiac injury, such as cardiac arrhythmias, cell contracture, and necrosis. During myocardial reperfusion, these ion movements are magnified because the return of blood flow decreases the extracellular H⫹ concentration, stimulating the Na⫹ /H⫹ antiport to extrude more intracellular H⫹ ions. This again leads

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to an intracellular Na⫹load and Ca2⫹ overload. To counteract these phenomenons, many investigators studied the ability of various NHE inhibitors, such as amiloride, analogs of amiloride, and other drugs such as HOE 694 or the most recent designed one, cariporide (HOE 642), to prevent cardiac ischemic–reperfusion damage. Numerous data obtained in animal models have revealed that most of these agents are indeed capable of attenuating the development of myocardial arrhythmias and/ or contracture during both ischemia and reperfusion. Although NHE1 inhibitors have to be tested and their efficacy proven in patients by clinical trials, these inhibitors look promising in the treatment of ischemic heart disease. They may also be of use in situations in which low-flow coronary rates are followed by an immediate recovery of flow, as in coronary artery bypass graft surgery, percutaneous transluminal coronary angioplasty, thrombolytic therapy, and coronary arterial vasospasm (Levitsky et al., 1998). One pathology has been associated with a decrease in the activity of cardiac NHE1. A low Na⫹ /H⫹ exchange has indeed been well characterized in diabetes (LagadicGossmann et al., 1988) and has been proposed as the origin of protection of the diabetic heart after an episode of ischemia/reperfusion. Le prigent et al. (1997) investigated in more detail the mechanisms underlying the low Na⫹ /H⫹ exchange in diabetic hearts. They found that a lower intracellular Ca2⫹ concentration associated with lower Ca2⫹ calmodulin-dependent kinase II activity may account for the depressed cardiac Na⫹ /H⫹ exchange in diabetes (Le Prigent et al., 1997). Interestingly, one of the gene responsible for diabetes suceptibility mapped to a loci of mouse chromosome 4 where the NHE1 gene may also be located, showing a putative link between NHE1 and this pathology, although an increase in NHE1 activity has been found in lymphocytes of these insulin-resistant diabetic mine (Morahan et al., 1994).

III. SUMMARY Since the cloning of the NHE1 gene, much has been learned about the structure of the Na⫹ /H⫹ antiporter and the mechanistic regulation of NHE1 expression and activity. This chapter summarized those progresses related more specifically to cardiac function. The neurohumoral regulation of NHE1 activity and the protein kinases cascades involved in this modulation are outlined. One of the most exciting fields, of investigation in NHE1 function deals with the possible clinical use of specific NHE1 inhibitors. Taking advantage of the high sensitivity of NHE1 to amiloride derivatives or HOE compounds, the Na⫹ /H⫹ antiporter indeed provides a

promising therapeutic target in order to prevent the deleterious effects of sichemia/reperfusion injuries.

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24 Transport in Nucleus CARMEN M. PEREZ-TERZIC,*,†, A. MARQUIS GACY,‡ and ANDRE TERZIC† *Division of Cardiovascular Diseases and Department of Internal Medicine, Department of Molecular Pharmacology and Experimental Therapeutics, †Department of Physical Medicine and Rehabilitation, and ‡Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota 55905

I. INTRODUCTION

bile phase. The stationary phase involves the actual translocation of the molecule across the nuclear membrane. The mobile phase is discussed first in the context of the two distinct classes of nuclear transport: diffusiondriven passive transport and energy-dependent active transport. The stationary phase is also discussed, but primarily in the context of the nuclear envelope structure.

A defining feature of eukaryotes is the presence of a nucleus, a compartment that confines genetic material in each cell (Fig. 1). Replication of DNA and transcription into RNA occur within the nucleus. Then, the RNA is exported from the nucleus into the cytosol where translation of RNA into protein occurs. Compartmentalization of such vital molecular processes obligates an extensive nucleocytoplasmic exchange of critical constitutive and regulatory proteins. This chapter provides a synopsis on the function and structure of the nucleus as the mediator of nucleocytoplasmic transport required for the regulation of genetic processes in eukaryotic cells, including cardiomyocytes, in which intracellular processes, including synthesis of proteins and nuclear transport, are controlled by extracellular and intracellular stimuli.

A. Passive Diffusion Smaller proteins (⬍60 kDa) may enter the nucleus by passive diffusion. However, the majority of small proteins are transported by signal-mediated mechanisms that control their localization rather than by proper diffusion. Proteins mobilized in/out the nucleus by diffusion are transported at rates that suggest that only their size, not sequence, is limiting. Although such small proteins can equilibrate between the nucleus and the cytoplasm, only nuclear proteins accumulate in the nucleus against a concentration gradient. This suggests that selective nuclear accumulation is mediated by the free diffusion of small proteins into the nucleus with selective retention of nuclear proteins through binding to nondiffusible components within the nucleoplasm. No regulatory mechanism for the diffusion of small proteins or ions into nuclei has been identified. However, it has been suggested that a Ca2⫹-sensitive lumenal effector(s) is the major regulator of overall nucleocytoplasmic transport (Greber and Gerace, 1995; StehnoBittel et al., 1995; Perez-Terzic et al., 1996). Gp210, a

II. NUCLEAR TRANSPORT The two-way exchange between the cytoplasm and the nucleus is an essential signaling mechanism responsible for the regulation of gene expression by hormones and growth factors and for the control of DNA synthesis and cell entry into mitosis. Transport occurs across the nuclear envelope in two steps: a mobile phase and a stationary phase. The delivery of a molecule to the nuclear envelope through its interactions with specific nuclear and cytoplasmic components is considered the mo-

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transport is saturable and requires energy. In principle, four major processes in bidirectional nuclear transport (import/export) are distinguished (Fig. 2): (1) a fast binding of the protein to be transported to the transport protein (receptor) and interactions of the complex protein/receptor to components of the envelope, (2) a relatively slower, energy/temperature-dependent translocation of the protein/receptor, (3) release of the protein into the nucleopasm or cytoplasm, and (4) recycling of the transporters or receptors. A number of factors have been recognized to play a critical role in the transport processes.

C. Signals Directing Nuclear Transport

FIGURE 1 Schematic of membrane structures in a cell. A single membrane separates the cytoplasm from the extracellular space. Organelles have single membranes (e.g., vacuoles) or stacks of membranes (e.g., Golgi). Mitochondria comprise a defining outer membrane and an invaginated larger inner membrane. Similarly, the nucleus comprises a defining inner membrane and a large outer membrane, continuous with the endoplasmic and sarcoplasmic reticulum (ER/SR). Transport into and out of the nucleus occurs through the nuclear pore complex (NPC). Approximate calcium concentrations for each cellular compartment are indicated.

nuclear protein, possesses an N-terminal domain located in the nuclear cisterna with a putative Ca2⫹-binding site and could be such a Ca2⫹-sensitive lumenal effector. Passive diffusion is decreased when the antibody RL27, raised against the lumenal domain of gp210, is expressed in the endoplasmic reticulum (Greber and Gerace, 1992). Furthermore, depletion of Ca2⫹ content of the endoplasmic reticulum and nuclear cisterna (by Ca2⫹ ionophores or chelators) inhibits passive nuclear transport (Greber and Gerace, 1995; Stehno-Bittel et al., 1995; Perez-Terzic et al., 1996).

B. Active Transport Active transport is responsible for bidirectional movement of the majority of macromolecules (⬎앑60 kDa), as well as for transport of smaller proteins that possess a nuclear import (NLS) or export (NES) signal (Gorlich and Mattaj, 1996; Corbett and Silver, 1997; Cole and Hammel, 1998; Pennisi, 1998). All nuclear proteins are imported from the cytoplasm, their site of synthesis, to the nucleus. Transfer RNAs (tRNAs) and mRNA, however, are exported from the nucleus to their site of function, the cytosol. Active

A signal sequence encoded in a protein that causes nuclear import is recognized as the nuclear localization signal (NLS), whereas the sequence that causes nuclear export is called the nuclear export signal (NES). Several different forms of each type of targeting signals have now been identified that show no apparent homology to each other and appear to be recognized by different receptors (Gorlich and Mattaj, 1996; Pemberton, 1998). The conventional NLS that directs protein import is characterized by either one or two short stretches of positively charged amino acids that can be localized anywhere in the primary sequence of the protein. Some proteins have two runs of basic amino acids separated by a spacer region. Another important signal is the M9 domain (30 amino acids) found in chromatin-associated RNA-binding proteins (hnRNP). M9 also functions as a signal for export in mammalian cells. In addition, other protein import signals, which do not resemble either M9 or classic NLS signals, have been recognized (Goldfarb, 1997). The first NES were identified in the HIV REV protein (Fornerod et al., 1997b). This signal is characterized by a short leucine-rich sequence motif (approximately 10 amino acids sequence). Similar sequences have been identified in proteins, including TFIIIA (amphibian transcription factor IIIA) and I␬B움. It has been found that NES interact with FG repeats of several nucleoporins, and it was suggested that translocation requires sequential binding of the NES to FG repeats present in the nucleoporins. However, the NES of REV is able to competitively inhibit translocation of U1 snRNA and 5S RNA, but does not affect the export of mRNA and tRNA (Fornerod et al., 1997a). This suggests that REV has a similar cellular export pathway that is normally used by U1 snRNA and 5S RNA, but not by mRNA and tRNA.

D. Receptor/Carrier The classical NLS-directed import is employed by proteins that display a basic NLS. In this mode of import,

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FIGURE 2 Major steps in the active nuclear import and export pathways across the nuclear envelope. See text for a description of the pathways.

the NLS motif is recognized by a cytoplasmic NLS– receptor complex consisting of two subunits: importin 움 or karyopherin 움 (앑60 kDa) and importin 웁 or karyopherin 웁 (앑95 kDa). Evidence suggests that multiple members of importin or karyopherin 움 exist in higher eukaryotes. These appear to have some substrate specificity, not only due to differential localization, but also due to different affinities toward substrates (Pemberton et al., 1998). The family of importin 웁 homologous consists of proteins of similar molecular weight and a high level of conservation at the amino-terminal domain, which interacts with Ran-GDP or Ran-GTP. They possess a carboxy-terminal that interacts with importin 움 and an overlapping domain that interacts with nucleoporins (Pemberton et al., 1998). The classic model describes importin 움 or karyopherin 움 as primarily respon-

sible for binding to NLS-containing proteins and importin 웁 or karyopherin 웁 as responsible for targeting the complex toward the envelope. Proteins with repeat domains GLFG and FXFG (nucleoporins) have been shown to interact with importin 웁. A new pathway has been discovered with the identification of receptors that mediate nuclear import of the shuttling hnRNP. In this case, the 38 amino acid stretch involved in the import of hnRNP has been characterized as the M9 signal. A factor that binds the M9 signal has also been identified and named transportin. The yeast homologous of transportin is Kap104. Both transportin and Kap104 show homology to importin 웁, but, unlike importin 웁, directly bind their import substrates (hnRNP) without an adaptor such as importin 움 (Goldfarb, 1997). A nuclear export receptor for leucine-rich NES has

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been identified and named CRM1 or exportin (Fornerod et al., 1997a; Ossareh-Nazari et al., 1997). It exhibits similarities with importin 웁 and is located as soluble or associated to an NPC. CMR1 forms a complex that include the NES-containing substrate and RanGTP. CRM1 is involved in the export of U1 snRNA and 5S RNA. At least one nucleoporin with which CRM1 interacts has been identified: CAN/Nup214. To date, different receptors have been identified that mediate the nuclear export of tRNA, tRNA-processing proteins, mRNA-binding proteins, and ribosomal proteins. CRM1-independent nuclear export has also been described for importin 움, which also contains a leucinerich NES. Leptomycine B (LMB) is a metabolite generated by Streptomyces that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein REV (Ossareh-Nazari et al., 1997). It was shown that LMB inhibits the signal-mediated nuclear export by direct binding to CRM1. LMB has, therefore, been used as a tool to further investigate nuclear export.

E. Energy Dependence of Nuclear Transport A small guanosine triphosphatase (GTPase), Ran, plays, an essential role in the nuclear transport of macromolecules (Moore, 1998). Ran is an abundant soluble protein localized predominantly in the nucleus. In its nucleotide-bound state (Ran-GTP/Ran-GDP), Ran is regulated by the Ran-Specific, GTPase-activating protein Ran-GAP1 and the specific guanine nucleotide exchange factor RCC1 (or Prp20p). Ran-GAP1 is cytoplasmic or is attached to the cytoplasmic margin of NPC. In contrast, RCC1 is exclusively nuclear and bound to chromatin. The asymmetric distribution of both Ran regulators is thought to result in a low concentration of Ran-GTP in the cytoplasm versus a high concentration of Ran-GTP into the nucleus. Such a Ran-GTP gradient is believed essential for the formation and dissociation of complexes involved in nuclear import and export. Import complexes associate in the presence of RanGDP (Cytoplasm) and disassemble in the presence of Ran-GTP (nucleus). Conversely, export complexes assemble in the presence of Ran-GTP (nucleus) and disassemble in the presence of Ran-GDP (Cytoplasm) (Moore, 1998). Although a guanosine triphosphatase (GTPase) is key in the transport processes, it seems that the translocation step does not require GTP hydrolysis (Moore, 1998). It seems that Ran acts primarily to facilitate cooperative binding of signal-containing proteins and its cargo and perhaps also mediates specific interactions of these complexes with nucleoporins. However, nuclear transport appears to require energy utilization in intact

cells (Greber and Gerace, 1995; Corbett and Silver, 1997; Nigg, 1997; Perez-Terzic et at., 1999). The source of energy for the movement of signal-carrier proteins is still unknown.

F. Other Proteins/Factors Involved in Nuclear Transport Ran-binding proteins—RanBP1, RanBP2, RamBP3a, and RanBp3-b—interact with the GTP-bound form of Ran tightly and are required to modulate Ran-mediated GTP hydrolysis. These proteins can function as a GAPactivating protein and have been shown to stabilize a trimeric complex of RanBP1, importin 웁, and Ran. RanBP2 is localized at cytoplasmic filaments and is thought to serve as a docking element accepting the complex to be transported. Moreover, these Ran-binding proteins have an FXFG motif, which is typical for certain nucleoporins (Moore, 1998). Another protein involved in nuclear transport is p10 (Pante and Aebi, 1996; Nehrbass and Blobel, 1996). This is a Ran-interacting protein of 10 kDa, which, in conjunction with Ran, translocates the import–ligand complex across the central channel. It could function as a regulator of the RanGTPase cycle when Ran is confined to the NPC. In addition to these proteins, others nucleoporins have been identified that interact with the nuclear import/ export signals, receptors, and RanGTP/GDP. These proteins may facilitate the bidirectional transport of macromolecules (Corbett and Silver, 1997; Nigg, 1997; Ohno and Fornerod, 1998; Talcott and Moore, 1999).

G. Receptor Recycling and Shuttling When transport of the import substrate into the nucleus has been completed, importin 움 and 웁 have to return to the cytoplasm. Reexport of importin 움 appears to be very rapid and requires the regeneration of RanGTP. However, the reexport of importin 웁 seems to be much faster than that of importin 움.

III. NUCLEAR STRUCTURE A. Nuclear Envelope The Nuclear envelope is a double membrane system separating the nucleus from the cytoplasm (Dingwall, 1992) (Fig. 3). Outer and inner nuclear membranes are separated themselves by a restricted space termed the nuclear cisterna. Whereas the inner membrane defines the boundary of the nucleus, the outer membrane is in continuation with the endoplasmic/sarcoplasmic

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FIGURE 3 The nuclear pore complex in a cardiomyocyte. A scanning electron micrograph of a fixed cardiomyocyte with the cytoplasmic membrane partially stripped to reveal the nucleus. (Insets clockwise from top left) A scanning electron micrograph of nuclear pore complexes on the surface of the nucleus. A three-dimensional surface reconstruction of a single nuclear pore complex using atomic force microscopy. Transmission electron microscopy of a cross section through a nuclear pore complex showing the pore opening and the double membrane of the nuclear envelope.

reticulum (Fig. 3). However, the absence of nuclear membranes of enzymes present in the endoplasmic/ sarcoplasmic reticulum, such as phosphocholine cytidyltransferase, and the uneven distribution of cytochrome P450 isoenzymes between nuclear and endoplasmic/ sarcoplasmic reticulum membranes distinguish the two membrane systems (Fahl et al., 1978). Associated with the nucleoplasmic face of the inner nuclear membrane, a 100-nm-thick filamentous karyoskeletal structure formed by lamins and named nuclear lamina exists (Moir and Goldman, 1993). Nuclear lamina display a dynamic behavior and have been implicated in the reas-

sembly of the nucleus after mitosis (Moir and Goldman, 1993).

B. Nuclear Pore Complex (NPC) The outer and inner nuclear membranes fuse together at nuclear pore complex (NPC) sites, assuring direct communication between the cytoplasm and the nucleoplasm (Figs. 3 and 4). The NPC appears to be the structure responsible for mediating all types of transport across the nuclear envelope. NPCs are distinctive structures of the nuclear envelope. The NPC has a molecular

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NPC, its frequent absence in microscopy has given rise to he notion that it may represent material in transit rather than a constitutive component of the NPC (Akey, 1995). Cytoplasmic filaments (30–50 nm long) protrude from the cytoplasmic ring of the NPC. The nucleoplasmic face of the NPC also exhibits long filaments (50–100 nm long) that extend from the nucleoplasmic ring and are joined distally by a 30- to 50-nm-diameter terminal ring, forming a basket structure or ‘‘fish trap’’ (Fig. 4). Such cytoplasmic filaments, and the nucleoplasmic basket, make the NPC distinctly asymmetric relative to the plane of the nuclear envelope. It is believed that they play a role in docking material destined for trafficking across the NPC. The three-dimensional structure of yeast NPC has been constructed, and comparisons with vertebrate NPC were done to provide insight into the function and evolution of NPCs (Yang et al., 1998). FIGURE 4 Exploded view schematic of the nuclear pore complex. The three primary components, cytoplasmic ring and filaments, the membrane-spanning body, and the nuclear basket, each maintain an octameric axial symmetry. The transporter, or central plug, is situated in the center of the membrane-spanning body.

mass of 앑125 MDa. These megacomplexes contain multiple copies of over 50 different polypeptids called nucleoporins. Only 15% of constitutive proteins of an NPC have been cloned and characterized. The majority of known nucleoporins are O-glycosylated by N-acetyl glucosamine and contain characteristic FG repeats (Talcott and Moore, 1999). The NPC structure is highly dynamic and must disassemble prior to mitosis and reassemble after cell division. In addition, new NPCs must be added continuously to maintain proper density during interphase as the nuclear envelope expands. The number of NPCs present within the nuclear envelope appears to be related to the metabolic activity of a cell and may vary form about 1–5 to 20–50 pore/애m2. The NPC structure has been most studied in nonmammalian systems, such as Xenopus oocytes and yeast, using electron microscopy. It appears to have tripartite structure that spans the nuclear envelope and consists of a central cylindrical body embedded between cytoplasmic and nuclear octagonal rings of 32 and 21 MDa, respectively. Each ring consists of eight particles positioned in a symmetrical array around a central axis spanning the cytoplasmic and nucleoplasmic surfaces of the nuclear envelope (Akey, 1993; Fig. 4). The central body contains a channel with a central granule or plug and eight radiating spokes connecting the particles of the rings to the central granule with an eightfold symmetry (Fig. 4). The central plug is controversial component of the NPC structure. Although it has been postulated to represent the actual transporter of molecules across the

C. Mechanism of Translocation The actual movement of import substrates through the NPC, a distance approximately of 200 to 300 nm, has not been clearly defined. Two general mechanisms have been proposed. In the first, movement through the pore would occur by random nondirected diffusion, but upon reaching a check-point, an ‘‘irreversible’’ gate would open, committing the substrate to enter the nucleus. In the second, a series of association and dissociation of the substrate with the transport channel would occur as the substrate moves down from one binding site to the next of increased affinity in a stepwise manner (Moore and Blobel, 1993; Gorlich and Mattaj, 1996; Corbett and Silver, 1997; Nigg, 1997; Ohno et al., 1998).

D. Nuclear Transport Systems in the Nuclear Envelope: Ca2⫹ Channels and Ca2⫹ Pump Ca2⫹ is important in nuclear function: nuclear vesicle fusion requires Ca2⫹ mobilization and ca2⫹ regulates the expression of immediate-early response genes (Hardingham et al., 1997). In addition, several kinases, some of them specific for the nucleus, such as PKC-L and calmodulin-dependent kinase II, are targets for Ca2⫹ – calmodulin-dependent signaling. Although the NPC is considered to be permeable to ions, evidence shows that the Ca2⫹ concentration in the nucleoplasm differs form that in the cytosol. Such an apparent difference has been recorded after the receptor stimulation of cells when cytosolic Ca2⫹ may rise without affecting the nuclear concentration of Ca2⫹. In this regard, the nucleoplasm appears ‘‘insulated’’ form large fluctuations in cytosolic Ca2⫹ (Hernandez-Cruz et al., 1990; Brinni et al., 1994). Ca2⫹ enters the nuclear cisterna as it does in the lumen of the endoplasmic reticulum. The outer nuclear

24. Transport in Nucleus

membrane contains an ATP-dependent transporter that efficiently pumps Ca2⫹ into the nuclear cisterna. Inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2⫹-permeable channels release Ca2⫹ form the nuclear cisterna into the cytoplasm, and perhaps into the nucleoplasm. The nucleus has its own phosphoinositide catalytic system. This may produce InsP3, but its activation mechanism is unknown. InsP3-dependent receptor channels in nuclei have similar conductance properties and kinetics, as well as sensitivity to heparin, as those of the InsP3 found in the membrane of the endoplasmic reticulum. An endogenous nuclear protein kinase C type II isoenzyme specifically phosphorylates the nuclear InsP3 receptor, which increases receptor sensitivity. Nuclear membrane receptors for inositol 1,3,4,5-tetrakisphosphate (InsP4) and cyclic ADP-ribose (cADPR) have also been proposed. Moreover, potassium and chloride-selective, as well as nonselective, cation channels have been identified in the outer nuclear membrane by patch clamp methods applied to isolated nuclei (Perez-Terzic et al., 1997; DeFelice and Mazzanti, 1998).

E. Role of Ca2⫹ in the Regulation of Transport across the NPC Diffusion of small molecules, as well as active transport, is decreased when the Ca2⫹ concentration of the endoplasmic reticulum is depleted. Using intact isolated nuclei and nuclear ‘‘ghost’’ preparations (containing intact nuclear membranes but no nucleoplasm), the role of Ca2⫹ in the Nucleoplasm and Ca2⫹ in the nuclear cisterna in controlling passive diffusion across the NPC has been defined. The use of pharmacological tools (such as Insp3) to deplete Ca2⫹ from the nuclear cisterna is isolated nuclei showed that nuclear cisternal Ca2⫹ levels regulate the diffusion of intermediate-sized molecules across the NPC. One exciting possibility is that nuclear phosphoinositides and InsP3-sensitive channels present in the nuclear envelope, and involved in the regulations of Ca2⫹ stores in the cisterna, may play a central signaling role. Active transport is also regulated by Ca2⫹ depletion. In intact mammalian cells, including cardiomyocytes, depletion on intracellular Ca2⫹ downregulates the transport of histones and proteins carrying an NLS (Greber and Gerace, 1995; Sweitzer and Hanover, 1996; Perez-Terzic et al., 1999).

F. Conformational Changes in the NPC and Regulation of Nuclear Transport Control mechanisms responsible for coordinating specific conformational transitions in the NPC are not fully understood. Field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were em-

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ployed to study the conformation of the unclear pore complex (Akey, 1995; Perez-Terzic et al., 1996; 1999; Rakowska et al., 1998; Wang and Clapham, 1999). When nuclear Ca2⫹ stores are full, the central channel of the nuclear pore complex appears empty under FESEM (앑10-nm depth resultion), but AFM revealed that the plug has recessed (perez-Terzic et al., 1996). Depletion of nuclear Ca2⫹ stores results in an apparent upward shift of the plug to a level even with the outer rim of the NPC (using both FESEM and AFM). Using laser confocal and atomic force microscopy, nuclear transport and the structure of individual nuclear pores under different cellular conditions were visualized in cardiomyocytes (Perez-Terzic et al., 1999). In response to depletion of Ca2⫹ stores or ATP/GTP pools, the cardiac nuclear pore complex adopts two distinct conformations leading to different patterns of nuclear import regulation. Depletion of Ca2⫹ indiscriminately prevents nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocks facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, allowing other molecules to pass into the nucleus through peripheral routes (Perez-Terzic et al., 1999). Moreover, the addition of ATP has been shown to induce conformational changes, specifically to increase the height of the NPC (Rakowska et al., 1998). These results suggest that the filling state of the nuclear Ca2⫹ store, as well as the presence/absence of ATP/GTP, governs conformational changes in the nuclear pore complex, which may control the transport of molecules through the NPC. A number of mechanisms might explain how the nuclear transport apparatus, located on the nuclear/ cytoplasmic surface of nuclear membranes, could be affected by Ca2⫹ depletion of the nuclear cisterna. Gp210, a major transmembrane glycoprotein associated with the NPC, possesses a single transmembrane domain and a short carboxy-terminal domain, which may form part of the lumenal domain of the NPC. The aminoterminal portion of the protein, which resides in the nuclear cisterna, contains a putative EF hand Ca2⫹-binding domain. It is tempting to speculate that the depletion of Ca2⫹ from the nuclear cisterna may be sensed by gp210. This or other related molecules could then trigger rearrangement within the lumenal domain and result in a conformational change in the NPC.

IV. REGULATION OF NUCLEAR TRANSPORT IN CARDIAC CELLS Little is known about the nuclear structure and transport in cardiac cells (Bloom, 1970). In the majority of cell types, the cutoff for nuclear import varies between 45 and 60 kDa. In cardiomyocytes, a lower apparent

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size limit, between 10 and 40 kDa, has been estimated (Perez-Terzic et al., 1999). In this regard, the cardiac NPC appears to have a smaller pore than that estimated in other cell types. However, the NPC of cardiomyocytes overall displays a structure similar to that reported for lower eukaryotic cell types. Cardiac cells exhibit a characteristic toroid structure, of 150 nm in diameter, including a characteristic multimeric cytosolic ring surrounding a central pore (Perez-Terzic et al., 1999). Cardiac NPCs respond to changes in the cellular metabolic state. Depletion of intracellular Ca2⫹ in cardiomyocytes induces closure of the cytosolic ring of the NPC and reduction of the apparent depth of he central pore (Fig. 5). This inhibits nuclear transport of both histone H1 (active transport) and 10-kDa dextrans (pas-

sive diffusion). Moreover, depletion of intracellular ATP/GTP in cardiomyocytes induces a combined closure/relaxation of NPCs (Fig. 5). This selectively inhibits nuclear transport of H1 (active transport), but not dextrans (passive diffusion) (Perez-Terzic et al., 1999). These results indicate that a cardiac NPC can adopt distinct conformational changes, induced by depletion of Ca2⫹ or ATP/GTP stores, which are associated with distinct patterns of nuclear import regulation. The importance of these results rests in the fact that in ischemic heart disease or heart failure, disturbances in both ion and energy homeostasis accompany aberrant patterns of gene expression, raising the possibility that altered nuclear transport contributes to the progression of disease.

FIGURE 5 Model of nuclear transport regulation showing active translocation of histones (fl-H1) and 10kDa dextrans (10-kDa) under control conditions (left), a nonselective block caused by Ca2⫹ depletion (middle), and a selective block caused by the depletion of ATP/GTP (right). Depletion of Ca2⫹ and ATP/GTP reduces the opening of the central pore (open arrows), whereas depletion of ATP/GTP relaxes the complex (hatched arrows), which leads to the opening of alternate pathways for transport (star-like opening).

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FIGURE 6 Schematic of pathways that utilize the nuclear pore complex for import and export across the nuclear envelope. Passive, unfacilitated transport requires no external factors whereas active or facilitated transport pathways require additional factors, such as karyopherins or transportins, and energy utilization through ATP/GTP and RAN cycling. Other pathways not currently known may also use the NPC for transport across the nuclear membrane.

V. SUMMARY The complete complex picture of the bidirectional nuclear transport of molecules has only begun to be revealed (Fig. 6). In addition to the classical ones, other subtypes of transport need to be uncovered. Characterization of all factors playing a role in the process, as well as the full definition of the molecular architecture of NPCs and the identification of all nucleoporins, is necessary to precisely define how molecules move across the nuclear membrane. Extensive research performed to date indicates that the same complex apparatus efficiently and selectively coordinates different types of bidirectional transport of many molecules and that these transports are governed by different cellular conditions.

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Perez-Terzic, C., Gacy, A. M., Bortolon, R., Dzeja, P. P., Puceat, M., Jaconi, M., Prendergast, F. G., and Terzic, A. (1999). Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import. Circ. Res. 84, 1292–1301. Peterson, O. L., Gerasimenko, O. V., Gerasimenko, J. V., Mogami, H., and Tepikin, A. V. (1998). The calcium store in the nuclear envelope. Cell Calcium 23, 87–90. Rakowska, A., Danker, T., Schneider, S., and Oberleithner, H. (1998). ATP-induced shape change of nuclear pores visualized with the atomic force microscope. J. Membr. Biol. 163, 129–136. Stehno-Bittel, L., Perez-Terzic, C., and Clapham, D. E. (1995). Diffusion across the nuclear envelope inhibited by depletion of the nuclear calcium store. Science 270, 1835–1838. Sweitzer, T. D., and Hanover, J. A. (1996). Calmodulin activates nuclear protein import: A link between signal transduction and nuclear transport. Proc. Natl. Acad. Sci. USA 93, 14574– 14579. Talcott, B., and Moore, M. S. (1999). Getting across the nuclear pore complex. Trends Cell Biol. 9, 312–318. Wang, H., and Clapham, D. E. (1999). Conformational changes of the in situ nuclear pore complex. Biophys. J. 77, 241–247. Yang, Q., Rout, M., and Akey, C. W. (1998). Three-dimensional architecture of the isolated yeast nuclear pore complex: Functional and evolutionary implications. Mol. Cell. 1, 223–234.

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25 Sarcoplasmic Reticulum Ca2⫹ Transport ISTVAN EDES

GUOXIANG CHU and EVANGELIA G. KRANIAS

Department of Heart and Lung Diseases University Medical School HU-4032 Debrecen, Hungary

Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine Cincinnati, Ohio 45267

The rising cytosolic Ca2⫹ concentration induces contraction through binding to troponin C, which activates a chain of conformational changes, allowing the thin and thick filaments to interact. Subsequently, Ca2⫹ is dissociated from troponin C and is removed rapidly from the cytosol by various systems, resulting in relaxation. At least three processes are responsible for the removal of Ca2⫹ to end contraction (Fig. 1): (a) the SR Ca2⫹ pump, which actively translocates Ca2⫹ at the cost of ATP into the SR system; this is thought to be the most important process in mediating relaxation; (b) the Na⫹ /Ca2⫹ exchanger, which transports Ca2⫹ out of the cell during diastole; and (c) the sarcolemmal Ca2⫹ATPase, which also extrudes Ca2⫹ from the cell. The SR is a tubular network, which serves as a sink for Ca2⫹ ions during relaxation and as a Ca2⫹ source during contraction. In cardiac muscle, about 60–70% of the intracellular Ca2⫹ released during systole is taken up by the SR, and the remaining amount is extruded from the cell by the Na⫹ /Ca2⫹ exchanger and the sarcolemmal Ca2⫹-ATPase. The SR in both skeletal and cardiac muscles also contains an acidic protein, calsequestrin (Fig. 1), which binds 40–50 mol of Ca2⫹ /mol of protein. The binding and release of Ca2⫹ by calsequestrin is believed to be an integral step of excitation– contraction coupling, but the details of this process are still not fully understood. Sarco/endoplasmic reticulum Ca2⫹-ATPases (SERCA), encoded by three different genes (SERCA1, SERCA2, and SERCA3), are essential for maintaining Ca2⫹ homeostasis in the cell by sequestering Ca2⫹ from the cytosol into the lumen of the sarco/endoplasmic reticulum. The functional significance of the SERCA2

I. INTRODUCTION An important role of Ca2⫹ in muscle contraction was first indicated a century ago by Ringer (1883), who demonstrated that a frog heart would not contract in the absence of extracellular Ca2⫹. Since then, it has been shown that Ca2⫹ is a physiological regulator for contractile proteins and several other enzymes and processes in muscle. This chapter focuses on the role of various Ca2⫹-ATPases in maintaining Ca2⫹ homeostasis in the cell, with special emphasis on the cardiac sarcoplasmic reticulum (SR) Ca2⫹-ATPase, which is the primary regulator of Ca2⫹ levels and thus contractility in the mammalian heart. During the cardiac action potential, Ca2⫹ enters the cell via Ca2⫹ channels, which also act as dihydropyridine receptors (Fig. 1). This Ca2⫹ can stimulate the release of Ca2⫹ from the intracellular calcium organelle, the SR. The SR Ca2⫹-release channel in cardiac and skeletal muscle, which is the ryanodine receptor, spans the gap between the transverse tubule and the SR (‘‘foot’’ protein). Furthermore, the outer cell membrane Ca2⫹ channel is located close to the SR Ca2⫹ channel. Thus, excitation–contraction coupling involves the sarcolemmal Ca2⫹ channel and the SR Ca2⫹-release channel, with the Ca2⫹ current through the sarcolemmal channel being responsible for the initiation of Ca2⫹ release from the SR (Fig. 1). In skeletal muscle, the sarcolemmal membrane depolarization is responsible for the induction of SR Ca2⫹ release. The relative importance of release from the SR in activation of cardiac muscle contraction varies from preparation to preparation, but in the heart of mammals it usually accounts for 40–70% of the Ca2⫹ required.

Heart Physiology and Pathophysiology, Fourth Edition

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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FIGURE 1 Schematic diagram of Ca2⫹ fluxes in cardiac cell. Na-CaX, Na⫹ /Ca2⫹ exchanger; Calseq., calsequestrin; ICa, slow inward Ca2⫹ current; SR, sarcoplasmic reticulum.

pump in the regulation of Ca2⫹ homeostasis and contractility has been elucidated by the generation of mutant mice with (a) overexpression of SERCA2 in the cardiac compartment, using transgenesis; and (b) ablation of SERCA2 using gene targeting in embryonic stem cells. This chapter summarizes the structural properties and tissue distribution of the various SERCA isoforms, signaling pathways involved in the regulation of SERCA activity, and the physiological or pathophysiological relevance of alterations of SERCA expression/activity in genetically modified mouse models and in cardiac disease.

II. STRUCTURE AND TISSUE DISTRIBUTION OF SR Ca2⫹-ATPase The major protein in the SR membrane in the Ca2⫹ATPase (Mr 100,000), representing about 40% of the total protein in cardiac SR. Recombinant DNA studies revealed that the SR or endoplasmic reticulum (ER) Ca2⫹-ATPase family (SERCA) is the product of at least three alternatively spliced genes (Misquitta et al., 1999)

(Table I). SERCA1 is expressed in fast-twitch skeletal muscle, and alternative splicing of the 3⬘ end of the primary transcript gives rise to two mRNA forms, which are expressed at different stages of development (Brandl et al., 1986). Alternatively, spliced forms of SERCA 2 have been detected in cardiac muscle and slow skeletal muscle (SERCA2a) and in adult smooth muscle and nonmuscle tissues (SERCA2b). SERCA3 is expressed in a selective manner, with the highest mRNA levels in intestine, spleen, lung, uterus, and brain. SERCA2 is about 85% identical to SERCA1, whereas SERCA3 is about 75% identical to either SERCA1 or SERCA2. The human SERCA2 gene is localized on chromosome 12 and maps to position 12q23-q24.1. The proposed general model of the enzyme has three cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow extramembrane pentahelical stalk (Brandl et al., 1986). The cytoplasmic region includes a nucleotide-binding site, or a domain to which the MgATP substrate binds, and a phosphorylation domain, which contains an aspartic acid residue (Asp-351) to which the phosphate is covalently bound (Fig. 2). The third cytoplamic domain is the 웁-strand domain,

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TABLE I SERCA Isoforms and Tissue Distribution Gene SERCA1 SERCA2

SERCA3

Locus 16p12.1 12q23–q24.1

17p13.3

Isoform SERCA1a SERCA1b SERCA2a SERCA2b

Exons

SERCA3a

1–23 1–21, 1–21, 1–22 1–23, 1–25 1–20,

SERCA3b SERCA3c

1–20, 21 (partial), 22 1–22

23 25

Tissue distribution Adult fast skeletal muscle Neonatal fast skeletal muscle Cardiac/slow skeletal muscle Smooth muscle, nonmuscle (e.g., cerebellum, cerebrum)

25 22

Platelets, lymphoid cells, Purkinje neurons, islets of Langerhans, salivary glands, endothelial cells Kidney, pancreas, endothelial cells Kidney, pancreas

whose function is still not fully understood. In skeletal muscle, 2 mol of Ca2⫹ is transported per mole of ATP hydrolyzed. In cardiac muscle, a similar stoichiometry is expected, but this ratio has been generally found to be lower (0.4–1.0 mol Ca2⫹ /mol ATP). Ca2⫹ has been shown to bind to a region involving several of the membrane-spanning 움 helices (M4, M5, and M6) on the cytoplasmic side. The important amino acid residues constituting the Ca2⫹-binding sites are Glu-309 on the

M4 transmembrane segment, Glu-771 on the M5 transmembrane segment, and Asn-796, Thr-799, and Asp800 on the M6 transmembrane segment. It has been proposed that Glu-771 and Thr-799 are associated with the first Ca2⫹-binding site (site 1), whereas Glu-309 and Asn-796 are associated with the binding of the second Ca2⫹ ion (site 2). Asp-800 was suggested to donate ligands to both Ca2⫹-binding sites (MacLennan et al., 1997). During the Ca2⫹ transport cycle, the ezyme undergoes a transition from a high-affinity state to a lowaffinity state for Ca2⫹, and the ions are translocated from the binding sites into the lumen of the SR (Fig. 2). This reaction pathway is characterized by the covalent phosphorylated Ca2⫹-ATPase form (E1앑P) when the energy of ATP is transferred to an acylphosphoprotein intermediate (Fig. 3). E1앑P rapidly becomes E2-P when the energy contained originally in the acylphosphoprotein is transduced into the translocation of bound Ca2⫹ into the SR (‘‘marionette’’ model, see Fig. 2). Subse-

FIGURE 2 Model illustrating Ca2⫹ translocation by SERCA type Ca2⫹ pumps. In E1앑P conformation, Ca2⫹ binds to the high-affinity binding sites in the cytosol. The energy of the hydrolyzed ATP triggers a series of conformational changes and transforms the E1앑P intermediate to the E2-P intermediate. These conformational changes are directly coupled to alterations in the orientation of the transmembrane regions leading to Ca2⫹ release into the lumen of the sarcoplasmic reticulum.

FIGURE 3 Reaction scheme of sarcoplasmic reticular Ca2⫹-ATPase.

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quently, the acid-labile intermediate (E2-P) decomposes to enzyme (E2) and inorganic phosphate (MacLennan et al., 1997). The Ca2⫹-free form of the enzyme exists in two different conformational states: one with low affinity for Ca2⫹ (E2) and one with high affinity for Ca2⫹ (E1) (Fig. 3). The conversion of E2 to E1 is proposed to be the ratelimiting step in the cycle. Thapsigargin (a plant sesquiterpene lactone) has been shown to interact specifically with the M3 transmembrane segment of the E2 form of all members of the SR Ca2⫹-ATPase family and to inhibit enzyme activity even at subnanomolar concentrations. The E1 form of the enzyme has been stabilized and crystallized in the presence of lanthanide (La3⫹) or Ca2⫹ ions (Dux et al., 1985). However, vanadate ions in the absence of Ca2⫹ induced the formation of E2-type crystals. E1-type crystals consist of single chains of Ca2⫹ATPase molecules spaced evenly on the surface of the SR. E2-type crystals consist of dimer chains of ATPase molecules forming an oblique surface lattice. The transition between E1 and E2 conformation may involve a shift in the monomer–oligomer equilibrium (Dux et al., 1985).

spectroscopy revealed that phospholamban is present primarily as pentamers in the SR membrane. However, in the dephosphorylated state, 앑20% of phospholamban is present as monomers and phosphorylation promotes pentameric assembly due to changes in the isoelectric point (from 10 to 6.7) of phospholamban (Simmerman and Jones, 1998). The complete amino acid sequence of phospholamban has been determined for various tissues and species. There is currently no evidence for the existence of any isoforms for this protein and the phospholamban gene has been mapped to human chromosome 6. The calculated molecular weight of phospholamban is 6080, and the protein has been proposed to contain two major domains (Fig. 4): a hydrophilic domain (domain I), which has two unique phosphorylatable sites (Ser-16 and Thr-17), and a hydrophobic C-terminal domain (domain II), which is anchored into the SR membrane. The hydrophilic domain (amino acids 1–30) has been further divided into two subdomains: domain Ia (amino acids 1–20) and Ib (amino acids 21–30). Domain Ia has a net positive charge in the dephosphorylated form and consists of an 움 helix followed by a proline residue at

III. REGULATION OF SR Ca2⫹-ATPase Regulation of SR Ca2⫹-ATPase activity by phospholamban has been well documented by in vitro and in vivo studies. Other regulatory mechanisms, including sarcolipin and direct phosphorylation of SERCA protein by Ca2⫹ /calmodulin-dependent protein kinase II or insulin/tyrosine kinase, have also been proposed.

A. Phospholamban 1. Structure of Phospholamban In cardiac muscle, slow-twitch skeletal muscle, and smooth muscle, the SR contains a low molecular weight protein, called phospholamban, which can be phosphorylated by various protein kinases. The phosphorylation and dephosphorylation of phospholamban, which comprises 3–4% of the SR membrane protein, regulate Ca2⫹ATPase activity in the SR membrane. However, the exact stoichiometry of phospholamban to SR Ca2⫹ATPase is not known. In early studies, a stoichiometric relationship of 1 or 2 mol phospholamban per 1 mol of Ca2⫹-ATPase was proposed for cardiac SR membranes. In reconstituted systems, a molar ratio of 3 : 1 of phospholamban/Ca2⫹-ATPase was necessary to obtain maximal regulatory effects. Furthermore, the ‘‘functional unit’’ of phospholamban is not currently clear. Experiments using electron paramagnetic resonance

FIGURE 4 Molecular model of the structure of phospholamban. The cytoplasmic 움-helix (domain Ia; residues 8–20) is interrupted by Pro21 (heavy circles). Residues 22–32 (domain Ib) are relatively unstructured and may interconvert between transient conformations, and residues 33–52 constitute the transmembrane domain II (움-helix). Ser-16 and Thr-17 (black circles) are the adjacent phosphorylation sites. Shaded circles indicate the leucines (Leu-37, Leu-44, and Leu51), which are important for phospholamban subunit interactions (pentamer formation).

25. SR Ca2⫹ Transport

position 21 (stalk region). Domain Ib has been suggested to be relatively unstructured (Simmerman and Jones, 1998), whereas the hydrophobic domain (amino acids 31–52) forms a 움 helix in the SR membrane (Fig. 4). Phospholamban migrates as a 24- to 28-kDa pentamer in sodium dodecyl sulfate (SDS) gels and dissociates into dimers and monomers upon boiling in SDS before electrophoresis. Spontaneous aggregation of phospholamban into pentamers was also observed upon expression of this protein in bacteria or in mammalian cells. Site-specific mutagenesis experiments identified cysteine (Cys-36, Cys-41, and Cys-46), leucine (Leu-37, Leu-44, and Leu-51), and isoleucine (Ile-40 and Ile-47) residues in the hydrophobic transmembrane domain as essential amino acids for phospholamban pentamer formation (Simmerman et al., 1996). Leucine and isoleucine amino acids are suggested to form zippers in the membrane, which stabilize the pentameric form of the protein with a central pore (Fig. 5), defined by the surface of hydrophobic amino acids (Simmerman et al., 1996). Based on this pentameric self-association of phospholamban, a channel function for this protein has been proposed. Monoclonal antibodies, raised against phospholamban, stimulate SR Ca2⫹ uptake in vitro. Furthermore, removal of phospholamban from the SR or uncoupling phospholamban from the Ca2⫹-ATPase (using detergents, high ionic strength solutions, or polyanions such as heparin sulfate) markedly increase the affinity of the

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SR Ca2⫹ pump for Ca2⫹. These findings suggest that the dephosphorylated form of phospholamban is an inhibitor of the SR Ca2⫹-ATPase. This ‘‘depression hypothesis’’ has been confirmed by studies using purified Ca2⫹ATPase and purified or recombinant phospholamban in reconstituted systems. Inclusion of phospholamban resulted in inhibition of the SR Ca2⫹-ATPase activity in reconstituted vesicles or cells (Kim et al., 1990). Cyclic AMP phosphorylation of phospholamban reversed its inhibitory effect on the Ca2⫹ pump. The inhibitory role of phospholamban on SR and cardiac function has been directly confirmed using genetically engineered mouse models. Overexpression of the protein (phospholamban-overexpressing mice) was associated with the inhibition of SR Ca2⫹ transport, Ca2⫹ transient, and depression of basal left ventricular function (Kadambi et al., 1996). However, partial (phospholamban-heterozygous mice) or complete ablation (phospholamban-deficient mice) of the protein was associated with increases in SR Ca2⫹ transport and enhanced cardiac function (Luo et al., 1994, 1996). Actually, a close linear correlation between the levels of phospholamban and cardiac contractile parameters was observed, indicating that phospholamban is a prominent regulator of myocardial contractility. These findings suggest that changes in the level of this protein will result in parallel changes in SR function and cardiac contraction. The region of phospholamban interacting with the Ca2⫹-ATPase has been proposed to involve amino acids

FIGURE 5 Heptad repeat model of the transmembrane domain of phospholamban monomer (left) and pentamer (right). Residues 31–52 of monomeric phospholamban are configured as a 3.5 residues/360⬚ turn helix with positions from ‘‘a’’ to ‘‘g’’ of heptad repeat squared (left). Leucine and isoleucine residues constituting zippers are localized to positions ‘‘a’’ and ‘‘d’’, respectively. Darkly shaded circles represent mutations that enhance the inhibitory function of phospholamban by enhanced monomer formation (destabilization of pentamer structure). Lightly shaded circles represent mutations that reduce inhibitory function. These ‘‘lossof-function’’ mutants are all located on the exterior face of each helix in a pentamer (positions ‘‘b’’, ‘‘e’’, and ‘‘f ’’). The phospholamban pentamer model (right) shows the interaction between monomers at positions ‘‘a’’ and ‘‘d’’ to form the leucine zipper (heavy squares). Adapted from Simmerman et al. (1996) and MacLennan et al. (1998).

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2–18 (Toyofuku et al., 1994b). Based on these reports, the simplest model for the interaction between the phospholamban cytoplasmic domain and the SR Ca2⫹ATPase is one in which the highly positively charged region of phospholamban (residues 7–16) interacts directly with a negatively charged region on the surface of the Ca2⫹-ATPase (Lys-Asp-Asp-Lys-Pro-Val402) to modulate the functional association between the two proteins (Fig. 6) (MacLennan et al., 1998). This interaction is disrupted by the phosphorylation of Ser-16 or Thr-17 in phospholamban because the positive charges of the phospholamban cytosolic domain are partially neutralized by the phosphate moiety in this vicinity. Phosphorylation of phospholamban by the cAMPdependent protein kinase at Ser-16 is associated with local unwinding of the 움 helix at position 12–16, resulting in conformational changes in the recognition unit of the protein. Interestingly, phospholamban peptides, corresponding to the hydrophobic membrane-spanning domain, also affect Ca2⫹-ATPase activity by lowering its affinity for Ca2⫹. The importance of the membrane-spanning region of phospholamban in inhibiting SR Ca2⫹-ATPase activity has been clarified by Kimura et al. (1997). It was shown that substitution of pentamer-stabilizing residues (Leu-37, Leu-44, Leu-51, Ile 40, and Ile 47) in the membrane-spanning region (domain II) by alanine resulted in monomeric mutants, which were more effective inhibitors of SR Ca2⫹-ATPase activity than wild-type phospholamban. These phospholamban monomeric mutants

were called ‘‘supershifters’’ because they decreased the apparent affinity of SR Ca2⫹-ATPase more effectively than wild-type phospholamban. Thus, it was proposed that monomeric phospholamban is the active form, which is involved in the interaction with SR Ca2⫹ATPase. Furthermore, the scanning alanine-mutagenesis studies (Kimura et al. 1997) have identified the amino acid residues in the transmembrane domain of phospholamban (Leu-31, Asn-34, Phe-35, Ile-38, Leu-42, Ile48, Val-49, Leu-52), which are associated with ‘‘loss of function.’’ These amino acids are located on the exterior face of each helix in the pentameric assembly of phospholamban (opposite from the pentamer-stabilizing face) (Fig. 5). A schematic representation of the interaction of phospholamban with SR Ca2⫹-ATPase is shown in Fig. 6. Based on in vitro expression studies, it has been proposed that the phospholamban monomer is the active species for interaction with the SR Ca2⫹-ATPase and that the pentamers are regarded as functionally inactive forms of phospholamban (Kimura et al., 1997; MacLennan et al., 1998). Phosphorylation of phospholamban monomers promotes association into inactive pentamers. Thus, two important steps for SR Ca2⫹-ATPase inhibition have been suggested: (1) dissociation of monomeric phospholamban from dephosphorylated pentamers (Kd1) and (2) binding of phospholamban monomers to the SR Ca2⫹-ATPase (Kd2). These dissociation constants (Kd1 and Kd2) will control both the concentration of phospholamban monomers and the

FIGURE 6 Model for regulation of sarcoplasmic reticular Ca2⫹-ATPase by phosphorylated and nonphosphorylated phospholamban. Phosphorylation of phospholamban disrupts the interaction between the two proteins so that the inhibition of Ca2⫹-ATPase is relieved. Note that both the cytosolic domain and the membrane-spanning region of phospholamban are involved in the phosphorylationmediated conformational change to relieve the inhibition. Phosphorylation of phospholamban monomers promotes association into inactive phosphorylated pentamers.

25. SR Ca2⫹ Transport

concentration of units in which monomers are associated with the SR Ca2⫹-ATPase (Kimura et al., 1997; MacLennan et al., 1998). There are at least two interaction sites between phospholamban and the SR Ca2⫹-ATPase (Fig. 6): one in the cytoplasmic domains of the two proteins and another one within the transmembrane sequences. The interaction between the hydrophobic membrane-spanning regions is associated with inhibition of the apparent affinity of SR Ca2⫹ATPase for Ca2⫹ (KCa). The interaction between cytosolic phospholamban domain Ia and SR Ca2⫹-ATPase modulates the inhibitory interaction in the transmembrane region (domain II) through long-range coupling. Disruption of the cytosolic interactions (domain Ia) by the phosphorylation of phospholamban or binding of a phospholamban antibody results in disruption of the inhibitory intramembrane interactions. However, resolution of the exact molecular mechanism by which phospholamban inhibits SR Ca2⫹-ATPase Ca2⫹ affinity and the concomitant regulation of SR Ca2⫹ transport will have to await the development of new methodology, which allows the detection of protein–protein interactions in a membrane environment. 2. In Vitro Studies on Regulation of SR Ca2ⴙ-ATPase In the early 1970s, it was suggested that the effects of various catecholamines on cardiac function might be partly attributed to phosphorylation of the SR by the cAMP-dependent protein kinase(s). It soon became clear that the substrate for the protein kinase (PK) was not SR Ca2⫹-ATPase but phospholamban. Various other high and low molecular weight SR proteins were also identified as minor substrates for cAMP-dependent PK, but only the changes in the phosphorylation of phospholamban were associated with functional alterations of the cardiac SR. Cardiac SR membranes contain an endogenous cAMP-dependent PK and a Ca2⫹ /CaM-dependent PK that have been shown to phosphorylate phospholamban independently of each other (Kranias, 1985a). Phosphorylation by cAMP-dependent PK occurred on Ser16, whereas Ca2⫹ /CaM-dependent PK catalyzed the phosphorylation of Thr-17 exclusively (Simmerman et al., 1986). Phosphorylation by either kinase was shown to result in stimulation of the initial rates of SR Ca2⫹ATPase activity and Ca2⫹ transport. Stimulation was associated with an increase in the apparent affinity of the SR Ca2⫹-ATPase for Ca2⫹ (KCa). A ‘‘state of filling’’ (SOF) kinase, which is sensitive to Ca2⫹ concentration within the SR lumen, has also been proposed to phosphorylate phospholamban. This kinase is activated on Ca2⫹ depletion from the SR and it phosphorylates Ser-

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16 in phospholamban, thereby facilitating refilling of the SR (Bhogal et al., 1998). In vitro, phospholamban is phosphorylated by two additional PKs: PK-C and a cGMP-dependent PK. Protein kinase C (Ca2⫹ /phospholipid-dependent PK) phosphorylates the protein at a site distinct from those phosphorylated by either cAMP-dependent PK or Ca2⫹ /CaM-dependent PK (Movsesian et al., 1984). Phosphorylation was shown to stimulate SR Ca2⫹ATPase activity and it was suggested that this activity plays a role in the action of agents known to stimulate phosphoinositide (PI) hydrolysis, as one product of PI hydrolysis, diacylglycerol, is an activator of PK-C. Cyclic GMP-dependent PK was found to phosphorylate phospholamban on the same residue (Ser-16) as that phosphorylated by cAMP-dependent PK (Raeymakers et al., 1988). This phosphorylation stimulated cardiac SR Ca2⫹ transport similar to the effects of cAMP-dependent PK. Furthermore, the stimulatory effects on Ca2⫹ transport, mediated by the cGMP-dependent phosphorylation of phospholamban, were also observed in smooth muscle, which may be of particular interest because some vasodilators act by increasing cGMP levels in vascular smooth muscle. The presence of endogenous PKs in cardiac SR necessitates the presence of phosphoprotein phosphatase(s) for reversible regulation of the Ca2⫹ pump. Protein phosphatases have been generally classified into type 1 and type 2. Type 1 phosphatase is inhibited by nanomolar concentrations of the protein inhibitor-1 and inhibitor-2, whereas type 2 phosphatases are unaffected. In heart muscle, both types of phosphatase have been reported to be present and both can dephosphorylate phospholamban (Kranias and Di Salvo, 1986). A type I protein phosphatase was shown to be associated with cardiac SR membranes, and this activity could catalyze the dephosphorylation of both cAMP-dependent PK and Ca2⫹ /CaM-dependent PK phosphorylated sites (Ser-16 and Thr-17) on phospholamban. Dephosphorylation was associated with a reduction in the stimulatory effects of PKs on the SR Ca2⫹ pump (Kranias, 1985b). SR phosphatase is similar to the skeletal muscle protein phosphatase IG (PPIG), which is composed of a catalytic (C) subunit and a G subunit. The G subunit may become phosphorylated by cAMP-dependent PK, which causes release of the C subunit from SR vesicles or glycogen particles into the cytosol, rendering phospholamban in the phosphorylated state and thus capable of stimulating SR Ca2⫹ transport. In vivo studies have also shown that the phosphatase activity associated with cardiac SR membranes may be regulated by cAMPdependent processes. 웁-adrenergic stimulation of intact beating hearts was associated with inhibition of the SR

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phosphatase activity, and this inhibition correlated with increases in the phosphorylation status of the phosphatase inhibitor, inhibitor-1. Thus, regulation of SR phosphatase activity may be one of the mechanisms by which cells achieve amplification of the cAMP-dependent processes. 3. In Vivo Studies on Regulation of SR Ca2ⴙ-ATPase The phosphorylation of SR proteins and their regulatory effects on the SR Ca2⫹-ATPase activity have been studied in perfused hearts from various animal species whose ATP pool was labeled with [32P]orthophosphate. Microsomal fractions enriched in SR were prepared from hearts freeze-clamped during stimulation with different agonists (catecholamines, forskolin, phosphodiesterase inhibitors, phorbol esters) and analyzed by gel electrophoresis and autoradiography for the degree of 32 P incorporation. 웁-adrenergic agonist (isoproterenol) stimulation of perfused hearts produced an increase in 32 P incorporation into phospholamban (Kranias and Solaro, 1982). The stimulation of 32P incorporation into phospholamban was associated with an increased rate of Ca2⫹ uptake into SR membrane vesicles and an increased SR Ca2⫹-ATPase activity. These biochemical changes were associated with increases in left ventricular functional parameters (contractility and relaxation). The in vivo phosphorylation of phospholamban was specific to inotropic agents that increased the cAMP content of the myocardium (웁-adrenergic agonists, forskolin, and phosphodiesterase inhibitors). However, positive inotropic interventions, which increased the intracellular Ca2⫹ level by cAMP-independent mechanisms (움-adrenergic agonists, ouabain, and elevated [Ca2⫹]), failed to stimulate phospholamban phosphorylation and relaxation. Calmodulin inhibitors (fluphenazine) attenuated the isoproterenol-induced phosphorylation of phospholamban, and it was shown that phospholamban contains equimolar amounts of phosphoserine (pSer-16) and phosphothreonine (pThr-17) under steady-state isoproterenol exposure. However, phosphorylation of Ser-16 correlated most closely with changes in cardiac function in beating hearts (Talosi et al. 1993). Based on these results and findings in transgenic animals (Luo et al. 1997), it was proposed that (1) prevention of Ser-16 phosphorylation (Ser-16 씮 Ala mutation) results in attenuation of the 웁-adrenergic response in mammalian hearts and (2) phosphorylation of Ser-16 is a prerequisite for Thr-17 phosphorylation. The muscarinic agonist acetylcholine attenuated the increases in cAMP levels, phosphorylation of phospholamban, and the SR Ca2⫹-ATPase activity produced

either by 웁-adrenergic stimulation or by phosphodiesterase inhibition (using isobutylmethylxanthine) (Lindemann and Watanabe, 1985). Protein kinase C and cGMP-dependent PK, which have been shown to phosphorylate phospholamban in vitro, failed to demonstrate similar effects in beating guinea pig hearts in response to stimuli that activate PK-C or elevate the cGMP levels (Edes and Kranias, 1990). Thus, the physiological relevance of PK-C and PK-G in beating hearts is not clear at present. Functional alterations in SR Ca2⫹-ATPase activity may explain, at least partly, the activating and relaxing effects of 웁-adrenergic agents in cardiac muscle (Figs. 7 and 8). The cAMP-dependent phosphorylation of phospholamban under either in vitro or in vivo conditions increases the rate of SR Ca2⫹ transport and SR Ca2⫹-ATPase activity. Such an increase in Ca2⫹ transport is expected to contribute primarily to the relaxing effects of catecholamines (Fig. 7). An additional mechanism, which contributes to the increased phosphorylation of phospholamban on 웁-adrenergic stimulation, is the phosphorylation of the phosphatase inhibitor protein by the stimulated cAMP-dependent kinase. This

FIGURE 7 Schematic diagram of possible relaxing and activating effects of 웁-adrenergic agents in the heart. PP1, protein phosphatase 1.

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troponin I has also been shown to decrease the sensitivity of myofilaments for Ca2⫹ in both intact myocardium and skinned fibers. The desensitization of myofibrills is accompanied by an increased Ca2⫹ off-rate from troponin C, which could contribute to faster relaxation (Fig. 8). In addition, phosphorylation of the SR Ca2⫹ release channel (ryanodine receptor) by Ca2⫹ /CaMdependent protein kinase may stimulate Ca2⫹ release from the SR vesicles and contribute to the elevation of intracellular Ca2⫹ levels during systole. Thus, the enhanced Ca2⫹ influx across the sarcolemma, together with the increased Ca2⫹ levels to be released from the SR, may result in an elevation of the Ca2⫹ available for the contractile machinery, leading to an increase in the amplitude of contraction (Fig. 8).

B. Other Regulatory Mechanisms

FIGURE 8 Effects of 웁-adrenergic agents on protein phosphorylation in cardiac cells. Increased intracellular cAMP levels activate the cAMP-dependent protein kinase(s), which phosphorylates various proteins (phospholamban, inhibitor-1, Ca2⫹ channel, and myofibrillar proteins) and increases the rates of SR Ca2⫹ uptake and release.

phosphorylation results in inactivation of protein phosphatase 1 and, thus, inhibition of dephosphorylation of phospholamban during the action of catecholamines (Fig. 7). The increased phosphorylation of phospholamban and the increased Ca2⫹ levels accumulated by the SR would lead to the availability of higher levels of Ca2⫹ to be subsequently released for binding to the contractile proteins (Fig. 7). The critical and prominent role of phospholamban in the mediation of 웁-adrenergic functional responses was also confirmed in transgenic animal studies. Cardiac myocytes or work-performing heart preparations from phospholamban-deficient mice exhibited largely attenuated responses to 웁-adrenergic stimulation (Luo et al., 1996; Wolska et al., 1996), indicating that phospholamban is a key phosphoprotein in the heart’s responses to 웁-adrenergic agonists. Phosphorylation of other myocardial phosphoproteins has also been suggested to be involved in the mediation of positive inotropic and lusitropic effects of 웁-adrenergic agonists. Cyclic AMP-dependent protein kinase-mediated phosphorylation of the 움1 subunit of the Ca2⫹ channel (Fig. 8) is associated with an increase in the voltage-dependent Ca2⫹ current (ICa), which enhances the Ca2⫹ levels available in the cytosol during 웁-adrenergic agonist stimulation. Phosphorylation of

Sarcolipin, a 31 amino acid peptide, has a structure similar to phospholamban but is expressed predominantly in fast-twitch skeletal muscle. Like phospholamban, sarcolipin inhibits SERCA1 or SERCA2 pump activity in vitro. The regulatory effects of sarcolipin are under the control of intracellular Ca2⫹ concentration: sarcolipin decreases the Ca2⫹ affinity (Km) of SERCA at low Ca2⫹ concentrations but stimulates Ca2⫹ uptake rate (Vmax) at saturating Ca2⫹ concentrations (Odermatt et al., 1998). However, the signaling pathways regulating the sarcolipin effects on the Km and Vmax of SERCA at different Ca2⫹ concentrations remain unclear. Ca2⫹ /calmodulin-dependent protein kinase II has also been shown to phosphorylate Ser-38 in SERCA2 (not SERCA1) and increase the Vmax of SERCA2 (Toyofuku et al., 1994a). The endogenous Ca2⫹ /calmodulin-dependent protein kinase II was reported to be sufficient in activating the SERCA2 pump, although some investigators were unable to demonstrate this activation. Insulin and tyrosine kinases may also modulate SERCA activity. Studies indicate that the insulin receptor substrates (IRS1 and IRS2) can bind to SERCA1 and SERCA2 in an insulin-regulated manner and that insulin stimulates this association (Algenstaedt et al., 1997). Because insulin receptor substrates can be phosphorylated by tyrosine kinase, this observation raises the possibility of a novel regulatory mechanism for SERCA activity. However, the molecular nature and specificity of the interactions among insulin, tyrosine kinase, and the SERCA pump remain to be determined.

IV. SR Ca2⫹-ATPase AND CARDIAC FUNCTION During the last decade, advances in the technology applied to genetic manipulation have provided novel

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approaches to gain insights into the role of SR Ca2⫹ handling proteins in cardiac physiology. The functional role of SERCA2 and phospholamban (see earlier discussion) in the regulation of Ca2⫹ homeostasis and contractility in the mammalian heart has been elucidated by the development of genetically modified mice with either overexpression or ablation of these proteins. Overexpression of SRCA2a in the cardiac compartment was associated with increased rates of contraction and relaxation in single cardiomyocytes isolated from transgenic mice overexpressing SERCA2a (He et al., 1997; Baker et al., 1998). The increases in SERCA2a levels in the transgenic hearts also reflected increases in contractile parameters assessed in work-performing heart preparations and in vivo using cardiac catheterization. Consistent with the increases in SERCA2a protein levels, the maximal velocity of SR Ca2⫹ transport was enhanced and the Ca2⫹ kinetics in isolated cardiomyocytes were accelerated. These findings indicate that the SERCA2 pump plays a critical role in refilling the SR with Ca2⫹ and that increased SERCA2 expression enhances cardiac contraction and relaxation parameters. This hypothesis was further supported by reports using gene transfer techniques. In neonatal rat myocytes infected with adenovirus-SERCA2a, the protein levels of SERCA2a were increased, which led to a higher Ca2⫹ transient amplitude, lower diastolic Ca2⫹ concentration, faster decline of Ca2⫹ transients, and greater cell shortening (Hajjar et al., 1998). In contrast, in vitro gene transfer of phospholamban was shown to reduce Ca2⫹ kinetics and myocyte relaxation in rats (Hajjar et al., 1998). It is also interesting to note that transgenic mice with ectopic expression of SERCA1a in the heart showed an enhanced maximum velocity of SR Ca2⫹ uptake and cardiac contractile parameters (Loukianov et al., 1998), similar to overexpression of SERCA2a, indicating that SERCA1a can functionally substitute for the endogenous SERCA2a. Reduction in SERCA2a levels, obtained by targeting the gene in embryonic stem cells, resulted in depressed rates of cardiac contraction and relaxation. In SERCA2 heterozygous mutant mouse hearts, which express 65% of the protein levels of SERCA2 compared to wild types, the reduction in SERCA levels was associated with decreases in systolic ventricular pressure, in vivo rates of cardiac contraction, and mean arterial blood pressure (Periasamy et al., 1999). Maximally stimulated cardiac contractile parameters by 웁-adrenergic agonists were also lower in SERCA2 heterozygous hearts than in wild-type hearts, consistent with the reduced SERCA2 levels and diminished maximal velocity of the SR Ca2⫹ transport system. Thus, SERCA2 is a major determinant of contractility in the mammalian heart.

V. SR Ca2⫹-ATPase IN CARDIAC DISEASES The complex regulation of the SR function clearly indicates that even small disturbances in SR Ca2⫹ handling may result in profound changes and deterioration of normal myocardial function. The fast removal of Ca2⫹ by the SR Ca2⫹-ATPase during diastole and the subsequent rapid release through the SR Ca2⫹ channel (ryanodine receptor) at the beginning of contraction are prerequisites for normal diastolic and systolic function. The next section briefly outlines the current state of research on the alterations in the SR Ca2⫹-ATPase and their potential implications in major cardiac diseases.

A. SR Ca2⫹-ATPase in Heart Failure In heart failure, alterations of intracellular Ca2⫹ handling are thought to be a major contributor to impaired contraction and relaxation. Ca2⫹ kinetics is characterized by diminished amplitude, elevated diastolic Ca2⫹ levels, and prolonged decay of the Ca2⫹ transient in failing cardiac myocyte. Thus, gene expression and function of SERCA in failing myocardium have been an area of intensive investigation in an attempt to better understand the pathophysiology of heart failure. Quantitative analysis of SERCA gene expression with correlation to functional alterations has been reported in both experimental animal models and in human failing hearts, and they indicate that abnormal Ca2⫹ handling in heart failure is due, at least in part, to a reduction in SERCA pump activity. Dilated cardiomyopathy is a frequent form of cardiac muscle disease and is characterized by an impaired systolic function and dilatation of both ventricles (systolic pump failure). In various animal models of primary and secondary dilated cardiomyopathy, it was shown that both SR Ca2⫹-binding capacity and uptake were depressed because of the decreased activity and protein level of the SR Ca2⫹-ATPase (Edes et al., 1991). In some studies of human end stage idiopathic-dilated cardiomyopathy, decreases were noted for SERCA2 protein levels, SR Ca2⫹ uptake rates, and Ca2⫹-ATPase activity as well as myocardial Ca2⫹ handling (Hasenfuss et al., 1997; Hajjar et al., 1998). Examination of mRNA levels in left ventricular biopsies from patients with dilated cardiomyopathy revealed a significant decrease in mRNA content for the SR Ca2⫹-ATPase relative to other mRNA forms (Mercadier et al., 1990; Arai et al., 1993). Another type of cardiomyopathy, hypertrophic cardiomyopathy, has only been recognized in clinical practice since the early 1970s. The characteristics of this disease are asymmetric interventricular septal hypertrophy and narrowing of the left ventricular outflow tract,

25. SR Ca2⫹ Transport

with or without outflow obstruction (outflow tract pressure gradient). It was shown that in the familial form of hypertrophic cardiomyopathy, which accounts for about 60% of all cases, mutations in myofibrillar protein genes (웁-myosin heavy chain, troponin T, 움-tropomyosin, and C protein) are associated with the disease (Schwartz et al., 1996). Additionally, prolongation of the Ca2⫹ transient, abnormal Ca2⫹ handling, and a decline in SR Ca2⫹ATPase mRNA levels are reported to be characteristic for human hypertrophic cardiomyopathy (Hasenfuss et al., 1997; Mercadier et al., 1990), which may explain the diastolic functional impairment in this disease. In chronic heart failure due to hemodynamic overload, irrespective of the specific etiology (valvular heart disease, cardiomyopathy, chronic ischemic heart disease, or hypertension), a reduction was observed in both the number and the activity of the SR Ca2⫹ pump (Hasenfuss et al., 1997). Furthermore, a close correlation was obtained between SR Ca2⫹-ATPase mRNA or protein levels and myocardial function (Mercadier et al., 1990; Hasenfuss et al., 1997). Using a rat model with postinfarction heart failure, an increase in SERCA expression levels through long-term growth hormone therapy was shown to enhance the contractile reserve in cardiac myocytes (Tajima et al., 1999). These findings indicate that a defect in SR function is one of the key abnormalities in heart failure. However, some investigators were unable to detect a decrease in SR Ca2⫹ uptake activity or the immunodetectable levels of the SR Ca2⫹-ATPase protein in the left ventricular myocardium from patients with idiopathicdilated cardiomyopathy. Furthermore, a controversy still exists regarding the altered SR function and the accompanied changes in the expression of cardiac SR Ca2⫹-ATPase at mRNA or protein level. In the left ventricular myocardium of patients undergoing heart transplantation, due to idiopathic-dilated cardiomyopathy or ischemic cardiomyopathy, the protein levels of SERCA or phospholamban were not changed, whereas the mRNA levels of SERCA and phospholamban were reduced significantly (Linck et al., 1996). Discrepancies between mRNA levels and protein levels may be due to mRNA processing, posttranslational modification, or structural changes in SERCA in relation to other SR proteins. It is also interesting to note that Na⫹ –Ca2⫹ exchanger gene expression was reported to be increased in failing human hearts, and it was hypothesized that the upregulation of this protein may compensate for the depressed SR function. Furthermore, the gating mechanism of the SR Ca2⫹ release channel was reported to be abnormal in dilated cardiomyopathy, and it was suggested that defective excitation–contraction coupling is involved in the pathogenesis of this disease.

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B. SR Ca2⫹-ATPase in Myocardial Ischemia A brief period of ischemia (10–20 min) induces reversible tissue damage in cardiac muscle, resulting in the ‘‘stunned’’ myocardium. This condition is characterized by regional contractile abnormalities (declines in both systolic and diastolic function), which persist for several hours, despite the absence of necrosis. These hemodynamic changes are associated with a reduction in SR Ca2⫹ transport (Krause et al., 1989). The maximal activity of the SR Ca2⫹-ATPase was found to be depressed, and the Ca2⫹ sensitivity of this enzyme was decreased (Krause et al., 1989). In isovolumic rat heart preparations, slowing of the Ca2⫹ transient decline led to slowed relaxation during myocardial ischemia. Interestingly, the slowed decline of the Ca2⫹ transient was also observed using cyclopiazonic acid to inhibit SR Ca2⫹-ATPase, suggesting that the impaired Ca2⫹ uptake rate is a major injury causing slowed relaxation during ischemia (Halow et al., 1999). Furthermore, a decrease in the coupling ratio (mol Ca2⫹ /mol ATP) was observed in SR membranes isolated from the stunned myocardium, which was suggested to be the result of an increase in the Ca2⫹ permeability of the SR membrane. The SR Ca2⫹ release process was also found to be impaired in the stunned myocardium due to the reduction of the number of ryanodine receptors. These data suggest that complex modifications of the SR function occur in the stunned myocardium, which are at least partly responsible for the contractile impairment found in this condition. In long-lasting myocardial ischemia, gradual declines in both SR Ca2⫹-ATPase activity and Ca2⫹ uptake were found, which may be due to degradation of the SR Ca2⫹-ATPase (Schoutsen et al., 1989). Ischemia was also shown to result in a gradual decrease in the phosphorylation status of phospholamban under both in vitro (Schoutsen et al., 1989) and in vivo conditions, which correlated with a decrease in SR Ca2⫹-ATPase activity. Thus, it has been postulated that the long-lasting ischemia-induced progressive inactivation of the SR Ca2⫹ pump not only is a consequence of the specific loss of enzyme activity, but may also be related to the altered characteristics of phospholamban (Schoutsen et al., 1989). However, some investigators failed to detect any changes in the mRNA or protein levels of SERCA or phospholamban during ischemia followed by reperfusion, whereas the contractile function was depressed (Luss et al., 1998). These results suggest that mechanisms other than altered expression of SERCA and/or phospholamban may be involved in cardiac dysfunction observed during acute ischemia, short-term hibernation, and stunning. A combination of various pathogenic factors has been suggested to be responsible for the re-

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duced SR function and the final tissue necrosis in the ischemic myocardium. These pathological factors include pH reduction (acidosis), activation of intracellular proteolytic enzymes, and increased generation of free radicals.

C. SR Ca2⫹-ATPase in Hyperthyroidism and Hypothyroidism Thyroid hormones are important regulators of myocardial contractility and relaxation. Chronic increases in thyroid hormone levels lead to cardiac hypertrophy, with increases in the heart rate and cardiac output as well as left ventricular contractility and velocity of relaxation. However, opposite effects are associated with a hypothyroid condition. Mechanisms underlying these changes have been the subject of numerous investigations. It is assumed that in hypo- and hyperthyroid hearts the altered gene expression of cardiac SR proteins, and hence the changes in intracellular Ca2⫹ transients, is the most important determinant of the altered myocardial function. It was shown that the velocity of ATP-dependent Ca2⫹ transport and the Ca2⫹-ATPase activity are specifically increased in SR vesicle preparations from hyperthyroid compared with euthyroid hearts (Beekman et al., 1989). Opposite changes were noted for hypothyroid animals compared with euthyroid ones (Beekman et al., 1989). Examination of the steady-state mRNA levels of the cardiac SR Ca2⫹-ATPase and the ryanodine receptor revealed a significant increase (140–190%) in hyperthyroid animals and a marked decline (40–50%) in hypothyroid animals (Arai et al., 1991). The changes in mRNA levels for Ca2⫹-ATPase in hypothyroid and hyperthyroid conditions also reflected changes in the protein amounts of the enzyme in these hearts (Kiss et al., 1994, 1998). Interestingly, in the case of phospholamban, the regulator of Ca2⫹-ATPase, and calsequestrin there was no coordinated regulation with respect to Ca2⫹-ATPase. In fact, both the relative mRNA level and the protein content of phospholamban were reported to decrease in hyperthyroid animals, whereas there was no change noted in the calsequestrin mRNA level upon L-thyroxine treatment. In hypothyroid hearts, an opposite trend was noted, as the protein amount of phospholamban was found to be increased as compared to euthyroid or hyperthyroid animals (Kiss et al., 1994, 1998). Consequently, the phospholamban/Ca2⫹-ATPase protein ratio was highest in hypothyroid animals, followed by euthyroid and hyperthyroid animals. Alterations in the phospholamban/Ca2⫹-ATPase ratio were associated with coordinate alterations in SR Ca2⫹ uptake, affinity of the SERCA2 for Ca2⫹, and myocardial function (Kiss et al., 1994; Kimura et al., 1994).

These changes indicate that the SR proteins responsible for Ca2⫹ uptake and release (Ca2⫹-ATPase and ryanodine receptor) are coordinately regulated in hypothyroid and hyperthyroid hearts and provide a simple explanation for the altered Ca2⫹ release and reuptake capacity, and hence the myocardial function under these conditions.

VI. SUMMARY Ca2⫹ acts as an intracellular second messenger, relaying information within the cardiac cells to regulate the strength, velocity, and frequency of cardiac contraction and relaxation. To coordinate all these activities, Ca2⫹ signals need to be extremely flexible yet precisely regulated. Tight control and flexibility of Ca2⫹ signaling are achieved through multiple regulatory mechanisms as illustrated in this chapter, which make it possible for Ca2⫹ to act in the various combinations of space (extracellular, intracellular or within the lumen of the SR), time (‘‘frequency modulation signaling’’), and amplitude (‘‘amplitude modulation signaling’’) in the heart to fit its physiology and pathophysiology. Ca2⫹ levels in muscle are primarily regulated by the sarcoplasmic reticulum network, which serves as a sink for Ca2⫹ ions during relaxation and as a Ca2⫹ source during contraction. In cardiac muscle, most of the intracellular Ca2⫹ released during systole is taken up by the SR through its Ca2⫹-ATPase. This translocation of Ca2⫹ from the cytosol into the SR lumen uses ATP as the energy source, and it is characterized by the formation of a phosphorylated intermediate (E1앑P) for the Ca2⫹-ATPase. One of the most important regulatory proteins of SR Ca2⫹-ATPase pump activity (SERCA1 and SERCA2 but not SERCA3) is phospholamban, which is present in cardiac muscle, slow-twitch skeletal muscle, and smooth muscle. In its dephosphorylated form, phospholamban is an inhibitor of Ca2⫹-ATPase and phosphorylation relieves this inhibition. Phosphorylation of phospholamban occurs by cAMP-dependent, cGMP-dependent, Ca2⫹ /calmodulin-dependent, and Ca2⫹ /phospholipid-dependent protein kinases in vitro. In vivo, phospholamban is phosphorylated only by cAMP-dependent and Ca2⫹ /calmodulin-dependent protein kinases in intact beating hearts. Phospholamban phosphatase activity has been reported to be present in SR membranes, which can dephosphorylate this regulatory protein and reverse its stimulatory effects on Ca2⫹ATPase. However, the molecular mechanisms by which phospholamban regulates SR Ca2⫹-ATPase and how phospholamban and SR Ca2⫹-ATPase interact in concert to fine-tune intracellular Ca2⫹ levels, and thus myocardial contractility, are not fully understood. Other

25. SR Ca2⫹ Transport

regulatory mechanisms of SERCA function, including sarcolipin and direct phosphorylation of SERCA protein by Ca2⫹ /calmodulin-dependent kinase II or tyrosine kinase, have also been proposed, which merit further investigation. Alterations in the expression levels of SR Ca2⫹ATPase and its regulator phospholamban have been linked to altered Ca2⫹ homeostasis and cardiac dysfunction, ischemia, hypertrophy, and heart failure. Studies also indicate that SERCA activity and phospholamban expression levels may be implicated in the cardiac phenotypes of hyperthroidism and hypothroidism. In most instances, alterations in SR Ca2⫹-ATPase protein levels and activity correlated with alterations in myocardial function. However, it still needs to be determined whether alterations in the expression levels of SR Ca2⫹ATPase are etiological for heart failure or simply represent an adaptive response in the course of the disease. The primary structure of the various Ca2⫹ pumps has been known, and there is growing interest in the further use of molecular biological approaches and specifically site-directed mutagenesis for these enzymes to obtain more information about their structural–functional relationships. The ultimate question is: what is the precise mechanism by which Ca2⫹ is transported across ATPases? Site-directed mutagenesis studies and construction of molecular models have already given some information along these lines and hopefully will provide further data that will finally answer this question. Furthermore, in the absence of appropriate crystallographic data, a deeper understanding of the molecular mechanisms involved in the regulation of SR Ca2⫹-ATPases under normal and pathological conditions may elucidate the structural–functional relationships in these enzymes and their role in maintaining Ca2⫹ homeostasis in the cell.

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lation of cardiac sarcoplasmic reticulum. Biochim. Biophys. Acta 844, 193–199. Kranias, E. G. (1985b). Regulation of calcium transport by protein phosphatase activity associated with cardiac sarcoplasmic reticulum. J. Biol. Chem. 260, 11006–11010. Kranias, E. G., and Di Salvo, J. (1986). A phospholamban protein phosphatase activity associated with cardiac sarcoplasmic reticulum. J. Biol. Chem. 261, 10029–10032. Krause, S. M., Jacobus, W. E., and Becker, L. C. (1989). Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic ‘‘stunned’’ myocardium. Circ. Res. 65, 526–530. Linck, B., Boknik, P., Eschenhagen, T., Muller, F. U., Neumann, J., Nose, M., Jones, L. R., Schmitz, W., and Scholz, H. (1996). Messenger RNA expression and immunological quantification of phospholamban and SR Ca2⫹-ATPase in failing and nonfailing human hearts. Cardiovasc. Res. 31, 625–632. Lindemann, J. P., and Watanabe, A. M. (1985). Muscarinic cholinergic inhibition of 웁-adrenergic stimulation of phospholamban phosphorylation and Ca2⫹ transport in guinea pig ventricles. J. Biol. Chem. 260, 122–133. Loukianov, E., Ji, Y., Grupp, I. L., Kirkpatrick, D. L., Baker, D. L., Loukianova, T., Grupp, G., Lytton, J., Walsh, R. A., and Periasamy, M. (1998). Enhanced myocardial contractility and increased Ca2⫹ transport function in transgenic hearts expressing the fasttwitch skeletal musclesarcoplasmic reticulum Ca2⫹-ATPase. Circ. Res. 83, 889–897. Luo, W., Grupp, I. L., Harrer, J., Ponniah, S., Grupp, G., Duffy J. J., Doetschman, T., and Kranias, E. G. (1994). Targeted ablation of phospholamban gene is associated with markedly enhanced myocardial contractility and loss of 웁-adrenergic stimulation. Circ. Res. 75, 401–409. Luo, W., Wolska, B. M., Grupp, I. L., Harrer, J. M., Haghighi, K., Ferguson, D. G., Slack, J. P., Grupp, G., Doetschman, T., Solaro, R. J., and Kranias, E. G. (1996). Phospholamban gene dosage effect in the mammalian heart. Circ. Res. 78, 839–847. Luo, W., Chu, G., Sato, Y., Zhou, Z., Kadambi, V., and Kranias, E. G. (1997). Transgenic approaches to define the functional role of dual site phosphorylation of phospholamban. J. Biol. Chem. 273, 4734–4739. Luss, H., Boknik, P., Heusch, G., Muller, F. U., Neumann, J., Schmitz, W., and Schulz, R. (1998). Expression of calcium regulatory proteins in short-term hibernation and stunning in the in situ porcine heart. Cardiovasc. Res. 37, 606–617. MacLennan, D. H., Rice, W. J., and Green N. M. (1997). The mechanism of Ca2⫹ transport by sarco(endo)plasmic reticulum Ca2⫹ATPases. J. Biol. Chem. 272, 28815–28818. MacLennan, D. H., Kimura, Y., Toyofuku T. (1998). Sites of regulatory interaction between Ca2⫹-ATPases and phospholamban. Ann. N. Y. Acad. Sci. 853, 31–42. Mercadier, J. J., Lompre, A. M., Duc, P., Boheler, K. R., Fraysse, J. B., Wisnewsky, P., Allen, P. D., Komajda, M., and Schwartz, K. (1990). Altered sarcoplasmic reticulum Ca2⫹-ATPase gene expression in the human ventricle during end-stage heart failure. J. Clin. Invest. 85, 305–309. Misquitta, C. M., Mack, D. P., and Grover, A. K. (1999). Sarco/ endoplasmic reticulum Ca2⫹ (SERCA)-pumps: Link to heart beats and calcium waves. Cell Calcium 25, 277–290.

Movsesian, M. A., Nishikawa, M., and Adelstein, R. S. (1984). Phosphorylation of phospholamban by calcium-activated, phospholipid-dependent protein kinase. J. Biol. Chem. 259, 8029–8032. Odermatt, A., Becker, S., Khanna, V. K., Kurzydlowski, K., Leisner, E., Pette, D., and MacLennan, D. H. (1998). Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2⫹-ATPase. J. Biol. Chem. 273, 12360–12369. Periasamy, M., Reed, T. D., Liu, L. H., Ji, Y., Loukianov, E., Paul, R. J., Nieman, M. L., Riddle, T., Duffy, J. J., Doetschman, T., Lorenz, J. N., and Shull, G. E. (1999). Impaired cardiac performance in heterozygous mice with a null mutation in the sarco/ endoplasmic reticulum Ca2⫹-ATPase isoform 2 (SERCA2) gene. J. Biol. Chem. 274, 2556–2562. Raeymakers, L., Hofmann, F., and Casteels, R. (1988). Cyclic GMPdependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle. Biochem. J. 252, 269–273. Ringer, S. A. (1883). A further contribution regarding the influence of different constituents of the blood on the contraction of the heart. J. Physiol. 4, 29–42. Schoutsen, B., Blom, J. J., Verdouw, P. D., and Lamers, J. M. (1989). Calcium transport and phospholamban in sarcoplasmic reticulum of ischemic myocardium. J. Mol. Cell. Cardiol. 21, 719–727. Schwartz, K., and Mercadier, J.-J. (1996). Molecular and cellular biology of heart failure. Curr. Opin. Cardiol. 11, 227–236. Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986). Sequence analysis of phospholamban: Identification of phosphorylation sites and two major structural domains. J. Biol. Chem. 261, 13333–13341. Simmerman, H. K. B., Jones, L. R. (1998). Phospholamban: Protein structure, machanism of action, and role in cardiac function. Physiol. Rev. 78, 921–947. Simmerman, H. K. B., Kobayashi, Y. M., Autry, J. M., and Jones, L. R. (1996). A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J. Biol. Chem. 271, 5941–5946. Tajima, M., Weinberg, E. O., Bartunek, J., Jin, H., Yang, R., Paoni, N. F., and Lorell, B. H. (1999). Treatment with growth hormone enhances contractile reserve and intracellular calcium transients in myocytes from rats with postinfarction heart failure. Circulation 99, 127–134. Talosi, L., Edes, I., and Kranias, E. G. (1993). Intracellular mechanism mediating the reversal of 웁-adrenergic stimulation in intact beating hearts. Am. J. Physiol. 264, H791–H797. Toyofuku, T., Kurzydlowski, K., Narayanan, N., MacLennan, D. H. (1994a). Identification of Ser38 as the site of cardiac sarcoplasmic reticulum Ca2⫹-ATPase that is phosphorylated by Ca2⫹ /calmodulin-dependent protein kinase. J. Biol. Chem. 269, 26492–26496. Toyofuku, T., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1994b). Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca2⫹-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 269, 3088– 3094. Wolska, B. M., Stojanovic, M. O., Luo, W., Kranias, E. G., and Solaro, R. J. (1996). Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2⫹. Am. J. Physiol. 271, C391–C397.

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26 Calcium Release from Cardiac Sarcoplasmic Reticulum GERHARD MEISSNER Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599

by Ca2⫹. This finding led to the formulation of two different mechanisms of E-C coupling: the mechanical coupling hypothesis in vertebrate skeletal muscle and the Ca2⫹-induced Ca2⫹ release hypothesis in heart (Rios and Pizarro, 1991; Franzini-Armstrong and Protasi, 1997). In mammalian skeletal muscle, voltage-sensing L(long-lasting)-type Ca2⫹ channels/dihydropyridine receptors (DHPRs) located in tubular infoldings (T tubule) of the surface membrane open SR Ca2⫹ release channels through direct protein–protein interactions without functioning as Ca2⫹-conducting channels. In contrast, in cardiac muscle, dihydropyridine-sensitive L-type Ca2⫹ channels (DHPRs) in the surface membrane and T tubule mediate the influx of Ca2⫹ during an action potential, which in turn triggers the massive release of Ca2⫹ by opening SR Ca2⫹ release channels (Fig. 1). Intracellular Ca2⫹ transients arise as the sum of localized Ca2⫹ release events called Ca2⫹ sparks. In cardiac muscle, the opening of a single DHPR may be sufficient to evoke a Ca2⫹ spark by activating a functional Ca2⫹ release unit, which may consist of one or more RyRs (Santana et al., 1996). SR Ca2⫹ release channels, also known as ryanodine receptors (RyRs) or feet, span the gap between the junctional T tubule and SR membranes (Fig. 1). In mammalian ventricle, most RyRs are in close justaposition to L-type Ca2⫹ channels, which provides a morphological basis for Ca2⫹-induced Ca2⫹ release. In contrast, in mammalian atrium, which lacks a well-developed T tubule system, many RyRs are located on the SR away from L-type Ca2⫹ channels (Carl et al., 1995), which suggests differences in the mechanism of SR Ca2⫹ release in ventricle and atrium.

I. INTRODUCTION In mammalian cells, the release of Ca2⫹ from the endo/sarcoplasmic reticulum (SR) occurs through two large Ca2⫹ channel protein complexes: a ryanodinesensitive Ca2⫹ release channel (ryanodine receptor, RyR) and the related inositol 1,4,5-trisphosphate receptor (IP3R) Ca2⫹ channel. The RyR ion channel is activated by a surface membrane excitatory electrical signal, known as excitation–contraction (E-C) coupling. The IP3R ion channel releases Ca2⫹ in response to a chain of voltage-independent events that involve the hormoneinduced formation of inositol 1,4,5-trisphosphate (IP3). E-C coupling predominates in skeletal and cardiac muscle, whereas the IP3-dependent, voltage-independent mechanism is active in smooth muscle. Immunocytochemical and subcellular fractionation studies indicate that the IP3 receptor is present at low levels in ventricular and atrial cardiomycytes, absent from the SR, and confined to regions near the intercalated discs (Kijima et al., 1993).

II. MECHANISMS OF EXCITATION–CONTRACTION COUPLING In cardiac and skeletal muscle, an action potential initiates the release of Ca2⫹ from the SR; however, there are important differences in skeletal and cardiac RyR activation. One distinguishing feature is that skeletal muscle E-C coupling is not dependent on extracellular Ca2⫹, whereas cardiac muscle E-C coupling is regulated

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FIGURE 1 Schematic representation of a segment of a ventricular cardiac muscle cell. The drawing depicts the surface membrane and its invaginations, the transverse (T) tubule, and an intracellular membrane system, the sarcoplasmic reticulum (SR). Ca2⫹ release is mediated by SR Ca2⫹ release channels that span the gap between the SR and T tubule membranes and form junctional couplings with the T tubule. Ca2⫹ sequestration is mediated by the SR Ca2⫹ pump or Ca2⫹-ATPase. Not shown are SR Ca2⫹ release channels that form junctional couplings with the surface membrane.

Other mechanisms that may contribute to cardiac E-C coupling include a partial regulation of the cardiac RyR by the DHPR via a voltage-dependent mechanism comparable to that in skeletal muscle. In addition, all mammalian heart cells contain two additional, wellcharacterized Ca2⫹ entry pathways, T(transient)-type Ca2⫹ channels and the Na⫹ –Ca2⫹ exchanger. T-type Ca2⫹ channels activate and inactivate at potentials more negative than L-type Ca2⫹ channels and are relatively insensitive to blockers of L-type channels. The Na⫹ –Ca2⫹ exchanger is voltage dependent by exchanging three Na⫹ for one Ca2⫹. Its major function is to extrude Ca2⫹ and hence to contribute to muscle relaxation. However, the

exchanger also functions in the reverse mode and mediates the entry of Ca2⫹ into cardiac cells during an action potential. Both the T-type Ca2⫹ channel and the Na⫹ – Ca2⫹ exchanger can trigger SR Ca2⫹ release; however, there is little evidence that they have a significant role in initiating SR Ca2⫹ release in normal hearts. The contribution of extracellular Ca2⫹ entry to cardiac contraction varies with species and age. In adult hearts, up to 90% of the Ca2⫹ for contraction can be provided by the SR. In neonatal hearts, the SR is only partially developed. Therefore, Ca2⫹ fluxes across the surface membrane take on a greater role in neonatal hearts in regulating muscle contraction and relaxation.

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Sequestration of Ca2⫹ into the SR by a Ca2⫹-transporting ATPase and extrusion by a surface membrane Ca2⫹-transporting ATPase and the Na⫹ –Ca2⫹ exchanger restore the myofibrillar Ca2⫹ concentration from 10⫺6 – 10⫺5 to 앑10⫺7 M, causing muscles to relax. Five distinct Ca2⫹-ATPase isoforms encoded by three different genes have been identified. Heart muscle expresses primarily the cardiac/slow-twitch skeletal muscle (SERCA2a) isoform, which transports two Ca2⫹ into the SR by a mechanism that involves the formation of an acid-stable phosphoenzyme intermediate and the hydrolysis of one ATP. In normal beating hearts, lumenal ionized SR Ca2⫹ has been estimated at about 1.0 mM and the ionized cytosolic Ca2⫹ at about 0.1 애M, yielding a 10,000-fold Ca2⫹ gradient across the SR membrane (Chen et al., 1998). Storage of Ca2⫹ by SR is facilitated by a number of lumenal Ca2⫹-binding proteins, including calsequestrin and calreticulin. In addition to causing muscle to relax, SR Ca2⫹ATPase influences the amount of SR Ca2⫹ that is available for release during the next beat. Its activity and hence the SR Ca2⫹ content is regulated by phosphorylation of phospholamban, a 6-kDa intrinsic SR membrane protein (Luo et al., 1994). The dephospho form of phospholamban inhibits Ca2⫹-ATPase via tight association with the enzyme. Phosphorylation causes dissociation and enzyme activation, primarily by increasing the affinity for Ca2⫹. Both cAMP-dependent protein kinase and Ca2⫹ /calmodulin-dependent protein kinase phosphorylate phospholamban in intact ventricles in response to 웁-adrenergic stimulation (Wegener et al., 1989).

and brain (RyR3) RyR. RyR2 is the predominant isoform in cardiac muscle; however, the cardiac isoform is also expressed in brain and other tissues at low levels (Sutko and Airey, 1996). The brain RyR is expressed as a minor component in cardiac muscle; however, unlike the cardiac isoform, it apparently is not essential for muscle function (Takeshima et al., 1996, 1998). The predicted amino acid sequence similarity among the three mammalian RyRs is 65–70%. The C terminus represents the most hydrophobic region of the RyRs and accordingly has been proposed to form the Ca2⫹ channel pore (Takeshima et al., 1989). Studies with tryptic fragments and deletion mutants of the skeletal muscle RyR have confirmed that this region represents the Ca2⫹-conducting region. The remaining amino acid sequences are highly hydrophilic and form the ‘‘foot structure’’ that projects into the cytosol toward T tubule DHPRs (Fig. 2). In negatively stained electron micrographs, the cardiac RyR exhibits a fourfold symmetry with a quatrefoil, four-leaf structure (Anderson et al., 1989). Cryo-electron microscopic and image analysis reveals that RyRs are composed of a large, loosely packed 29 ⫻ 29 ⫻ 12-nm cytosolic foot region and a smaller transmembrane region that extends 앑7 nm toward the SR lumen (Wagenknecht and Radermacher, 1997). Various regulatory sites are localized on the large cytosolic foot structure, such as those for calcium, magnesium, ATP, and calmodulin. Two SR junctional proteins, triadin and junctin, interact physically with the RyR and may modulate channel activity (Zhang et al., 1997). RyRs also contain phosphorylation sites and reactive thiols, which suggest that receptor phosphorylation and reactive nitrogen and oxygen species have a role in the in vivo regulation of channel activity.

III. STRUCTURE OF Ca2⫹ RELEASE CHANNEL/RyR The molecular properties and function of the cardiac muscle RyR have been studied most extensively using SR membrane fractions isolated from canine and rabbit hearts. Fragmentation of the SR during homogenization and subsequent fractionation by differential and sucrose density gradient centrifugation yields microsomal membrane fractions that are enriched in Ca2⫹ release and [3H]ryanodine-binding activities. The RyR is purified as a 30S protein complex composed of four 560-kDa subunits (Meissner, 1994) in tight association with four 12.6-kDa (FK506 binding protein) subunits (Timerman et al., 1996). While much has been learned about the function of the large RyR subunits, the precise role of the small subunits is unknown. Mammalian cells express three structurally and functionally related 560-kDa RyR polypeptides, referred to as the skeletal muscle (RyR1), cardiac muscle (RyR2),

IV. REGULATION OF RyR BY ENDOGENOUS EFFECTORS RyRs are cation-selective channels capable of multiple interactions with other molecules. Their in vitro function has been studied extensively in SR vesicle– Ca2⫹ flux and single channel measurements. A third more indirect method relies on the use of ryanodine, a plant alkaloid that binds with high affinity and specificity to RyRs in skeletal and cardiac muscle, brain, and other tissues. Ryanodine modifies the conductance and gating of the RyR ion channels by forming an open subconductance state at nanomolar to micromolar concentrations and by fully closing channels at elevated concentrations (Fig. 3). [3H]Ryanodine is widely used as an indicator of channel activity due to its preferential binding to open channels.

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FIGURE 2 Effectors of the cardiac Ca2⫹ release channel/ryanodine receptor.

FIGURE 3 Effects of ryanodine on a single purified canine cardiac Ca2⫹ release channel reconstituted in a planar lipid bilayer. Channel currents, shown as upward deflections (–, closed channel), were recorded in symmetric 0.25 M KCl, pH 7.35, medium containing 10 애M free cytosolic Ca2⫹. The upper trace shows the abrupt appearance of a subconductance state with a conductance of about half of that of the unmodified channel and a channel open probability (Po) of 앑1, several minutes after the addition of 2 애M cytosolic ryanodine. The lower trace shows the transition from the subconductance state to a fully closed state about 1 min after the addition of 100 애M cytosolic ryanodine (from Xu et al., 1998b).

RyRs conduct monovalent ions more efficiently than Ca2⫹ (앑750 pS with 250 mM K⫹ vs 앑150 pS with 50 mM Ca2⫹ as the current carriers) (Meissner, 1994). They are Ca2⫹-gated channels that are activated by micromolar cytosolic Ca2⫹ and inhibited by millimolar cytosolic Ca2⫹. As shown in single channel measurements of Fig. 4, the cardiac RyR ion channel can be nearly maximally activated by Ca2⫹. Bimodal Ca2⫹ dependence of channel activity suggests the presence of high-affinity activating and low-affinity inhibitory Ca2⫹binding sites. These are presumed to be located on the large cytosolic foot region of RyRs. In addition to cytosolic Ca2⫹, SR lumenal Ca2⫹ regulates skeletal and cardiac muscle RyRs (Xu and Meissner, 1998). Two different channel sites have been proposed: binding of Ca2⫹ to lumenal channel sites and access of lumenal Ca2⫹ to cytosolic Ca2⫹ activation and inactivation sites. At the present time, the reasons for the different results are not clear, but they may reflect a predominance of one of the two mechanisms, depending on the experimental conditions. Other possible Ca2⫹-dependent mechanisms include regulation of RyR by SR lumenal (calsequestrin) and cytosolic (calmodulin) Ca2⫹-binding proteins. In cardiac muscle, an action potential causes RyR2 to open and close rapidly. Photorelease of Ca2⫹ from caged compounds and rapid solution changes show that Ca2⫹ activates the cardiac RyR on a time scale of milliseconds. Ca2⫹ is also thought to be important for closing the cardiac Ca2⫹ release channel; however, the

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mechanism of channel inactivation remains to be established. The cardiac RyR ion channel is regulated by various endogenous and exogenous effector molecules, in addition to Ca2⫹ (Meissner, 1994; Fig. 2). These include small diffusible endogenous molecules, such as Mg2⫹ and ATP, and the drugs caffeine and ryanodine. Among the adenine nucleotides, cyclic ADP-ribose is most effective in activating the cardiac RyR, although a physiological role of this compound in cardiac muscle is controversial (Guo et al., 1996). Other adenine nucleotides (ATP, ADP, AMP) that strongly activate the skeletal muscle RyR in the absence of Ca2⫹ are less effective in activating the cardiac isoform at low Ca2⫹ concentrations. Millimolar concentrations of Ca2⫹ and Mg2⫹, and nanomolar concentrations of calmodulin, inhibit channel opening (Meissner, 1994). In the presence of Mg2⫹ and ATP, the cardiac channel is rendered less sensitive to inhibition by Ca2⫹, which raises the question of whether Ca2⫹ can effectively inactivate the channel in the myocardium (Xu et al., 1996).

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Moreover, complex regulation of cardiac RyR activity by endogenous and exogenous kinases has been reported. In vitro phosphorylation of serine 2809 by a Ca2⫹ /calmodulin-dependent protein kinase activates the calmodulin-inhibited cardiac RyR (Witcher et al., 1991). The phosphorylation site is close to a calmodulin-binding site, suggesting an interaction between the two regulatory mechanisms. In other studies it was found that phosphorylation modulates inhibition of the cardiac channel by Mg2⫹ (Hain et al., 1995). Mg2⫹ blocked the channel and this block was removed by phosphorylation of the cardiac channel by either cAMP-dependent protein kinase or Ca2⫹ /calmodulindependent protein kinase II. Furthermore, calmodulin was shown to block the channel in the dephosphorylated state, which was overcome by treatment with Ca2⫹ /calmodulin-dependent protein kinase but not by cAMP-dependent protein kinase. These findings suggest that the cardiac RyR apparently must be phosphorylated in the active state at physiological Mg2⫹ concentrations (앑1 mM).

FIGURE 4 Single channel recording of a purified 30S canine cardiac Ca2⫹ release channel. Single channel currents, shown as upward deflections (–, closed channel), were recorded in symmetric 0.25 M KCl medium with the indicated concentrations of free Ca2⫹ at a holding potential of ⫹35 mV (A) and ⫺35 (䊐) and 35 (䊏) mV (B). Data show that channel open probability (Po) is dependent on cis (SR cytoplasmic) Ca2⫹ but not membrane potential (from Xu et al., 1996).

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V. REGULATION OF RyR BY EXOGENOUS EFFECTORS Because of their multiple ligand interactions, RyRs constitute an abundant target for controlling cellular functions. A large number of drugs have been found to affect RyR function, including ryanoids, xanthines, toxins, local anesthetics, and polycationic reagents (Zucchi and Ronca-Testoni, 1997; Xu et al., 1998b; Fig. 2). However, their use as potential therapeutic agents for controlling intracellular Ca2⫹ release from SR has been limited due to a lack of target specificity, with dantrolene (a muscle relaxant used in the prevention and treatment of malignant hyperthermia) being a notable exception. Because of their high selectivity, ryanoids have the potential to serve as therapeutic agents in controlling intracellular Ca2⫹ concentrations. However, naturally occurring ryanoids essentially irreversibly either activate or inactivate RyRs and therefore are too potent as modulators of SR Ca2⫹ release. To be suitable as therapeutic agents, it will be necessary to synthesize ryanoids that reversibly modify the activity of RyRs and are specific for RyR2.

VI. Ca2⫹ RELEASE CHANNEL/RyR FUNCTION IN ISCHEMIC MYOCARDIUM Elimination of the flow of oxygenated blood to the myocardium leads to a variety of electrical, metabolic, and structural changes that may result from the cumulative effects of lack of oxygen and substrate supply, accumulation of metabolites, imbalances in intracellular and extracellular ionic mileu, and protein and lipid modifications. While the changes in H⫹, Ca2⫹, Mg2⫹, and ATP in general are not sufficient to cause substantial impairment of RyR activity within a few minutes of ischemia (Xu et al., 1996), SR Ca2⫹ release is likely affected by pronounced changes that occur during sustained ischemia. These include a fall of intracellular pH to values as low as pH 6.0, depletion of the high-energy adenine nucleotide pool, and increases in cytosolic-free Ca2⫹ and Mg2⫹ concentrations. A change in each of these parameters affects SR Ca2⫹ release, as cardiac RyR activity depends on cytosolic Ca2⫹ concentration, is modulated by Mg2⫹ and ATP, and decreases when the pH is lowered to 6 (Xu et al., 1996). A major effect of decreasing pH is a lowered Ca2⫹ sensitivity of the channel so that higher Ca2⫹ concentrations are required to activate the channel (Fig. 5). Additional factors that might alter SR Ca2⫹ release in ischemic myocardium are changes in RyR2 phosphorylation and redox state; however, these are not well defined.

FIGURE 5 Dependence of [3H]ryanodine binding to cardiac SR vesicles on Ca2⫹ and pH. Specific [3H]ryanodine binding to cardiac SR vesicles was determined in the presence of 5 mM Mg2⫹ and 5 mM AMPPCP (a nonhydrolyzable ATP analogue) at pH 7.3, pH 6.5, and pH 6.2 in 0.1 M KCl medium containing the indicated concentrations of free Ca2⫹ (from Xu et al., 1996).

SR Ca2⫹ flux in normal and ischemic rabbit heart preparations was examined with the use of a SR membrane-permeable Ca2⫹ buffer (Chen et al., 1998). In the normal beating perfused preparation, lumenal-ionized SR Ca2⫹ was about 1.5 mM. SR Ca2⫹ decreased by about 30% at the start of systole. Energetic calculations taking into account SR lumenal and cytosolic Ca2⫹ and ATP phosphorylation potential indicated that the Ca2⫹ gradient across the SR membrane reached about 80% of its thermodynamic limit. Application of isoproterenol yielded a Ca2⫹ gradient closer to its thermodynamic limit, presumably by causing phospholamban phosphorylation and thus allowing the SR Ca2⫹-ATPase to compete more effectively for cytosolic Ca2⫹ and increase SR Ca2⫹ load. In nonpreconditioned and preconditioned ischemic hearts, the SR Ca2⫹ gradient decreased in accordance with a decrease in ATP phosphorylation potential. The decrease was due to an increase in cytosolic Ca2⫹ rather than a decrease in SR Ca2⫹.

VII. S-NITROSYLATION AND OXIDATION OF Ca2⫹ RELEASE CHANNEL/RyR RyRs are excellent targets for nitric oxide (NO) in the normal heart and reactive oxygen molecules formed in the postischemic heart because they contain a large number of free sulfhydryls. The tetrameric mammalian

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RyR2 has 364 cysteines, which includes 89 cysteines per 560-kDa RyR subunit and 2 per FK506-binding protein subunit. In the purified tetrameric RyR2, 앑80 cysteines (20 per subunit) are free (Xu et al., 1998a). Accordingly, in the normal heart, a large number of thiols are likely in a reduced state because cells maintain a reducing environment through thiol-reducing compounds, with the most abundant being glutathione. Reactivity of RyR sulfhydryls has been studied extensively using heavy metals, anthraquinones such as doxorubicin or adriamycin, and specific sulfhydryl-reacting compounds such as 4-(chloro-mercuri)phenylsulfonic acid, thimerosal, 4,4⬘-dithiodipyridine, diamide, and N-ethylmaleimide (Zucchi and Ronca-Testoni, 1997). Lower concentrations of these compounds in general activate, whereas higher concentrations inhibit RyRs. An early model postulated a complex redox site on the skeletal muscle RyR composed of several SH groups (Abramson and Salama, 1989). The reversible oxidation and reduction of these sulfhydryl groups may aid in the opening and closing of RyRs. Reactive oxygen intermediates are formed extensively during the reoxygenation of ischemic tissue and include superoxide (O2⫺), hydroxyl radicals (OH ⭈ ), and hydrogen peroxide (H2O2). Early studies indicated that SR Ca2⫹ uptake in response to reactive oxygen species decreased. Subsequent studies have suggested that reactive oxygen species accomplish this primarily by affecting the activity of the RyRs. Both superoxide and hydrogen peroxide activated skeletal and cardiac muscle RyRs. More recently, it has been suggested that calmodulin is, to a large part, responsible for the increased release of Ca2⫹ from the SR. Sulfhydryl oxidation decreased the inhibition of RyRs by calmodulin. In turn, calmodulin may protect RyRs from oxidative modifications during periods of oxidative stress (Zhang et al., 1999). NO is a ubiquitous regulator of cellular functions and fulfills many of the criteria of a physiological modulator of cardiac and skeletal muscle excitation–contraction coupling (Reid, 1998; Eu et al., 1999). Cardiac muscle cells express all three NO synthase isoforms (eNOS, nNOS, iNOS) and are lined by an endothelium and exposed to several circulating cell types, all of which liberate NO. However, the mechanism of NO regulation is less clear. NO increases cGMP levels in muscle and such increases may alter RyR activity, possibly involving the phosphorylation of RyRs by cGMP-dependent protein kinase or by changing the cytosolic levels of RyR effectors such as cyclic ADP ribose. Another possibility is that NO directly affects RyRs via covalent modifications of thiol groups. Activation and inhibition have been reported, suggesting that NO-generating molecules may modify RyRs in multiple ways (Eu et al.,

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1999). Activation of single RyR2s by the NO-generating molecule nitrosoglutathione (Fig. 6) resulted in nitrosylation and oxidation (two to three and approximately three sites/RyR subunit, respectively) that was reversed by the sulfhydryl-reducing agent dithiothreitol (DTT), whereas oxidation of a greater number of thiols (approximately seven per RyR subunit) by two other NO-generating molecules (nitrosocysteine, 3-morpholinesydnonimine) produced activation that was not reversed by DTT (Xu et al., 1998a). These results suggest that NO-generating molecules can affect the cardiac RyR via covalent modifications of thiol groups, leading to either a reversible or an irreversible alteration of RyR2 ion channel activity. S-Nitrosylation of RyR may be of physiological significance in the normal heart, whereas excess oxidation during periods of oxidative stress can be deleterious, leading to loss of control.

FIGURE 6 Activation of a single purified and reconstituted cardiac Ca2⫹ release channel by nitrosogluthathione (GSNO). Single channel currents, shown as upward deflections (c, closed channel), were recorded in symmetric 0.25 M KCl, pH 7.4, medium containing 2 애M free cytosolic Ca2⫹ before (top trace) and after (middle trace) the successive addition of 1 mM GSNO and 10 mM dithiothreitol (DTT) (bottom trace) to the cis side (SR cytosolic) of a bilayer. Holding potential was 35 mV. Channel open probability (Po) increased 1 min after the addition of 1 mM GSNO and returned close to that of the untreated channel after the addition of 10 mM DTT (from Xu et al., 1998a).

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NO may influence myocardial ischemia–reperfusion injury; however, its function is controversial, as both protective and deleterious effects have been observed. Zweier and Wang (1996) reported that in the postischemic heart during the early period of reflow, NO production increases and reacts with superoxide (O2⫺) to form the highly cytotoxic compound peroxynitrite (OONO⫺), which causes amino acid nitration and cellular injury.

VIII. SR Ca2⫹ TRANSPORT PROTEINS IN HYPERTROPHIC AND FAILING HEARTS Cardiac hypertrophy is an adaptive process that occurs in response to multiple stimuli, including hypertension, increased mechanical load, and myocardial infarction. While an increased growth of the heart can initially compensate for an increased workload, sustained exposure ultimately leads to heart failure. Cellular changes thought to contribute to heart failure include remodeling of the contractile apparatus, prolongation of the cardiac action potential, decreased responsiveness to 웁-adrenergic stimulation, and depressed and delayed relaxation. Alterations in intracellular Ca2⫹ handling are thought to be largely responsible for the repressed and delayed relaxation of failing hearts. However, the mechanisms that are responsible for or lead to the observed changes in intracellular Ca2⫹ homeostasis are less well known. Numerous processes contribute to the rise and fall of intracellular Ca2⫹, and abnormalities can be expected to have profound effects on function and gene expression as Ca2⫹ ions affect multiple signaling pathways. Other difficulties in identifying the underlying mechanisms of an altered Ca2⫹ homeostasis are that the etiology of hypertrophy and heart failure is temporal, depends on the animal model studied, and cellular changes may not occur uniformly throughout the myocardium. Studies with failing human hearts are complicated by drug treatments and ischemic episodes that may introduce additional variables difficult to evaluate. Variable changes in the expression of mRNA and protein levels, as well as activities of Ca2⫹ transport proteins, have been reported in both failing human hearts and experimental animal models (Balke and Shorofsky, 1998; Hasenfuss, 1998). A decrease in RyR2 and SR Ca2⫹-ATPase (SERCA2) message levels has been reported with and without a decrease in protein levels and activities. Similarly, the mRNA level of the SERCA2a regulatory protein phospholamban was downregulated without a clear consensus whether a decrease occurs in the protein level. However, increased mRNA levels and transport activities have been reported for the plasmalemmal Na⫹ /Ca2⫹ exchanger, the other major Ca2⫹ removal system of the heart.

The functional consequences of a decreased SR Ca2⫹ uptake relative to increased Ca2⫹ removal by the Na⫹ / Ca2⫹ exchanger have been investigated in failing human hearts (Dipla et al., 1999) and in a canine tachycardiainduced heart failure model (O’Rourke et al., 1999). Results suggest that an altered expression of Ca2⫹ transport proteins diminishes Ca2⫹ cycling by the SR. Ca2⫹ influx via the Na⫹ /Ca2⫹ exchanger was increased during the action potential, whereas SR Ca2⫹ release and uptake were reduced. Modeling studies suggest that the changes are sufficient to account for the reduced amplitude, altered shape, and slowed Ca2⫹ transients in the failing canine heart. Abnormal Ca2⫹ handling may occur in the absence of apparent changes in the expression of key Ca2⫹ transport proteins. In mildly hypertrophic ventricular cells from spontaneously hypertensensitive rats, contractility, time to relaxation, and average Ca2⫹ spark amplitude were increased without a change in L-type Ca2⫹ channel current density and kinetics or changes in the density of several SR proteins, including the cardiac RyR, Ca2⫹ATPase, and phospholamban (Shorofsky et al., 1999). In single myocytes derived from Dahl salt-sensitive rats with more severe hypertrophy or spontaneously hypertensensitive rats, calcium release was reduced without a change in L-type Ca2⫹ channel current or RyR2 density (Gomez et al., 1997). 웁-adrenergic stimulation largely overcame the defects in hypertrophic but not failing hearts. These results suggest that a change in the phosphorylation state or perhaps a structural rearrangement between L-type Ca2⫹ channels and SR Ca2⫹ release channels may be responsible for the alterations in Ca2⫹ handling observed in cardiac hypertrophy and failure. Several knockout and transgenic mouse models have been developed to clarify the function of SR in normal and failing hearts. Deletion of RyR2 resulted in embryonic lethality and altered cardiomyocytes (Takeshima et al., 1998). Effects of an increased activity of the two major Ca2⫹ removal systems in the heart were investigated in transgenic mice overexpressing the fast-twitch SR skeletal muscle (SERCA1a) or cardiac (SERCA2a) Ca2⫹-ATPase isoforms (Baker et al., 1998) by deleting phospholamban (Luo et al., 1994) or by overexpressing the Na⫹ /Ca2⫹ exchanger (Adachi-Akahane et al., 1997). An increase in Ca2⫹ transient amplitude and cardiac contractility, as well as faster rates of contraction and relaxation, were observed in transgenic hearts that overexpressed the two SR Ca2⫹-ATPases. Targeted ablation of the phospholamban gene increased contractility and the Ca2⫹ affinity of SR Ca2⫹-ATPase to levels equal to those observed in hearts stimulated maximally by the 웁-adrenergic agonist isoproterenol. In cardiac myocytes overexpressing the Na⫹ /Ca2⫹ exchanger, an enhanced Ca2⫹ influx and efflux were measured. L-type Ca2⫹ chan-

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nel current and SR content essentially remained the same. In the mouse models described previously, no significant increases in heart size and gross morphology were observed. However, overexpression of cardiac calsequestrin is associated with cardiac hypertrophy in transgenic mice (Jones et al., 1998; Sato et al., 1998). In isolated transgenic cardiomyocytes, SR Ca2⫹ release was strongly depressed, leading to depressed contractility. Depressed Ca2⫹-induced SR Ca2⫹ release was observed despite an increased SR content. The cellular mechanisms underlying the changes in Ca2⫹ handling in transgenic mice are unclear, as conflicting data were obtained regarding the expression of several SR proteins. Jones et al. (1998) reported that the cardiac RyR and its two associated proteins, triadin and junction, were downregulated, whereas Ca2⫹-ATPase and phospholamban were unchanged or only slightly increased. In contrast, Sato et al. (1998) found that the protein levels of Ca2⫹-ATPase and phospholamban were increased in transgenic hearts, whereas protein levels of the RyR, triadin, and junction were unchanged relative to control mice. To summarize, changes in SR Ca2⫹ uptake and release are seen in hypertrophic and failing hearts. However, the changes in SR Ca2⫹ cycling do not necessarily arise from an altered expression of individual SR proteins. A second major unresolved question is whether the changes in Ca2⫹ handling have a primary or secondary role in inducing cardiac hypertrophy and heart failure. Many signaling molecules that respond to changes in intracellular Ca2⫹ have been implicated in the transition of the normal heart to the hypertrophic heart, and ultimately failing heart. Two prominent Ca2⫹-signaling molecules are calmodulin (Gruver et al., 1993) and calcineurin (Walsh, 1999); however, their precise functions in cardiac hypertrophy and heart failure remain to be established.

IX. SUMMARY Ryanodine receptors are calcium channels that control the levels of intracellular Ca2⫹ by releasing Ca2⫹ from intracellular calcium-storing organelles. They were named ryanodine receptors because of the specific binding of the plant alkaloid ryanodine, which has facilitated their purification and characterization. Mammalian tissues express three structurally and functionally related RyRs (RyR1, RyR2, RyR3), also known as skeletal, cardiac, and brain RyRs, because they were first identified in and isolated from the three respective tissues. The cardiac RyR2 isoform has been isolated as a large protein complex of four 560-kDa (RyR polypeptide)

and four 12.6-kDa (FK506-binding protein) subunits and shown to be regulated by multiple endogenous effectors, including Ca2⫹, Mg2⫹, H⫹, and ATP. In ischemic hearts, a change in each of these parameters affects SR Ca2⫹ release. The cardiac RyR contains phosphorylation sites and reactive thiols, which suggests that protein kinases and phosphatases and reactive nitrogen and oxygen species may influence channel activity in normal, ischemic, and postischemic hearts. Changes in SR Ca2⫹ release and uptake are thought to contribute to the repressed and delayed relaxation of failing hearts; however, the mechanisms responsible for these changes remain to be determined. Recently described transgenic mouse models might help in clarifying the role of specific SR proteins in the Ca2⫹ handling of normal and failing hearts.

Acknowledgment I thank Daniel Pasek for providing Figs. 1 and 2.

Bibliography Abramson, J. J., and Salama, G. (1989). Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum. J. Bioenerg. Biomembr. 21, 283–294. Adachi-Akahane, S., Lu, L., Li, Z., Frank, J. S., Philipson, K. D., and Morad, M. (1997). Calcium signaling in transgenic mice overexpressing cardiac Na⫹-Ca2⫹ exchanger. J. Gen. Physiol. 109, 717–729. Anderson, K., Lai, F. A., Liu, Q. Y., Rousseau, E., Erickson, H. P., and Meissner, G. (1989). Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2⫹ release channel complex. J. Biol. Chem. 264, 1329–1335. Baker, D. L., Hashimoto, K., Grupp, I. L., Ji, Y., Reed, T., Loukianov, E., Grupp, G., Bhagwhat, A., Hoit, B., Walsh, R., Marban, E., and Periasamy, M. (1998). Targeted overexpression of the sarcoplasmic reticulum Ca2⫹-ATPase increases cardiac contractility in transgenic mouse hearts. Circ. Res. 83, 1205–14. Balke, C. W., and Shorofsky, S. R. (1998). Alterations in calcium handling in cardiac hypertrophy and heart failure. Cardiovasc. Res. 37, 290–299. Carl, S. L., Felix, K., Caswell, A. H., Brandt, N. R., Ball, Jr., W. J., Vaghy, P. L., Meissner, G., and Ferguson, D. G. (1995). Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium. J. Cell Biol. 129, 672–682. Chen, W., London, R., Murphy, E., and Steenbergen, C. (1998). Regulation of the Ca2⫹ gradient across the sarcoplasmic reticulum in perfused rabbit heart: A 19F nuclear magnetic resonance study. Circ. Res. 83, 898–907. Dipla, K., Mattiello, J. A., Margulies, K. B., Jeevanandam, V., and Houser, S. R. (1999). The sarcoplasmic reticulum and the Na⫹ / Ca2⫹ exchanger both contribute to the Ca2⫹ transient of failing human ventricular myocytes. Circ. Res. 84, 435–444. Eu, J. P., Xu, L., Stamler, J. S., and Meissner, G. (1999). Regulation of ryanodine receptors by reactive nitrogen species. Biochem. Pharmacol. 57, 1079–1084.

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Franzini-Armstrong, C., and Protasi, F. (1997). Ryanodine receptors of striated muscles: A complex channel capable of multiple interactions. Physiol. Rev. 77, 699–729. Gomez, A. M., Valdivia, H. H., Cheng, H., Lederer, M. R., Santana, L. F., Cannell, M. B., McCune, S. A., Altschuld, R. A., and Lederer, W. J. (1997). Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science 276, 800–806. Gruver, C. L., DeMayo, F., Goldstein, M. A., and Means, A. R. (1993). Targeted developmental overexpression of calmodulin induces proliferative and hypertrophic growth of cardiomyocytes in transgenic mice. Endocrinology 133, 376–388. Hain, J., Onoue, H., Mayrleitner, M., Fleischer, S., and Schindler, H. (1995). Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. J. Biol. Chem. 270, 2074–2081. Hasenfuss, G. (1998). Alterations of calcium-regulatory proteins in heart failure. Cardiovasc. Res. 37, 279–289. Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998). Regulation of Ca2⫹ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. J. Clin. Invest. 101, 1385–1393. Kijima, Y., Saito, A., Jetton, T. L., Magnuson, M. A., and Fleischer, S. (1993). Different intracellular localization of inositol 1,4,5-trisphosphate and ryanodine receptors in cardiomyocytes. J. Biol. Chem. 268, 3499–3506. Luo, W., Grupp, I. L., Harrer, J., Ponniah, S., Grupp, G., Duffy, J. J., Doetschman, T., and Kranias, E. G. (1994). Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ. Res. 75, 401–9. Meissner, G. (1994). Ryanodine receptor/Ca2⫹ release channels and their regulation by endogenous effectors. Annu. Rev. Physiol. 56, 485–508. O’Rourke, B., Kass, D. A., Tomaselli, G. F., Kaab, S., Tunin, R., and Marban, E. (1999). Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure. I. Experimental studies. Circ. Res. 84, 562–570. Reid, M. B. (1998). Role of nitric oxide in skeletal muscle: Synthesis, distribution and functional importance. Acta Physiol. Scand. 162, 401–409. Rios, E., and Pizarro, G. (1991). Voltage sensor of excitation-contraction coupling in skeletal muscle. Physiol. Rev. 71, 849–908. Santana, L. F., Cheng, H., Gomez, A. M., Cannell, M. B., and Lederer, W. J. (1996). Relation between the sarcolemmal Ca2⫹ current and Ca2⫹ sparks and local control theories for cardiac excitationcontraction coupling. Circ. Res. 78, 166–171. Sato, Y., Ferguson, D. G., Sako, H., Dorn, G. W., II, Kadambi, V. J., Yatani, A., Hoit, B. D., Walsh, R. A., and Kranias, E. G. (1998). Cardiac-specific overexpression of mouse cardiac calsequestrin is associated with depressed cardiovascular function and hypertrophy in transgenic mice. J. Biol. Chem. 273, 28470– 28477. Shorofsky, S. R., Aggarwal, R., Corretti, M., Baffa, J. M., Strum, J. M., Al-Seikhan, B. A., Kobayashi, Y. M., Jones, L. R., Wier, W. G., and Balke, C. W. (1999). Cellular mechanisms of altered contractility in the hypertrophied heart: Big hearts, big sparks. Circ. Res. 84, 424–434.

Sutko, J. L., and Airey, J. A. (1996). Ryanodine receptor Ca2⫹ release channels: Does diversity in form equal diversity in function? Physiol. Rev. 76, 1027–1071. Takeshima, H., Ikemoto, T., Nishi, M., Nishiyama, N., Shimuta, M., Sugitani, Y., Kuno, J., Saito, I., Saito, H., Endo, M., Iino, M., and Noda, T. (1996). Generation and characterization of mutant mice lacking ryanodine receptor type 3. J. Biol. Chem. 271, 19649–19652. Takeshima, H., Komazaki, S., Hirose, K., Nishi, M., Noda, T., and Iino, M. (1998). Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2. EMBO J. 17, 3309–3316. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and Numa, S. (1989). Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339, 439–445. Timerman, A. P., Onoue, H., Xin, H. B., Barg, S., Copello, J. Wiederrecht, G., and Fleischer, S. (1996). Selective binding of FKBP12.6 by the cardiac ryanodine receptor. J. Biol. Chem. 271, 20385– 20391. Wagenknecht, T., and Radermacher, M. (1997). Ryanodine receptors: Structure and macromolecular interactions. Curr. Opin. Struct. Biol. 7, 258–265. Walsh, R. A. (1999). Calcineurin inhibition as therapy for cardiac hypertrophy and heart failure: Requiescat in pace? Circ. Res. 84, 741–743. Wegener, A. D., Simmerman, H. K., Lindemann, J. P., and Jones, L. R. (1989). Phospholamban phosphorylation in intact ventricles: Phosphorylation of serine 16 and threonine 17 in response to betaadrenergic stimulation. J. Biol. Chem. 264, 11468–11474. Witcher, D. R., Kovacs, R. J., Schulman, H., Cefali, D. C., and Jones, L. R. (1991). Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J. Biol. Chem. 266, 11144–11152. Xu, L., Eu, J. P., Meissner, G., and Stamler, J. S. (1998a). Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279, 234–237. Xu, L., Mann, G., and Meissner, G. (1996). Regulation of cardiac Ca2⫹ release channel (ryanodine receptor) by Ca2⫹, H⫹, Mg2⫹, and adenine nucleotides under normal and simulated ischemic conditions. Circ. Res. 79, 1100–1109. Xu, L., and Meissner, G. (1998). Regulation of cardiac muscle Ca2⫹ release channel by sarcoplasmic reticulum lumenal Ca2⫹. Biophys. J. 75, 2302–2312. Xu L, Tripathy, A., Pasek, D. A., and Meissner, G. (1998b). Potential for pharmacology of ryanodine receptor/calcium release channels. Ann. N. Y. Acad. Sci. 853, 130–148. Zhang, J. Z., Y. L., Wu, B. Y., Williams, G., Rodney, F., Mandel, G. M., Strasburg, and Hamilton, S. L. (1999). Oxidation of the skeletal muscle Ca2⫹ release channel alters calmodulin binding. Am. J. Physiol. Cell Physiol. 45, C46–C53. Zhang, L., Kelley, J., Schmeisser, G., Kobayashi, Y. M., and Jones, L. R. (1997). Complex formation between junction, triadin, calsequestrin, and the ryanodine receptor: Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J. Biol. Chem. 272, 23389–23397. Zucchi, R., and Ronca-Testoni, S. (1997). The sarcoplasmic reticulum Ca2⫹ channel/ryanodine receptor: Modulation by endogenous effectors, drugs and disease states. Pharmacol. Rev. 49, 1–51.

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27 Function of Vascular Endothelium STEPHANIE H. WILSON and AMIR LERMAN Mayo Clinic and Foundation Rochester, Minnesota 55905

I. INTRODUCTION

lead to the development and progression of atherosclerosis. In view of its key role in early atherogenesis, the endothelium may represent an important early therapeutic target for treatment strategies against coronary artery disease (CAD). This chapter focuses on ‘‘endothelial dysfunction’’ in hypercholesterolemia and diabetes and on the potential avenues or options for therapy in these two well-known risk factors for CAD.

For many years the endothelium was thought of as the vascular tree’s smooth ‘‘cellophane wrapper,’’ with no other specific functions than selective permeability to water and electrolytes. However, enormous advances since the 1980s have led to an understanding of the complex functions of this large endocrine organ. We now know that the endothelium embodies a wide range of homeostatic functions (1), with the ability to act in both sensory and effector capacities. Endothelial cells can sense changes in blood flow and blood pressure, as well as inflammatory and hormonal signals from the bloodstream. In addition, these cells release a variety of vasoactive, anti-inflammatory, and thromboregulatory substances, which potentially play an important role in the prevention of atherosclerosis. Thus, the endothelium may be considered the ‘‘gatekeeper’’ of the vascular wall. The earliest stages of coronary atherosclerosis are characterized by abnormal endothelium-dependent vasorelaxation (2) and are associated with increased endothelial adhesion and subsequent transendothelial migration of circulating monocytes (3). Endothelial ‘‘dysfunction’’ can therefore be characterized not only by impaired vasoreactivity with decreased local bioavailability of the vasodilator substance nitric oxide (NO) (4), but also by abnormal inflammatory cell– endothelial interactions and increased expression of adhesion molecules. Endothelial dysfunction appears to precede even the earliest development of atherosclerosis in experimental models (5). Thus, endothelial dysfunction, understood as an altered vascular response, should be regarded as a precursor of events that

Heart Physiology and Pathophysiology, Fourth Edition

II. NORMAL ENDOTHELIAL FUNCTION A. Endothelium as a Vasoregulator The importance of the endothelium as a modulator of vascular tone was first recognized in 1980. Furchgott and Zawadski (6) postulated the existence of an endothelial relaxing factor, noting that rabbit aortic rings relaxed in response to acetylcholine only in the presence of an intact endothelium. It was subsequently discovered in 1987 that endothelial cells release a soluble-relaxing factor, initially called endothelium-derived relaxing factor, found to be identical to NO (7). Although NO has a short half-life of less than a few seconds, it traverses the subendothelial space rapidly to increase cyclic guanosine monophosphate, leading to smooth muscle relaxation (8). The basal release of NO from endothelial cells produces a constant, active vasodilator tone that antagonizes a variety of vasoconstrictor substances also released from the endothelium. NO release is stimulated by increased flow and by a variety of circulating agents, such as bradykinin, acetylcholine, and thrombin, via activation of specific endothelial cell membrane receptors.

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FIGURE 1

L-Arginine–NO pathway. Depiction showing synthesis of nitric oxide from via the enzyme nitric oxide synthase (NOS). NO promotes relaxation of smooth muscle cells and inhibits vascular smooth muscle cell proliferation and platelet aggregation.

L-arginine

L-Arginine has been identified as the biological precursor of NO (9) (Fig. 1) and is converted to NO by the enzyme nitric oxide synthase (NOS) (10). NOS releases NO from the terminal guanidino nitrogen group of L-arginine, producing L-citrulline as a by-product. The generation of NO from L-arginine can be specifically blocked by analogues of arginine, such as N-monomethyl-L-arginine (L-NMMA), proven a useful tool in clinical research, allowing investigation of the role of NO in vivo. Although NO plays a major role in the regulation of vascular tone and growth, other endothelium-derived relaxing factors should be considered. The endothelium is capable of releasing other vasodilator substances, such as prostacyclin, endothelium-derived hyperpolarizing factor, and natriuretic peptides (11, 12). Moreover, the symmetry between endothelium-derived relaxing and contracting factors creates a balance that results in vasoregulation (Table I). Endothelin-1 (ET-1), a peptide that was first sequenced in 1988 (13), is the most potent known vasoconstrictor in humans. Its physiological role

TABLE I Endothelium: Balance of Opposing Factors Vasodilators Nitric oxide Prostacyclin Endothelium-derived hyperpolarizing factor Bradykinin

Vasoconstrictors Endothelin-1 Angiotensin II

includes maintenance of basal arterial vasomotor tone and it is present in low concentrations in healthy individuals. The role of ET-1 in pathophysiological states will be discussed later. Angiotensin II is also a powerful vasoconstrictor that plays an important role in vascular tone and structure (14).

B. Endothelium as an ‘‘Anti-inflammatory’’ Barrier In healthy subjects, the endothelium provides a barrier against the infiltration of circulating inflammatory cells, such as monocytes and lymphocytes. In addition to its role in regulating vascular tone, NO has been described as an endogenous ‘‘antiatherogenic’’ molecule. NO interferes with the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and E-selectin. This leads to decreased leukocyte adhesion and migration in the vessel wall, key events in early atherosclerosis (15, 16). NO also inhibits vascular smooth muscle cell proliferation and migration (17, 18), and alters platelet–vessel wall interactions and platelet aggregation (Table II). Many of these events in early atherogenesis may be coordinated via the transcriptional factor, nuclear factor-␬B (NF-␬B). In the inactive state, NF-␬B is present as a dimer in the cytosol of cells, bound to an inhibitory protein, I␬B (19). Certain stimuli present in the atherogenic environment, including cytokines and oxidants (20), lead to the activation

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TABLE II Some Functions of Nitric Oxide Vasodilator Inhibitor of vascular smooth muscle cell proliferation Inhibitor of platelet adherence/aggregation Inhibitor of leukocyte/endothelial interactions

of NF-␬B. This activation results in the translocation of NF-␬B into the nucleus and its subsequent binding to the specific promoter regions of target genes, including VCAM-1 and monocyte chemoattractant protein-1 (MCP-1). NO stabilizes the NF-␬B inhibitor, IKB움 (21), thus leading to decreased activated NF-␬B. This process may represent a central mechanism by which NO, in a coordinated fashion, can inhibit the gene expression of a variety of inflammatory mediators involved in atherosclerosis. Whereas vasodilator substances such as NO appear to have antiatherogenic properties, evidence suggests that ET-1, in addition to its vasoconstrictor action, is proatherogenic. ET-1 is a strong chemoattractant for circulating monocytes and causes macrophage activation (22). ET-1 also stimulates vascular smooth muscle cell proliferation, probably via the ET-A receptor (23). In view of this, varying levels of ET-1 could play a role in the development and progression of atherosclerosis.

III. PATHOPHYSIOLOGY OF THE ENDOTHELIUM Endothelial ‘‘dysfunction’’ is characterized by an imbalance of endothelium-derived relaxing and contracting factors. It occurs with a variety of cardiovascular risk factors prior to the development of overt atherosclerotic plaque or structural changes in the vascular tree, implying that a common pathway may be involved. Endothelial dysfunction appears to result from ‘‘endothelial injury,’’ with either physical damage or more subtle cellular damage brought on by CAD risk factors (24). This endothelial damage may cause a decrease in NO bioavailability with ensuing detrimental changes in monocyte adhesion, smooth muscle cell migration and proliferation, and platelet adhesiveness. In addition, altered endothelial function may serve as a marker for this complex process (Fig. 2). Mechanisms for decreased NO bioavailability that occur with a variety of atherosclerotic risk factors are not fully understood. In vivo, the activity of the L-arginine–NO pathway is a balance between synthesis and breakdown of NO. A decreased formation of NO may relate to lack of substrate (i.e., L-arginine) or abnormal endothelial NOS activity. L-Arginine is taken up actively by cells—this process may be abnormal in atherosclerosis. However, the intracellular concentration of L-arginine far exceeds the apparent Km of NOS, making it less likely that lack of an extracellular substrate is a

FIGURE 2 Endothelial dysfunction. Various CAD risk factors can bring on endothelial damage, which may result in a decrease in nitric oxide activity and an increase in angiotensin II and other substances. This imbalance in endothelium-derived factors results in deleterious changes in monocyte adhesion, smooth muscle cell proliferation, and platelet aggregation.

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rate-limiting step. A decrease in transcription of the endothelial NOS enzyme or an increase in circulating concentrations of asymmetrical dimethyl-arginine (ADMA) (25), an endogenous inhibitor of NOS, may lead to a decrease in NO in pathophysiologic states. Exogenous L-arginine may overcome this competitive inhibition of NOS by ADMA. In addition, a possible increased breakdown of NO due to inactivation by oxygen-free radicals may also be an important cause of decreased biological NO activity (26). Oxidative stress, or the tissue damage caused by free radicals, is present in multiple CAD risk factor states, including hypercholesterolemia, hypertension, and diabetes. Oxidative end products with an important role in the pathogenesis of atherosclerosis include oxidized low-density lipoprotein (LDL) in hypercholesterolemia and advanced glycation end products in diabetes. Oxidative stress may thus be a common pathway in endothelial ‘‘dysfunction’’ for a variety of risk factors. In contradistinction to the decreased bioavailability of NO found in endothelial dysfunction, increased levels of ET-1 have been demonstrated in humans in early atherosclerosis and endothelial dysfunction (27). These higher levels may contribute to an increase in vasomotor tone. In addition, the proatherogenic properties of ET-1 mentioned previously may contribute to the overall atherogenic milieu.

A. Clinical Measurement of Endothelial Function In view of the short biological half-life of NO, endothelium-dependent vasorelaxation in response to a variety of stimuli has been employed extensively as a marker of NO activity in the vasculature. Numerous endothelium-dependent agonists have been identified, including acetylcholine and bradykinin (28), and increased blood flow has also been used as a stimulus to NO release. Vasomotor responses have been measured in both coronary and peripheral circulations (29, 30). Changes in both large vessel diameter and small vessel blood flow have been utilized as a measure of endothelial function. Endothelial function was initially assessed in the cardiac catheterization laboratory in patients with established CAD. Coronary artery diameter can be measured by quantitative angiography before and after intracoronary infusion of acetylcholine. In normal arteries, acetylcholine stimulates the release of NO, with a vasodilatory response. In patients with endothelial dysfunction, a vasoconstrictor effect occurs, due to a direct effect of acetylcholine on the vascular smooth muscle and reduced endothelial NO activity (31). Using coronary doppler flow wires, abnormalities have also been detected in response to endothelium-dependent vasodila-

tors in the microcirculation (32). As early events in endothelial function may occur at the microcirculation level, an emphasis should be placed on techniques that can detect changes at this level. Moreover, because most coronary vascular tone regulation occurs at the small vessel level, endothelial dysfunction may result in alterations in myocardial perfusion in patients with CAD risk factors (33). More recently, peripheral circulation testing has extended these findings of ‘‘endothelial dysfunction’’ to patients with risk factors for CAD, including hypertension, hyperlipidemia, smoking, and hyperhomocystinemia, but no overt signs of clinical atherosclerosis (34). Two types of peripheral arterial testing have been utilized. Noninvasive detection of endothelial dysfunction in the brachial artery was first described in 1992 (35). In this technique, the brachial arterial diameter is measured in response to reactive hyperemia following blood pressure cuff occlusion, leading to endotheliumdependent vasodilator. In contrast, intraarterial infusion of endothelium-dependent and -independent vasodilation substances into the forearm circulation, followed by measurement of forearm blood flow using plethysmographic techniques, examines microcirculatory endothelial function (36). These techniques have been employed as surrogate measures of coronary endothelial function. Several studies suggest that abnormal brachial flowmediated dilatation (FMD) reflects coronary endothelial dysfunction. However, other studies indicate that a ‘‘normal’’ brachial FMD does not rule out coronary disease (37–40).

IV. ENDOTHELIAL DYSFUNCTION IN HYPERCHOLESTEROLEMIA AND POTENTIAL REVERSIBILITY A. Alterations of Vasoregulation Hypercholesterolemia is an acknowledged risk factor for atherosclerosis. It is associated with abnormal endothelium-dependent vasodilation and decreased NO bioavailability in both animal and human studies, independent of the presence of atherosclerosis (41). Studies suggest that cholesterol reduction is associated with decreased cardiac morbidity and mortality, possibly due, at least in part, to improved endothelial function. Multiple studies have shown an improvement in endothelial function following lipid-lowering therapy with HMG-CoA reductase inhibitors, occurring as early as 1 month after commencement of treatment (42–44), in patients with and without coronary disease. LDL cholesterol is an important determinant of endothelial function in hypercholesterolemia. Increased

27. Function of Vascular Endothelium

oxidized LDL and oxidative stress have also been postulated as potential mechanisms for abnormal vasomotor function and early atherosclerotic lesion formation in hypercholesterolemia. The use of antioxidants in hypercholesterolemic animal models preserves endothelial function. In humans, the results have been variable; the combination of lipid-lowering and antioxidant therapy with probucol improved endothelial function to a greater extent than lipid-lowering therapy alone (42). However, vitamin antioxidants, although effective in short-term therapy (45), have not produced beneficial long-term effects on endothelial function in the setting of hypercholesterolemia (46, 47). L-2-Oxothiazolidine4-carboxylic acid (OTC), a substance that augments cellular glutathione levels and alters the oxidative state of the cell, improves endothelium-dependent vasodilation in patients with CAD (48). Although recent results appear promising, the long-term effects of OTC have not been studied. The ability of antioxidants to modulate the intracellular oxidative state in the vessel wall, rather than simply decrease LDL oxidation, may prove crucial for their effectiveness in altering endothelial dysfunction and early atherosclerosis. As mentioned previously, abnormal NO bioavailability is an early event in atherosclerosis. Tetrahydrobiopterin is an essential cofactor for NOS production. A human study demonstrated that tetrahydrobiopterin infusion improved the abnormal endothelial function of familial hypercholesterolemia (49). Similar to OTC, the long-term effects of this factor have not been studied. It has been postulated in animal models that L-arginine, the physiological substrate for NO, may increase NO synthesis, thus improving endothelial function (50). Studies have shown an improvement in endotheliumdependent vasodilation in asymptomatic hypercholesterolemic subjects (51), young men with CAD (52), and patients with endothelial dysfunction (53). The balance between endothelium-dependent vasorelaxing and constricting factors is altered in hypercholesterolemia. ET-1 levels are increased in experimental hypercholesterolemia (54) and in humans with hypercholesterolemia (55). Hypercholesterolemia animal studies have shown a reversal of endothelial dysfunction following treatment with endothelin-receptor antagonists, providing an exciting possibility for future treatment (56). However, human studies utilizing endothelin antagonists in atherosclerosis or endothelial dysfunction are still awaited.

B. Alterations in the ‘‘Anti-inflammatory’’ Barrier Hypercholesterolemia leads to an increased expression of endothelial cell adhesion molecules, with oxi-

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dized LDL upregulating the expression of adhesion molecules as well as NF-␬B (20) in experimental studies. NF-␬B is a redox-sensitive factor, and in vitro studies have shown that antioxidants may decrease its activation. Animal models of hypercholesterolemia also show an increased expression of the cell adhesion molecule VCAM-1 on aortic endothelium after only 1 week of diet (57) with the subsequent development of a mononuclear cell infiltrate. Studies by Adams et al. (58) demonstrated a decrease of in vitro human monocyte adhesion and expression of endothelial-adhesion molecules following treatment with L-arginine. Additionally, in young men with known CAD, treatment for 3 days with L-arginine decreased monocyte adhesion to endothelial cells in vitro. Longterm studies on the effect of L-arginine have not been performed thus far. However, alteration of monocyte adhesion represents a potential area of therapy in the future.

V. ENDOTHELIAL DYSFUNCTION IN DIABETES A. Alterations of Vasoregulation Vascular complications are the major cause of morbidity and mortality in persons with diabetes mellitus (59). Extensive evidence exists for vasodilatory abnormalities in diabetes in animal and human models and in both type 1 and type 2 diabetes (60–62). Most studies evaluating endothelial function in type 1 diabetes found impaired endothelium-dependent vasodilation (63), utilizing plethysmography of the forearm microcirculation. The degree of abnormality is probably related to the duration of diabetes and the presence of vascular complications. Interestingly, a similar impairment with acute rather than chronic hyperglycemia has been demonstrated (64), suggesting that oxygen radicals formed during hyperglycemia may interfere with NO interaction at the vascular smooth muscle level (65). Functional NO activity has been studied less extensively in NIDDM, but again appears to be abnormal. However, type 2 diabetes is a complex disease, and multiple components of this insulin resistance syndrome may contribute to abnormalities in endothelial function, including hypertension, oxidized LDL, low HDL, and hypertriglyceridemia. Increased oxidative stress is almost certainly involved in the pathogenesis of both type 1 and type 2 diabetes. Pieper et al. (66) demonstrated that tetrahydrobiopterin, a necessary cofactor for NOS, reversed abnormalities in endothelium-dependent relaxation in diabetic rat vasculature, but had no effect on normal rat vasculature. These data suggest that tetrahydrobiop-

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terin availability may play an important role in the regulation of NO, a finding that needs to be tested in human studies. Acute parenteral administration of vitamin C has been reported to reverse endothelial dysfunction in the forearm resistance vessels of persons with both type 1 and type 2 diabetes (67–69). The mechanism of this effect is probably via the oxygen radical-scavenging action of the antioxidant vitamin C, leading to increased availability of NO. To date, however, no clinical study confirms the long-term benefits of radical scavenging in diabetic endothelial dysfunction, although antioxidant therapy appears to hold potential. Studies examining the vasodilator and antioxidant effects of the antidiabetes drug troglitazone, which has similarities in structure with vitamin E, show that this agent increased the resistance of LDL to oxidation in healthy subjects and patients with type 2 diabetes (70–73). Further studies are needed to elucidate the potential benefits of this agent. A report from Giugliano et al. (74) in humans demonstrated that the hemodynamic disturbances induced by hyperglycemia were reversed by L-arginine, implicating impaired availability of NO. Administration of angiotensin-converting enzyme inhibitors has had variable effects on endothelial function in human subjects with type 1 diabetes (75, 76). Whether these improvements in endothelial function will have an impact on cardiovascular morbidity and mortality has yet to be determined.

B. Alterations in the ‘‘Anti-inflammatory’’ Barrier As mentioned previously, monocyte–endothelial cell adhesion is a key early event in atherogenesis and appears to be dysfunctional in diabetes. Experimental studies have shown increased monocyte adhesion to human endothelial cells with chronic exposure to high glucose condition (77). Flow cytometry studies have revealed increased adhesion molecule expression on monocytes isolated from diabetic subjects, with a parallel rise in monocyte adhesion to endothelial cells (78). This increase has been found in both type 1 and type 2 diabetes. Although these in vitro data are intriguing, it is not possible at present to extend these data by measuring these endothelial adhesion molecules in vivo. It is also unclear whether these abnormalities are potentially reversible with good glycemic control or other therapeutic modalities.

VI. SUMMARY The endothelium appears to play a critical role in the development of atherosclerosis, affecting vasomotor tone, monocyte and platelet adhesion, and vascular

smooth muscle cell proliferation, in addition to regulating myocardial perfusion. Major advances have been made in understanding the mechanisms involved in endothelial dysfunction and its potential areas of reversibility. There is promising evidence from animal models that improving endothelial function may slow the progression of atherosclerosis. Because endothelial function may serve as a marker for the initiation and progression of atherosclerosis, a more aggressive approach is warranted for early detection and treatment of abnormal endothelial function. In addition, although many agents can lessen endothelial dysfunction in the short term, more long-term reversibility studies need to be performed in humans. This will undoubtedly provide an exciting challenge for future research and development.

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28 Ion Channels in Vascular Endothelium BERND NILIUS and GUY DROOGMANS Department of Physiology Catholic University of Leuven B-3000 Leuven, Belgium

I. INTRODUCTION

such as tissue plasminogen activator (tPA), tissue factor pathway inhibitor (TFPI), and Von Willebrand factor (vWF) (for reviews, see Inagami et al., 1995; Nilius and Casteels, 1996; Nilius et al., 1997e). EC not only respond to humoral substances, which bind to receptors, but also to mechanical forces, due to changes in flow rate (shear stress) or blood pressure (biaxial tensile stress) (Daut et al., 1988; Davies, 1995; Davies and Barbee, 1994; Davies and Tripathi, 1993; Malek and Izumo, 1994). These responses are at least partially mediated by ion channels and will be discussed in detail. Finally, secretory signals also arise from cell–cell contacts with other cells, such as blood cells, and from interaction between EC and extracellular matrix proteins (Davies et al., 1988; Nilius and Casteels, 1996; Nilius et al., 1997e).

Endothelial cells (EC) form an ideal anticoagulative surface for blood flow by secreting heparin sulfates and expressing ectonucleotidases and thrombomodulin. However, it is now well established that they also play a central role in regulating a plethora of vital processes such as blood clotting, blood flow, blood pressure, vessel growth, wound healing by initiating angiogenesis and vessel repair, and controlling the migration of cells between blood and tissue. They are able to prevent blood clotting but can also trigger it in response to various signals, and therefore act thrombolytic as well as thrombogenic. They are antigen-presenting cells being involved in immune responses. The permeability of the blood–tissue interface is controlled by changes in their contractile state and their ability to modulate cell–cell contacts. One of the most important functions is their interaction with the underlying vascular smooth muscle cells (SMC) to adjust the vessel diameter to the hemodynamic needs (Davies et al., 1988; Inagami et al., 1995; Nilius and Casteels, 1996). These multiple functions are mediated by the production and release of a variety of vasoactive agents, which affect the cells in the vessel wall or in its immediate vicinity, including the endothelial cells themselves. These substances include nitric oxide (NO or endothelium-derived relaxing factor, EDRF), endotheliumderived hyperpolarizing factor (EDHF), various prostaglandins, endothelins (ET), natriuretic peptide, small signaling molecules, such as substance P, ATP, growth factors, steroids, and even larger proteins, such as receptors and proteins involved in the blood clotting cascade,

Heart Physiology and Pathophysiology, Fourth Edition

II. ROLE OF ION CHANNELS IN VASCULAR ENDOTHELIUM A. The Unique Role of Ca2⫹ in EC Signaling These various extracellular signals are integrated at the EC surface and elicit the described and often functionally opposite cell responses via specific second messengers. Among these second messengers, cytosolic Ca2⫹ is one of the most important signals, which is very well conserved throughout the phylogenetic tree (Parekh and Penner, 1997). Because of the importance of Ca2⫹ signals and their connection to secretory events in EC, this chapter focuses on mechanisms involved in the regulation of Ca2⫹ signaling. Two types of Ca2⫹ signals can be differentiated by their phenotype: (1) a bi-

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phasic response of the intracellular Ca2⫹ concentration, [Ca2⫹]i, consisting of a fast peak and a long-lasting plateau and (2) oscillations of [Ca2⫹]i (Jacob, 1990; Jacob et al., 1988; Nilius, 1998; Nilius et al., 1997e, 1998a). Figure 1 shows Ca2⫹ signals evoked by a vasoactive agonist, UTP, a compound that mimics the effects of ATP by binding to P2Y2 (P2U) receptors in these cells (Viana et al., 1997). Other agonists, such as histamine, bradykinin, and acetylcholine, induced similar responses. At higher concentrations of the vasoactive agonists, the response consists of an initial peak rise in [Ca2⫹]i followed by a more-or-less pronounced plateaulike increase (Fig. 1A). At low concentrations of the agonist, oscillations of [Ca2⫹]i can often be observed (Fig. 1B). These oscillations appear only in a small window of agonist concentrations and fuse to a Ca2⫹ plateau at higher agonist concentrations (Jacob et al., 1988; Oike et al., 1994). Application of vasoactive agonists can also induce Ca2⫹ waves, which travel through the cell at a speed between 5 and 40 애m/sec, depending on the agonist concentration (Missiaen et al., 1996). These different types of Ca2⫹ signals depend on various mechanisms. Like most eukaryotic cells, EC utilize two ways to increase [Ca2⫹]i: (1) release of Ca2⫹ from intracellular stores, which is mainly responsible for the fast Ca2⫹ peak, and (2) activation of Ca2⫹ influx from the extracellular space (Parekh and Penner, 1997), which mainly accounts for the sustained rise in [Ca2⫹]i. In ECs, like in most nonexcitable cells, the activation of plasmalemmal ion channels serves two purposes: (1) providing influx routes for Ca2⫹ (Nilius, 1991; Nilius et al., 1993, 1997e) and (2) regulating and fine-tuning of the inwardly driving force for Ca2⫹ influx (Nilius, 1991, 1998; Nilius and Droogmans, 1995; Nilius et al., 1997, 1998a). The plateau phase shown in Fig. 1A depends strongly on the driving force for Ca2⫹ and on the presence of

extracellular Ca2⫹. Withdrawal of Ca2⫹ from the extracellular medium induced a decline of [Ca2⫹]i, showing that the Ca2⫹ plateau depends on the influx of Ca2⫹ from the extracellular space. Figure 2 demonstrates the obvious impact of membrane potential on the Ca2⫹ signal in two different types of endothelial cells. Cells derived from the pulmonary artery (CPAE cells, Figs. 2A and 2B) are characterized by a low plateau during agonist stimulation. In contrast, another cell type derived from human umbilical vein endothelium (EA cells, Figs. 2C and 2D) shows an accentuated plateau under similar conditions. Adjusting the driving force for inward Ca2⫹ transport in voltage-clamped cells shows that [Ca2⫹]i is increased at more negative potentials and decreased if the driving force is reduced. In contrast, the release phase (peak) is voltage independent. Obviously, the membrane potential can modulate the amount of inwardly transported Ca2⫹ during the stimulation of EC. Importantly, the sustained plateau is a necessary condition for essential endothelial functions, such as secretion of prostacyclin (PGI2), NO, tPA, plateletactivating factor (PAF), vWF, and TFPI (Inagami et al., 1995; Carter and Ogden, 1992; Iouzalen et al., 1995; Lantoine et al., 1998).

B. Electrogenesis in Endothelium The membrane potential, which is controlled by K⫹, Cl , and nonselective cation channels (see later), is the most important regulator of the driving force for transmembrane Ca2⫹ fluxes. It is, therefore, of interest to know how the membrane potential of EC is generated at rest and during stimulation. Values of the resting membrane potential range between approximately 0 and ⫺70 mV. This variability depends on the different expression levels and the background or ‘‘housekeep⫺

FIGURE 1 Ca2⫹ patterns during agonist stimulation. Human endothelial cells derived from umbilical vein stimulated by 10 애M UTP (left) and 0.5 애M UTP (right). The plateau phase disappears after the withdrawal of extracellular Ca2⫹, which does not influence the Ca2⫹ peak. In contrast, Ca2⫹ oscillations are maintained under Ca2⫹-free conditions.

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FIGURE 2 Calcium responses to agonist stimulation depend on the driving force and the type of endothelial cells. (A and C) Ca2⫹ response of different types of endothelial cells (CPAE cells: cultured bovine pulmonary artery cells in A and C. EA cells: HUVEC-derived cell line Ea926.hy in B and D) to stimulation with various vasoactive agonists. Cells are not voltage clamped. Note the differences in the plateau Ca2⫹ level. (B and D) Modulation of [Ca2⫹]i by the membrane potential in voltage-clamped cells. [Ca2⫹]i is measured at various potentials in voltage-clamped cells (nystatin-perforated patches). The holding potential (VM) was changed as indicated. ATP was applied as indicated by the horizontal bar. CPAE cells in B and EA cells in D. Note the elevation in [Ca2⫹]i by increased driving forces for Ca2⫹ at a more negative membrane.

ing’’ activity of various ion channels. These ‘‘housekeeping’’ channels have been described in detail: (1) an inwardly rectifying K⫹ channel, (2) an outwardly rectifying Cl⫺ channel, and (3) a nonselective, background cation channel (Voets et al., 1996; see also Fig. 3). The expression of K⫹ channels varies greatly between different EC types and even within the same strain of cultured EC. For example, inwardly rectifying K⫹ channels (IRK), which determine the resting potential in most cell types, are mainly expressed in macrovascular EC. This variability may be linked to progression through different stages of the cell cycle, but may also depend on culture conditions and the procedure of cell isolation (for a detailed discussion, see Daut et al., 1994; Nilius et al., 1997e). Heterogeneous expression levels will contribute to the large variability in the resting potential of EC. The N-shaped, current–voltage relationship observed in some ECs may induce bistability of the membrane potential (Daut et al., 1994; Mehrke et al., 1991). The resting potential of EC will shift toward a range between ⫺70 and ⫺60 mV when the inwardly rectifying K⫹ channel is dominant. In case the dominance is taken over by Cl⫺ channels, EC have a resting potential between ⫺40 and ⫺10 mV, which is close to

ECl, or even more positive if the nonselective background current is preactivated to a larger extent (for a detailed discussion, see Nilius et al., 1997a; Voets et al., 1996).

C. Membrane Potential during Cell Stimulation Any stimulation, receptor mediated or mechanical, may affect the membrane potential of EC. Vasoactive stimuli, such as acetylcholine, bradykinin, and histamine, hyperpolarize most EC. The concomitant changes in [Ca2⫹]i will shift the membrane potential to values near EK if the cell expresses Ca2⫹-activated K⫹ channels (see later), but to a value near the equilibrium potential for Cl if they express Ca2⫹-activated Cl⫺ channels (such as CPAE cells) (Figs. 4A and 4B). A large hyperpolarization will therefore occur only in cells that functionally express Ca2⫹-dependent K⫹ currents (Figs. 4C and 4D). Often, hyperpolarization is followed by a sustained depolarization of the membrane due to the activation of nonselective cation channels (Daut et al., 1988; Kamouchi et al., 1997; Luckhoff and Busse, 1990; Marchenko and Sage, 1993; Mehrke et al., 1991).

FIGURE 3 Electrogenesis in a nonstimulated (resting) endothelial cell (CPAE). (A) The total current–voltage relationship is shown in trace 1 (obtained from linear voltage ramps). After application of 1 mM Ba2⫹, trace 2 is left. Adding 100 mM mannitol to shrink the endothelial cell (for details, see Voets et al., 1996) results in trace 3 (dotted line). (B) The difference current (1-2), e.g., the Ba2⫹-blocked current, is the IRK current through Kir2.1. (Kamouchi et al., 1997). (C) After shrinking of the CPAE cell, the current through VRAC channels is inhibited (Nilius et al., 1997a) and is plotted as difference current (2-3). Note that the resting membrane potential reflects the respective contribution of the conductance of these three ionic channels. The remaining current (A, dotted line) is a background, nonselective cation channel (modified after Voets et al., 1996).

FIGURE 4 Response of the membrane potential in two different types of endothelial cells after agonist stimulation. (A and B) Response of CPAE cells to stimulation by 1 애M ATP. CPAE cells do not express BKCa, only Ca2⫹-activated Cl⫺ channels. Only a small hyperpolarization is seen, which depends on the Cl⫺ equilibrium potential, ECl (Kamouchi et al., 1997). (C and D) Ca2⫹ and membrane potential response after agonist stimulation (10 애M UTP) in an EA cell. These cells respond with a much larger increase in [Ca2⫹]I than CPAE cells and strongly hyperpolarize beyond the ECl. This hyperpolarization is due to activation of BKCa.

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III. Ca2⫹ INFLUX PATHWAYS During stimulation of endothelial cells, the influx of Ca2⫹ is one of the most important sources for elevation of [Ca2⫹]i. Multiple entry pathways for Ca2⫹ exist in ECs (for a review, see Nilius, 1998; Nilius et al., 1997a, 1998a). Two pathways have been described that might be of special interest for both agonist-induced and mechanically induced Ca2⫹ entry. Figure 5 gives an overview of the possible entry pathways for Ca2⫹.

A. Nonselective Cation Channels Mechanical stimulation endothelium activates a nonselective cation channel with a conductance of 34 pS for monovalent cations and 6 pS for Ca2⫹, which thus provides an influx pathway for Ca2⫹ (Marchenko and Sage, 1996a, 1997). A similar entry pathway for Ca2⫹ is upregulated in hypertensive rats (Hoyer et al., 1996). Another nonselective cation channel, with a conductance of 44 pS for monovalent cations, is also permeable for Ca2⫹ and activated by intracellular Ca2⫹ (Baron et al., 1996). Typical receptor-operated Ca2⫹ channels (ROC), such as the P2x receptor, are not present in endothelial cells. A Ca2⫹-permeable, nonselective cation channel (NSC) has been described (Nilius, 1990, 1991; Nilius et al., 1993 a,b). This channel has a conductance of approximately 25 pS and is permeable for Na⫹ ⬎ Cs⫹ ⬎ Ca2⫹ with a permeation ratio PCa /PNa ⫽ 0.03 (Kamouchi et al., 1998; Nilius, 1998). This current is slowly activated

485

and has been observed only in the presence of [Ca2⫹]i. Buffering of [Ca2⫹]i with 10 mM BAPTA always completely prevented activation of this current. However, loading the cells with Ca2⫹ via a patch pipette is not sufficient to activate the current. Interestingly, application of a store-depleting inhibitor of SERCa pumps, such as tert-butylbenzohydrochinone (tBHQ) or thapsigargin can also activate this NSC. The influx of Ca2⫹ through this NSC is coupled to agonist stimulation and depends on Ins(1,4,5)P3 production. Block of PLC with U73122—a pyrrole-dione derivative—rapidly inhibits the Ca2⫹ influx, whereas the pyrrolidine-dione derivative U73343, which does not inhibit PLC, is ineffective. NPPB, Ni2⫹, ecanozole, and SKF 96365 also inhibit the agonist-induced Ca2⫹ entry (Kamouchi et al., 1998). In most of the nonexcitable cells, capacitative Ca2⫹ entry (CCE), which is (partially) controlled by the filling state of intracellular Ca2⫹ stores, is presumed to be the major pathway for Ca2⫹ influx after agonist stimulation (Parekh and Penner, 1997). This pathway is provided by highly selective Ca2⫹ channels, the so-called CRAC channels (Hoth et al., 1993; Hoth and Penner, 1993). CCE or CRAC currents are also present in endothelial cells (Fasolato and Nilius, 1998; Gericke et al., 1993, 1994; Oike et al., 1994). Figure 6 shows a representative example of a CRAC current activated by the depletion of intracellular Ca2⫹ stores in CPAE cells dialyzed with 10 mM BAPTA to buffer [Ca2⫹]i at very low concentrations. Inclusion of Ins(1,4,5)P3 in the patch pipette, extracellular application of ionomycin, or administration

FIGURE 5 Influx pathways for extracellular Ca2⫹ in vascular endothelium (NSC, nonselective cation channel, trpC channels encoded by trp genes; CRAC, Ca2⫹ release-activated Ca2⫹ channels; CIF, Ca2⫹ influx factor as a possible gating signal for Ca2⫹-selective CRACs). For further explanations, see text.

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of the SERCa blockers thapsigargin and tBHQ slowly activate a tiny inward current (Fig. 6A). The density of this current is approximately ⫺0.5 pA/pF at 0 mV. Its permeation sequence is Ca2⫹ Ⰷ Na⫹ ⬎ Cs⫹ and Ca2⫹ ⬎ Ba2⫹ (Fasolato and Nilius, 1998). This channel shows inward rectification (Fig. 6B) and is blocked by micromolar concentrations of La3⫹, ecanozal, NPPB, and Ni2⫹. However, a selective tool still does not exist. Elevation of extracellular Ca2⫹ increases the current. Removal of extracellular divalent ions induces a large, inactivating Na⫹ current that is blocked by micromolar concentrations of lanthanum (Fig. 6C). The density of this current is between 10 and 20 times smaller than reported for Jurkat and peritoneal mast cells (Parekh and Penner, 1997). This endothelial current shares well-described properties of the typical CRAC current present in a variety of nonexcitable cells (Hoth et al., 1993; Hoth and Penner, 1993; Parekh and Penner, 1997; Penner et al., 1993).

B. TRPs: Ca2⫹ Entry Channels? A gene family—trp from ‘‘transient receptor potential’’ discovered in photoreceptors of drosophila—has

been described that might be related to Ca2⫹ entry (Birnbaumer et al., 1996; Zhu et al., 1996). An important component of endothelial cell stimulation is the activation of the 웁-type phospholipase C (PLC). Via the PLC pathway, Ins(1,4,5)P3 is produced, which initiates store depletion. Possibly, trp channels are involved in PLCmediated Ca2⫹ entry. Mammalian trp homologues have been expressed in a variety of mammalian cells, including endothelium (Groschner et al., 1998; Kamouchi et al., 1999), and are found to form Ca2⫹ influx channels (for review, see Holda et al., 1988; Zhu and Birnbaumer, 1998). The trp family is still expanding. So far, at least four human isoforms, six from mice, two from rat, and four from bovine have been described (Birnbaumer et al., 1996; Philipp et al., 1996; Zhu and Birnbaumer, 1998). Trp proteins consist of between 700 and 1000 amino acids. In all isoforms, three ankyrin motifs are conserved in the N terminus. All Trp proteins are characterized by six transmembrane-spanning helices and a putative pore region between helix TM5 and TM6. TM4 lacks the charges that are typical for voltage-gated ion channels (Bennett et al., 1995; Birnbaumer et al., 1996; Philipp et al., 1996; Zhu and Birnbaumer, 1998). How the channels gate is unknown. One hypothesis refers to

FIGURE 6 Activation of a store-depletion dependent current (CRAC) (A) Time course of CRAC activation at a 0-mV holding potential. The current is activated by application of the SERCa inhibitor BHQ. Cells were perfused with a different external standard solution containing 20 mM CaCl2 or 0 mM as indicated. Cells were dialyzed with a standard internal solution containing 12 mM BAPTA. Currents were blocked by 1 애M La3⫹ added to either the standard or the Ca2⫹free bath solution. (B) I–V relationship in high Ca2⫹ medium, obtained by subtracting ramps recorded in (a) and (b). (C) I–V relationship in divalent-free medium, obtained by subtracting ramps recorded in the same medium containing 1 애M La3⫹ (d) from ramps at the peak Na⫹ current (c). Note the block of the current by 1 애M LaCl3 (for details, see Fasolato and Nilius, 1998). Modified after Nilius (1998); and Nilius et al. (1998a).

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FIGURE 7 Scheme of three members of the trp family, all identified in endothelial cells. For explanations, see text.

a possible physical contact between the trp’s and the Ins(1,4,5)P3 receptor of intracellular Ca2⫹ stores, possibly via the ankyrin-like motifs at the N terminus in trpencoded proteins (Bennett et al., 1995; Berridge, 1995). Figure 7 gives a structural scheme of the trp members that have been identified so far in endothelial cells (for a review, see also Holda et al., 1988; Zhu and Birnbaumer, 1998). Trps are expressed endogenously in endothelial cells; however, the pattern of expression seems to be cell specific. CPAE cells only express trp1 and 4, but not trp3, whereas HUVEC-derived endothelial cells (EA cells) express trp1,3, and 4 (Kamouchi et al., 1999). In both cell types, typical CRAC currents have been identified (Fasolato and Nilius, 1998; Groschner et al., 1998; Kamouchi et al., 1999); however, a Ca2⫹-permeable nonselective cation channel NSC is not present in CPAE cells (Kamouchi et al., 1999). If trp3-lacking CPAE are stimulated, they respond to vasoactive agonists only with a small Ca2⫹ plateau as already shown in Fig. 2. However, if these cells are transfected with htrp3, the agonist response is characterized by large Ca2⫹ plateaus (Kamouchi et al., 1999).

IV. ION CHANNELS CONTROLLING THE DRIVING FORCE FOR Ca2⫹ ENTRY Endothelial ion channels play a crucial role in the regulation of Ca2⫹ signaling, not only as pathways for Ca2⫹ entry, but also for the fine-tuning of electrochemical driving forces for Ca2⫹. The membrane potential of endothelial cells is controlled by (1) K⫹ channels, among which inwardly rectifying K⫹ channels and Ca-activated K⫹ channels play a dominant role, and (2) Cl⫺ channels, which belong to the family of volume-regulated anion channels (VRAC) and Ca2⫹-activated Cl⫺ channels (CaCC). Interestingly, CFTR channels have also been

detected in endothelial cells (Tousson et al., 1998) and nonselective cation channels (Voets et al., 1996, Kamouchi et al., 1999).

A. Potassium Channels 1. Inwardly Rectifying Kⴙ Channels One of the most important channels for the control of the resting potential in nonstimulated cells is the K⫹inward rectifier, IRK, which conducts inward currents at potentials more negative than the K⫹ equilibrium potential but permits much smaller currents at potentials positive to that potential. This conductance, together with the basal activity of the volume-regulated anion channel and a background nonselective cation current, determines the resting membrane potential of endothelial cells (Campbell et al., 1991; Fransen and Sys, 1997; Voets et al., 1996). As already discussed, the activity of these channels is often counteracted by Cl⫺ channels and background NSC channels, which explains the huge variability of the EC resting potential between ⫺10 and ⫺70 mV. The single channel conductance of endothelial Kir ranges from 23 to 30 pS in symmetrical K⫹ solutions (Elam and Lansman, 1995; Nilius and Droogmans, 1995; Nilius et al., 1993, 1997e; Pennefather and Decoursey, 1994; Silver and Decoursey, 1990; Takeda et al., 1987; Kamouchi et al., 1997). Typically its conductance increase with the square root of the extracellular K⫹ concentration (Nilius and Riemann, 1990; Silver et al., 1994; Zunkler et al., 1995). The permeation profile of this endothelial channel is PK⬎PRb⬎PCs (Pennefather and Decoursey, 1994; Silver et al., 1994). Extracellular Ba2⫹, TEA, TBA, and Cs⫹ block Kir (Nilius and Droogmans, 1995; Pasyk et al., 1992; Revest and Abbott, 1992; Voets et al., 1996; von Beckerath et al., 1996). At negative potentials, the channel shows time-dependent inactivation, which is largely due to a

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block by extracellular Mg2⫹, and this effect is antagonized by extracellular K⫹ (Elam and Lansman, 1995). Figure 3 has already depicted an example of IRK and its block by Ba2⫹, which is a very convenient tool to select IRK in endothelium. Inward rectification is attributed to a time- and voltage-dependent block by intracellular Mg2⫹ (Elam and Lansman, 1995). However, Kir in bovine pulmonary artery endothelial cells still exhibited voltage- and time-dependent gating and small outward currents in nominal Mg2⫹-free internal pipette solutions (Silver and Decoursey, 1990), which may reflect an intrinsic gating property of IRK channels in these cells or might be attributed to technical difficulties due to cell dialysis. Interestingly, shear stress can evoke hyperpolarization and enhanced outward whole cell K⫹ currents, which can be blocked by Cs⫹, another typical inhibitor of IRK. Shear stress also enhanced the open probability of an IRK channel measured in luminal cell-attached patches (Olesen et al., 1988) and in inside-out patches (Jacobs et al., 1995). IRK seems to be metabolically regulated. Intracellular ATP but not its nonhydrolyzable analogues ATP웂S and adenylyl imidodiphosphate (AMP-PNP) prevented its rundown in the whole cell mode (Kamouchi et al., 1997). Reduction of intracellular ATP and induction of hypoxic conditions downregulates IRK dramatically (Kamouchi et al., 1997). The phosphatase inhibitor okadaic acid also prevented rundown, but protamine, an activator of phosphatase 2A (PP2A), enhanced the rate of rundown. Phosphorylation of the channel molecule therefore seems to be essential for maintaining its activity, with its rundown probably being due to dephosphorylation by PP2A (Kamouchi et al., 1997). An interesting observation is the possible involvement of IRK in the regulation of the membrane potential during EC activation by vasoactive agonists. Angiotensin II, vasopressin, VIP, ET-1, and histamine inhibit IRK in capillary and macrovascular endothelial cells (Hoyer et al., 1991; Nilius et al., 1993; Pasyk et al., 1992; Zhang et al., 1994). A similar inhibition has been observed by application of GTP웂S via a patch pipette (Hoyer et al., 1991; Kamouchi et al., 1997). This inhibition is probably due to activation of a PTX-insensitive G-protein, which may mediate these inhibitory actions by modulating PPA2. IRK is a member of the Kir family, Kir2.1 (Forsyth et al., 1997; Kamouchi et al., 1997). This channel is a strongly inwardly rectifying channel with a single channel conductance of 30 pS. The two transmembrane regions are highly conserved. The channel consists of 427 amino acids, two membrane-spanning regions, and a highly conserved. TIGYG-H5 motif in the pore region. The M84 site confers pH insensitivity, which also charac-

terizes the endothelial channel. Mg2⫹ and spermine, spermidine, putrescine block appears to be connected to D172. The C terminus contains a phosphorylation motive, RRESEI, in which S425 seems to be important for channel regulation (Forsyth et al., 1997; Ruppersberg and Fakler, 1996). 2. Ca2ⴙ-Activated K Channels Stimulation of endothelial cells activates Ca2⫹ influx pathways, which would depolarize these cells, thereby reducing the inwardly driving force for Ca2⫹. This can be compensated by the activation of Ca2⫹-dependent K⫹ channels (KCa). The open probability of these channels is increased by the elevation of cytosolic Ca2⫹ to cause membrane hyperpolarization. Different types of Ca2⫹activated K⫹ channels have been described, e.g., BKCa or maxi-K channels, IKCa or intermediate conductance, and SKCa or small conductance channels. However, expression of these channels is also very heterogeneous between different types of endothelial cells (Nilius et al., 1997e). High-conductance Ca2⫹-activated K⫹ channels, BKCa , are expressed in most endothelial cells and are assumed to link chemical signals to electrical responses. They have a conductance between 165 and 240 pS (Hoyer et al., 1994; Ling and Oneill, 1992; Nilius and Riemann, 1990; Rusko et al., 1992). An example of endothelial cells is shown in Fig. 8. It is an approximately 230-pS channel when measured under conditions of symmetrical K⫹ concentrations. Application of vasoactive agonists induces a fast increase in the probability of the channel being open. The open probability increases further with more positive potentials. The apparent Ca2⫹ affinity of the channel is increased at positive potentials and decreased at negative potentials. Its open probability depends on both intracellular Ca2⫹ and voltage: an increase in [Ca2⫹]I shifts the activation curve of the channels toward more negative potentials (Baron et al., 1996; Daut et al., 1994; Haburcak et al., 1997). Pharmacologically, the endothelial BKCa is blocked by charybdotoxin (IC50 approximately 50 nM), iberiotoxin, TEA (IC50 approximately 1 mM, 100% block at 10 mM), d-tubocurarine, and quinine. Mg2⫹ blocks voltage dependently from outside (Baron et al., 1996; Daut et al., 1994; Rusko et al., 1992). BKCa channels can be activated by the benzimidazolone compounds NS004 and NS1619 (in the micromolar range), which also induced an increase in the open probability of BKCa and also a shift toward more negative potentials (Gribkoff et al., 1996). Stimulating effects of BKCa are extremely important for endothelial cells because such compounds may help release NO and support vasorelaxation due to the increased driving force for Ca2⫹ influx. Indeed, it has been

28. Ion Channels in Vascular Endothelium

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FIGURE 8 Single channel currents through a Ca2⫹-activated K⫹ channel evoked by ATP in EA cells. (A) Increase in n*popen (open probability times number of channels, probably 1 in this example) of the BKCa after application of 10 애M ATP (cell-attached patch, holding potential of 60 mV, symmetrical K⫹ conditions). (B) Amplitude histogram. Single channel amplitude is 13.9 pA at 60 mV, which refers to a single channel conductance of 231 pS. (C) No channel is active before application of the agonist. (D) Single channels traces obtained during superfusion of the cell with ATP (10 애M) (modified after Nilius, 1998; Nilius et al., 1998a).

shown in many assays that BKCa interferes with NO release (Busse et al., 1993). Ca2⫹-activated K⫹ channels in vascular smooth muscle cells are targets for various physiological factors released from the endothelium, including NO (Bolotina et al., 1994), and EDHF. The latter has been likely identified as a cytochrome P450derived arachidonic acid metabolite (Popp et al., 1996) under which epoxyeicosatrienoic acids (EETs). These compounds exert an autocrine effect on BKCa recorded in inside-out patches of primary cultured pig coronary artery endothelial cells (Baron et al., 1997). The direct activation of BKCa by NO could provide another autocrine activation of BKCa in endothelial cells and therefore may induce a positive feedback on NO release. However, in cultured endothelial cells, the NO donor S-nitrosocysteine neither directly activated BKCa nor modulated BKCa channels activated by increased [Ca2⫹]i (Haburcak et al., 1997). Interestingly, the K⫹ efflux via BKCa might induce the activation of K⫹-sensitive smooth muscle IRK channels, which in turn also induce hyperpolarization. Thus, K⫹ itself may act as EDHF (Edwards et al., 1998). From RT-PCR (reverse transcriptase-polymerase chain reaction) analysis, BKCa in human endothelial EA cells has been identified as hslo (Kamouchi et al., 1997).

The structure of this channel is not completely known. At the N terminus, six or even seven transmembrane helices have been suggested. A segment 0 in the Nterminal is probably a coupling site for the 웁 subunit. The unique C terminus contains possibly four additional helices, H7–H10. The channel has approximately 1200 amino acids (Dworetzky et al., 1994; Tseng Crank et al., 1994). Interestingly, hslo is not expressed in all endothelial cells. Cultured pulmonary artery endothelium (CPAE) does not express functional hslo channels. Transfection of CPAE cells with hslo induced elevated Ca2⫹ levels during the plateau phase. The increased [Ca2⫹]i correlated well with the corresponding effects of vasoactive agonists on the membrane potential, indicating that the expression of cloned hslo–BKCa exerts a positive feedback on Ca2⫹ signals in endothelial cells. This effect counteracts the negative (depolarizing) effect of the stimulation of Ca2⫹-activated Cl⫺ channels. Figure 9 shows an example of the effect of expression of hslo in cells that normally lack this channel (Kamouchi et al., 1997). In addition, the 웁 subunit seems to be absent in cell types where BKCa channels are expressed. A functional indication for the absence of the 웁 subunit is the lack of effects of the BKCa opener DHS-I, which

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FIGURE 9 Responses of [Ca2⫹]i and the membrane potential, VM, of CPAE cells after expression of hslo, a Ca2⫹-activated K⫹ channel, the BKCa. (A) Membrane potential and [Ca2⫹]i measured in CPAE cells during stimulation (unclamped cell, loaded with Fura AM, 0.1 mM EGTA in a patch pipette, 1 애M ATP, Cl⫺ equilibrium potential, ECl in this experiment is 0 mV). Note the very low plateau and the low plateau of the Ca2⫹ signal and the depolarization toward positive potentials. (B) Response of a CPAE cell transfected with hslo. Note the increased Ca2⫹ plateau and the large transient hyperpolarization beyond the Cl⫺ equilibrium potential (ECl ⫽ 0 mV). For more details, see Kamouchi et al. (1997).

only works in the presence of the 웁 subunit. Expression of the 웁 subunit in HUVEC-derived endothelium induced a leftward shift of the current–voltage relationship, which induces a sensitizing effect on BKCa (Papassotiriou et al., 1999). Intermediate IKCa are activated by Ca2⫹, are inwardly rectifying, and have a conductance between 30 and 80 pS in symmetrical K⫹ and 15 pS at physiological extracellular K⫹. Charybdotoxin, as well as quinine, TBA are efficient blockers of these IK channels (Ling and Oneill, 1992; Sauve et al., 1990; Van Renterghem et al., 1995). A 30-pS channel in cultured bovine aortic endothelial cells is clearly Ca2⫹ dependent ands seems to be Gprotein regulated (Vaca et al., 1992). IKCa channels with a conductance between 40 and 80 pS are activated by Ins(1,4,5)P3-sensitive Ca2⫹ release induced by agonists, such as bradykinin, acetylcholine, and ATP, and by ET1 and ET-3 (for a review, see Nilius et al., 1997e). The biophysical and pharmacological profiles of these channels in endothelial cells are consistent with those of the cloned hIK channel (Ishii et al., 1997; Jensen et al., 1998). Small conductance K channels with a conductance of about 10 pS in asymmetrical conditions have also been observed in ECs. These channels lack voltage depen-

dence and are blocked by extracellular TBA, apamin, and d-tubocurarine (Groschner et al., 1992; Marchenko and Sage, 1996a; Muraki et al., 1997). 3. ATP-Sensitive K Channels Intracellular dialysis of ATP, application of glucosefree/NaCN solutions, or the KATP channel opener pinacidil increased whole cell and single channel currents in endothelial cells from rat aorta and brain microvessels and could be reversibly blocked by glibenclamide or by ATP in inside-out patches (Janigro et al., 1993). Those channels have a conductance of approximately 25 pS and are activated by lowering the intracellular ATP concentration: Activation also occurs by application of the K⫹ channel activator levcromakalim. The sulfonylurea derivative glibenclamide or an increase of the ATP concentration in inside-out patches inhibits the channel (Katnik and Adams, 1995, 1997). The K channel openers HOE 234, diazoxide, and pinacidil, as well as substituting L-glucose for D-glucose, hyperpolarized guinea pig coronary capillaries to a potential close to EK, as measured with the voltage-sensitive dye bisoxonol. This effect was reversed by the addition of

28. Ion Channels in Vascular Endothelium

glibenclamide (Langheinrich and Daut, 1997). Interestingly, these channels may also be activated by shear stress (Hutcheson and Griffith, 1994; Kuo and Chancellor, 1995).

B. Cl⫺ Channels 1. Endothelial Volume-Regulated Anion Channel (VRAC) Endothelial cells are constantly exposed to shear forces and also to biaxial mechanical stress induced by stretching or bending of the cell membrane. Both forces might be important for unfolding of the plasma membrane. Such an unfolding has been discussed as a possible signal to activate anion channels that are important for the regulation of cell volume after swelling (Okada, 1997). This unfolding mechanism could be of specific importance in endothelial cells because of the abundant presence of caveolae. Similar, if not identical, anion channels are found in nearly every cell type. These anion channels are under physiological conditions mainly permeated by Cl⫺. They are referred to as volume-regulated anion channels because cell volume is the signal that most closely correlates with channel activation (Nilius et al., 1997a; Okada, 1997). VRAC is activated in EC under different conditions. A reduction of intracellular ionic strength due to cell swelling (Voets et al., 1999) and unfolding of part of the cell membrane are the main triggers that activate this

491

channel (Nilius et al., 1997a; Okada, 1997). Under hypotonic conditions, the current density of ICl, swell at 100 mV ranges between 100 and 150 pA/pF for cultured endothelial cells (Nilius et al., 1997a). In endothelial cells, VRAC is not only activated by changes in cell volume, but also by changes in cell shape and by shear stress. A typical VRAC current is shown in Fig. 10. It is activated by dialyzing the cell with an intracellular hypertonic solution, by challenging it with a extracellular hypotonic solution or by shear stress, or by dialyzing the cell with GTP웂S or a solution of reduced ionic strength. Figure 10A shows VRAC currents and the time course of the current measured at –80 mV during a challenge with an extracellular 27% hypoosmotic solution. It is obvious from the responses to voltage steps that the current is already partially activated in resting cells (Fig. 10C) and is therefore important for the electrogenesis of the resting potential in nonstimulated CPAE cells. Voltage steps (from ⫺80 to 100 mV, increment 20 mV) evoked only small currents in resting cells (Fig. 10C), but much larger currents during cell swelling (Fig. 10D). These currents show outward rectification and are time independent, except at potentials positive to 60 mV where they slowly inactivate. Cell shrinking reduced the currents below their value in control conditions, indicating that preactivation of VRAC under isotonic conditions. I-V curves, obtained either from voltage ramps or from the step protocol, show modest outward rectification. The reversal potential is close to the theoretical Cl⫺ equilibrium potential, ECl (Fig. 10B).

FIGURE 10 Activation of VRAC in cultured pulmonary artery endothelial cells. (A) Time course of the activation and deactivation of ICl, swell (measured at ⫺80 mV). The solid bar indicates application of a 25% HTS. (B) I–V curves obtained at the time points indicated in A. Current traces under isotonic (C) and hypotonic (D) conditions in response to a step-voltage protocol (holding potential is 0 mV, 2-sec steps ranging from ⫺80 to 100 mV, spaced 20 mV).

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IV. Vascular Endothelium

VRAC is mainly carried by Cl⫺, but amino acids such as aspartate, which is a constituent of the pipette solution, also contribute to the current. This may account for the reversal potential more positive than ECl. For freshly isolated endothelial cells and cells in primary culture, the density is less (40 pA/pS), (Nilius et al., 1994). Permeation of VRAC is characterized by the following sequence: SCN⫺ ⬎ I⫺ ⬎ NO3⫺ ⬎ Br⫺ ⬎ Cl⫺ ⬎ HCO3⫺ ⬎ F⫺ ⬎ gluconate ⬎ glycine ⬎ taurine ⬎ aspartate, glutamate. Thus, amino acids and organic osmolytes also permeate through VRAC (Nilius et al., 1994; Okada, 1997; Strange et al., 1996). The single channel conductance is approximately 40–50 pS at positive potentials and 10–20 pS at negative potentials (for a review, see Nilius et al., 1997a; Okada, 1997; Strange et al., 1996). Rectification is a property of the open channel. VRAC is further characterized by inactivation at positive potentials and a voltage-dependent recovery from inactivation. Its kinetic properties are modulated by extracellular divalent cations, extracellular pH, the permeating anion itself, and various channel blockers. Inactivation is accelerated by acidic extracellular pH and by an increase in the extracellular Mg2⫹ concentration, [Mg2⫹]e (Nilius et al., 1997a; Okada, 1997; Strange et al., 1996). Because VRAC is a ‘‘housekeeping’’ Cl⫺ channels in EC, its block induces changes in the resting membrane potential of EC. If those Cl⫺ channels are blocked, K⫹ channels may become the dominating channels for the

resting cells and the membrane is strongly hyperpolarized. This might also induce beneficial effects on agonist or shear stress-induced Ca2⫹ influx. Probably, the block of Cl⫺ channels seems to be a still underestimated tool to modulate the driving force for Ca2⫹ entry in endothelial cells and thus Ca2⫹ signaling. Figure 11 shows a striking example of the hyperpolarizing effect of the compound mibefradil, which blocks VRAC (Nilius et al., 1997b). 2. Ca2ⴙ-Dependent Clⴚ Channels, CaCC Under conditions of cell stimulation that induce an increase in [Ca2⫹]i , mainly two types of responses can be observed. It has been shown already that some ECs, such as HUVEC cells, respond with a striking hyperpolarization. Other endothelial cells, such as pulmonary artery cells (CPAE) cells respond with only small changes in the resting potential during an increase in [Ca2⫹]i . In these EC, Ca2⫹-dependent Cl⫺ channels are activated (Groschner et al., 1994; Himmel et al., 1993). They inactivate rapidly at negative potentials and activate slowly at positive potentials. Typical examples of the salient kinetic properties of CaCC currents are shown in Fig. 12. Agonists, such as histamine, ATP, and thrombin, activate CaCC. Outward tail currents are slowly decaying, whereas inward tail currents decay much faster (Nilius et al., 1997d). They show strong

FIGURE 11 VRAC inhibitors hyperpolarize the membrane potential of ECs. (A) Membrane potential was measured in current clamp mode in CPAE cells. In these cells a bimodal distribution has been described with one peak at ⫺80 mV and the other at approximately ⫺25 mV (Voets et al., 1996). Mibefradil (10 애M) induced a fast and reversible hyperpolarization of cells that have a less negative membrane potential. For details, see Nilius et al. (1997b). (B) VRAC blockers sometimes result in bistable behavior of the membrane potential. Spontaneous hyperpolarizations after application of the VRAC blocker, K977093, a carboxamid derivative (kindly provided by Dr. Lang, Hoechst, Frankfurt, Germany) are shown.

28. Ion Channels in Vascular Endothelium

493

FIGURE 12 Kinetic properties of the endothelial Ca2⫹-activated Cl⫺ current. (A) Time course of the changes in intracellular Ca2⫹ after breaking into the cell with a pipette solution buffered at 500 nM Ca2⫹. The arrow indicates the point at which the membrane in the patch was disrupted. (B) Membrane currents measured during linear voltage ramps from ⫺150 to 100 mV applied every 5 sec during the rising phase in [Ca2⫹]i marked by the open bar in A. The appearance of a strongly outwardly rectifying current illustrates the activation of a current during this rising phase. (C) Current traces recorded during voltage steps at the new steadystate level of [Ca2⫹]i , marked by the filled horizontal bar in A. Various voltage steps, VT , were applied from a holding potential, VH , of ⫺50 mV. Steps ranged from ⫺100 to 100 mV in increments of 20 mV. Note the slow activation at positive potentials and the fast deactivation at negative potentials and after returning to the holding potential VH (pipette Cs solution). (D) Deactivation of ICl, Ca , fully activated by a step to 100 mV, at various potentials, Vtail , ranging from ⫺140 to 80 mV (spaced 20 mV). Deactivation is monoexponential and clearly voltage dependent. The instantaneous current–voltage relationship is reconstructed from the initial amplitude of these tail currents and is shown in E. (E) Instantaneous I–V curve determined by the voltage protocol described in A. Data points represent mean ⫾ SEM of five cells and are normalized per unit membrane capacitance. Thus, rectification of endothelial CaCC is a time-dependent process (from Nilius et al., 1997d).

outward rectification. The permeability ratio of the Ca2⫹-activated conductance for the anions iodide : chloride : gluconate is 1.7 : 1 : 0.4 (Nilius et al., 1997c). The single channel conductance is approximately 7 pS at 300 mM extracellular Cl⫺, but only about 3 pS in physiological intra- and extracellular Cl⫺ concentrations (Nilius et al., 1997c). The channel open probability is high at positive potentials, but very small at negative potentials. Activation by Ca2⫹ follows at least a twostep binding. [Ca2⫹]i for half-maximal activation of ICl,Ca is voltage dependent and suggests that the apparent binding constant for Ca2⫹ decreases with depolarization. Its value at 0 mV is approximately 500 nM, and the binding site is 12% within the electrical field from the cytoplasmic side. The Hill coefficient, nH , of the binding was larger than 1 and increased with depolarization (Nilius et al., 1997d). Typical densities of these Ca2⫹activated Cl⫺ currents range from 10 to 30 pA/pF at

100 mV and are much smaller than those of volumeactivated Cl⫺ currents. Activation of these currents requires intracellular ATP (Watanabe et al., 1994). Their pharmacology is characterized by a block by NPA, DIDS, and Zn2⫹ and by calmodulin antagonists (Groschner et al., 1994; Nilius et al., 1997d). DIDS and niflumic acid inhibit ICl,Ca in a voltage-dependent manner, i.e., they exert a more potent block at positive potentials. The block by NPA, NPPB, and tamoxifen is voltage independent. Niflumic acid and tamoxifen are the most potent blockers.

C. Mechanosensitive Channels in Endothelium As already discussed, several ion channels in ECs are mechanosensitive. These channels have been described in detail previously (Nilius et al., 1997e).

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D. Channels Activated by Tensile Stress Membrane stretch induced by negative pressure applied via a patch pipette to ECs activates a channel that is permeable for monovalent cations (approximately 50) but also for Ca2⫹ (19 pS) (Lansman et al., 1987). This channel senses tensile stress. Stretch-activated nonselective cation channels with a conductance of 20–30 pS for monovalent cations and 10–20 pS for Ca2⫹ and Ba2⫹ have also been described in endocardial endothelium and microvascular ECs (Hoyer et al., 1994; Popp et al., 1992). Activation of these channels induces an increase in the intracellular Ca2⫹ concentration, [Ca2⫹]i. This increase is sufficient to activate BKCa and to hyperpolarize the membrane, thereby inducing a positive feedback on Ca2⫹ entry by increasing its driving force (Nilius, 1991; Nilius and Droogmans, 1995; Nilius et al., 1997e). Direct pressure-activated Ca2⫹-permeable ion channels have been described (Hoyer et al., 1996; Marchenko and Sage, 1996b, 1997).

E. Channels Activated by Shear Stress Changes in shear stress due to an alternating frequency of pulsatile flow activate small conductance potassium channels (SKCa) as well as big conductance K⫹ channels (BKCa). A variation of the viscosity also affects BKCa channels (Davies, 1995; Davies and Barbee, 1994; Davies and Tripathi, 1993; Davis et al., 1992; Hutcheson and Griffith, 1994; Jacobs et al., 1995). Opening of these K⫹ channels induces hyperpolarization, which may increase Ca2⫹ influx and NO release (Nilius, 1998; Nilius et al., 1998) and may also modulate the membrane potential in electrically coupled smooth muscle cells (Daut et al., 1994; Davies, 1995; Davies and Barbee, 1994; Davies et al., 1988). An inwardly rectifying, 30-pS K⫹ channel has been described as a possible mechanosensor of shear stress (Jacobs et al., 1995; Olesen et al., 1988). Its single channel conductance, its degree of rectification, and its Ca2⫹ sensitivity in excised patches suggest that this channel may belong to the class of medium conductance K⫹ channels. Shear stress also activates cation channels, which might be somewhat more permeable for Ca2⫹ than Na⫹ (Schwarz et al., 1992b), which is insensitive to activators of protein kinase C and is reversibly blocked by La3⫹ and nonsteroid antiinflammatory drugs such as mefenamic acid. This channel might be involved in shear stress-activated Ca2⫹ influx and shear stress-mediated Ca2⫹ transients (Schwarz et al., 1992, a,b). Tensile forces most likely induce small changes in cell surface, thereby folding or unfolding membrane invaginations or caveolae. Such a process seems to be responsible for the gating of volume-regulated Cl⫺ chan-

nels possibly via an involvement of the small G-protein RhoA (Nakao et al., 1999; Nilius et al., 1998b; Okada, 1997)

V. SUMMARY Ion channels are important for a variety of endothelial cell functions, with the regulation of intracellular Ca2⫹ signals probably being the most crucial one. This function requires a cooperation between channels providing Ca2⫹ entry pathways and channels stabilizing the inwardly driving force for Ca2⫹. Ca2⫹ entry channels comprise nonselective cation channels, which are often regulated by intracellular Ca2⫹ itself, Ca2⫹ releaseactivated Ca2⫹ channels, and agonist-activated nonselective Ca2⫹-permeable cation channels. The molecular nature of these channels is not yet known. Importantly, the driving force for Ca2⫹ entry modulates Ca2⫹ signaling. It is mainly controlled by Ca2⫹dependent K⫹ channels, the large conductance BKCa channels, the inwardly rectifying K⫹ channel (Kir2.1), and at least two Cl⫺ channels, i.e., the Ca2⫹-activated Cl⫺ channel and the volume-regulated, housekeeping anion channel. The expression pattern of ion channels in individual EC types determines the electrical response of these cells to activation by mechanical stimuli or by chemical agonists. A high degree of variability within the vessel tree can be anticipated.

Acknowledgments We thank Professor Jan Eggermont for the constructive and pleasant collaboration, Felix Viana (Alicante, Spain), Thomas Voets (Leuven), Rudi Vennekens (Leuven), and Masahiro Kamouchi (Fukuoka, Japan) for helpful discussion and providing unpublished data.

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Missiaen, L., Lemaire, F. X., Parys, J. B., De Smedt, H., Sienaert, I., and Casteels, R. (1996). Initiation sites for Ca2⫹ signals in endothelial cells. Pflu¨g. Arch. 431, 318–24. Muraki, K., Imaizumi, Y., Ohya, S., Sato, K., Takii, T., Onozaki, K., and Watanabe, M. (1997). Apamin-sensitive Ca2⫹-dependent K⫹ current and hyperpolarization in human endothelial cells. Biochem. Biophys. Res. Commun. 236, 340–343. Nakao, M., Ono, K., Fujisawa, S., and Iijima, T. (1999). Mechanical stress-induced Ca2⫹ entry and Cl⫺ current in cultured human endotheo¨lial cells. Am. J. Physiol. 276, C238–C249. Nilius, B. (1990). Permeation properties of a non-selective cation channel in human vascular endothelial cells. Pflu¨g. Arch. Eur. J. Physiol. 416, 609–611. Nilius, B. (1991). Regulation of transmembrane calcium fluxes in endothelium. News Physiol. Sci. 6, 110–114. Nilius, B. (1998). Signaltransduction in vascular endothelium: The role of intracellular calcium and ion channels. Verhandelingen van de Koninklijke Academie voor Geneeskunde van Belgie 60, 215–250. Nilius, B., and Casteels, R. (1996). Biology of the vascular wall and its interaction with migratory and blood cells. In ‘‘Comprehensive Human Physiology’’ (R. Gerger and U. Windhorts, eds.), pp. 1981– 1994. Springer Verlag, Berlin. Nilius, B., and Droogmans, G. (1995). Ion channels of endothelial cells. In ‘‘Physiology and Pathophysiology of the Heart’’ (N. Sperelakis, ed.), pp. 961–973. Kluwer Academic, Dordrecht/Norwell, MA. Nilius, B., Droogmans, G., Gericke, M., and Schwartz, G. (1993). Nonselective ion pathways in human endothelial cells. In ‘‘Nonselective Cation Channels: Physiology and Biophysics’’ (D. Siemen and J. Hescheler, eds.), pp. 269–280. Birkha¨user Verlag, Basel. Nilius, B., Eggermont, J., Voets, T., Buyse, G., Manolopoulos, V., and Droogmans, G. (1997a). Properties of volume-regulated anion channels in mammalian cells. Progr. Biophys. Mol. Biol. 66, 69–119. Nilius, B., Oike, M., Zahradnik, I., and Droogmans, G. (1994). Activation of a Cl⫺ current by hypotonic volume increase in human endothelial cells. J. Gen. Physiol. 103, 787–805. Nilius, B., Prenen, J., Kamouchi, M., Viana, F., Voets, T., and Droogmans, G. (1997b). Inhibition by mibefradil, a novel calcium channel antagonist, of Ca2⫹-and volume-activated Cl⫺ channels in macrovascular endothelial cells. Br. J. Pharmacol. 121, 547–555. Nilius, B., Prenen, J., Szucs, G., Wei, L., Tanzi, F., Voets, T., and Droogmans, G. (1997c). Calcium-activated chloride channels in bovine pulmonary artery endothelial cells. J. Physiol. 498, 381–396. Nilius, B., Prenen, J., Voets, T., Vandenbremt, K., Eggermont, J. and Droogmans, G. (1997d). Kinetic and pharmacological properties of the calcium activated chloride current in macrovascular endothelial cells. Cell Calcium 22(1), 53–63. Nilius, B., and Riemann, D. (1990). Ion channels in human endothelial cells. Gen. Physiol. Biophys. 9, 89–112. Nilius, B., Schwarz, G., and Droogmans, G. (1993a). Modulation by histamine of an inwardly rectifying potassium channel in human endothelial cells. J. Physiol. 472359-371, 371. Nilius, B., Schwarz, G., Oike, M., and Droogmans, G. (1993b). Histamine-activated, non-selective cation currents and Ca2⫹ transients in endothelial cells from human umbilical vein. Pflu¨g. Arch. Eur. J. Physiol. 424, 285–293. Nilius, B., Viana, F., and Droogmans, G. (1997e). Ion channels in vascular endothelium. Annu. Rev. Physiol. 59, 145–170. Nilius, B., Viana, F., Kamouchi, M., Fasolato, C., Eggermont, J., and Droogmans, G. (1998a). Ca2⫹ signalling in endothelial cells: Role of ion channels. Kor. J. Physiol. 2, 133–145. Nilius, B., Voets, T., Prenen, J., Barth, H., Aktories, K., Kaibuchi, K., Droogmans, G. and Eggermont, J. (1998b). Role of RhoA and

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29 Electromechanical and Pharmacomechanical Coupling in Vascular Smooth Muscle Cells GUY DROOGMANS, BERND NILIUS, HUMBERT DE SMEDT, JAN B. PARYS, and LUDWIG MISSIAEN Department of Physiology, Catholic University of Leuven, B-3000 Leuven, Belgium

I. INTRODUCTION

the T type (transient or low voltage activated; single channel conductance 7–15 pS) has also been observed in a number of smooth muscle cell preparations (Benham et al., 1987b; Friedman et al., 1986; Loirand et al., 1989; Simard, 1991; Wang et al., 1989; Yoshino et al., 1988). Expression of the L-type Ca2⫹ channel depends on the differentiated state of vascular smooth muscle cells, as the current was decreased significantly in dedifferentiated A7r5 cells and increased upon differentiation with retinoic acid. Expression of the L-type Ca2⫹ channel 움1C subunit is also highly coordinated with the expression of smooth muscle specific proteins, such as 움-actin and myosin heavy chain (Gollasch et al., 1998). L-type Ca2⫹ channels, which are responsible for the upstroke of the action potential and for Ca2⫹ influx activated by membrane depolarization, show a threshold for activation around ⫺40 mV and are fully activated around 0 mV. Inactivation of these channels is clearly voltage dependent, but also shows a component dependent on intracellular Ca2⫹. This latter component might be important during agonist stimulation as the rise in [Ca2⫹]i due to Ca2⫹ release from internal stores might down regulate the channels (Komori and Bolton, 1991; Schneider et al., 1991). McCarron et al. (1992) also described a positive feedback of intracellular Ca2⫹, i.e., an enhancement of the Ca2⫹ current via a Ca2⫹-dependent activation of calmodulin-dependent protein kinase II. Elevation of [Ca2⫹] at the cytoplasmic side of insideout patches inhibits Ca2⫹ channels by reducing their open probability but does not affect their availability, suggesting an interaction of intracellular Ca2⫹ with a single membrane-associated site that may reside on the channel protein itself (Schuhmann et al., 1997).

The contractile response of smooth muscle cells is triggered by an increase in free cytoplasmic calcium concentration ([Ca2⫹]i). This change in [Ca2⫹]i can be brought about by an increased influx of Ca2⫹ ions from the extracellular medium or by a mobilization of Ca2⫹ ions from intracellular pools. Vasoconstrictive agonists induce a contractile response that is poorly correlated with the concomitant changes in membrane potential. This has led to the introduction of the term pharmacomechanical coupling, as opposed to electromechanical coupling (caused by depolarization of the cell membrane), and to a subdivision of Ca2⫹ entry into voltage-gated and receptor-mediated mechanisms (Somlyo and Somlyo, 1967). The membrane potential is not only important for the gating of voltage-dependent channels, but is also a major determinant of the driving force for Ca2⫹ influx through receptor-mediated channels. This chapter reviews these various Ca2⫹ entry pathways and agonist-induced Ca2⫹ release mechanisms.

II. Ca2⫹ ENTRY MECHANISMS A. Voltage-Gated Ca2⫹ Channels 1. General Characteristics The L type (long-lasting or high voltage activated; single channel conductance 20–28 pS) is the most predominant Ca2⫹ channel in smooth muscle cells, although

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V. Excitation–Contraction Coupling

Voltage ranges for steady-state activation and inactivation overlap in a region between ⫺30 and ⫺20 mV (Droogmans and Callewaert, 1986). The window current at these potentials may account for the tonic component of Ca2⫹ influx and contraction during a sustained depolarization of the cell membrane, e.g., during K⫹ depolarization. L-type Ca2⫹ channels are voltage dependently blocked by dihydropyridines (DHPs). The block is less pronounced if the channels are activated from a more hyperpolarized potential, which suggests a much higher affinity of DHPs for the inactivated state of the channel (Bean, 1984; Bean et al., 1986; Benham et al., 1987b; Yatani et al., 1987). DHPs block the vascular smooth muscle L-type Ca2⫹ channel at lower concentrations than the cardiac Ca2⫹ channel, although their 움1 subunit (움1C), which binds DHPs, is derived from the same gene. Welling et al. (1997) showed that the different DHP sensitivities of cardiac and vascular L-type Ca2⫹ channels are caused, at least partially, by the tissue-specific expression of alternatively spliced IS6 segments of the 움1C gene. Changes in extracellular pH strongly affect the amplitude of the peak Ca2⫹ channel current in isolated smooth muscle cells from the basilar artery of the guinea pig (West et al., 1992). An alkaline extracellular pH reversibly increases and an acidic pH reduces the Ca2⫹ current (ICa) in vascular smooth muscle cells from bovine pial and porcine coronary arteries (Klo¨ckner and Isenberg, 1994a), suggesting that an acidic extracellular pH shifts the voltage-dependent gating due to the screening of negative surface charges and reduces the single Ca2⫹ channel conductance (웂Ca) due to the competition of protons and Ca2⫹ for a binding site that modulates 웂Ca . Changes in intracellular pH affect neither single channel conductance nor the lifetime of the open state, but do affect the channel availability (increased frequency of nonblank sweeps at alkaline pH), suggesting that deprotonation of cytosolic-binding sites shifts the channels from a sleepy to an available state (Klo¨ckner and Isenberg, 1994b). A reduction of PO2 (hypoxia) produces a rapid and reversible voltage-dependent inhibition of the macroscopic L-type Ca2⫹ current in enzymatically dispersed cells from rabbit cerebral, celiac, femoral, and main pulmonary arteries, as well as from the porcine coronary artery. Hypoxia selectively slows activation kinetics, but channel deactivation and inactivation are unaltered by low PO2. In addition, hypoxia produces a reversible shift of the Ca2⫹ conductance–voltage curve toward positive membrane potentials (Franco Obregon and Lopez Barneo, 1996; Franco Obregon et al., 1995). This O2 sensitivity of Ca2⫹ channels, which has also been observed for the 움1C subunit of the human cardiac L-type

Ca2⫹ channel (Fearon et al., 1997), may contribute to the well-known hypoxic dilatation of systemic and main pulmonary arteries. Application of positive or negative pressure to the pipette increases or decreases peak inward Ca2⫹ current in isolated rat basilar arterial myocytes. Changes in cell volume on application of hypo- or hyperosmotic superfusing solutions mimic these effects (Langton, 1993), suggesting that membrane stretch rather than applied pressure per se determines the changes in peak current. 2. L-Type Ca2ⴙ Channel Phosphorylation Intracellular ATP dose dependently enhances the L-type Ca2⫹ current without altering its voltagedependent features (Ohya et al., 1987; Ohya and Sperelakis, 1989a,b). These effects cannot be mimicked by stable ATP derivatives (AMP-PNP or AMP-PCP) and are abolished by nonspecific or specific protein kinase C (PKC) inhibitors but not by tyrosine kinase (tyr-PK) inhibitors, suggesting that the ATP-induced stimulation of L-type Ca2⫹ channels requires functional activity of a PKC isozyme (McHugh and Beech, 1997). However, both in whole cell and in cell-attached recordings from rat portal vein, the tyr-PK inhibitor genistein, but not its inactive analogue daidzein, inhibits the L-type Ca2⫹ current, suggesting a modulation by endogenous tyr-PK activity (Liu et al., 1997a; Liu and Sperelakis, 1997). Exposure of the cytoplasmic side of excised membrane patches to the purified catalytic subunit of protein phosphatase 2A (PP2A) reduces the open probability of Ca2⫹ channels, an effect that is completely prevented by okadaic acid, suggesting that a PP2A-sensitive regulatory site controls the gating of L-type Ca2⫹ channels (Groschner et al., 1996). Likewise, application of purified protein phosphatase 2B (PP2B) to the cytoplasmic side of inside-out patches from the same cell type also inhibits Ca2⫹ channel open probability and availability, suggesting a PP2B-mediated dephosphorylation process (Schuhmann et al., 1997). It is also interesting to note that phosphatase inhibitors reduce ICa in gastric and colonic smooth muscle (Ward et al., 1991), but enhance it in guinea pig taenia coli (Usuki et al., 1991), suggesting that phosphorylation may either inhibit or stimulate L-type Ca2⫹ channels. 3. Modulation by Cyclic Nucleotides Ohya et al., (1989a) reported that cyclic AMP (cAMP), unlike cardiac cells, has no effect on the L-type Ca2⫹ current in smooth muscle cells of guinea pig mesenteric artery, whereas 웁-adrenergic stimulation and forskolin both enhance the Ca2⫹ current in pig coronary artery (Fukumitsu et al., 1990) and rat portal vein

29. Electro- and Pharmacomechanical Coupling

(Liu et al., 1997b) through a cAMP-dependent process. In contrast, the vasodilating action of the parathyroid hormone, which was correlated with the inhibition of voltage-gated Ca2⫹ channels (Wang et al., 1991a), is mediated by a cAMP-dependent mechanism (Wang et al., 1991b). Also, L-type Ca2⫹ channel currents in rat tail artery are inhibited by the activation of cAMPdependent protein kinase (PKA) using 8-bromo-cAMP (Chik et al., 1996). Ruiz Velasco et al. (1998) showed that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca2⫹ channel activity in rabbit portal vein myocytes. Similar results were observed in rat mesenteric artery (Taguchi et al., 1997) and portal vein (Ishikawa et al., 1993). In cell-attached patches from a basilar artery of the guinea pig, 8-bromo-cAMP increases the probability of L-type Ca2⫹ channel opening, whereas the voltage dependence, the number of channels, the number of open states, the time constants of the open states, and the proportion of time spent in each open state are unchanged (Tewari and Simard, 1994). Molecular biological studies suggest that the Thr165 residue on the 웁 subunit of the L-type Ca2⫹ channel is the cAMP-dependent phosphorylation site, a residue that is not present in the smooth muscle 웁3 subunit (Zong et al., 1995). 4. Modulation by Vasoconstrictors Linked to G-Protein-Coupled Phospholipase C (PLC) Activation The modulation of smooth muscle L-type Ca2⫹ channels by these excitatory agonists is a puzzling and controversial issue. In some tissues ICa is enhanced by agonists, such as noradrenaline (Benham and Tsien, 1988; Nelson et al., 1988; Pacaud et al., 1989), angiotensin II (A-II, Bkaily et al., 1988; Ohya and Sperelakis, 1991), histamine (Oike et al., 1992), endothelin (Goto et al., 1989), acetylcholine (Matsuda et al., 1990), and 웁 agonists (Fukumitsu et al., 1990). In other tissues these agonists inhibit ICa (Droogmans et al., 1987; Galizzi et al., 1987; Pacaud et al., 1987; Tomita, 1988; Xiong et al., 1991). Even in the same tissue and under comparable experimental conditions, it has been reported that noradrenaline either enhances (Benham and Tsien, 1988) or inhibits L-type Ca2⫹ channels (Droogmans et al., 1987). The latter effect occurred through the activation of 움1-adrenergic receptors, whereas the former was not related to either 움 or 웁 stimulation. In most tissues, agonist-induced effects consist in a change of the peak amplitude of ICa without significant effects on the voltage-dependent characteristics of the channels. A shift in the voltage dependence of activation has been observed occasionally (Nelson et al., 1988). Most of the reported effects are probably mediated via

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the stimulation of PLC and activation via diacylglycerol (DG) of PKC, which is thought to phosphorylate a number of endogenous proteins, including ion channels. A variety of DG analogues mimic this PKC-activating action of DG and have been used as tools to delineate the role of PKC activation in agonist-mediated responses. The inhibitory effect of the peptides vasopressin and bombesin on ICa in aortic smooth muscle (cell line A7r5) can be mimicked by DG and phorbol esters (Galizzi et al., 1987). However, it has also been reported that 12-O-tetradecanoylphorbol-13-acetate (TPA) increases ICa in the same cells (Fish et al., 1988). The stimulating effect of acetylcholine on ICa in smooth muscle cells of toad stomach can be mimicked by analogues of DG that are able to activate PKC (Vivaudou et al., 1988). The reduction of ICa induced by noradrenaline in rabbit ear artery becomes irreversible if the GTPbinding proteins, which link receptor occupation to the stimulation of PLC, are permanently activated by GTP웂-S (Droogmans et al., 1987). The stimulation of ICa by A-II in guinea pig portal vein (Ohya and Sperelakis, 1991) and by histamine in rabbit saphenous artery (Oike et al., 1992) is also mediated by GTP-binding proteins. In contrast, the stimulation of ICa with A-II in A7r5 cells can be prevented by lavendustin-A, a selective inhibitor of Tyr-PK, and by LY-294002, an inhibitor of PI-3-kinase, but not by H-7, an inhibitor of PKC, suggesting that A-II may stimulate Ca2⫹ channels through Tyr-PK and PI-3-kinase without participation of PKC (Seki et al., 1999). Schuhmann and Groschner (1994) found that low concentrations (⬍30 nM) of TPA caused inhibition of Ca2⫹ channels, whereas higher concentrations of TPA (⬎100nM) elicited a transient stimulation, followed by a sustained inhibition of Ca2⫹ channel activity in cellattached patches of human umbilical vein smooth muscle, which they interpret by two distinct PKC-dependent mechanisms of L-type Ca2⫹ channel regulation. PKC inhibitors decrease channel availability and long open events, but do not affect the voltage dependence of the open probability and the single channel conductance in A7r5 vascular smooth muscle cells (Obejero Paz et al., 1998). Activation of PKC with phorbol 12,13-dibutyrate and inhibition of protein phosphatases by intracellular dialysis of okadaic acid are both ineffective in modulating whole cell and single channel currents, suggesting a high level of PKC activity in resting, nonstimulated A7r5 cells that modulates the gating of L-type Ca2⫹ channels. Arachidonic acid causes a gradual depression of the Ca2⫹ current in rabbit ileum, which cannot be prevented by the PKC inhibitor staurosporine and cannot be mimicked by phorbol esters (Shimada and Somlyo, 1992). Because Ca2⫹ activates phospholipase A2 , it is possible that the inhibition of the Ca2⫹ current in response to

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Ca2⫹-mobilizing agonists is mediated by arachidonic acid. It has also been shown that endothelin augments the current through L-type Ca2⫹ channels without modifying the voltage-dependent parameters of the current (Goto et al., 1989). It is not clear whether this is a direct action on the channels or whether it is mediated via second messengers.

B. Receptor-Mediated Ca2⫹ Entry These Ca2⫹ influx pathways are quite diverse with regard to their mode of activation and their biophysical and pharmacological properties. Some are activated by agonist–receptor interaction, either directly (receptoroperated channels, ROCs) or via generation of second messengers (second messenger–operated channels, SMOCs), whereas others are activated by the depletion of internal inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2⫹ pools (store-operated channels, SOCs). Stimulation of P2X purinergic receptors in rabbit ear artery activates a rapidly developing and desensitizing cationic current with a reversal potential near 0 mV. The single channels underlying this current are nonselective cation channels that discriminate poorly between monovalent cations and have a three- to four fold higher selectivity for Ca2⫹ over Na⫹ (Benham et al., 1987a; Benham and Tsien, 1987). The primary action of these channels is most likely depolarization of the cell membrane and activation of voltage-gated Ca2⫹ channels, but it has also been proposed that Ca2⫹ influx through these channels significantly contributes to the rise in [Ca2⫹]i (Benham, 1989; Schneider et al., 1991). It has also been reported that Ca2⫹ influx through this channel may initiate a subsequent large Ca2⫹ release from ryanodine-sensitive stores (Loirand and Pacaud, 1995; Pacaud et al., 1994). Most agonist-activated cation channels (Amede´e et al., 1990; Inoue and Kuriyama, 1993; Loirand et al., 1991; Oike et al., 1993; Wang et al., 1993; Wang and Large, 1991; Xiong et al., 1991) activate much slower than the fast ligand-activated channel described earlier and are therefore most likely activated by cytoplasmic second messengers linked to the receptor–transducer system. These SMOCs are coupled to pertussis-toxin sensitive G-proteins in most cell types, as internal perfusion with GDP웁S prevents their activation, whereas internal perfusion with GTP웂S activates them in the absence of an agonist. Although activation of these channels is often associated with Ca2⫹ release from internal stores, they are not directly activated by either IP3 or Ca2⫹. The concept of store-operated Ca2⫹ entry, which originated partly as a result of studies in smooth muscle

(Casteels and Droogmans, 1981; van Breemen, 1977) and which assumes a close interaction between internal stores and the plasma membrane, has evolved into a variety of models to explain agonist-induced Ca2⫹ entry in excitable and nonexcitable cells. Until now, no convincing electrophysiological evidence has been presented for the existence of a store depletion-activated Ca2⫹ entry channel in smooth muscle cells, similar to that in mast cells (Hoth and Penner, 1992), which is highly selective for Ca2⫹ and is inactivated by physiological intracellular Ca2⫹ concentrations. Also, the cellular mechanisms that link emptying of the pools to opening of SOCs in the plasma membrane are far from resolved.

C. Modulation of the Driving Force for Ca2⫹ Influx It is obvious that all factors that cause a change in membrane potential of vascular smooth muscle cells will not only affect gating of voltage-operated Ca2⫹ channels, but also the flow of Ca2⫹ through all Ca2⫹ entry pathways. It has been shown that local increases in [Ca2⫹]i due to spontaneous Ca2⫹ release from ryanodine-sensitive stores generate transient outward K⫹ currents through maxi-KCa channels, which cause hyperpolarization and relaxation (Nelson et al., 1995), indicating that Ca2⫹activated K⫹ channels may play a role in the control of myogenic tone in arteries. Nitric oxide, the major endothelium-derived relaxing factor (EDRF), is thought to relax smooth muscle cells by stimulation of guanylate cyclase, accumulation of its product cGMP, and cGMP-dependent modification of several intracellular processes, including activation of K⫹ channels through PKG. However, it has also been reported that both exogenous nitric oxide and native EDRF can directly activate single Ca2⫹-dependent K⫹ channels (Bolotina et al., 1994). It has been shown that the endothelium-derived hyperpolarizing factor (EDHF) in small resistance arteries is K⫹ that effluxes from endothelial cells, which increases the myoendothelial K⫹ concentration, hyperpolarizes, and relaxes adjacent smooth muscle cells by activating inwardly rectifying K⫹ channels (KIR, Edwards et al., 1998). Extracellular K⫹-induced hyperpolarizations involving KIR channels and concomitant dilatations have also been observed in rat coronary and cerebral arteries (Knot et al., 1996). KIR channels are mainly expressed in small-diameter cerebral and coronary arteries and may have functional consequences for the regulation of cell membrane potential and tone in these arteries (Quayle et al., 1996). KIR channels in smooth muscle do not appear to be as widely distributed as KATP channels. The latter

29. Electro- and Pharmacomechanical Coupling

channels, which contribute to the resting membrane conductance of some types of vascular smooth muscle and open under situations of metabolic compromise (Dart and Standen, 1995), are targets of a wide variety of vasodilators and constrictors. Adenosine and calcitonin gene-related peptide activate KATP channels through PKA (Kleppisch and Nelson, 1995; Quayle et al., 1994). Several vasoconstrictor receptors coupled to PKC inhibit KATP channels (Bonev and Nelson, 1996; Kubo et al., 1997; Wakatsuki et al., 1992) whereas Ogata et al. (1997) have reported a role for Tyr-PK in the regulation of KATP channel activity. Similarly, activation of nonselective cation channels (described in Section II,B) either directly gated by agonists or indirectly via agonist-induced Ca2⫹ release or agonist-generated second messengers not only cause Ca2⫹ influx through these channels, but also depolarize the cell membrane and thereby activate voltagedependent Ca2⫹ channels and reduce the driving force for Ca2⫹ entry. Finally, activation of Ca2⫹-activated Cl⫺ currents due to the agonist-induced release of internal Ca2⫹ (Droogmans et al., 1991; Kamouchi et al., 1998; Lamb et al., 1994; Pacaud et al., 1989, 1991; Soejima and Kokubun, 1988; Van Helden, 1988; Yuan, 1997) or of Cl⫺ currents activated by cell swelling (Greenwood and Large, 1998; Yamazaki et al., 1998) may also depolarize the cell membrane. This depolarization activates voltage-gated Ca2⫹ channels, thereby causing a concomitant influx of extracellular Ca2⫹. The resulting depolarization decreases the driving force for Ca2⫹ and thus reduces the influx of Ca2⫹ through receptor-mediated entry mechanisms.

III. PHARMACOMECHANICAL COUPLING The general principles of the signal-transducing pathway leading to contraction of vascular smooth muscle are well known: binding of an agonist to cell surface receptors stimulates inositol lipid breakdown, thereby producing IP3, which then releases Ca2⫹ from intracellular stores leading to contraction. IP3 mobilizes Ca2⫹ from nonmitochondrial Ca2⫹ pools by interacting with the IP3 receptor (IP3R) in the membrane of these internal stores (Berridge, 1993). IP3 seems to be the major physiological messenger for the Ca2⫹ release component of pharmacomechanical coupling (Kobayashi et al., 1989). Indeed, the ability of IP3 to mobilize Ca2⫹ has now been demonstrated in many different types of smooth muscle. In addition the time course of the tension development upon flashing the photolabile inactive precursor of IP3 (caged IP3) was comparable to the tension rise in intact muscle after agonist stimulation (Walker et al., 1987). Smooth muscle

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cells also express ryanodine receptors, and they too contribute to the shaping of the intracellular Ca2⫹ signal. The latter channels are reviewed in other parts of this book. In addition, other Ca2⫹-releasing messengers in smooth muscle, such as sphingosine-1-P (Ghosh et al., 1994), have been described, but their role in pharmacomechanical coupling is less clear. This classical view of pharmacomechanical coupling also has to take into account that changes in [Ca2⫹]i just underneath the plasma membrane can be much higher than in the bulk of the cytoplasm (Etter et al., 1994). Also, rises in [Ca2⫹]i can be very localized (sparks) and have little direct effect on the mean cytosolic [Ca2⫹]i, which regulates contraction (Nelson et al., 1995). Frequency modulation of Ca2⫹ sparks is involved in the regulation of arterial diameter (Porter et al., 1998). This part of the chapter focuses on the role of IP3Rs in pharmacomechanical coupling in vascular smooth muscle.

A. IP3R Heterogeneity IP3Rs are homo- or heterotetramers composed of polypeptide subunits with molecular masses of 220 to 260 kDa. There is evidence for at least three different genes encoding IP3Rs designated as type 1 (Furuichi et al., 1989; Mignery et al., 1990; Kume et al., 1993; Yamada et al., 1994), type 2 (Su¨dhof et al., 1991; Yamamoto-Hino et al., 1994), and type 3 (Blondel et al., 1993; Maranto, 1994). Further isoform diversity arises from alternative splicing of the mRNAs. Two alternatively spliced domains SI and SII were described for IP3R1 (Mignery et al., 1990; Danoff et al., 1991; Nakagawa et al., 1991a,b). The SI domain consists of 45 nucleotides coding for 15 amino acids and is located near the IP3-binding region. This SI domain is represented to a variable extent among the IP3R1 messengers in different cell types, including vascular smooth muscle cells (Nakagawa et al., 1991a,b; De Smedt et al., 1994). The SII domain consists of 120 nucleotides (40 amino acids) and is localized in the large cytosolic domain between two PKA phosphorylation sites. The SII subtype is brain specific (Danoff et al., 1991; Nakagawa et al., 1991a). It remains to be determined if similar splice mechanisms also generate tissue-specific forms of IP3R2 or IP3R3. IP3R2 shares 68–69% sequence identity with IP3R1 (Yamamoto-Hino et al., 1994). IP3R3 (Blondel et al., 1993) has 62 and 65% identity with the entire amino acid sequence of IP3R1 and IP3R2, respectively (YamamotoHino et al., 1994). Based on dot matrix analysis and sequence alignments, a pattern of 13 conserved regions separated by 13 short variable regions was found (Maranto, 1994). There is a considerable degree of simi-

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larity between the homologous conserved regions of all known IP3Rs. The variable regions may represent regions with less functional significance or regions that confer functional differences among IP3R isoforms.

B. Expression of IP3R Messengers in Vascular Smooth Muscle Cells Regional distribution of IP3R1 mRNA in various tissues was examined by Northern blot analysis and by in situ hybridization (Furuichi et al., 1990, 1993; Fujino et al., 1995). A considerable amount of IP3R1 mRNA was located in smooth muscle cells, such as those of arteries, bronchioles, oviduct, and uterus (Furuichi et al., 1990). Differences in the contractile responses of the cerebral vasculature were shown to result from differences in IP3R density and/or affinity (Zhou et al., 1997). A screening for IP3R2 and IP3R3 in different mouse tissues revealed that these mRNAs showed a much more cell- or tissue-restricted expression than the ubiquitous IP3R1, and they seemed to be mostly expressed in cell types that have a secretory function (Fujino et al., 1995). In a study where relative levels of the three different IP3R mRNAs were compared by ratio polymerase chain reaction, it was found that A7r5 cells, which are derived from rat aorta, express 75% IP3R1 and 25% IP3R3 with no indication for IP3R2 (De Smedt et al., 1994). Expression levels in cell lines may, however, deviate from the native tissue, as IP3R3 was expressed abundantly in proliferating cell types (De Smedt et al., 1997). IP3R3 was the predominant subtype in developing vascular smooth muscle and there was a switch to IP3R1 in developed smooth muscles (Tasker et al., 1999). IP3R1 and IP3R3 also seem to be the predominant isoforms expressed in renal glomerular mesangial and smooth muscle cells (Yang et al., 1995; Monkawa et al., 1998). In addition, it was shown that the expression levels of IP3R1 in renal vascular smooth muscle cells were downregulated in streptozotocin-induced diabetic rat and mice, in association with renal hypertrophy (Sharma et al., 1999). Although IP3R2 was present in nonvascular smooth muscle (Newton et al., 1994; Morgan et al., 1996), being the major isoform in cardiac myocytes (Ramos-Franco et al., 1998), there is as yet no report about the expression levels of this isoform in vascular smooth muscle.

C. Molecular Structure of IP3Rs The different IP3R isoforms are similar in their overall topology with a large hydrophilic region comprising 80% of the protein at the NH2-terminal, followed by a cluster of hydrophobic segments forming the channel domain and a short hydrophilic COOH-terminal.

Quick-freeze deep-etch replica electron microscopy revealed that the IP3R tetramer is a square-shaped assembly that, in comparison with the ryanodine receptor, was characterized by a very compact structure (Katayama et al., 1996). This compact character was supported by controlled proteolysis, which revealed that each subunit is constituted by noncovalent interactions of five major well-folded components that are not susceptible to mild trypsinolysis (Yoshikawa et al., 1999b). Functionally, IP3R can be divided into three parts: an IP3-binding domain, a coupling domain, and a channel domain. The IP3-binding domain of IP3R1 comprises the 650 amino-terminal residues (Mignery and Su¨dhof, 1990; Miyawaki et al., 1991). Photoaffinity labeling with an IP3 analogue localized IP3 binding between amino acids 476 and 501 (Mourey et al., 1993). Deletion mutation analysis of IP3R1 has shown that residues 225–578 are sufficient for specific IP3 binding and thus form the IP3-binding ‘‘core’’ (Yoshikawa et al., 1996, 1999a,c). The complete IP3-binding pocket consists of two noncovalently but tightly associated structural domains. The C-terminal part has low affinity for IP3, whereas the Nterminal one alone is not sufficient for binding but is capable of potentiating the binding affinity (Yoshikawa et al., 1999a). The critical region of 351 amino acids of IP3R1 shares 44% identity and 61% similarity with the corresponding regions in the other IP3R isoforms. One of the alternative splice variants of the type 1 isoform contains a 15 amino acid deletion in this region. The IP3R1 SI(⫺) splicing variant had a similar affinity as the IP3R1 SI(⫹) variant (Newton et al., 1994). The rank order of the affinity for IP3 is IP3R2 ⬎ IP3R1 ⬎ IP3R3 (Su¨dhof et al., 1991; Newton et al., 1994; Parys and Bezprozvanny, 1995; Yoneshima et al., 1997; Wojcikiewicz and Luo, 1998; Vanlingen et al., 1999), although deviations of this order were reported (Wojcikiewicz and Luo, 1998; Cardy and Taylor, 1998). The coupling domain (residues 651–2275) separates the ligand-binding domain from the Ca2⫹ channel domain and links IP3 binding to channel gating (Mignery and Su¨dhof, 1990). This region also integrates the various signals that control the channel opening and closing. Analysis of the IP3R1 sequence revealed the presence of several putative regulatory sites, e.g., several Ca2⫹binding sites (Mignery and Su¨dhof, 1990; Sienaert et al., 1996, 1997), two ATP-binding sites (Ferris et al., 1990), calmodulin-interaction domains (Yamada et al., 1995; Patel et al., 1997; Cardy and Taylor, 1998; Sipma et al., 1999), and an FKBP12-binding site (Cameron et al., 1995a,b, 1997). Also, phosphorylation sites for various protein kinases (reviewed in Joseph, 1996), including PKA, PKG, Tyr-PK, and Ca2⫹ /calmodulin-dependent protein kinase II, have been reported in this domain. The cross-talk among these diverse signaling pathways

29. Electro- and Pharmacomechanical Coupling

results in a complex regulation of IP3-induced Ca2⫹ release. The homology among the different IP3R isoforms is the lowest in the coupling domain, suggesting differential regulation in different isoforms. The channel domain contains six putative transmembrane domains, including a luminal loop with two N-glycosylation sites and a putative pore-forming sequence between the fifth and sixth transmembrane domain (Michikawa et al., 1994). The two glycosylation sites are preserved in the type 1 and 2 IP3R, whereas IP3R3 contains only one consensus site for N-glycosylation (Furuichi et al., 1994). The conservation of this consensus site suggests that all isoforms may be glycosylated and implies a common domain architecture for all IP3R isoforms. The intraluminal loop segment can be divided into a variable region (residues 2463–2523) and a region that is very conserved among the three isoforms (residues 2524–2569). The variable region contains a cluster of negatively charged (Glu and Asp) residues, the two consensus sites for N-glycosylation, and a highaffinity Ca2⫹-binding site (Sienaert et al., 1996). The conserved region includes a hydrophobic segment (residue 2530–2552) and is thought to form the putative pore, embedded in the endoplasmic reticulum membrane from the luminal side. All IP3Rs are tetrameric structures. From analysis of deletion mutants, it is apparent that transmembrane domains, in addition to forming the ion channel, are also essential for receptor anchoring and oligomerization (Mignery and Su¨dhof, 1990; Miyawaki et al., 1991; Joseph et al., 1997). Functional IP3Rs are tetrameric structures consisting of either homomeric or heteromeric interactions between the different isoform subunits (Monkawa et al., 1995; Wojcikiewicz and He, 1995; Joseph et al., 1995).

D. Purification and Characterization of Smooth Muscle IP3R 1. IP3R1 Smooth muscles are a rich source of IP3R1 from which this isoform can be easily purified. An up to 1000fold purification of the IP3R1 from aortic smooth muscle was reported (Chadwick et al., 1990). After detergent solubilization, purification was obtained using heparin– agarose chromatography, a lectin-based chromatography step, and either a double sucrose density gradient centrifugation step (Chadwick et al., 1990) or an additional chromatography step based on the interaction of IP3R1 with calmodulin (Koga et al., 1994; Islam et al., 1996). Similar procedures, leading to similar results, were used for the purification of IP3R1 from other smooth muscle types such as vas deferens (Mourey

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et al., 1990). The yield of a typical preparation varied, depending on the technique, between 30 (Chadwick et al., 1990) and 200 애g (Islam et al., 1996) purified IP3R1/kg bovine or porcine aorta as starting material. IP3R1 from aortic smooth muscle has the same properties and the same structure as the type 1 receptor from other tissues (Chadwick et al., 1990; Marks et al., 1990). Its monomeric molecular mass, as determined after gel electrophoresis, is, however, significantly smaller than the neuronal IP3R1 (261 versus 273 kDa) (Parys et al., 1995). Indeed, as in other peripheral tissues, it does not contain the S2 splice site and therefore represents a shorter splice isoform (Danoff et al., 1991; Nakagawa et al., 1991a). The splicing of the first splice site (S1) is very variable in peripheral tissues (Nakagawa et al., 1991a; De Smedt et al., 1994). In porcine aorta and in the much used A7r5 vascular smooth muscle cell line, S1 was included in 54% (Islam et al., 1996) and in 76% (Parys et al., 1995) of the receptors, respectively. As reported for the IP3R1 in other tissues, smooth muscle IP3R1 binds IP3 with high affinity and specificity and this binding is antagonized by heparin (IC50 1.5 애g/ml) and is stimulated by alkalinization, with optimal binding at pH 9.5 (Chadwick et al., 1990; Marks et al., 1990; Islam et al., 1996). Single channel analysis was performed after incorporation of either aortic smooth muscle microsomes (Ehrlich and Watras, 1988) or purified aortic smooth muscle IP3R (Mayrleitner et al., 1991) in lipid bilayers. Smooth muscle IP3R forms an IP3-dependent, ATP-stimulated, and heparin-sensitive Ca2⫹ channel of relatively low conductance (10–32 pS). These results are in accordance with the functional work performed in flux measurements (see further) and are similar to those obtained with cerebellar IP3R1. 2. IP3R3 In contrast to IP3R2, IP3R3 is expressed in vascular smooth muscle, but no technique allowing its purification has yet been described. Therefore the properties of smooth muscle IP3R3 can only be inferred from its properties, as described in other tissues. In those studies, it was demonstrated that IP3R1 and IP3R3 differ in numerous characteristics, including sensitivity toward IP3, ATP, and redox activity (Missiaen et al., 1998a).

E. Subcellular Localization of IP3Rs in Vascular Smooth Muscle 1. IP3R1 In aortic smooth muscle the localization of IP3R1 is predominantly central, which corresponds to the

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localization of the sarcoplasmic reticulum (SR) (Nixon et al., 1994). In vas deferens, IP3R1 also localized to the SR, which is more peripherally located, and also colocalized with the luminal Ca2⫹-binding protein calsequestrin (Nixon et al., 1994), although calsequestrin preferentially clustered at discrete sites (Villa et al., 1993). Calsequestrin, however, is expressed more strongly in phasic than in tonic smooth muscles and was not described in aortic smooth muscle (Nixon et al., 1994). In both aortic and vas deferens smooth muscle cells, a similar localization was found for the ryanodine receptor as for IP3R1 (Lesh et al., 1998). 2. IP3R3 The localization of IP3R3 corresponded with the distribution of the SR in neonatal portal vein and aorta. In fully developed portal vein IP3R1 localized to the SR and in fully developed aorta both type 1 and 3 IP3Rs localized to the SR, although with a slightly different pattern (Tasker et al., 1999).

F. Kinetics of the Release Photoreleasing a high [IP3] in guinea pig portal vein smooth muscle resulted in a [Ca2⫹]i rise within approximately 10 msec (Somlyo et al., 1992), which points to the existence of a fast release component. 45Ca2⫹ flux experiments in permeabilized A7r5 vascular smooth muscle cells in addition revealed a slow component (Missiaen et al., 1992a; Sugiyama and Goldman, 1995), which lasted up to 100 min (Missiaen et al., 1997a). This slow release was not related to IP3 metabolism, to a slow dissociation of Ca2⫹ from luminal-binding proteins, nor to a spontaneous inactivation of the IP3R. The slow release component might represent a partially open state of the IP3R occurring when luminal-free [Ca2⫹] is low (Missiaen et al., 1992b). Evidence shows that Ca2⫹ is taken up in a structurally different compartment than the site from where it is released (Satoh et al., 1990; Menniti et al., 1991). The slow kinetics can therefore be partly explained by luminal communications between stores, through which Ca2⫹ might move from store units with IP3Rs that are not sensitive to a low [IP3] to regions of the endoplasmic reticulum that are sufficiently sensitive. Ca2⫹ transfer through these communication pathways in smooth muscle cells is blocked completely and reversibly by palmitoyl-CoA (Rys-Sikora et al., 1994). Palmitoyl-CoA partly inhibited the slow release phase in A7r5 vascular smooth muscle cells, implying that the transfer of Ca2⫹ through luminal connections contributes to the slow IP3-induced Ca2⫹ release in A7r5 cells (Missiaen et al., 1997a).

G. Control by Cytosolic Ca2⫹ A biphasic effect of cytosolic Ca2⫹ on IP3R was first observed in smooth muscle (lino, 1990; lino and Endo, 1992). Increasing the [Ca2⫹] to 300 nM augments the effectiveness of IP3 in releasing Ca2⫹. IP3R can therefore be considered as a Ca2⫹-induced Ca2⫹ release channel. This positive feedback exerted by Ca2⫹ could be very important and lead to a very rapid release from internal stores (Hirose et al., 1998). The stimulatory effect of cytosolic Ca2⫹ on vascular smooth muscle IP3R can be demonstrated more easily when the pools contain less Ca2⫹ (Missiaen et al., 1992b). High Ca2⫹ concentrations inhibit the release in smooth muscle, including vascular smooth muscle (Suematsu et al., 1984; lino, 1990; Missiaen et al., 1992b; Sienaert et al., 1997). The inhibition in vascular smooth muscle especially occurred at lower IP3 concentrations (Missiaen et al., 1994a; Bootman et al., 1995). This inhibition by Ca2⫹ presents a negative feedback on the release. Negative feedback could underlay the ceiling to agonistinduced [Ca2⫹]i rises in single smooth muscle cells (Williams et al., 1987). IP3R consists of an amino-terminal IP3-binding domain, a carboxy-terminal Ca2⫹ channel, and a coupling domain between this binding site and the channel (Mignery and Su¨dhof, 1990). Seven Ca2⫹-binding sites have been mapped on the cytosolic side of IP3R1 (Sienaert et al., 1996, 1997). It is possible that Ca2⫹ exerts its effect by acting on one of these Ca2⫹-binding sites. Ca2⫹ fluxes indicated that Ca2⫹ must at least interact with three different sites (Sienaert et al., 1997). An alternative possibility is that the effects of Ca2⫹ are exerted via associated proteins. A threefold shift in IP3 affinity was observed in aortic smooth muscle microsomes in the presence of 2 애M Ca2⫹, but not on the purified receptor, suggesting that a Ca2⫹-sensitizing factor, which is separated from IP3R1 during the purification procedure, is involved (Benevolensky et al., 1994). Moreover, Ca2⫹ sensitivity was restored by the addition of a side fraction obtained during purification and that was devoid of IP3-binding activity. A factor with similar properties has been suggested in other tissues as well, but until now remained elusive (Danoff et al., 1988). The Ca2⫹-binding protein calmodulin shifts the inhibitory part of the biphasic Ca2⫹-activation curve toward lower Ca2⫹ concentrations in A7r5 vascular smooth muscle cells (Missiaen et al., 1999a). This finding is difficult to reconcile with previous studies demonstrating that calmodulin caused a Ca2⫹-independent inhibition of IP3 binding to the type 1 IP3R (Cardy and Taylor, 1998; Sipma et al., 1999) and to the recombinant IP3-binding domain of IP3R1 (Sipma et al., 1999). The apparent discrepancy between functional data and

29. Electro- and Pharmacomechanical Coupling

IP3-binding data may be due to the existence of multiple calmodulin-binding or Ca2⫹-binding sites that play a role and/or to the fact that IP3 binding, which is only one, albeit crucial, step for channel opening, is not equivalent to the channel activity itself (Missiaen et al., 1999a). Although both cerebellar and aortic IP3R1 bind to calmodulin in a Ca2⫹-dependent way, there is evidence for a stronger binding of aortic IP3R1 as compared to its cerebellar equivalent (Islam et al., 1996). The fact that the binding site for Ca2⫹ /calmodulin is localized in the proximity of the S2-splicing site (Yamada et al., 1995) may be relevant in this respect (Islam et al., 1996).

H. Control by Luminal Ca2⫹ The Ca2⫹ content of the store controls the release process in A7r5 vascular smooth muscle cells (Missiaen et al., 1992a). Store depletion shifts the dose–response relationship for IP3-induced Ca2⫹ mobilization to the right. Luminal Ca2⫹ may interact directly with the channel or via some associated protein. Iino and Endo (1992) suggested that the loading dependence of the IP3-dependent Ca2⫹ release could represent the stimulatory effect of cytosolic Ca2⫹. However, the properties of the stimulation by cytosolic and luminal Ca2⫹ in A7r5 vascular smooth muscle cells were different, indicating that the effect was exerted at different sites (Sienaert et al., 1997).

I. Partial Ca2⫹ Release Even a prolonged stimulation with a submaximal [IP3] is unable to release all the Ca2⫹ accumulated in the IP3-sensitive store in vascular smooth muscle (Missiaen et al., 1992a). It is therefore said that low doses of IP3 only induce a partial release, implying that the release in response to a submaximal IP3 stimulus suddenly stops, despite the fact that there is still a large amount of Ca2⫹ left in the IP3-sensitive store. One hypothesis to explain this partial release is that individual stores present a heterogeneous IP3 sensitivity and that a low [IP3] completely discharges the most sensitive stores, while leaving the less sensitive pools more or less untouched (an all-or-none release) (Muallem et al., 1989; Oldershaw et al., 1991; Ferris et al., 1992). The other hypothesis is that a low [IP3] releases Ca2⫹ from the whole population of stores and that the release mechanism somehow inactivates (a steady-state mechanism) (Irvine, 1990; Tregaer et al., 1991). The IP3 threshold was not correlated with the extent of Ca2⫹ release in permeabilized A7r5 vascular smooth muscle cells. In contrast, the maximum rate of release, which was changed either by varying the level of IP3R activation or by changing the concentration of IP3Rs at a constant level of IP3R activation, was directly related

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to the extent of Ca2⫹ release (Missiaen et al., 1999b). These findings seem to suggest that IP3-induced Ca2⫹ release represents a partial emptying of the stores and not an all-or-none Ca2⫹ release of separate quanta.

J. Effect of Nucleotides 1. Adenine Nucleotides IP3-induced Ca2⫹ release in permeabilized smooth muscle cells requires ATP (Smith et al., 1985), but IP3 can also release Ca2⫹ in the absence of adenine nucleotides (Iino, 1987, 1990; Ehrlich and Watras, 1988). At least 50 different adenine nucleotides activated IP3R in vascular smooth muscle (Missiaen et al., 1997b). Some of them, such as 3⬘-phosphoadenosine 5⬘-phosphosulfate, coenzyme A, di(adenosine-5⬘)tetra-, and pentaphosphate, were more effective than ATP. Stimulatory ATP concentrations had no effect on the threshold [IP3] needed for initiating Ca2⫹ release, but stimulated Ca2⫹ release in the presence of suprathreshold IP3 concentrations by increasing the cooperativity of the release process. The interaction sites have been located in the transducing domain of IP3R (Maes et al., 1999). They increase the efficiency of transmitting IP3 binding to channel opening. Whereas low concentrations of adenine nucleotides stimulate the release, higher concentrations inhibit it (Iino, 1991). This inhibition was associated with a further increase in cooperativity but also by a shift in threshold toward higher IP3 concentrations (Missiaen et al., 1997b). The inhibitory interaction site is probably the IP3-binding site itself, as ATP competitively inhibits the Ca2⫹ release (Guillemette et al., 1987; Maeda et al., 1991). 2. Cyclic ADP-Ribose (cADPR) Cyclic ADP-ribose inhibited IP3-induced Ca2⫹ release in permeabilized A7r5 vascular smooth muscle cells with an IC50 of 20 애M (Missiaen et al., 1998b). 8-Amino-cADPR antagonized this effect of cADPR. Inhibition was prevented by a whole series of inositol phosphates that did not affect IP3-induced Ca2⫹ release and by pyrophosphate and various nucleotide di- or triphosphates. cADPR therefore seems to interact with a novel regulatory site on IP3R distinct from the IP3binding domain or the stimulatory adenine nucleotidebinding site. 3. Caffeine Caffeine and theophylline inhibit IP3R in A7r5 vascular smooth muscle cells (Missiaen et al., 1994b). Caffeine

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may interact with one of the ATP-binding sites on IP3R, as ATP prevented the inhibition (Missiaen et al., 1998b) and as caffeine displaced ATP from recombinant adenine nucleotide-binding sites of IP3R1 (Maes et al., 1999).

fore a pharmacological tool that, at least at low concentrations, can rather specifically activate IP3R in smooth muscle.

IV. SUMMARY 4. Guanine Nucleotides Guanine nucleotides have little effect on smooth muscle IP3R (Iino, 1991; Missiaen et al., 1997b), although they may enlarge the capacity of the IP3-sensitive Ca2⫹ stores by connecting them with IP3-insensitive Ca2⫹ stores (Ghosh et al., 1989). GTP-binding proteins might regulate IP3-mediated Ca2⫹ release in smooth muscle (Saida and van Breemen, 1987; Saida et al., 1988; Neylon et al., 1992, 1998). Exposure to pertussis toxin attenuated the initial rate of IP3-induced Ca2⫹ release in vascular smooth muscle (Neylon et al., 1998). cGMP has no direct effect on IP3R, but it phosphorylates IP3R in vascular smooth muscle (Komalavilas and Lincoln, 1994, 1996). Phosphorylation by PKG occurs with somewhat faster kinetics for aortic IP3R1 as compared to cerebellar IP3R1 (Islam et al., 1996). This phosphorylation may be related to the known effect of cGMP on smooth muscle relaxation and was therefore investigated further (Yoshida et al., 1991; Koga et al., 1994; Komalavilas and Lincoln, 1994, 1996). The phosphorylation site involved was determined to be serine-1755, a residue that can also be phosphorylated by PKA (Komalavilas and Lincoln, 1994). In this respect it is important to note that PKG— not PKA—is associated with the SR in aortic smooth muscle cells (Cornwell et al., 1991). The preferential phosphorylation of serine-1755 is conflicting with a previous report giving evidence that only cerebellar IP3R1 (containing the S2 splice site) but not peripheral IP3R1 (lacking the S2 splice site) is phosphorylated on this residue (Danoff et al., 1991). This may indicate a difference in regulation between various splice isoforms of IP3R1.

K. Effect of the Sulfhydryl Reagent Thimerosal Low concentrations of thimerosal (⬍10 애M) sensitize the IP3R1 in vascular smooth muscle by shifting the threshold for Ca2⫹ release toward lower IP3 concentrations and by increasing the Hill coefficient (Missiaen et al., 1996). IP3R3 is not stimulated by thimerosal (Missiaen et al., 1998a). At higher concentrations, an inhibitory effect also becomes apparent. Stimulation of IP3R occurs at lower doses than those required to inhibit the endoplasmic reticulum Ca2⫹ pump (Parys et al., 1993). Still higher doses nonspecifically increase the passive Ca2⫹ leak. Thimerosal is there-

We have discussed the various Ca2⫹ entry pathways and intracellular Ca2⫹ release mechanisms that contribute to the increase in [Ca2⫹]i that triggers the contraction of vascular smooth muscle. The characteristics of voltage-dependent Ca2⫹ channels and the release mechanisms from IP3-sensitive Ca2⫹ stores are well documented. Although the concept of store-operated Ca2⫹ entry originates from smooth muscle, the link between channel activity and the filling degree of the store is still a matter of intense research. L-type voltage-dependent Ca2⫹ channels appear to be modulated not only by vasoactive agonists, but also by extracellular pH, PO2, and blood pressure. Vasoactive agonists also activate additional Ca2⫹ entry pathways, either directly or via generation of second messengers or via depletion of IP3sensitive Ca2⫹ stores. Some of these channels are highly Ca2⫹ selective, but others do barely discriminate between monovalent and divalent cations. The molecular nature of a number of these channels has been resolved, but the nature of store-operated Ca2⫹ entry is still unknown and is the subject of intense research in both excitable and nonexcitable cells. The elucidation of its molecular identity will ultimately identify the enigmatic nature of the signal transfer between store and Ca2⫹ channel. Evidence shows that Ca2⫹ is released from a different site than where it was initially accumulated, implying that the luminal transfer of Ca2⫹ between various compartments within the store must occur. IP3R seems to be the major pathway for Ca2⫹ release during pharmacomechanical coupling. Smooth muscle cells also express ryanodine receptors and they also contribute to the shaping of the intracellular Ca2⫹ signal. Although both Ca2⫹ channels exhibit the phenomenon of Ca2⫹-induced Ca2⫹ release, the relative importance of both Ca2⫹ release pathways still has to be assessed. There are three different types of IP3R and it is becoming increasingly evident that functional differences exist between these isoforms, including differences in redox sensitivity, in ATP sensitivity, and possibly also in Ca2⫹ sensitivity. It remains, however, to be determined whether these differences may lead to differences in pharmacomechanical coupling. Further work will also be needed to establish why Ca2⫹ release is only transient: it is not due to complete emptying of the stores, but either to an inhibition by the decreasing luminal [Ca2⫹] or by negative feedback exerted by cytosolic Ca2⫹. The relative

29. Electro- and Pharmacomechanical Coupling

importance of both phenomena still has to be established. In conclusion, although our knowledge of the transport processes involved in the [Ca2⫹]i rise during electroand pharmacomechanical coupling in vascular smooth muscle has increased dramatically since the early 1990s, progression in the molecular nature of IP3R isoforms and store-operated Ca2⫹ channels is less advanced and will present a major research area in the coming years.

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30 Mechanisms Regulating Cardiac Myofilament Response to Calcium R. JOHN SOLARO Department of Physiology and Biophysics University of Illinois at Chicago, College of Medicine Chicago, Illinois 60612

I. INTRODUCTION

the molecular motors responsible for active contraction. These motors consist of the heads of myosin molecules (crossbridges) that react with actin and promote force and shortening of the sarcomeres. A current picture of this force generating reaction is illustrated in Fig. 2, which shows a region of overlap between thin and thick sarcomeric filaments in diastole and in systole. No matter what the mechanism for altering force and shortening properties of the muscle cells, the actin–crossbridge reactions remains the end effector. Regulation of the level of cellular force generation and the ability to shorten against a load are related to the question: What regulates the number of actin–crossbridge reactions and the force generated per cycle? Regulation of rates of shortening and relengthening of the cells becomes related to the questions: What regulates the rate of switching on and switching off the actin–crossbridge reactions? In addition, what regulates the rate of the crossbridge cycle? This chapter discusses the general physiological mechanisms by which we believe the number of actin–crossbridge reactions, the force/ crossbridge, and the rate of cycling may be regulated during the common short-term physiological event of exercise.

Contraction and relaxation of heart muscle cells are not only essential to pumping of blood but are also exquisitely controlled to accommodate the manyfold changes in heart rate (HR) and venous return (VR) that occur with normal daily activities ranging from sleep to strong exercise. Figure 1 illustrates the change in left ventricular volume that occurs with an increase in VR and HR during exercise. In this case the HR has increased from 60 to 120/min, and stroke volume (SV) has increased by about a third. This common physiological event reveals two important aspects of the cardiac muscle regulation. The first is that the increase in cardiac output (CO) in response to the increase in VR has occurred with essentially no change in the end diastolic volume (EDV). The second is that rates of change in ventricular volume during ejection and during filling have increased, resulting in a reduction in the time for the completion of the contraction–relaxation cycle. This reduction in cycle time is clearly essential for the heart to have time to fill at this relatively fast frequency of activation. Inasmuch as recruitment is not a regulatory device in the heart, both of these changes reflect the engagement of regulatory mechanisms that control the dynamics and contractile intensity (contractility) of the cardiac muscle cells themselves. Although complex relations exist between the mechanics of the cells and ventricular mechanics, it is useful to relate the pressure generated in the ventricle to cell force or tension and volume in the ventricle to cell length. Regulatory mechanisms are transduced to alter function by affecting the sarcomeric proteins that possess

Heart Physiology and Pathophysiology, Fourth Edition

II. MYOFILAMENTS AND REGULATION OF CARDIAC OUTPUT IN EXERCISE Figure 3 depicts the relationships among ventricular volumes, pressures, and sarcomere lengths during a cardiac cycle. This cycle of changes in sarcomere length during a beat of the heart is triggered by coupling of

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FIGURE 1 Changes in dynamics of left ventricular volume changes at a resting heart rate (HR) of 60 beats per min (solid line) and a HR of 120 beats per minute (dashed line) during exercise. Arrows indicate stimulation during the rest state. Data illustrate changes in left ventricular dynamics that occur with exercise. The duration of systole at rest is 0.37 sec and of diastole is 0.63 sec. During exercise, the duration of systole is 0.26 sec and of diastole is 0.14 sec.

electrical excitation of the cells to the release of Ca2⫹. Details of the excitation–contraction coupling mechanism are described elsewhere in this volume. This section emphasizes the mechanism by which the Ca2⫹ signal is transduced to activate the molecular motors of the heart. This is shown in Fig. 2, which indicates that Ca2⫹ triggers activation by binding to troponin C (TnC), one of three proteins in the thin filament Tn complex. Binding of Ca2⫹ to TnC sets into motion a series of protein– protein interactions that reverses a prevailing inhibition of the actin–crossbridge reaction during diastole. As reviewed in Solaro and Rarick (1998), during diastole the actin–crossbridge reaction is inhibited by a steric block imposed by tropomyosin (Tm) and possibly by Tn. This steric block is associated with a relative inflexibility of both Tm and Tn on a thin filament that appears to be related to a tethering action of TnI, the inhibitory component of Tn, and TnT, the Tm-binding component of the Tn complex. An essential component of this state is binding of the C terminus of TnI to actin. Binding of Ca2⫹ to TnC releases TnI from this tether on actin and releases the steric hindrance. Tm is now flexible and moves toward the groove of the double helix of actin monomers. The crossbridges may now react with actin. An important aspect of the activation of the thin filament is that the actin–crossbridge reaction is able to promote the activation of near-neighbor crossbridges by moving the now flexible Tm further into the groove. Thus, activation of the myofilaments is perceived as a highly complex process involving allosteric, steric, and cooperative mechanisms. The complexity of the activation provides a rich array of mechanisms for the modulation of sarcomeric activity. One important mechanism of modulation of myofilament activation by Ca2⫹ is its dependence on sarcomere length (Allen and Kentish, 1985). This is illustrated

in Fig. 4. Figure 4 shows results of an experiment in which tension is measured as a function of Ca2⫹ concentration surrounding a preparation of cardiac cells in which the membranes have been removed with detergent. The medium surrounding the myofilaments is a solution that mimics the intracellular condition. The muscle cell or bundle of cells has been held isometric at two sarcomere lengths. As illustrated in Fig. 3, at the longer sarcomere length of 2.3 애m, tension generation is more sensitive to Ca2⫹ than at a sarcomere length of 1.8 애m. Although there have been a number of theories speculating why Ca2⫹ sensitivity is length dependent (reviewed in Solaro and Rarick, 1998; Solaro and VanEyk, 1996), an appealing idea is that the variation in interfilament spacing that occurs with changes in length alters crossbridge-dependent activation of the myofilaments. For example, at long sarcomere lengths with an associated decrease in interfilament spacing, there is an increase in the local concentration of crossbridges in the vicinity of the thin filament. Thus, the cooperative activation of the actin–myosin reaction is promoted at a given Ca2⫹ concentration. Data shown in Fig. 5 relate length-dependent activation to the Volume–end systolic pressure (Vol-ESP) relation. As indicated, the Vol-ESP relation was generated by a rapid infusion of volume into the cardiovascular system. This generated a series of pressure–volume loops with different ESP points. This concept is described more fully by Suga (1993), who clearly presented this idea as a means of determining the cardiac contractile state. Figure 5 depicts how these ESP points are related to the Ca2⫹ –tension relation and illustrates how the steepness of the Vol-ESP pressure relation is related to length-dependent activation at submaximal levels of Ca2⫹ activation. This dependence of pressure on ventricular volume is, of course, also known as Starling’s law. It is critical to the understanding of how the myofilaments participate in the regulation of cardiac output to understand that excitation–contraction coupling in basal, resting physiological states provides enough Ca2⫹ to bind but a fraction (about 25%) of the TnC sites during a beat, as illustrated in Fig. 6. This leaves a reserve of TnC sites and thus actin–crossbridge reactions available for recruitment. In contrast, fast skeletal muscle cells are generally fully activated by tetanic stimulation and tension is varied by the recruitment of motor units. It is also important to understand that with submaximal Ca2⫹ activation that tension developed by cardiac muscle will be relatively steeply related to sarcomere length, when compared to skeletal muscle. The volume-ESP relation illuminates the significance of variations in myofilament activation by Ca2⫹, crossbridges, and length in regulation of cardiac output.

30. Myofilaments and Cardiac Output

FIGURE 2 Functional units of cardiac myofilament contractile machinery in diastole (A) and during systole (B). Myosin heads (S1), with associated light chains (MLC) protruding from the edge of the thick filament, and actin monomers assembled into the thin filament with associated regulatory proteins- tropomyosin (Tm) and the troponin (Tn) complex are shown. Myosin-binding protein C (MyBP-C) is shown situated at the head–neck region of myosin and is connected to titin, which eventually connects with the Z line indicated. In diastole, Tm is fixed in its position on the thin filament by the Tn complex, which is tethered to Tm through TnT and to actin via the C terminus of TnI, the inhibitory unit of the complex. The position of Tm is such that myosin crossbridges are impeded from reacting with actin. The force-generating actin–myosin reaction is triggered by Ca2⫹ binding to an N-terminal site on TnC, the Ca receptor. The Ca-binding signal is transmitted to Tm through TnT, the Tm-binding unit of the Tn complex, and to TnI, which is released from its tether on actin by promotion of a tight interaction between the C terminus of TnI and the N terminus of TnC. Tm is now free to move on the filament, removing the steric hindrance of the actin–crossbridge reaction. As indicated by the actin–crossbridge reaction on the left, strong binding of a crossbridge promotes the reaction of a near-neighbor crossbridge by actively pushing Tm away from it blocking position. Movements of one Tm are transmitted to a contiguous Tm by a stretch of overlapping amino acids. Thus, the crossbridge on the left is shown to react with actin without Ca2⫹ bound to Tn. With removal of cytosolic Ca2⫹ by the sarcoplasmic reticulum, Ca2⫹ is released from TnC and these reactions are reversed.

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FIGURE 3 Volume–pressure relation of left ventricle during a heart beat. The loop represents data obtained in human subjects in a resting condition. End diastolic pressure (EDP) and volume (EDV) and end systolic pressure (ESP) and volume (ESV) points are designated with a filled circle. The ESP point represents a state in which the cells of a ventricle are neither lengthening nor shortening and developing the peak pressure (tension) at that particular ventricular volume (sarcomere length). Thus this point is indicated as on a line describing the volume–ESP relation, which reflects the length tension properties of the muscle cells. A series of such points may be generated by the rapid infusion of volume into the cardiovascular system, thereby elevating afterload with little effect on contractility (see Fig. 5). The relationship between these points and a circumferential array of ventricular cells and the length of the sarcomere are shown schematically to illustrate that the P-V loop is rooted in a complex relation between sarcomere length and ventricular geometry. See text for further discussion.

The level of Ca2⫹ activation or contractility is reflected in the ESP in the pressure–volume loop as illustrated in Fig. 7. It is immediately apparent that the ability of the sarcomeres to shorten down on the volume of blood in the ventricle and to reduce the ventricular volume, i.e., their contractility, is related to the position of the volume-ESP relation in the volume–pressure plane. In the short term, as during a bout of exercise, determinants of the position of the volume-ESP relation are the number or density of crossbridges reacting with the thin filament and the force/crossbridge cycle. In short-term regulation, the myofilament density is constant; a prominent determinant of the number of reacting crossbridges is the level of thin filament activation. Thin filament activation is in turn governed by the amounts of Ca2⫹ available for binding to cTnC, the extent of crossbridge-dependent activation of the myofilaments, the prevailing chemical state (e.g., covalent phosphorylation, charge as determined by ambient pH, and bound and free products and nucleotide), and the structural state (e.g., isoform population, expression of mutant components, or proteolytically altered compo-

nents) of the myofilaments. Key components of Ca2⫹ delivery to the myofilaments include the amount of Ca2⫹ stored in the sarcoplasmic reticulum and the relative rates of Ca2⫹ influx and efflux during a steady-state period of cardiac activity. With exercise, there is an increase in 웁-adrenergic stimulation and activation of protein kinase A (PKA). PKA-dependent phosphorylation of membrane proteins, especially Ca2⫹ channel proteins, phospholamban, and ryanodine receptors of the sarcoplasmic reticulum (SR) determines the amounts of Ca2⫹ available for binding to cTnC. As illustrated in Figs. 2 and 6, with more Ca2⫹ bound to TnC, more crossbridges may react with the thin filament in forcegenerating cycles. However, the myofilaments are not passive elements in this regulatory scheme. It is now clear that the myofilament response to Ca2⫹ is physio-

FIGURE 4 Illustration of the steady-state relationship between Ca2⫹ concentration and tension generation of myofilaments at long and short sarcomere lengths. Data were generated from experiments on membrane-free myofilaments of ventricular muscle cells, mounted in a force-measuring setup, and immersed in various solutions mimicking intracellular conditions. As the Ca2⫹ ion concentration was increased, incrementally, Ca2⫹ binds to TnC, promoting tension generation by force-generating crossbridges. Even though there is a single binding site on TnC, the relation is steep as expected from the cooperative activation of crossbridges as indicated in Fig. 2. When TnC became saturated with Ca2⫹, tension generation came to a plateau. Plateau tension is reduced as sarcomere length is reduced according to the length–tension relation. The Ca2⫹ –tension relation is more sensitive to Ca2⫹ at relatively long sarcomere lengths and less sensitive to Ca2⫹ at relatively short sarcomere lengths. As indicated by the long sarcomere length, the distance between thick and thin filaments (interfilament spacing) is relatively short, whereas at shorter sarcomere lengths, interfilament spacing is relatively long. Current theories indicate that decreases in interfilament spacing, as occurs with longer sarcomere lengths, ease the crossbridge reaction with the thin filament and thus ease cooperative activation. Thus at a given Ca2⫹ ion concentration, more tension is generated as interfilament spacing is reduced. See text for further discussion.

30. Myofilaments and Cardiac Output

FIGURE 5 The slope and position of the volume–ESP relationship ultimately depend on the length–tension relationship of the myofilaments, which is itself determined by filament overlap (reflected in plateau of tension at the various sarcomere lengths) and by the response of the myofilaments to Ca2⫹. The volume–ESP pressure has been generated by the rapid infusion of saline or blood into the cardiovascular system, generating a series of beats with increasing afterload and little or no change in contractility. Note that at a given level of Ca2⫹, tension falls as sarcomere length shortens as a result of length Ca2-dependent activation. Note that the volume–ESP relation is relatively steep, which would not be the case without lengthdependent activation of myofilaments.

logically modulated and integrated into the overall regulation of cardiac output during exercise.

III. COVALENT AND NONCOVALENT MODULATION OF MYOFILAMENT RESPONSE TO Ca2⫹ A. Modulation of Volume–ESP Pressure Relation During short-term physiological changes, such as moderate exercise, the chemical state of the myofilaments is modified by covalent phosphorylation that is regulated through Ca2⫹ and cyclic nucleotide pathways. Three key proteins that are substrates for these cAMP and/or Ca2⫹-dependent kinases are cTnI of the thin filament and myosin-binding protein C (MyBP-C) and myosin light chain 2 (MLC2) of the thick filament. Sites for PKA phosphorylation are located at Ser-22 and Ser-23 in the unique NH2 extension of the cardiac variant of TnI. Sites for PKC-dependent phosphorylation of cTnI do not appear to change the phosphorylation state during 웁-adrenergic stimulation (Fentzke et al.

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1999). Sites of phosphorylation of MyBP-C are also located on a unique amino-terminal extension of MyBP-C (Winegrad, 1999). Using site-directed mutagenesis, Gautel et al. (1995) identified a key regulatory motif specific for the cardiac isoform of MyBP-C. The isoform-specific motif consists of LAGGGRRIS—a loop in the N-terminal region of cardiac MyBP-C, which was identified as the key substrate site for phosphorylation by both PKA and calmodulin (CaM) kinase. A novel finding was that phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site by CaM-Kinase (Bennett et al., 1999). In the case of MLC 2, there are conserved Ser and Thr residues located in the N-terminal region, which are substrates for myosin light chain kinase, a Ca2⫹ and CaM-dependent kinase. Phosphorylation of these sites is responsible for the activation of smooth muscle contraction, but serves a modulatory role in striated muscle (Moss, 1992). Phosphorylation of myofilaments by PKA induces a desensitization of the myofilament to Ca2⫹ with little effect on maximum tension generation (Solaro and Rarick, 1998). Studies using a mutant cTnI lacking the amino-terminal extension demonstrated that phosphorylation of TnI by PKA is both necessary and sufficient to induce the reduction in the Ca2⫹ sensitivity of myofilament activity (Guo et al., 1994; Wattanapermpool et al., 1995). Moreover, myofilaments from hearts of transgenic mice in which cTnI was replaced with slow skeletal TnI demonstrated no change in Ca2⫹ sensitivity following phosphorylation with PKA (Fentzke et al., 1999). It is evident therefore that the phosphorylation of cTnI may represent a negative feedback regulator of tension generation. However, it is apparent that the phosphorylation of MLC 2 may promote tension development at a given level of Ca2⫹ and thus serve to sensitize the myofilaments to Ca2⫹ (Moss, 1992). Interestingly, MLC 2 phosphorylation does not appear to change during brief episodes of a adrenergic stimulation, but only with sustained changes in heart rate and the associated increased frequency of Ca2⫹ entry (Silver et al., 1986). It is likely that this frequency dependence of MLC 2 phosphorylation is due to the titration of calmodulin with Ca2⫹, which acts to integrate the Ca2⫹ signal. Although not fully understood in terms of functional significance, a potentially important mechanism for regulation of the Vol-ESP relation is an interplay between length-dependent activation of the myofilaments and their state of phosphorylation. Mechanisms such as light chain phosphorylation that sensitize the myofilaments to Ca2⫹ would be expected to blunt lengthdependent activation. Increases in relative sarcomere

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FIGURE 6 The resting state and basal state of activation of thin and thick filaments of sarcomere. It is estimated that one thin filament has 25–30 functional units (represented by each oval) consisting of one troponin–tropomyosin complex (represented by triangles). (Top) The relaxed condition and (bottom) the active condition during a basal state of contractility in which about 25% of the troponins bind Ca2⫹. Active force-generating crossbridges are shown extending to and reacting with the thin filament. Note that in some cases near-neighbor crossbridges are depicted as active without Ca2⫹ bound to troponin. See text and Fig. 2 for further description.

length and increases in MLC 2 phosphorylation both appear to promote the reaction of crossbridges with actin, and thus, in the extreme, with full phosphorylation of MLC 2, it would be expected that a change in interfilament spacing would not have as great an impact on Ca2⫹ activation as in the case with no MLC 2 phosphorylation. It would be expected that the opposite would be the case with regard to cTnI phosphorylation. In this case, in the extreme with no phosphorylation of cTnI, the myofilaments would be most sensitive to Ca2⫹ and presumably less responsive to an increase in sarcomere length than with full phosphorylation of cTnI PKC sites. However, Komukai and Kurihara (1997) have presented data indicating that the desensitizing effect of isoproterenol treatment was more pronounced at shorter muscle lengths than at optimal sarcomere length at Lmax . These studies were done in ferret papillary muscle preparations in which intracellular Ca2⫹ was measured by the aequorin technique. Thus, following stimulation of the preparations with isoproterenol, the lengthdependent change in the Ca2⫹ –tension relation was amplified. Although not clearly defined in these studies, it seems likely that the state of phosphorylation of cTnI rather than C protein was responsible for this effect.

To illustrate how the modulation of length-dependent activation may affect the steepness of the VolESP relation, Fig. 8 shows a Vol-ESP relation in which length-dependent activation has been modulated by phosphorylation of cTnI. Figure 8 illustrates the effects of desensitization of the myofilaments to Ca2⫹ as well as an amplification of length-dependent activation predicted by the study of Komukai and Kurihara (1997).

B. Modulation of Contraction/Relaxation Dynamics Phosphorylation of myofilament proteins by cAMPdependent protein kinase has been proposed to be an important mechanism for the increased rate of relaxation during 웁-adrenergic stimulation of the heart (Solaro, 1986, 1993). This proposal came about from data showing (1) that cTnI and C protein are phosphorylated in situ in association with 웁-adrenergic stimulation; (2) that in vitro the myofilament sensitivity to Ca2⫹ was reduced; (3) that the ‘‘off rate’’ for Ca2⫹ exchange with TnC increased when TnI was phosphorylated by PKA (Robertson et al., 1982); and (4) that unloaded shorten-

30. Myofilaments and Cardiac Output

ing velcocity, a reflection of the crossbridge turnover rate, was increased with myofilament protein phosphorylation (Saeki et al., 1990; Strang et al., 1994). Moreover, in myocytes of mutant mouse hearts in which phospholamban was knocked out, the relaxant effect of catecholamines was retained (Wolska et al., 1996). Whether or not this was due to cTnI phosphorylation is not clear, but it is apparent that the only other PKAdependent process that could account for this effect is cTnI phosphorylation. Unfortunately, experiments using a photolabile Ca2⫹ chelator were not able to resolve the issue of the role of cTnI phosphorylation in the increased rate of cardiac relaxation. Zhang et al. (1995) reported that myofilaments containing phosphorylated cTnI relax faster than dephosphorylated controls following release of the chelator, diazo-2. However, similar experiments by Johns et al. (1997) did not confirm these results. However, studies (Fentzke et al., 1999) with myofilaments from hearts of transgenic mice in which cTnI is fully replaced with slow skeletal TnI, which does not contain PKA phosphorylation sites, showed no change in crossbridge kinetics following PKA-dependent phos-

FIGURE 7 Increased contractility as reflected in the Ca2⫹ –force relation of myofilaments and in P-V loops. In the example shown, steadystate P-V loops are shown for one beat at rest and for one beat during exercise. (Top) The shift in the P-V relation occurred by an increase in amounts of Ca2⫹ released to the myofilaments. Small changes in the amounts of Ca2⫹ released are amplified in the ESP-V relation because of the steep relation between Ca2⫹ and myofilament force. During exercise, with an increase in HR from 60 to 120 bpm and an increase in venous return, cardiac output has increased substantially from about 4.5 to 12.0 liter/min with little change in EDV. This occurs because of the increase in contractility. See also Fig. 1 for dynamics (see text for further description).

525

FIGURE 8 Volume–end systolic (Vol-ESP) pressure relations indicating the effect of desensitization (long dashes) of the myofilament response to Ca2⫹ that occurs with phosphorylation through the 웁-adrenergic pathway. Desensitization is not the same at long and short sarcomere lengths and thus the Vol-ESP relation generated would be expected to be different from one in which the desensitization is the same as both long and short sarcomere lengths (dotted line). See text for further discussion.

phorylation. MyBP-C was phosphorylated in these preparation. Wild-type myofilaments showed an increase in crossbridge kinetics with PKA phosphorylation of cTnI and MyBP-C. It was concluded from these studies that the enhanced rate of relaxation noted in Fig. 1 is due in part to phosphorylation of the myofilaments, particularly at cTnI. With sustained increases in heart rate, phosphorylation of MLC 2 and of MyBP-C may also contribute to cardiac dynamics. In the case of MLC 2 phosphorylation, it is apparent that the phosphorylation may slow down crossbridge kinetics. Phosphorylation of MyBP-C is more complex in that the Ca2⫹ –calmodulin site must first be phosphorylated before the PKA sites are sensitive as substrates for PKA. The phosphorylation of MyBP-C has been suggested to enhance crossbridge kinetics (Winegrad, 1999). Thus, the picture that emerges is of a complex set of mechanisms that oppose and promote the increased rate of crossbridge cycling. More work needs to be done to fully understand how these mechanisms play out in various physiological states, but there is little doubt that the process involves integrated membrane control and myofilament control mechanisms. Whatever the case, it is clear that the cycle time of the contraction–relaxation cycle must be tuned to the frequency of stimulation of the cells as indicated in Fig. 1.

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IV. SUMMARY Membrane control of the amounts and rates of Ca2⫹ movements to and from the myofilaments is clearly a powerful mechanism for regulating cardiac output. This chapter focused on mechanisms controlling the myofilament response to Ca2⫹ and how these mechanisms are an integral part of the dynamics of the contraction– relaxation cycle and to the postion of the relation between ventricular volume and ESP. These mechanisms are significant in the intrinsic control of the heart by Starling’s law, which appears to involve lengthdependent myofilament activation. Extrinsic control by the autonomic nervous system also involves changes in the myofilament response to Ca2⫹. Moreover, although further discussion is beyond the scope of this chapter, ischemia, reperfusion injury, stunning, and heart failure appear likely to involve major changes in the myofilament response to Ca2⫹ (Solaro, 1999). Isoform shifts of myofilament proteins also modulate the response to Ca2⫹ in physiological and pathological states, and emerging evidence shows that familial hypertrophic cardiomyopathy is a ‘‘sarcomeric disease’’ genetically linked to missense mutations in the crossbridge and in the regulatory proteins TnT and Tm (Palmiter and Solaro, 1997). Finally, targeting the myofilaments for the action of inotropic agents, the so-called myofilament Ca sensitizers, remains an important research objective.

Bibliography Allen, D. G., and Kentish, J. C. (1985). The cellular basis of the length-tension relation in cardiac muscle. J. Mol. Cell Cardiol. 17, 821–840. Bennett, P. M., Furst, D. O., and Gautel, M. (1999). The C-protein (myosin binding protein C) family: Regulators of contraction and sarcomere formation? Rev. Physiol. Biochem. Pharmacol. 138, 203–234. Fentzke, R. C., Buck, S. H., Patel, J. R., Lin, H., Wolska, B. M., Stojanovic, M. O., Martin, A. F., Solaro, R. J., Moss, R. L., and Leiden, J. M. (1999). Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart. J. Physiol. (Lond.) 517, 143–157. Gautel, M., Zuffardi, O., Freiburg, A., and Labeit, S. (1995). Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: A modulator of cardiac contraction? EMBO J, 14, 1952–1960. Guo, X., Wattanapermpool, J., Palmiter, K. A., Murphy, A. M., and Solaro, R. J. (1994). Mutagenesis of cardiac troponin I: Role of the unique NH2-terminal peptide in myofilament activation. J. Biol. Chem. 269, 15210–15216.

Johns, E. C., Simnett, S. J., Mulligan, I. P., and Ashley, C. C. (1997). Troponin I phosphorylation does not increase the rate of relaxation following laser flash photolysis of diazo-2 in guinea-pig skinned trabeculae Pflu¨g. Arch. 433, 842–844. Komukai, K., and Kurihara, S. (1997). Length dependence of Ca(2⫹)tension relationship in aequorin-injected ferret papillary muscles. Am. J. Physiol. 273, H1068–H1074. Moss, R. L. (1992). Ca2⫹ regulation of mechanical properties of striated muscle: Mechanistic studies using extraction and replacement of regulatory proteins. Circ. Res. 70, 865–884. Palmiter, K. A., and Solaro, R. J. (1997). Molecular mechanisms regulating the myofilament response to Ca2⫹: Implications of mutations causal for familial hypertrophic cardiomyopathy. Basic Res. Cardiol. 92(Suppl. 1), 63–74. Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982). The effect of troponin I phosphorylation on the Ca2⫹-binding properties of the Ca2⫹regulatory site of bovine cardiac troponin. J. Biol. Chem. 257, 260–263. Saeki, Y., Shiozawa, K., Yanagisawa, K., and Shibata, T. (1990). Adrenaline increases the rate of cross-bridge cycling in rat cardiac muscle. J. Mol. Cell. Cardiol. 22, 453–460. Silver, P. J., Buja, L. M., and Stull, J. T. (1986). Frequency-dependent myosin light chain phosphorylation in isolated myocardium. J. Mol. Cell. Cardiol. 18, 31–37. Solaro, R. J. (1986). Protein phosphorylation and the cardiac myofilaments. In ‘‘Protein Phosphorylation in Heart’’ (R. J. Solaro, ed.), pp. 129–156. CRC Press, Boca Raton, FL. Solaro, R. J. (1993). Modulation of activation of cardiac myofilaments by beta-adrenergic agonists. In ‘‘Modulation of Cardiac Calcium Sensitivity’’ (D. A. G. Allen and J. A. Lee, eds.), pp. 160–177. Oxford Univ. Press, Oxford. Solaro, R. J. (1999). Troponin I, stunning, hypertrophy, and failure of the heart. Circ. Res. 84, 122–124. Solaro, R. J., and Rarick, H. M. (1998). Troponin and tropomyosin: Proteins that switch on and tune in the activity of cardiac myofilaments. Circ. Res. 83, 471–480. Solaro, R. J., and Van, Eyk. J. (1996). Altered interactions among thin filament proteins modulate cardiac function. J. Mol. Cell. Cardiol. 28, 217–230. Strang, K. T., Sweitzer, N. K., Greaser, M. L., and Moss, R. L. (1994). Beta-adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats. Circ. Res. 74, 542–549. Suga, H. (1993). Cardiac performance as viewed through the pressurevolume window. Circulation 88(Suppl. 4), I-C. Wattanapermpool, J., Guo, X., and Solaro, R. J. (1995). The unique amino-terminal peptide of cardiac troponin I regulates myofibrillar activity only when it is phosphorylated. J. Mol. Cell Cardiol. 27, 1383–1391. Winegrad, S. (1999). Cardiac myosin binding protein C. Circ. Res. 84, 1117–1126. Wolska, B. M., Stojanovic, M. O., Luo, W., Kranias, E. G., and Solaro, R. J. (1996). Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2⫹. Am. J. Physiol. 271, C391–C397. Zhang, R., Zhao, J., and Potter, J. D. (1995). Phosphorylation of both serines in cardiac troponin I is required to decrease the Ca2⫹affinity of cardiac troponin C. J. Biol. Chem. 270, 30773–30780.

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31 Vascular Smooth Muscle Contraction GARY J. KARGACIN and MICHAEL P. WALSH Smooth Muscle Research Group University of Calgary Faculty of Medicine Calgary, Alberta, Canada T2N 4N1

I. INTRODUCTION

lysis of ATP provides the energy for crossbridge cycling and shortening of the muscle. The contractile apparatus of smooth muscle consists of the contractile proteins (actin/tropomyosin and myosin, which are organized into thin and thick filaments, respectively) and associated regulatory proteins [myosin light chain kinase (MLCK), calmodulin (CaM), myosin light chain phosphatase (MLCP), caldesmon, and calponin]. Smooth muscles contain about one-third the amount of myosin and more actin and tropomyosin than striated muscles (Table I), yet are capable of generating similar stresses (force/cross-sectional area). There are estimated to be 8 thin filaments per thick filament in nonarterial smooth muscles and 15 thin filaments per thick filament in arteries. Smooth muscle tissue contents of the regulatory proteins have been estimated as follows: CaM, 30–40 애M; MLCK, 3–4 애M; MLCP, 1 애M; calponin, 125–230 애M; and caldesmon, 10–33 애M. In general, the mechanisms of regulation of motile processes can be divided into two main classes: actinor thin filament-linked regulation and myosin- or thick filament-linked regulation, depending on whether the regulatory mechanism is physically associated with the thin or thick filament, respectively. The best characterized thin filament-linked regulatory mechanism is the troponin–tropomyosin system exemplified by vertebrate striated muscles (Chalovich, 1992). The principal mechanism of regulation of smooth muscle contraction involves a thick filament-linked process, myosin phosphorylation–dephosphorylation (Somlyo and Somlyo, 1994). In addition, evidence supports thin filament-

The mechanism of contraction of smooth muscle is believed to be fundamentally the same as that of skeletal and cardiac muscles, i.e., contraction occurs according to the crossbridge cycling/sliding filament/swinging lever arm model whereby thick and thin filaments slide relative to one another, without a change in length of the filaments, at the expense of ATP hydrolysis (Geeves and Holmes, 1999). This conclusion has come from ultrastructural studies of a variety of smooth muscles, direct observation of actin filaments moving on myosin substrates, determination of the three-dimensional structures of actin and of myosin heads in various conformational states in combination with high-resolution X-ray crystallographic reconstructions of actomyosin complexes, and measurements of force and step length generated by a single myosin molecule interacting with a single actin filament. The myosin head consists of two distinct domains: a motor domain containing the MgATPase catalytic site and the actin-binding site, and the regulatory domain (neck region) containing the light chains and the 움-helical heavy chain to which they bind (see later). Crystallographic structures of the myosin head at the beginning and end of the power stroke support the swinging lever arm mechanism, involving a rotation of the light chain domain of the myosin head, which would account for the predicted filament displacement of 5–10 nm. Upon activation of the muscle, therefore, actin interacts with myosin to activate the intrinsic MgATPase activity of the myosin heads, and the hydro-

Heart Physiology and Pathophysiology, Fourth Edition

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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TABLE I Muscle Contents of Actin, Tropomyosin, and Myosin a Molar ratio

Tissue concentration (mM )

Muscle type

Actin

Tropomyosin

Myosin

Actin

Tropomyosin

Myosin

Arterial smooth Nonarterial smooth Skeletal

28.6 15.5 5

4.8 2.7 0.6

1 1 1

1.6 0.87 0.7

0.27 0.15 0.1

0.056 0.056 0.18

a

Values are taken from Hartshorne (1987).

linked regulation that can modulate or fine-tune the contractile state of smooth muscles and involves the actin-binding proteins, calponin and caldesmon. The purpose of the remainder of this chapter is to discuss the important properties of the contractile proteins and of the regulatory proteins that are involved in the principal mechanism controlling smooth muscle contraction, i.e., CaM, MLCK, and MLCP. The reader is referred to review articles on calponin (Winder et al., 1998) and caldesmon (Marston and Huber, 1996) for discussion of the potential roles of these thin filamentassociated proteins in the modulation of smooth muscle contractility.

II. CONTRACTILE PROTEINS A. Actin Thin filaments are composed primarily of actin arranged in a double helix of noncovalently associated monomers (Fig. 1), similar to the structural organization of the thin filaments of striated muscles. One complete turn of the helix occurs every 74 nm, corresponding to 앑13 actin monomers, and the width of the thin filament is 6–8 nm. The length of thin filaments in vivo is estimated to be 1.38 애m, compared to 1.05 애m in skeletal muscle thin filaments and 0.6–⬎1.1 애m in mammalian cardiac muscles. One end of the actin filament is attached to amorphous structures called dense bodies,

which are either free in the cytosol or associated with the plasma membrane. Cytoplasmic dense bodies contain 움-actinin whereas membrane-associated dense plaques contain 움-actinin and vinculin. Dense bodies are believed to be analogous to the Z lines of striated muscles. The other ends of the actin filaments interdigitate with myosin filaments. Membrane-associated dense bodies provide the ultimate anchorage of the contractile network at the level of the plasma membrane. Actin has a monomer molecular mass of 42 kDa (374 or 375 amino acids). Two principal actin isoforms are expressed in smooth muscle: smooth 움 and smooth 웂, which differ by only three N-terminal amino acids. The 움 variant is predominant in vascular smooth muscles, the 웂 variant in enteric smooth muscles, and approximately equal amounts of 움 and 웂 actins are found in other smooth muscles, e.g., uterus. Most smooth muscles also express cytoplasmic (nonmuscle) 웁 and 웂 actin isoforms. Thin filaments appear to be uniform with respect to actin isoform expression, i.e., distinct isoforms do not appear to be segregated in different thin filaments. Actin has three particularly important properties: (i) the ability to polymerize to form long filaments (conversion of monomeric G-actin to polymeric F-actin), (ii) the ability to bind myosin and activate its MgATPase activity, and (iii) the ability to bind tropomyosin and regulatory proteins. G-actin binds 1 mol of ATP/mol, which is hydrolyzed as the G-actin polymerizes to form F-actin. Actin also contains 1 mol of bound divalent cation/mol, probably Mg2⫹ in vivo.

FIGURE 1 Arrangement of tropomyosin along the thin filament.

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31. Vascular Smooth Muscle Contraction

B. Tropomyosin

C. Myosin

Tropomyosin is present in smooth muscle at a molar ratio to actin monomers of 1:7 and is located in the grooves between the two strands of the actin double helix (Fig. 1), as it is in striated muscles. Smooth muscle tropomyosin exists in two isoforms, 움 and 웁, expressed in approximately equal amounts. Each isoform contains 284 amino acids and has a molecular mass of 앑33 kDa. The native structure is dimeric, and both homodimers (움2 and 웁2) and heterodimers (움웁) have been detected. Dimeric tropomyosin is composed of two elongated, 움-helical polypeptides coiled around each other, with a total length of 41–42 nm, a width of 2 nm, and a helical pitch of 13.7 nm. Tropomyosin forms polymers by headto-tail aggregation, which likely reflects its organization on the actin filament. One strand of tropomyosin molecules is associated with each of the two actin strands in the thin filament. Skeletal and smooth muscle tropomyosins both exhibit the same sevenfold repeating sequence, corresponding to the seven actin-binding sites. One tropomyosin dimer spans seven actin monomers in the thin filament, consistent with the calculated stoichiometry in vivo. The function of smooth muscle tropomyosin has not been clearly established, although there is general agreement that it has a significant potentiating effect on actin-activated myosin MgATPase. It has been suggested that tropomyosin may play an important role in conjunction with caldesmon in regulating actin–myosin interaction in smooth muscle.

Several classes of myosins have been identified and are involved in diverse motile processes. The class of myosin involved in smooth muscle contraction, as in striated muscles, is myosin II, which will be referred to simply as myosin. Smooth muscle myosin is a hexamer composed of two heavy chains (앑205 kDa each) and two pairs of light chains [20-kDa regulatory light chains (LC20) and 17-kDa alkali light chains (LC17)] (Fig. 2) to give a native molecular mass of 앑484 kDa. Electron microscopy of rotary-shadowed myosin reveals the long rod-like tail (an 움-helical coiled-coil) and two-headed structure, similar to striated muscle and nonmuscle myosins. The light chains are located in the neck region of the molecule where the heads meet the tail. Myosin molecules aggregate into filamentous structures via interactions of parts of their tail regions (the LMM region; see later). The globular heads and part of the tail (HMM region) protrude away from the body of the thick filament, constituting the crossbridge to the thin (actin) filament. Mammalian smooth muscle myosin filaments are 앑15–18 nm in diameter and 2.2 애m long. Fragmentation of smooth muscle myosin with a variety of proteases has revealed much information about the domain structure of the molecule (Fig. 2). The globular heads are composed of the amino-terminal ends of the heavy chains and both pairs of light chains, and each motor domain contains an actin-binding site and a site of ATP hydrolysis. Myosin can be cleaved by 움-chymotrypsin into light meromyosin (LMM; composed of the last two-thirds of the tail) and heavy meromyosin

FIGURE 2 Schematic representation of smooth muscle myosin showing the two globular heads and the 움-helical coiled-coil tail. Motor domains contain the ATPase and actin-binding sites. One LC17 and one LC20 are bound to the neck region of the heads, with LC17 being closer to the motor domain. S1, subfragment 1 corresponding to the myosin head; S2, subfragment 2; HMM, heavy meromyosin; LMM, light meromyosin. Taken from Walsh et al. (1995) with permission of The National Research Council of Canada.

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TABLE II Phosphorylation-Induced Activation of Smooth Muscle Actomyosin MgATPase Activity a Myosin phosphorylation (mol Pi /mol myosin)

Reconstituted system Myosin alone

MgATPase rate (nmol Pi /mg myosin·min)

ⴙCa2ⴙ

ⴚCa2ⴙ

ⴙCa2ⴙ

ⴚCa2ⴙ

2.0 2.0

0.0 0.0

91.6 3.1

3.0 3.1

a Myosin (1 애M ) was incubated at 30⬚C in 25 mM Tris–HCl (pH 7.5), 60 mM KCl, 10 mM MgCl2 , 1 mM [웂-32P]ATP, 0.1 애M CaM, and 74 nM MLCK in the absence or presence of 6 애M actin and 2 애M tropomyosin and in the presence of either 0.1 mM CaCl2 or 1 mM EGTA. Reactions were started by the addition of ATP, and samples of reaction mixtures were withdrawn at t ⫽ 1, 2, 3, 4, 5, 6, and 7 min for quantification of myosin phosphorylation and ATP hydrolysis. ATPase rates were determined by regression analysis of the linear time courses of ATP hydrolysis. All proteins were isolated from smooth muscle.

(HMM; composed of the two globular heads and onethird of the tail). HMM can be cleaved with papain to S1 (free myosin heads), which retain actin binding and the ATPase site. Phosphorylation of smooth muscle myosin plays a central role in the regulation of smooth muscle contraction (see later). Myosin phosphorylation has been characterized thoroughly both in vitro and in vivo and is catalyzed by Ca2⫹ /CaM-dependent MLCK. Specific phosphorylation occurs at serine 19 in each of the two 20-kDa light chains. Under some circumstances, phosphorylation can also occur at the neighboring threonine 18. Phosphorylation is Ca2⫹ and CaM dependent and results in a substantial increase in actin-activated myosin MgATPase activity (Table II). Myosin in the absence of actin exhibits a low MgATPase activity that is unaffected by phosphorylation. Both the heavy and the light chains of smooth muscle myosin are expressed as several different isoforms: six heavy chain isoforms (four smooth muscle-specific isoforms that are alternatively spliced variants of a single gene and two cytoplasmic or nonmuscle variants), a smooth muscle and a nonmuscle variant of LC20 , and two LC17 isoforms (a and b, denoting their respective acidic and basic properties, that are alternatively spliced variants of a single gene that differ in 5 of the 9 Cterminal amino acids). The smooth muscle-specific heavy chain isoforms are distinguished by two different C termini (of 9 or 43 amino acids) and the absence or presence of a 7 amino acid insert that forms a flexible surface loop in the head region near the nucleotidebinding site. The presence of the 7 amino acid insert doubles the actin-activated MgATPase activity of the phosphorylated myosin isoform and actin filament velocity in the in vitro motility assay (described later). Insert-containing isoforms are expressed predominantly

in phasic smooth muscles and insert-lacking isoforms in tonic vascular smooth muscles. It is unclear whether the LC17 isoform pattern of expression affects the kinetics of contractility, but LC17a is expressed predominantly in phasic and LC17b in tonic smooth muscles. Current evidence suggests that shortening velocity in smooth muscles is controlled primarily by myosin phosphorylation (MLCK:MLCP activity ratio) rather than myosin isoform composition.

III. REGULATION OF SMOOTH MUSCLE CONTRACTION The work of Filo et al. (1965) with skinned (glycerinated) smooth muscle preparations established the importance of Ca2⫹ in the activation of smooth muscle contraction. Sobieszek (1977) demonstrated that phosphorylation of smooth muscle myosin is required for actin activation of its MgATPase activity, which led to formulation of the phosphorylation theory of the regulation of smooth muscle contraction (Fig. 3). Stimulation of the smooth muscle cell by neurotransmitters, hormones, growth factors, or membrane depolarization induces an elevation of cytosolic-free Ca2⫹ concentration ([Ca2⫹]i), the activating Ca2⫹ originating from intracellular stores [the sarcoplasmic reticulum (SR)] [via inositol 1,4,5-trisphosphate (IP3) receptor and possibly ryanodine receptor Ca2⫹ release channels], and the extracellular space (via voltage-gated or receptor-operated Ca2⫹ channels). The relative importance of these two sources of activating Ca2⫹ depends on the nature of the stimulus and the smooth muscle cell type. [Ca2⫹]i rises transiently from 0.12–0.27 애M in resting cell to 0.5–0.7 애M in the stimulated cell (Williams and Fay,

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31. Vascular Smooth Muscle Contraction

1986). This modest increase in [Ca2⫹]i results in Ca2⫹ binding to CaM and activation of MLCK. The activated kinase catalyzes phosphorylation at serine 19 in each of the two 20-kDa light chains of myosin. Phosphorylated myosin can interact with actin and hydrolyze ATP at a fast rate (Table II), which provides the energy for rapid crossbridge cycling and the development of tension. Relaxation follows Ca2⫹ removal from the sarcoplasm by the action of Ca2⫹ transport ATPases in the SR and sarcolemmal membranes and, to a lesser extent, the sarcolemmal Na⫹-Ca2⫹ exchanger (Casteels et al., 1986). This leads to dissociation of Ca2⫹ from CaM and inactivation of MLCK. As a consequence, myosin phosphorylation stops, and myosin that had been phosphorylated during the activation phase of the contractile cycle is dephosphorylated by MLCP. Myosin heads dissociate from actin and the muscle relaxes. There is a latency period of 앑500 msec following neural stimulation of smooth muscle prior to the onset of isometric force development, with the rate-limiting step probably being the activation of MLCK, i.e., the conformational change induced in the kinase by Ca42⫹CaM. This delay is longer in the case of agonist activation that does not involve a change in membrane potential. For example, the delay is 앑1.2 sec for agonists that

generate IP3 , with the time from agonist occupancy of the receptor to IP3 production being 0.5–1 sec. A substantial body of evidence has accumulated from biochemical and physiological experimentation that supports the phosphorylation theory. Several types of experimental systems have been used: systems reconstituted from purified contractile and regulatory proteins; systems composed of crude actomyosin or myofilament preparations that contain associated regulatory proteins; in vitro motile systems in which movement of myosin-coated beads over an actin cable network or movement of fluorescently labeled actin filaments over immobilized myosin is recorded; skinned (demembranated) or permeabilized smooth muscle strips and single cells; and intact smooth muscle strips and single cells. Some of the key findings from these approaches can be summarized as follows: i. Several investigators have observed a positive correlation between myosin phosphorylation and (a) the actin-activated MgATPase activity of smooth muscle myosin (Table II) and (b) tension development in skinned and intact smooth muscle strips (e.g., Hoar et al., 1979; Mita and Walsh, 1997). ii. The effect of myosin phosphorylation on actin

Relaxation

2Ca 2+ (Ca 2+)2-CaM-MLCK inactive

2Ca 2+

ATP

Myosin

PP1 M

(Ca2+)4-CaM-MLCK active ADP

Pi

Myosin-P + Actin

H2O

Actomyosin-P

ATP

ADP + Pi

Contraction FIGURE 3 Myosin phosphorylation–dephosphorylation as the primary mechanism of regulation of smooth muscle contraction. CaM, calmodulin; MLCK, myosin light chain kinase; PP1M , myosin light chain phosphatase; Myosin-P, phosphorylated myosin; Actomyosin-P, actophosphorylated myosin; Pi , inorganic phosphate.

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V. Excitation–Contraction Coupling

activation of myosin MgATPase was reversed following dephosphorylation catalyzed by MLCP and was restored following rephosphorylation with MLCK (Sellers et al., 1981). iii. Addition of MLCP to skinned smooth muscle strips induced relaxation (e.g., Shirazi et al., 1994). iv. Thiophosphorylation of myosin with ATP웂S [adenosine 5⬘-O-(3-thiotriphosphate)] resulted in loss of Ca2⫹ sensitivity of the actin-activated myosin MgATPase (Sherry et al., 1978). (ATP웂S is a good substrate for protein serine/threonine kinases but thiophosphorylated myosin is resistant to MLCP.) v. Incubation of skinned smooth muscle strips with ATP웂S in the presence of Ca2⫹ led to thiophosphorylation of myosin and Ca2⫹-insensitive contraction upon addition of ATP (necessary since ATP웂S will not support actomyosin ATPase activity) (Cassidy et al., 1979). vi. Several pharmacological agents, including phenothiazine and naphthalenesulfonamide derivatives, bind with high affinity to CaM in a Ca2⫹-dependent manner. These and other agents inhibit MLCK activity by binding to CaM and preventing its interaction with the kinase. They also inhibit Ca2⫹-dependent, actin-activated MgATPase activity and tension development in skinned or intact muscle strips (e.g., Asano et al., 1982). ML-9, a competitive inhibitor of MLCK with respect to ATP, inhibited K⫹-or 움1-adrenoceptor agonist-induced contractions of intact smooth muscle strips, and Ca2⫹-induced contractions of skinned smooth muscle preparations (Saitoh et al., 1987). vii. A Ca2⫹-independent form of MLCK can be produced by mild proteolysis of Ca2⫹ /CaM-dependent MLCK or by expression of a truncated MLCK. This enabled myosin phosphorylation to be achieved in the absence of Ca2⫹, allowing separation of the effects of myosin phosphorylation from those of other potential Ca2⫹-dependent regulatory mechanisms (Walsh et al., 1982). When myosin was phosphorylated by the Ca2⫹-independent MLCK in the absence of Ca2⫹, the Ca2⫹ sensitivity of the actin-activated myosin MgATPase was abolished. Furthermore, incubation of skinned tissue strips with Ca2⫹-independent MLCK and ATP in the absence of Ca2⫹ elicited tension development that was accompanied by specific phosphorylation of LC20 . Relaxation upon washout of the kinase was accompanied by dephosphorylation of the myosin. The amount of tension developed following myosin phosphorylation in the absence of Ca2⫹ was comparable to that observed in the presence of Ca2⫹. Maximum tension development could therefore be accounted for solely by myosin phosphorylation. Similar results were obtained using isolated, skinned smooth muscle cells or by microinjection of constitutively active MLCK into isolated single smooth muscle cells. viii. Synthetic peptide inhibitors of MLCK, which are more specific than the pharmacological agents re-

ferred to earlier, have also been used to demonstrate the functional significance of myosin phosphorylation in the regulation of smooth muscle contraction. Peptides corresponding to the CaM-binding or autoinhibitory domains of MLCK (see later) inhibited Ca2⫹-induced force when added to skinned smooth muscle strips or K⫹induced contractions when microinjected into single cells (e.g., Kargacin et al., 1990). ix. Phosphatase inhibitors, particularly okadaic acid, calyculin A, and microcystin, induced contraction of intact or permeabilized smooth muscle preparations concomitant with an increase in LC20 phosphorylation (Hartshorne et al., 1998). x. The in vitro motility assay is a useful model of unloaded shortening velocity in the intact muscle. Usually, this involves quantification by fluorescence videomicroscopy of the movement of fluorescently labeled actin filaments over myosin immobilized on a nitrocellulose membrane. For movement to occur, smooth muscle myosin must be phosphorylated at serine 19 of LC20 (Sellers et al., 1985). Much attention has been devoted toward an understanding of the molecular mechanism whereby myosin phosphorylation leads to actin activation of the myosin MgATPase. Results of these studies indicate that, in the absence of LC20 phosphorylation, interactions between the regulatory domains of the two heads of myosin, stabilized by the proximal part of the rod, maintain the off state. Phosphorylation of LC20 then leads to disruption of these interactions and a conformational change in the neck region of the molecule that is transmitted to the nucleotide- and actin-binding sites some 8–10 nm distant. This, in turn, permits sliding of actin and myosin filaments and the development of tension.

IV. REGULATORY PROTEINS A. Calmodulin Calmodulin belongs to a family of EF hand-containing Ca2⫹-binding proteins that include troponin C of striated muscles, parvalbumins of fast-twitch skeletal muscles, S-100 proteins such as calcyclin, the intestinal vitamin D-dependent Ca2⫹-binding protein, and many others (Kawasaki et al., 1998). CaM has been shown to regulate a wide variety of enzymatic activities and physiological processes, and as many as 80 target proteins have been identified. These include the enzyme MLCK of smooth muscle that binds CaM in a Ca2⫹dependent manner with 1:1 stoichiometry and an apparent Kd for CaM of 1 nM (Gallagher et al., 1997). Consistent with its diverse regulatory functions, CaM is ubiquitous in distribution and highly conserved structurally.

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Ca2⫹ regulatory proteins such as CaM respond to transient increases in cytosolic-free [Ca2⫹] that result from cell activation by extracellular signals, e.g., hormones, neurotransmitters, growth factors, or membrane depolarization. CaM possesses four structurally homologous Ca2⫹-binding sites with dissociation constants in the micromolar range (Vogel, 1994). Sarcoplasmic-free [Ca2⫹] ([Ca2⫹]i) in the resting smooth muscle cell is 앑0.1–0.2 애M and rises to 앑0.5–0.7 애M upon stimulation. In the presence of a peptide corresponding to the CaM-binding domain of MLCK, the affinity of the two C-terminal Ca2⫹-binding sites of CaM for Ca2⫹ is increased 앑10-fold, suggesting that Ca2⫹ is bound to these sites at resting [Ca2⫹]i (Johnson et al., 1996). With two Ca2⫹ ions bound, CaM binds to but does not activate MLCK. As a result of the rise in [Ca2⫹]i upon stimulation, Ca2⫹ binds to the two N-terminal sites of CaM, triggering a conformational change that results in activation of the kinase. Detailed understanding of the CaM–MLCK interaction and the mechanism of activation of the kinase was gained from determination of the three-dimensional

structure of free CaM and CaM bound to a synthetic peptide corresponding to the CaM-binding site of MLCK (Meador et al., 1992; Fig. 4, see color insert). The kinase peptide adopts a basic amphipathic 움-helical structure (common among Ca2⫹-dependent CaM-binding peptides), i.e., having a hydrophobic surface and a discrete positively charged surface. CaM wraps around the peptide with multiple side chain interactions.

B. Myosin Light Chain Kinase MLCK catalyzes the transfer of the terminal phosphoryl group of MgATP2⫺ to serine 19 (and sometimes threonine 18) in each of the two 20-kDa light chains of myosin (Gallagher et al., 1997). Catalysis by the smooth muscle enzyme occurs by an ordered sequential mechanism: MgATP binding occurs first followed by binding of the LC20 substrate. MLCK has been isolated, and its cDNA cloned, from a variety of smooth muscle tissues. The amino acid sequences of smooth muscle MLCKs are highly conserved except for an unusual region of repeated amino acids rich in lysine, proline, alanine,

A regulatory domain

actin binding domain uncII-1 1

42

147

240

uncII-2 288

catalytic domain

uncI

381/382

481

526

uncII-3 776 815 857

GSGKFGx16K ATP-binding site

B KDTKNAEAKKLSKDRMKKYM ARRKWQKTGHAV RAIGRLSS Autoinhibitory domain CaM-binding domain FIGURE 5 (A) Domain structure of smooth muscle MLCK. Numbering is derived from the sequence of the chicken gizzard enzyme (Olson et al., 1990). The regulatory domain includes autoinhibitory and CaM-binding domains (shown in B). MLCK also contains two classes of structural motifs (앑100 residues long), referred to as uncI and uncII, which are found in the unc-22 gene product (twitchin) of the nematode Coenorhabditis elegans and the giant skeletal muscle protein titin. The uncI motif is related to the fibronectin type III domain and the uncII motif to the immunoglobulin C-2 domain. (B) Regulatory domain of smooth muscle MLCK. The autoinhibitory domain (residues K776–V807) overlaps the CaM-binding domain (residues A796–S815). Residues important for auto inhibition are in bold italicized lettering (K776, K784, K788, R790, Y794, M795, and A796) and residues important for CaM binding and activation are in bold lettering (W800, G804, I810, G811, R812, and L813).

931 972

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V. Excitation–Contraction Coupling

and threonine located near the N terminus of the mammalian enzymes. The apoenzyme consists of a single polypeptide chain with the domain structure depicted in Fig. 5A. The catalytic domain is homologous to other protein serine/threonine kinases. The regulatory domain consists of overlapping CaM-binding and autoinhibitory domains (Fig. 5B). Smooth muscle MLCK contains an actin-binding site at the N terminus that serves to anchor the kinase to the thin filament, thereby localizing MLCK in proximity to its substrate myosin. As indicated earlier, CaM may be bound to MLCK at resting [Ca2⫹]i due to the presence of two Ca2⫹ ions at the C-terminal sites, but MLCK will not be in an active conformation. Activation would then occur following binding of Ca2⫹ to the N-terminal sites of CaM (Fig. 3). One attractive feature of this model is that a local increase in [Ca2⫹] in the vicinity of the myofilaments would be required to activate contraction, so that local changes in [Ca2⫹] in other regions of the cell, regulating other processes, could occur without affecting contractility. MLCK exhibits a high degree of substrate specificity: it phosphorylates only myosin and its proteolytic fragments (HMM and S1) or isolated LC20 . This high degree

of specificity indicates rigid structural requirements in the substrate. The N-terminal sequence of chicken gizzard LC20 is Ac-SSKRAKAKTTKKRPQRATS19NVFA. Arginine 16 on the N-terminal side of serine 19 and asparagine 20, valine 21, and phenylalanine 22 on the C-terminal side, as well as other residues in the N-terminal half of LC20 , are important for substrate recognition by smooth muscle MLCK. The mechanism of activation of MLCK by Ca2⫹ – CaM is depicted in Fig. 6 and is based on the fact that the kinase contains overlapping autoinhibitory and CaMbinding domains. MLCK at resting [Ca2⫹]i is folded so that the autoinhibitory domain is bound within the substrate-binding domain of the active site. This prevents access to the myosin substrate. Formation of the Ca42⫹ –CaM complex elicits a conformational change in CaM that is transmitted to the kinase, causing removal of the autoinhibitory domain from the myosin-binding site, allowing myosin binding and catalysis to occur. This intrasteric mechanism is supported by several studies with synthetic peptides and site-directed mutagenesis, including the observation that replacement of part of the autoinhibitory sequence with the LC20 substrate se-

N

N active site

Ca2+

autoinhibitory domain

C

CaM C

CaM

CaM

CaM

N CaM CaM

Inactive

Active 2⫹

C

2⫹

FIGURE 6 Mechanism of activation of MLCK by Ca . At resting [Ca ]i , CaM likely contains two Ca2⫹ ions bound to the C-terminal sites and is tethered to MLCK via this domain, but the enzyme is inactive as the autoinhibitory domain masks the active site. When [Ca2⫹]i rises in response to stimulation of the smooth muscle cell, Ca2⫹ binds to the N-terminal Ca2⫹-binding sites of CaM, causing a conformational change that removes the autoinhibitory domain from the active site, thereby allowing access to the myosin substrate. Modeling of the catalytic core of smooth muscle MLCK, based on the crystal structures of other protein serine/threonine kinases, suggests extensive contacts between the regulatory domain and the surface of the catalytic core that maintain the inactive conformation of the kinase. The autoinhibitory sequence appears to form ionic interactions with glutamic acid residues at positions 600 and 644 in the catalytic core (Fig. 5A), thus preventing LC20 binding. Removal of the autoinhibitory domain by CaM allows LC20 to bind in the catalytic site largely via ionic interactions between arginine 16 of LC20 and the same glutamate residues in the kinase. However, the ATP-binding pocket is readily accessible in the absence or presence of bound CaM.

31. Vascular Smooth Muscle Contraction

535

quence resulted in autophosphorylation in the absence of Ca2⫹ /CaM. It is important to note, however, that substitution of several basic residues within the autoinhibitory domain with alanine or glutamate, or reversal of a segment of the inhibitory sequence, failed to produce a constitutively active enzyme.

C. Myosin Light Chain Phosphatase The implication that myosin phosphorylation plays a central role in the regulation of smooth muscle contraction led to the isolation and characterization of MLCP (Hartshorne et al., 1998), a type 1 protein serinethreonine phosphatase that is composed of three subunits: a 38-kDa catalytic subunit (PP1C), a 130-kDa myosin-binding subunit (MYPT), and a 20-kDa subunit that, when added to skinned porcine renal arterial smooth muscle strips, increased the Ca2⫹ sensitivity of the contractile apparatus. MYPT serves to anchor the phosphatase to the thick filaments by binding to myosin. It also binds to PP1C and increases its activity toward phosphorylated myosin or phosphorylated LC20 . Several isoforms of MYPT are expressed due to alternative splicing of transcripts encoded by two genes. MLCP is commonly referred to as PP1M to indicate that it is a type 1 phosphatase that associates with myosin. In the early days of studies of the regulation of smooth muscle contraction it was assumed that regulation was achieved exclusively at the level of MLCK. This is clearly not the case and it is now evident that PP1M is subject to complex regulation, both activation and inhibition. 1. Ca2ⴙ Sensitization Interest in PP1M regulation was stimulated by the discovery that a variety of contractile agonists can increase LC20 phosphorylation and force without a change in [Ca2⫹]i , i.e., in addition to inducing contraction by elevating [Ca2⫹]i , excitatory agonists can induce contraction by increasing Ca2⫹ sensitivity. This was shown to be due to inhibition of PP1M rather than activation of MLCK and involves a GTP-binding protein, as addition of GTP웂S or GTP plus agonist (e.g., carbachol) to 움toxin-permeabilized smooth muscle strips increased both force and LC20 phosphorylation at a fixed, submaximal [Ca2⫹]. Figure 7 depicts a signaling pathway leading from seven-transmembrane domain receptors to inhibition of PP1M that could explain agonist-induced Ca2⫹ sensitization of force. The time lag between receptor stimulation and the onset of force production in this case is 앑10–15 sec. Agonist stimulation of the receptor leads to activation of the small GTPase RhoA. This likely involves exchange of GDP for GTP, catalyzed by a guanine nucleotide exchange factor, and dissociation

FIGURE 7 Mechanisms of Ca2⫹ sensitization of smooth muscle contraction. Inhibition of PP1M due to (i) MYPT phosphorylation by ROK, (ii) PKC-phosphorylated CPI-17, or (iii) arachidonic acid-induced dissociation leads to an increase in LC20 phosphorylation and force without a change in [Ca2⫹]i . ROK, Rho-associated kinase; MYPT, myosin targeting subunit of PP1M ; PP1M , myosin-associated type 1 protein serine/threonine phosphatase; MLCK, Ca2⫹ /calmodulin-dependent myosin light chain kinase; LC20 K, Ca2⫹-independent LC20 kinase; LC20 , 20-kDa light chain of myosin; PLA2 , phospholipase A2; aPKC, atypical protein kinase C isoenzyme; CPI-17, PKC-potentiated inhibitory protein of PP1M of 17 kDa.

of a guanine nucleotide dissociation inhibitor. Activated RhoA-GTP leads to the activation of Rho-associated kinase (ROK), which phosphorylates the MYPT subunit of PP1M , thereby inhibiting the phosphatase (Kimura et al., 1996; Uehata et al., 1997). This alters the PP1M :MLCK activity ratio in favor of the kinase, resulting in an increase in LC20 phosphorylation and force without a change in [Ca2⫹]i . A distinct kinase that phosphorylates the same site in MYPT (threonine 654) copurifies with PP1M (Ichikawa et al., 1996). Evidence suggests that a Ca2⫹-independent LC20 kinase, distinct from MLCK, may account in part for this increase in LC20 phosphorylation (Weber et al., 1999).

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V. Excitation–Contraction Coupling

Several aspects of the pathway in Fig. 7 remain to be clarified. (i) What are the intermediates between the receptor and RhoA? For example, is a heterotrimeric Gprotein involved? (ii) Does RhoA-GTP activate ROK directly? Activated RhoA translocates from the cytosol to the plasma membrane where it may interact with a membrane-bound effector that could be ROK or an upstream component of the pathway. (iii) Does ROK activation occur at the membrane? If so, presumably the activated kinase translocates to the myofilaments where it phosphorylates MYPT. (iv) Does ROK also phosphorylate myosin directly? ROK has been shown to phosphorylate LC20 in vitro at serine 19, but indirect evidence does not support this occurring in vivo. Protein kinase C (PKC) has also been implicated in the regulation of PP1M , although direct phosphorylation of PP1M by PKC appears unlikely (Fig. 7). A novel smooth muscle protein, CPI-17, has been identified as a PKC substrate (Li et al., 1998). When phosphorylated, it becomes a potent inhibitor of PP1M . Arachidonic acid, generated from the hydrolysis of membrane phospholipids by phospholipase A2 , may be involved in Ca2⫹ sensitization of smooth muscle contraction via activation of an atypical PKC isoenzyme (Gailly et al., 1997) or ROK (Feng et al., 1999). Alternatively, arachidonic acid may inhibit PP1M directly via binding to MYPT, causing dissociation of the holoenzyme (Gong et al., 1992). 2. Ca

2ⴙ

Desensitization

Vasodilating substances cause relaxation not only by reducing [Ca2⫹]i , but also by decreasing Ca2⫹ sensitivity. Two general methods whereby smooth muscle contraction can be desensitized to Ca2⫹ have been proposed: (i) phosphorylation of MLCK by Ca2⫹ /CaM-dependent protein kinase II (CaM kinase II) and (ii) activation of PP1M (Fig. 8). MLCK is phosphorylated by CaM kinase II at serine 815 at the C terminus of the CaM-binding site (Fig. 5B). This phosphorylation increases the KCaM (the CaM concentration required for half-maximal activation of MLCK) by 앑10-fold and is blocked by the presence of bound CaM. MLCK phosphorylation at this regulatory site and an increase in KCaM have been observed in intact smooth muscles contracted with a muscarinic agonist (via release of SR Ca2⫹) or KC1 (via Ca2⫹ influx). In permeabilized cells, MLCK is phosphorylated in a Ca2⫹-dependent manner. This phosphorylation is blocked by CaM kinase II inhibitors. MLCK phosphorylation requires higher [Ca2⫹] than LC20 phosphorylation, suggesting that MLCK phosphorylation will occur physiologically only when [Ca2⫹]i rises to high levels. Activation of the cGMP pathway may also lead to Ca2⫹ desensitization of smooth muscle contraction (Fig.

FIGURE 8 Mechanisms of Ca2⫹ desensitization of smooth muscle contraction. A decrease in LC20 phosphorylation at a fixed [Ca2⫹] can occur due to (i) inhibition of MLCK by CaM kinase II-catalyzed phosphorylation or (ii) activation of PP1M via PKG-catalyzed phosphorylation of telokin. GCS and GCM , soluble (cytosolic) and membrane-bound forms of guanylyl cyclase, respectively; cGMP, cyclic guanosine-3⬘, 5⬘-monophosphate; PKG, cGMP-dependent protein kinase; PP1M , myosin-associated type 1 protein serine/threonine phosphatase; LC20 , 20-kDa light chain of myosin; CaM kinase II, Ca2⫹ / calmodulin-dependent protein kinase II; MLCK, Ca2⫹ /calmodulindependent myosin light chain kinase.

8). This may occur via cGMP-dependent protein kinase (PKG)-catalyzed phosphorylation of telokin, an acidic protein that is encoded by the MLCK gene (Wu et al., 1998). Telokin corresponds to the C terminus of MLCK and is expressed due to an alternate promoter in the MLCK gene. Phosphorylation of telokin by PKG enhances its ability to activate PP1M , resulting in a decrease in LC20 phosphorylation and relaxation without a change in [Ca2⫹]i . While expressed at high levels (70–90 애M) in phasic muscles, only trace amounts of telokin have been detected in tonic smooth muscles. The site of PKG-catalyzed phosphorylation in telokin (serine 15) corresponds to the site in MLCK phosphorylated by CaM kinase II (serine 815; see earlier discussion).

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It is important to note that PKG has also been implicated in reducing [Ca2⫹]i via enhanced sequestration of Ca2⫹ by the SR, enhanced Ca2⫹ efflux, decreased Ca2⫹ influx as a result of decreased Ca2⫹ channel activity and hyperpolarization resulting from increased K⫹ channel activity, and decreased SR Ca2⫹ release through antagonism of IP3 formation and possibly phosphorylation of its receptor.

V. SUMMARY Detailed knowledge of the structure and function of isolated contractile proteins, combined with ultrastructural, kinetic, biochemical, and physiological studies, has led at least to reasonable hypotheses concerning how smooth muscle contracts and how this process is regulated. What emerges is the concept that smooth muscle contraction occurs via a sliding filament mechanism similar to that in striated muscles, and the details of this mechanism are becoming clearer thanks largely to very elegant X-ray crystallographic structure determinations. From the point of view of control, it is well established that contractile regulation is achieved by fluctuations in the concentration of sarcoplasmic-free Ca2⫹, and the mechanism whereby Ca2⫹, acting via CaM, activates smooth muscle contraction via the phosphorylation of myosin is well understood. A great deal is known about the kinase and phosphatase responsible for the phosphorylation and dephosphorylation of myosin, including their regulation. Several aspects of this control system remain the subject of extensive investigation, however, e.g., the hypothesis that CaM (with its C-terminal Ca2⫹binding sites saturated with Ca2⫹) is bound to MLCK at resting [Ca2⫹]i ; the mechanism whereby LC20 phosphorylation in the neck region of myosin affects the motor domain that is quite distant; and the elucidation of signaling pathways that terminate in activation or inhibition of MLCK/MLCP (e.g., the RhoA pathway leading to PP1M inhibition). It is becoming increasingly apparent that myosin phosphorylation–dephosphorylation is not the only mechanism for regulating smooth muscle contraction. There is substantial physiological and biochemical evidence for the existence of secondary control systems that can modulate the contractile state of the smooth muscle, e.g., functional roles of the thin filament-associated proteins caldesmon and calponin (that have not been addressed in this chapter) will require further definition. The importance of the latch state (the maintenance of force with myosin dephosphorylation) is subject to ongoing investigation (Hai and Murphy, 1988). There are complex relationships among force, velocity

of shortening, and LC20 phosphorylation in smooth muscle tissues that could be explained by the formation of latch bridges (attached, dephosphorylated crossbridges). Although LC20 phosphorylation is obligatory for crossbridge attachment and force generation, Hai and Murphy (1988) proposed that dephosphorylation could occur prior to detachment of the crossbridge from actin. The effect of dephosphorylation then is to slow the rate of detachment of attached crossbridges, presumably by reducing the rate of ADP release. Many of the mechanical properties of smooth muscle are undoubtedly a consequence of the structure and organization of the cytoskeleton. Increasing attention is being directed toward characterization of the cytoskeletal proteins: intermediate filament proteins and actin-binding proteins that presumably function in the spatial organization of the contractile machinery. As yet we know relatively little of the contribution these components make to the contractile properties of the muscle cell. Ultimately, it may turn out that the differences in mechanical properties observed in different smooth muscle tissues may be a function of cytoskeletal proteins rather than contractile proteins themselves. The answers to these and many other questions will come from innovative application of a host of experimental approaches and techniques of molecular biology, biochemistry, biophysics, and physiology. Contractile abnormalities of smooth muscle are major causes of disease, e.g., asthma, high blood pressure, coronary artery disease, and cerebral vasospasm following subarachnoid hemorrhage. An excess of excitatory agonists, such as histamine in allergy, causes bronchoconstriction and asthmatic symptoms. Enhanced responsiveness of vascular smooth muscles to normal stimuli results in vasoconstriction and increased blood pressure. Bathing of cerebral blood vessels in blood following subarachnoid hemorrhage leads to massive vasoconstriction (long-term narrowing of the cerebral artery). Clearly, a detailed understanding of the complex molecular mechanisms involved in regulating smooth muscle contraction will impact greatly on our ability to treat and prevent such diseases.

Acknowledgment We are very grateful to Lenore Youngberg for secretarial support.

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Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245–248. Li, L., Eto, M., Lee, M. R., Morita, F., Yazawa, M., and Kitazawa, T. (1998). Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. J. Physiol. 508, 871–881. Marston, S. B., and Huber, P. A. J. (1996). Caldesmon. In ‘‘Biochemistry of Smooth Muscle Contraction’’ (M. Ba´ra´ny, ed.), pp. 77–90. Academic Press, San Diego. Meador, W. E., Means, A. R., and Quiocho, F. A. (1992). Target ˚ structure of a calmodulinenzyme recognition by calmodulin: 2.4 A peptide complex. Science 257, 1251–1255. Mita, M., and Walsh, M. P. (1997). 움1-Adrenoceptor-mediated phosphorylation of myosin in rat tail arterial smooth muscle. Biochem. J. 327, 669–674. Olson, N. J., Pearson, R. B., Needleman, D. S., Hurwitz, M. Y., Kemp, B. E., and Means, A. R. (1990). Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. Natl. Acad. Sci. USA 87, 2284–2288. Rhoads, A. R., and Friedberg, F. (1997). Sequence motifs for calmodulin recognition. FASEB J. 11, 331–340. Saitoh, M., Ishikawa, T., Matsushima, S., Naka, M., and Hidaka, H. (1987). Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J. Biol. Chem. 262, 7796–7801. Sellers, J. R., Pato, M. D., and Adelstein, R. S. (1981). Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin. J. Biol. Chem. 256, 13137–13142. Sellers, J. R., Spudich, J. A., and Sheetz, M. P. (1985). Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J. Cell. Biol. 101, 1897–1902. Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., and Hartshorne, D. J. (1978). Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. Biochemistry 17, 4411–4418. Shirazi, A., Iizuka, K., Fadden, P., Mosse, C., Somlyo, A. P., Somlyo, A. V., and Haystead, T. A. J. (1994). Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme: The differential effects of the holoenzyme and its subunits on smooth muscle. J. Biol. Chem. 269, 31598–31606. Sobieszek, A. (1977). Vertebrate smooth muscle myosin: enzymatic and structural properties. In ‘‘The Biochemistry of Smooth Muscle’’ (N. L. Stephens, ed.), pp. 413–443. University Park Press, Baltimore, MD. Somlyo, A. P., and Somlyo, A. V. (1994). Signal transduction and regulation in smooth muscle. Nature 372, 231–236. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M., and Narumiya, S. (1997). Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990–994. Vogel, H. J. (1994). Calmodulin: A versatile calcium mediator protein. Biochem. Cell Biol. 72, 357–376. Walsh, M. P., Bridenbaugh, R., Hartshorne, D. J., and Kerrick, W. G. L. (1982). Phosphorylation-dependent activated tension in skinned gizzard muscle fibers in the absence of Ca2⫹. J. Biol. Chem. 257, 5987–5990. Walsh, M. P., Kargacin, G. J., Kendrick-Jones, J., and Lincoln, T. M. (1995). Intracellular mechanisms involved in the regulation of vascular smooth muscle tone. Can. J. Physiol. Pharmacol. 73, 565–573.

31. Vascular Smooth Muscle Contraction Weber, L. P., Van Lierop, J. E., and Walsh, M. P. (1999). Ca2⫹independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J. Physiol. 516, 805–824. Williams, D. A., and Fay, F. S. (1986). Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin-2. Am. J. Physiol. 250, C779–C791. Winder, S. J., Allen, B. G., Cle´ment-Chomienne, O., and Walsh, M. P.

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(1998). Regulation of smooth muscle actin-myosin interaction and force by calponin. Acta Physiol. Scand. 164, 415–426. Wu, X., Haystead, T. A. J., Nakamoto, R. K., Somlyo, A. V., and Somlyo, A. P. (1998). Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin: Synergism with cyclic nucleotide-activated kinase. J. Biol. Chem. 273, 11362– 11369.

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32 Myocardial Energy Metabolism PAUL F. KANTOR

GARY D. LOPASCHUK

LIONEL H. OPIE

McMaster University Hamilton, Canada

Cardiovascular Research Group University of Alberta Edmonton, Alberta, Canada T6G 2S2

University of Cape Town RSA 7925 Cape Town, South Africa

I. INTRODUCTION

oxygen to form water. Acetyl CoA is an activated twocarbon substrate fragment that begins the final common pathway of all of the three major substrates of the myocardium: free fatty acids, glucose, and lactate. This chapter reviews the pathways whereby each of these major substrates (or fuel sources) is broken down to acetyl CoA and the mechanisms that regulate this process.

Metabolism comes from the Greek word meaning ‘‘change,’’ referring to the transformation of potential energy stored in substrates to that of ATP. The previous decade has also seen a fundamental change in the understanding of cardiac metabolism, from the perspective of a science which measures quantity and flux of substrates to one that describes the subcellular molecular interactions and the genes that regulate them. The relevance of cardiac metabolism to the cardiologist and cardiac surgeon continues to increase, especially with the introduction of new drugs that directly alter energy metabolism (1) and with the development of nuclear magnetic resonance spectroscopy (NMR) and positron emission tomography (PET) techniques, enabling the noninvasive measurement of cardiac substrate metabolism. An overview of the process and regulation of myocardial energy metabolism, together with an emphasis on areas of clinical relevance, is included in this chapter. Metabolism is critical for normal cardiac contractility. Contractile function necessitates a high turnover rate of ATP in the myocardium, and hence a correspondingly high rate of mitochondrial ATP production. Within the mitochondria the Krebs cycle, or tricarboxylic acid (TCA) cycle, breaks down acetyl CoA to CO2 and the reducing equivalents NADH and FADH2 ; the latter are used by the electron transport chain to pump hydrogen atoms out of the mitochondria, forming a proton gradient across the mitochondrial membrane. This gradient is required for the synthesis of ATP by oxidative phosphorylation. During the process of oxidative phosphorylation, protons from NADH and FADH2 combine with

Heart Physiology and Pathophysiology, Fourth Edition

II. GLUCOSE A. Importance of Glucose for Metabolism of the Heart Although glucose is not the major fuel utilized by the myocardium during normal aerobic metabolism, it is of special interest. Historically, interest in glucose metabolism dates back to at least 1907, when Locke and Rosenheim (2) described glucose uptake by the isolated heart. In 1914, Evans (3) suggested that only one-third of the heart’s energy was supplied by carbohydrate oxidation. Cruickshank and associates (4) suggested that the ‘‘direct combustion’’ of fatty acids, probably the blood fatty acids, met the rest of the heart’s energy requirements. Thus, these early workers delineated carbohydrate and fatty acids as two of the most important myocardial fuels. From a biochemical point of view, glucose is of interest because factors controlling its uptake and utilization by glycolysis or glycogen synthesis have been studied extensively and an integrated scheme of the control of these processes has now been established. An overveiw of the metabolic pathways is summarized in Fig 1.

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

FIGURE 1 An overview of pathways of myocardial glucose and glycogen metabolism showing the reactions of glycolysis (left) and glycogen synthesis and breakdown (right). Intermediaries are shown in bold face, enzymes are italicized, and cofactors are indicated in regular print. GLUT-4 is the myocardial glucose transporter. MCT is the monocarboxylate transporter. Dotted lines indicate the position of reactions detailed in subsequent figures. Glucose phosphorylation by hexokinase is nonreversible in myocardium, and phosphofructokinase catalyzes the first nonreversible reaction of glycolysis. Two molecules of glyceraldehyde 3-phosphate and all subsequent intermediaries result from each glucose entering glycolysis and are denoted (2). The pyruvate dehydrogenase reaction is also not reversible. On the right hand side of the figure, all the reactions of glycogen metabolism are reversible. An immediate net yield of two ATP is derived from the glycolytic degradation of one exogenous glucose and one ATP if the source is from glycogen.

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Therapeutically, glucose is of interest for several reasons: (i) glycolysis provides an immediate source of cytosolic ATP in circumstances of acute oxygen deficit; (ii) glycolytically derived ATP may be a preferential and important source of ATP for membrane ion transport processes; and (iii) high glucose oxidation rates are critical to the restoration of normal myocardial function following ischemia–reperfusion (5,6).

B. Regulation of Glucose Uptake (Fig. 2) In the absence of insulin, glucose transport is generally rate limiting over any glucose phosphorylation process within the myocyte (6), with a steep inward transsarcolemmal gradient evident. Insulin alters glucose transport by inducing translocation to the sarcolemma of the monosaccharide transporters GLUT-4 and to a lesser extent GLUT-1. This process involves the established pathway of insulin signaling via insulin receptor activation of insulin receptor substrate 1, stimulation of phosphatidylinositol 3-kinase, and activation of Rab4 and Rab3C guanine nucleotide-binding proteins, facilitating the membrane translocation process of the

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GLUT-4 molecule (7). GLUT-4 is a large 500 amino acid protein, which spans the membrane with 12 helices, and is believed to form a hydrophilic glucose-transporting channel by conformational changes involving a tryptophan-rich domain (8–10). The GLUT-4 isotype, which is predominant in muscle and adipose tissue (9), is insulin sensitive. GLUT-4 overexpression will therefore induce a state of hypoglycemia and heightened insulin sensitivity, as demonstrated in transgenic mice expressing human GLUT-4 (11). Evidence suggests that insulin also stimulates GLUT-4 activity via a separate MAP kinase-mediated activation of transporters already recruited to the sarcolemma (12). GLUT-1, the transporter that has been better described in terms of its structural and spatial configuration, also exists in the heart. It is the dominant myocardial isotype during fetal life, undergoing a rapid regression with the onset of suckling, whereupon transcription and expression of the GLUT-4 isotype accelerate (13). Both transporters have a Km approaching the plasma glucose concentration, and because fluctuations in plasma glucose concentration will ordinarily not be very great, an increase in overall transmembrane flux

FIGURE 2 Regulation of myocardial glucose uptake. Glucose enters by facilitated diffusion through sarcolemmal glucose transporter proteins (GLUT-4). Following a carbohydrate meal, rising glucose stimulates pancreatic 웁 cells to secrete insulin. Insulin receptor stimulation results in a tyrosine kinase-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). IRS-1 binds to several kinases, predominantly phosphatidyl inositol 3kinase (PI-3K). This mediates the release of GLUT-4 units from intracytoplasmic stores and their translocation to the sarcolemma via one of several guanosine nucleoside phosphate (GNP)-binding proteins, designated RAB 3C and RAB 4. Glucose influx is driven by the concentration gradient, but is increased greatly by GLUT4 expression. Rapid phosphorylation of glucose by hexokinase preserves this gradient. Adrenergic stimulation, hypoxia, and ischemia also increase GLUT-4 expression. Transcriptional regulation of GLUT-4 synthesis (not shown) is also insulin dependent.

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requires an increase in GLUT-4 numbers on the sarcolemma. This is achieved rapidly by exocytosis of previously synthesized units, which are stored intracellularly, and by increased transcription of the transporter protein for longer-term requirements. Numerous influences are now known to alter this dynamic equilibrium (14), including circulating hormones, myocardial energy balance, oxygen supply, and the presence of nonglucose substrates (15). The effect of insulin, increased workload, and ischemia is to increase glucose uptake and both GLUT-1 and GLUT-4 translocation (17, 18), although the signal may be mediated through a pathway other than PI-3K during ischemia (19). Conversely, the diabetic state, decreasing cardiac work, and the presence of fatty acids as an alternate substrate (20) reduce glucose uptake.

C. Regulation of Glycolysis Following uptake, the major metabolic pathway of glucose is that of glycolysis (Fig. 1). This is the common pathway for both glucose and glycogen breakdown, which converts one molecule of glucose 6-phosphate to two molecules of pyruvate (see review by Depre et al, 6). Anaerobic glycolysis involves the further commitment of pyruvate to lactate formation and the generation of ATP and NADH when oxygen is unavailable and mitochondrial uptake is forestalled. Aerobic glycolysis implies the further activation of pyruvate and its uptake into mitochondria without the production of lactate. In reality, these mechanisms coexist, with the fate of pyruvate being determined by the prevailing supply of oxygen, acetyl CoA from fatty acids, NADH, and the need for glycolytic ATP production. Under anaerobic circumstances, glycolysis is the sole source of ATP and therefore becomes a critical reaction. The introduction of terms describing the presence (or lack thereof) of oxygen is important to the understanding of experimental glycolytic regulation. Although artificial, a helpful means of conceptualizing the control of glycolysis is by examining its component reactions in turn: the logical starting point is that of the metabolism of glucose 6-phosphate (G6p) by hexokinase, which is also the juncture of the pathways of glucose uptake and glycogen breakdown (Fig. 1). 1. Hexokinase Hexokinase catalyzes the initial phosphorylation of glucose to form glucose 6-phosphate. It is found in two isoenzymes in the heart (hexokinase I and II, with II being the predominant isoform) (21). Both have a similar Km of around 0.1 mM for glucose. Although hexoki-

nase is a cytosolic enzyme, it preferentially uses ATP generated in the mitochondria this phosphorylation reaction (22). It has been found to bind to cytosolic aspect of the mitochondria membrane at specific contact points where the inner and outer membranes come together (23). This is a cooperative process, which forms an oligomeric hexokinase enzyme complex, and lowers the Km for glucose. The inhibitory effect of G6p on hexokinase is also reduced. Phosphorylation is therefore accelerated and glycolytic flux increases (23, 24). In addition, hexokinase activity is increased by protein kinase A phosphorylation (25). Insulin is also able to regulate hexokinase II by inducing mRNA transcription, again through the PI-3K pathway (26) Glucose 6-phosphate, the reaction product, is in flux equilibrium to fructose 6-phosphate, which becomes the substrate for the primary ‘‘irreversible’’ reaction of the glycolytic pathway: the phosphorylation of fructose 6phosphate by phosphofructokinase (PFK). 2. Phosphofructokinase Randle and Morgan (20) in Cambridge and Morgan’s group (27) in Nashville were the first to induce anoxia in isolated perfused hearts and to establish that the ensuing increase in glycolytic flux was governed by a series of reactions, including acceleration of glucose transport into the cell and an increase in the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. This reaction is catalyzed by phosphofructo-1-kinase (PFK-1), a tetrameric enzyme molecule (28). As illustrated in Fig. 3, it is an ATP-requiring reaction, with ATP also acting as a negative allosteric effector. Citrate and H⫹ ions will also inhibit the activity of PFK, with AMP and fructose 2,6-bisphosphate (F2,6-BP) being the important positive allosteric effectors (29). PFK-1 shows near total inhibition in the absence of physiologic concentrations of fructose 2,6-bisphosphate (30). The concentration and regulation of the latter are therefore of significance and are discussed later. PFK-1 is therefore a pivotal enzyme in the glycolytic pathway. It operates at the junction of pathways of glucose degradation (glycolysis) or storage (glycogenesis). It is subject to direct, substrate-mediated allosteric effects and also to the regulatory effect of remote products of flux, in the form of citrate. Fructose 2,6-bisphosphate is now recognized as an important forward regulator of PFK activity, which it stimulates potently (29). F2,6-BP is produced from fructose 6-phosphate by the enzyme 6-phosphofructo-2-kinase (PFK-2), which is a bifunctional enzyme, also having the capability of a F2,6-BP phosphatase, and hence of controlling both synthesis and degradation of F2,6BP. In the heart, kinase activity dominates by 100-fold

32. Myocardial Energy Metabolism

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FIGURE 3 Regulation of phosphofructo-1-kinase (PFK-1). PFK-1 is the major regulatory point in the glycolytic pathway under aerobic conditions. It mediates the final energy-consuming reaction in glycolysis with the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. PFK-1 activity is diminished greatly in the presence of increased cytoplasmic ATP and citrate (indicating an adequate cellular energy level and an adequate supply of acetyl-CoA to the TCA cycle). End product inhibition by H⫹ ions is also important in severe ischemia. An alternate fate of fructose 6-phosphate is the ATP-requiring phosphorylation reaction producing fructose 2,6 -bisphosphate catalyzed by phosphofructo-2-kinase (PFK-2). Fructose 2,6-bisphosphate has a powerful forward induction effect on PFK-1. Increased cellular AMP levels and the action of protein kinase A stimulate PFK-2, whereas citrate inhibits it.

over phosphatase activity under simulated physiologic conditions (30), with the phosphatase having a Km an order of magnitude higher than the normal physiologic levels of FBP. PFK-2 is itself controlled by several mechanisms: the enzyme binds avidly to ATP and is equally sensitive to allosteric inhibition by citrate, as is demonstrated by the inverse relation between citrate and F2,6-BP concentrations in perfused rat hearts. Increasing concentrations of substrate (fructose 6-phosphate) will increase F2,6-BP levels, and increasing concentrations of citrate will tend to inhibit the production of F2,6-BP. Phosphofructokinase-2 is also under phosphorylation control by protein kinase A and the protein kinases involved in the insulin-signaling cascade (31–33). The powerful regulatory effect of F2,6-BP, and hence of PFK-2 as a forward modulator of PFK-1 activity, is thought to be critical in the integrated response of glucose metabolism to complex physiologic stimuli. 3. Glyceraldehyde 3-phosphate Dehydrogenase Control of glycolysis in the heart can pass from phosphofructokinase to other points down the line of the

reaction chain during conditions such as ischemia (34, 35) or an abrupt normoxic–anoxic transition (36). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which catalyzes the conversion of glyceraldehyde-3phosphate to 1,3-diphosphoglycerate, is a strategic point of glycolytic regulation for two reasons. First, it produces NADH molecules that are yielded during glycolysis. Second it is interchangeable with dihydroxyacetone phosphate, linking it to the metabolism of glycerol generated from triglyceride breakdown. Regulation of glycolysis via altered GAPDH activity is probably the most important regulatory point within the glycolytic pathway during ischemia. During severe total ischemia, glycolysis is inhibited rather than stimulated. In this situation, PFK-1 is inhibited by acidosis, whereas GAPDH is also inhibited by several of the reaction products of glycolysis (37, 38). In mild ischemia, however, glucose uptake increases along with glycolytic flux through GLUT-4 induction and the allosteric upregulation of PFK-1 activity. In this setting, the accumulation of NADH and lactate are important regulators of GAPDH, and of glycolysis overall. Similarly, in the diabetic heart, where glucose uptake is reduced and glycerol and fatty acid oxidation increase,

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significant inhibition of GAPDH has been demonstrated. This is readily reversed (with an accompanying increase in glycolytic flux) by inhibition of aldose reductase (38). Other workers have demonstrated a rise in GAPDH flux in the setting of postischemic recovery and with adrenergic stimulation, implicating an alteration in the cytosolic energy quotient as the underlying mechanism (39). Protons also inhibit GAPDH, as do high concentrations of NADH (38, 40). The molecular mechanisms involved are not yet described, but clearly anaerobic glycolysis will tend to slow down as tissue NADH and protons accumulate to inhibit GAPDH and PFK-1. a. Proton Production by Anaerobic Glycolysis During anaerobic glycolysis, the reduced cofactor, NADH⫹ H⫹ (which equals NADH2), formed by the enzyme GAPDH is reconverted to NAD⫹ during the formation of lactate. The overall reaction produces two molecules of ATP, independently of oxygen. Thus, during anaerobic glycolysis, protons are not formed. Why then is anaerobic glycolysis a potential source of intracellular acidosis? Gevers (40) and Dennis et al. (41) have examined this question in detail. When all the charges are written into the individual glycolytic reactions and allowance is made for the probable degree of interaction of ADP and ATP with Mg2⫹, the following equations are derived: Glucose ⫹ 2 MgADP⫺ ⫹ 2 Pi2⫺ 씮 2 lactate⫺ ⫹ 2 MgATP2⫺

(1)

Under anaerobic conditions, ATP will be broken down as fast as it is produced. Protons are produced by the hydrolysis of this ATP: 2 MgATP2⫺ 씮 2 MgADP⫺ ⫹ 2 Pi2⫺ ⫹ 2 H⫹

(2)

These equations are only approximations and depend on a number of assumptions, including the concentration of free Mg2⫹ in the cytosol and the intracellular pH (the latter influencing the phosphate charge). Thus the turnover of glycolytically derived ATP (and not the production of lactate) is the ‘‘source’’ of protons produced during anaerobic glycolysis.

D. Metabolism and Glycolysis: Critical Features 1. The rate of transport of glucose across the sarcolemma by the stereospecific glucose transporter, GLUT4, and to a lesser extent GLUT-1, is of major importance in controlling the rate of glucose uptake by the heart.

Even at extremely reduced rates of coronary flow during severe ischemia, glucose delivery can be rate limiting to glycolysis (42). 2. Translocation of the glucose transporter to the sarcolemmal membrane is increased greatly by insulin, hypoxia, and increased heart work, conditions that also increase the rate of flux through glycolysis by increasing the activity of the enzyme phosphofructokinase. 3. Following uptake, the majority of intracellular glucose is phosphorylated to glucose 6-phosphate and is then converted to fructose 6-phosphate, which is the substrate for the enzyme phosphofructokinase. 4. Phosphofructokinase activity is inhibited by the presence of high-energy phosphate compounds. An alternative phosphorylation product of fructose 6-phosphate is fructose 2,6-bisphosphate, which accelerates the activity of phosphofructokinase greatly. 5. Under aerobic conditions (normal oxygenation), the end point of glycolysis is pyruvate, which enters the tricarboxylic acid cycle for further aerobic metabolism to form NADH2 and FADH2 . 6. Under anaerobic conditions, the end point of glycolysis is lactate; the rates of anaerobic glycolysis are not high enough to provide sufficient energy for the contracting heart, although it may provide for the needs of the potassium-arrested heart. 7. In mild ischemia, adrenergic stimulation, falling ATP and NADH2 levels, with rising AMP levels, stimulate glucose uptake and glycolysis. In severe ischemia, the products of glycolysis accumulate, resulting in an inhibition of glycolytic flux at the level of phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. Hence, in severe ischemia, when glycolysis is needed most to produce anaerobic ATP, glycolysis is also least able to perform this vital function. 8. In the recently described condition of myocardial hibernation (discussed later), the myocardium remains viable but poorly contractile, while still able to extract and metabolize glucose. Evidence suggests (42) that glucose delivery in this setting is indeed the major ratelimiting step to glycolysis, as the glucose extraction ratio is in fact increased. Thus the prevailing theory that enzyme inhibition limits glycolysis during ischemia may yet be challenged. This holds new relevance given the resurgent interest in glucose–insulin–potassium (GIK) in the management of ischemic syndromes (43)

III. GLYCOGEN Glycogen is a polysaccharide (i.e., a combination of many molecules of glucose) that forms large storage granules (macroparticles up to 104 kDa in size) in the

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cytoplasm of the heart. Although frequently thought of as a ‘‘storage’’ carbohydrate, the glycogen molecule is in a constant state of turnover as a result of variable rates of synthesis and degradation. This cycling of glucose molecules through glycogen may act as a control mechanism for myocardial glucose utilization and is influenced by the presence or absence of alternate substrates such as fatty acids or lactate (44). In the regulation of synthesis, glycogen stores increase during fasting and fall in the fed state (45). These changes in the glycogen content can be attributed to changes in blood free fatty acids levels: when bloodfree fatty acid concentrations are high, as during fasting, then glycolysis is inhibited and glucose 6-phosphate levels rise, with increased synthesis of cardiac glycogen (45). In the fed state, blood-free fatty acid concentrations are low, and so is cardiac glycogen, despite the high rates of glucose uptake and the activity of insulin, which stimulates glycogen synthesis. This is explained by a high rate of glycogen turnover (high rates of synthesis and breakdown of glycogen), especially the outer glycogen chains in the fed state, an effect thought to be mediated by insulin (46).

A. Glycogen Synthesis The pathways of glycogen synthesis are separate from those of glycogen breakdown because there are two different enzyme systems. Advances in the understanding of glycogen synthesis (47, 48) point to the initiation of synthesis on a self-glycosylating protein, glycogenin. The addition of glucose residues to glycogenin results in a complex of molecular mass around 400 kDa and around 10% protein content, rendering it acid precipitable. This ‘‘proglycogen’’ eventually becomes a macromolecular glycogen particle following further glucose incorporation (48). Glucose becomes committed to glycogen synthesis with the conversion of glucose 6-phosphate to glucose 1-phosphate. The transfer of glucose 1-phosphate to the end of a preexisting glycogen chain, or to a glycogenin primer, is affected by glycogen synthase, an enzyme that exists in two forms depending on the recipient molecule. Glycogen synthase is highly regulated and is the chief enzyme in the synthesis process. In its active, dephosphorylated state (synthase a), it incorporates activated glucose 1-phosphate molecules (using uridine triphosphate, derived from ATP as an energy transfer molecule) onto the glycogen chain. Phosphorylation (inactivation) and dephosphorylation (activation) of glycogen synthase are, in turn, controlled by several protein kinases (49), with protein kinase A, phosphorylase kinase, and protein kinase C being the most important.

Substantial glycogen synthesis does not take place in a state of energy depletion, as high-energy phosphates are required. However, in the presence of sufficient energy, and insulin, glycogen synthesis can proceed readily. The mode of action of insulin in stimulating glycogen synthesis is complex (50). Insulin increases the percentage of glycogen synthase in the dephosphorylated active state by increasing specific protein phosphatase activity in relation to glycogen synthase. In contrast, glycogen synthesis diminishes as the activity of the synthase phosphatase falls (51). In addition to the direct effect of insulin, the other major factor stimulating glycogen synthesis is a high cellular content of glucose 6phosphate. Conditions increasing the cardiac contents of glucose 6-phosphate are (a) high circulating insulin and glucose, as encountered after a high carbohydrate meal, and (b) inhibition of glycolysis, as may occur when the heart is using fatty acids during fasting or due to insulin deficient diabetes mellitus. In these situations, the continued buildup of cardiac glycogen will eventually inhibit its own synthesis, which explains why a high level of glycogen is accompanied by a low rate of turnover. In contrast, during provision of glucose and insulin, glycolysis is accelerated and not inhibited, and the outer chains of glycogen that are formed are rapidly broken down so that the overall level of glycogen does not rise despite the increased rate of synthesis. Direct measures of glycogen turnover in the isolated perfused heart have confirmed that cardiac glycogen is oxidized rapidly and resynthesized continuously under normal aerobic conditions (52, 53).

B. Glycogen Breakdown The two major mechanisms for stimulating glycogen breakdown are increased by cyclic AMP or by a fall in high-energy phosphate levels. Cyclic AMP promotes the cascade that eventually converts the inactive glycogen phosphorylase b to the active phosphorylase a. Cyclic AMP activates protein kinase A (PKA), which in turn activates phosphorylase b kinase, converting phosphorylase b to phosphorylase a. Cleavage of glycogen 1,4 bonds and the incorporation of Pi- result in the yield of glucose 1-phosphate. The latter is in equilibrium with glucose 6-phosphate and enters the glycolytic pathway as described earlier. This pathway requires no ATP: as a result, the conversion of glucose to glycogen and its subsequent breakdown are highly efficient, yielding 97% of the original energy potential. Calmodulin, an intracellular calcium-binding protein, is one of the subunits of phosphorylase b kinase; hence, calcium ions are required for the formation of phosphorylase b.

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Another mechanism that can promote glycogenolysis is an increase in phosphorylase b activity, without its conversion to the a form. This process, which occurs independently of adrenergic stimulation, is set in motion by the formation of adenosine monophosphate and inorganic phosphate from the breakdown of ATP (54). Hence, in ischemia, glycogenolysis is enhanced by both the cyclic AMP-dependent formation of phosphorylase a and the enhanced activity of phosphorylase b during ATP breakdown and AMP and Pi accumulation.

C. Function of Cardiac Glycogen Cardiac glycogen is a potential source of glucose for glycolysis and therefore a potential source of anaerobic ATP. However, myocardial energy demands are such that the glycogen content would have to be extremely high and glycogenolysis extremely rapid for glycogen to function as an important glycolytic fuel source. Nevertheless, very rapid glycogen breakdown may protect the heart from an acute lack of external fuel when myocardial energy demands increase suddenly (55). Glycogen may also act as a storage ‘‘buffer’’ for carbon from glucose taken up from the circulation. In the fasted state in humans, the major portion of glucose taken up is not oxidized immediately, but is probably converted to glycogen with oxidation delayed (56). When glycogen is mobilized, its glucosyl moeities are oxidized preferentially rather than converted to lactate (57). Thus, with acute increases in cardiac work, glycogen-derived pyruvate is selectively routed into glucose oxidation, with lactate efflux regulating the cellular redox state (58).

IV. LACTATE AND PYRUVATE Lactate is a significant source of oxidative fuel for the heart under resting conditions in vivo. Furthermore, as blood lactate rises with vigorous exercise, it may actually exceed the contribution of glycolysis as a source of pyruvate (59). Carbon-labeled tracer studies in humans reveal avid uptake and flux of lactate through the TCA cycle. At least two isoforms of a transporter protein mediating the uptake of lactate by the heart are known to exist (60). These carriers are designed to transport short chain monocarboxylates (C2-C5 chain length), including lactate, pyruvate, and the simple ketoacids acetoacetate and -hydroxybutyrate. A 45-kDa lactate transporter, called the monocarboxylate transporter-1 (MCT-1), and a closely homologous MCT-2 have been

cloned and characterized (61, 62). MCT-1 cotransports protons across the sarcolemma along with lactate and appears to regulate both the influx and the efflux of lactate. Once taken up, intracellular lactate is converted by lactate dehydrogenase to pyruvate, thereby joining the pyruvate derived from glycolysis that is destined for oxidative decarboxylation.

A. Lactate Dehydrogenase (LDH) LDH is a tetrameric unit composed of four subunits of the H or M type, where H is the form predominant in the heart and M is the form predominant in skeletal muscle. The distribution of these subunits conveys specific electrophoretic mobility to this 34-kDa protein. Lactate dehydrogenase catalyzes the reversible reaction: lactate ⫹ NAD⫹ 씮 pyruvate ⫹ NADH⫹ ⫹ H⫹

(3)

Under conditions of adequate oxygenation and a high rate of lactate uptake, the equation proceeds toward pyruvate. During oxygen debt, when NAD and H⫹ accumulate and pyruvate cannot be metabolized further, the reaction proceeds toward lactate. The myocardial activity of lactate dehydrogenase is high enough to make it unlikely that it could be a rate-limiting enzyme. It is important to note that the flux of substrate in fact bidirectional from lactate to pyruvate and vice versa during normal physiologic function. This is confirmed by the observation that a substantial proportion of exogenous glucose extracted actually contributes to immediate lactate release, despite the concurrent uptake of some 50% of the blood lactate presented to the heart and its conversion to pyruvate (63). The circulating concentration of pyruvate is usually very low so that it only accounts for a small part of the myocardial oxygen uptake of the normal heart. The major pathways of pyruvate derived from either glucose uptake or glycogen breakdown are either oxidative decarboxylation via pyruvate dehydrogenase and Kreb’s cycle or conversion to lactate. The latter is traditionally thought of as a process specific to anaerobic glycolysis, helping to convert back to NAD⫹, the NADH ⫹ H⫹ accumulated in earlier steps. However, convincing evidence shows that under physiologic conditions, as well as during hyperglycemia, that pyruvate from exogenous glucose may be routed to lactate rather than oxidized further (64). A third fate of pyruvate is that of anaplerotic (supplementary) diversion to ‘‘top up’’ specific TCA cycle intermediaries pyruvate can also be converted to alanine with the cotransamination of glutamate to form 움-ketoglutarate or oxaloacetate and then malate as part of the malic acid shuttle. However, the most important

32. Myocardial Energy Metabolism

fate of pyruvate is its entry into the mitochondrial matrix.

B. Pyruvate Dehydrogenase (PDH) The pyruvate dehydrogenase complex is a strategic regulatory enzyme complex situated on the inner mitochondrial membrane. This multimeric complex comprises three enzymes and five coenzymes clustered on the inner mitochondrial membrane, with a combined molecular mass of 6000 kDa. Mediating decarboxyl-

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ation, acetylation, and dehydrogenation of pyruvate in its passage into the mitochondrial matrix, it constitutes the rate-limiting step for oxidation of glucose by the TCA cycle, catalyzing the reaction: pyruvate ⫹ CoA ⫹ NAD⫹ 씮 acetyl-CoA ⫹ CO2 ⫹ NADH ⫹ H⫹

(4)

The regulation of PDH is illustrated in Fig. 4. PDH is inhibited competitively by its end products acetyl-CoA and NAD and is also subject to covalent activation and inactivation. Pyruvate dehydrogenase complex kinase

FIGURE 4 Pyruvate metabolism. Under aerobic conditions with a reduced fatty acid substrate, pyruvate is activated by the addition of coenzyme A, decarboxylated and dehydrogenated, to form acetyl-CoA by a complex of three closely linked enzymes known as the pyruvate dehydrogenase complex (PDH). Located in the mitochondrial membrane, they also translocate the intermediary into the matrix for entry into the TCA cycle. Because the PDH complex is inhibited powerfully by end product accumulation, most of the pyruvate produced is simply converted to lactate and removed during ischemia (excess unoxidized NADH) or aerobic metabolism with normal fatty acid levels (sufficient acetyl CoA levels). This regenerates NAD⫹, allowing glycolysis to continue. The complex is downregulated by kinase-mediated phosphorylation and inactivation and can be reactivated by phosphatase-mediated cleavage (sensitive to Ca2⫹) as illustrated. Thus the ratios of acetyl-CoA/CoASH, NADH/NAD, and ATP/ADP all inhibit the access of pyruvate (and therefore glucose) to final oxidation in the TCA cycle.

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(PDHK) is an ATP-dependent kinase that phophorylates (incorporating up to three phosphate residues) the subunit of the carboxylase component of PDH and thereby inactivates the enzyme complex. This kinase is activated by the reaction end products, acetyl-CoA and NADH, resulting in an inactivation of pyruvate dehydrogenase. Furthermore, the active form of PDH itself is subject to end product inhibition by acetyl-CoA and NADH. Thus when NADH rises, such as during ischemia, flux through pyruvate dehydrogenase is inhibited (65). Reactivation of PDH is achieved by the dephosphorylation of pyruvate dehydrogenase by a pyruvate dehydrogenase phosphatase (PDHP). PDHP is in turn activated by Mg2⫹ and Ca2⫹. In the fed state, only about 20% of PDH is in the active form, suggesting that the presence of alternative oxidation substrates to glucose may be important in regulating PDH activity. Randle’s group (66) and later Hansford et al. (67) showed elegantly that palmitoylcarnitine oxidation downregulates the activity of PDH in rat heart mitochondria by mechanisms involving the modulation of the acetyl-CoA/CoASH, ATP/ADP, and NADH/NAD⫹ ratios. With rapid increases in cardiac work, where glycolysis is stimulated, mechanisms involving altered mitochondrial redox potential, and possibly increased intramitochondrial calcium (65, 68), can, however increase the activity of PDH by 60–90% (69) [for reservations, see Kobayashi and Neely (70)]. While the provision of pyruvate from glycolysis increases PDH activity, fatty acids in the perfusate decrease glucose oxidation dramatically (71).

V. FATTY ACIDS The importance of fatty acids as a fuel source for the heart was initially reported by Cruikshank and Kosterlitz (72), who demonstrated their utilization by hearts perfused in the absence of glucose. Thereafter, component enzymes of the oxidation pathway were found to be present in the myocardium (73), confirming that fatty acid oxidation occurs in the heart. Bing et al. (74) then demonstrated that carbohydrate oxidation accounted for only a minor part of myocardial oxygen consumption, with the majority being consumed in fatty acid oxidation. Shipp et al. (75) also demonstrated that the heart perfused with both glucose and fatty acids preferentially oxidized fatty acids. The myocardial preference for fatty acids as an aerobic substrate has since been confirmed in the experimental setting, as well as in the human heart (76), accounting for the relatively low respiratory quotient of the myocardium. This is especially true in the fasted state, where up to 90% of oxidative energy is derived from fatty acids.

A. Fatty Acid Uptake Fatty acids are hydrophobic molecules, which must be metabolized in an aqueous environment. They are rendered soluble in transit from the gut or liver to the myocardium by being linked covalently to different carrier proteins. The steps in this process are illustrated in Fig. 5. In plasma, they are bound to albumin, transported as triglycerides bound to apolipoproteins in VLDL, or contained in chylomicrons. 1. Lipoprotein Lipase The hydrolysis of VLDL and chylomicrons by lipoprotein lipase releases fatty acids from the one and three positions of the triacylglycerol moieties, making them available along with glycerol for direct uptake. Alternatively, fatty acids may cross the endothelium bound to albumin by diffusion or by facilitated uptake. Lipoprotein lipase is secreted from the capillary endothelium, and possibly from myocytes. Its activity requires the presence of apolipoprotein CII and is reviewed in detail elsewhere (77). The heart extracts fatty acids efficiently, either from triacylglycerols or from albumin, but the precise mechanism of this process is poorly understood. It appears to have two separate components: (1) a diffusion process that is a linear function of the free fatty acid concentration (the molar ratio of fatty acid to albumin) and (2) a saturable component that shows features of a proteinfacilitated process (78). Transendocytosis of fatty acid– albumin complexes through the capillary endothelium, lateral diffusion through the abluminal interstitial space, and binding by a sarcolemmal fatty acid binding protein have all been proposed (79) (Fig. 5). Although considerable interest has focused on the molecular characterization of a number of sarcolemmal fatty acid transport proteins (80), the role of these proteins in regulating the rate of myocardial fatty acid uptake has yet to be established. 2. Fatty Acid-Binding Proteins (FABPs) Once in the cytoplasm, fatty acids are attached to one of several low molecular weight (14–15 kDa) transport ligands known collectively as fatty acid-binding proteins (81, 82). At least five of these proteins exist and were originally named for the tissue in which they were first characterized. A strong correlation between the FABP content and the fatty acid oxidative capacity of any given tissue has been demonstrated (82). In the heart, an additional 10-kDa aqueous cytoplasmic-binding protein is present, which binds specifically to CoA-activated acyl groups. This acyl-CoA-binding protein (ACBP) is

32. Myocardial Energy Metabolism

553

FIGURE 5 Fatty acid uptake. Fatty acids are presented to the myocyte for uptake and are bound to albumin as monomers or as triacyl units bound to glycerol (TG) in lipoproteins and chylomicrons (circles). Fatty acyl chains are released from triglycerides by lipoprotein lipase, which is secreted from the endothelial surface. They then move through the interstitial space by diffusion. In the case of albumin, a facilitated uptake process mediated by an endothelial transporter protein (T) is postulated. At the sarcolemma, fatty acids are bound by one of several fatty acid-binding proteins, and they are then moved into the cytosol where they undergo activation (addition of CoA) as the primary step in all further metabolism. Activation requires two high-energy phosphates from ATP and is catalyzed at the mitochondrial outer membrane by acyl-CoA synthetase.

believed to facilitate the intracellular shuttling of acyl moieties once uptake has occurred (83). The control of fatty acid uptake is therefore dependent on several factors: (1) the presence of an adequate supply of triacylglycerol or of albumin-bound fatty acids, (2) the presence of lipoprotein lipase on the endothelial surface, and (3) the presence of fatty acid transport-binding proteins near the sarcolemma. Fatty acid chain length and molecular structure also influence the uptake process (82), probably because the affinity of each species for the albumin carrier varies and because of differences in the intracellular disposition for long, medium, and short chain acyl moieties.

B. Fatty Acid Metabolism

a. Activation Fatty acid activation is a prerequisite step for all subsequent metabolic processes involving fatty acids. It involves the conversion of the carboxylic acid terminus to a CoA thioester, enhancing the reactivity of the molecule. The reaction consumes ATP and is catalyzed by one of a family of acyl-CoA synthetases, which differ in their chain length specificity and in their subcellular location (84). Long-chain acyl-CoA synthetase has been cloned and sequenced (85) and is of principal importance in the myocardium. It is situated at the cytoplasmic side of the mitochondrial membrane and functions in the activation of palmitate prior to entry into the mitochondria. Activation is a nonreversible step and commits the fatty acid to either triglyceride resynthesis or oxidation.

1. Cytoplasmic Pathways and Regulation

b. Triacylglycerol Turnover

The reaction pathways of intracytoplasmic fatty acids are illustrated in Fig. 6.

It is known that triacylglycerol can be stored as cytoplasmic droplets or as membrane-associated lipid parti-

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FIGURE 6 Mechanism of mitochondrial fatty acid uptake. Fatty acids are activated by acyl-CoA synthetase to produce fatty acyl-CoA. Carnitine palmitoyl transferase I (CPT I) links carnitine to the fatty acyl chain, releasing CoASH and forming acyl-carnitine in the intermembrane space. A carnitine-acyl translocase on the inner mitochondrial membrane then translocates acyl-carnitine into the mitochondrial matrix, where it is reconstituted to acyl-carnitine by carnitine palmitoyl transferase II (CPT II) with the release of carnitine back into the intermembrane space. Fatty acylCoA then enters the 웁-oxidation sequence of reactions.

cles (86, 87). This is thought to constitute a store of fatty acids, which is in constant turnover (88). Akin to the relationship of glycogen and glucose, this pool provides a source of fatty acids for oxidation when the exogenous supply is low. Thus the endogenous triglyceride pool can account for up to 50% of the myocardial energy requirement in the absence of exogenous fatty acids (88). Enzymes required for triglyceride synthesis are clearly present in the heart, but only a minority of the fatty acid taken up is cycled through the triacylglycerol pool prior to being utilized in oxidation. This futile cycle is energetically wasteful, as 3 mol of ATP would be consumed (at the step of fatty acid activation) for each mole of triglyceride synthesized. Under ischemic

conditions, the H⫹ ion load generated by the fatty acid activation step would be detrimental. The cycle of fatty acids to triglycerides and subsequent lipolysis to yield FFA again is a topic dealt with in more depth in a separate chapter. 2. Mitochondrial Uptake Following activation, the acyl-CoA residue is converted to acyl carnitine by carnitine palmitoyl transferase (CPT I) translocated across the inner mitochondrial membrane by carnitine acyl translocase and is then converted back to acyl-CoA by carnitine palmitoyl transferase II (CPT II), now within the mitochondrial

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32. Myocardial Energy Metabolism

matrix. L-Carnitine is an essential cofactor to this process: as observed prophetically by Fritz (89) in the 1950s, the ‘‘presence of carnitine in muscle and other tissues may facilitate the transfer of long-chain fatty acids to the enzymatically active intramitochondrial sites for fatty acid oxidation.’’ L-Carnitine is a simple compound of widespread distribution, with some properties resembling those of a vitamin. The structure is (CH3)3N⫹ CH2 CH(OH) CH2 COO⫺ The current conceptual framework of how the CPT translocation system works has been the subject of much debate: the widely accepted model is illustrated in Fig. 6. The reader is referred to the comprehensive review of McGarry and Brown (90) of the evolving understanding and current knowledge of this important system. CPT I and CPT II are distinct proteins that interact closely in vivo. CPT I exists in at least two isoforms (91), dominant in either liver [L-CPT I, 88 kDa, and inhibited by malonyl-CoA (92)] or muscle [M-CPT I, 82 kDa, and 100 times more sensitive to malonyl-CoA inhibition, with a much lower affinity for carnitine (93)]. Both species contain an inhibitor binding and a catalytic domain within the same peptide chain. Both are detergent labile, as they are integral proteins within the outer mitochondrial membrane. CPT I functions to catalyze the reaction of acyl-CoA and carnitine at the outer mitochondrial membrane. The resulting products are acyl-carnitine and free CoA. The reaction is written as CPT I

Carnitine ⫹ acyl-CoA ——씮 acyl-carnitine ⫹ CoA-SH

(5)

The acyl-carnitine product is able to move to the mitochondrial matrix, possibly by means of a translocase, where it is acted on by CPT II. CPT II is a 71-kDa protein that is loosely associated with the inside of the inner mitochondrial membrane. It is insensitive both to detergent solubilization and to inhibition by malonylCoA and exists in a single isoform. It catalyzes the reaction reconstituting acyl-CoA within the mitochondrial matrix: CPT II

acyl-carnitine ⫹ CoA-SH ——씮 acyl-CoA ⫹ carnitine

ner mitochondrial membrane. It is a 32-kDa protein that transports carnitine and acyl-carnitine across the membrane in a complementary fashion, maintaining the balance of acyl-CoA substrate for both CPT II and CPT I (94). It was Fritz and Yue (95) who originally proposed that acyl-CoA existed in two pools, one within the mitochondria and one in the cytoplasm. The relatively impermeable inner mitochondrial membrane separates these pools, and they proposed that acyl-carnitine, but not acyl-CoA, could cross that barrier. Acetyl-CoA can also combine with carnitine in a reaction catalyzed by intramitochondrial CAT to form acetyl-carnitine. Transfer of acetyl-carnitine out of the mitochondria helps couple the rates of cytosolic fatty acid activation to mitochondrial oxidation rates (96). Thus the end result of provision of excess fatty acids is that the intramitochondrial acetyl-CoA/CoASH ratio rises, and acetyl-carnitine is formed by the action of CAT on mitochondrial acetyl-CoA and free carnitine. Acetyl-carnitine is then translocated out of the mitochondria, resulting in a rise in cytosolic acetyl-carnitine levels. The acetyl groups on acetyl-carnitine are then transferred by a cytosolic CAT onto CoA to form acetylCoA, reducing the cytosolic-free CoA levels. Hence there is less CoA available for fatty acid activation. This sequence is illustrated in Fig. 7. The activity of CPT I is the most tightly regulated step in the metabolic pathway of fatty acids in the heart. It controls the access of long chain fatty acids to the mitochondria and therefore comprises the ratelimiting step in fatty acid oxidation. CPT I is a tightly regulated enzyme, with the most important regulatory mechanism being the powerful inhibitory effect of malonyl-CoA. 3. Malonyl-CoA and the Regulation of Fatty Acid Oxidation Malonyl-CoA is a three-carbon activated compound, formed by the action of acetyl-CoA carboxylase (ACC) on acetyl-CoA: ACC

(6)

The intramitochondrial acyl-CoA then enters the fatty acid oxidation spiral, and the carnitine formed intramitochondrially is transported outward by a carnitine– acyl-carnitine exchange mechanism known as carnitine acyl-translocase (CAT). CAT is a less well-characterized, but equally important component of this system. CAT is a specific carnitine/acyl-carnitine cotransporter located in the in-

Acetyl-CoA ⫹ HCO3⫺ ⫹ ATP ——씮 Malonyl-CoA ⫹ H2O ⫹ ADP

(7)

Although first recognized in lipogenic tissues such as liver, it is now known to play an important role in the regulation of fatty acid catabolism. It functions as a powerful inhibitor of CPT I and reduces the influx of long chain acyl-CoA into the mitochondria, reducing fatty acid oxidation. The regulation of malonyl-CoA in the myocardium is illustrated in Fig. 7.

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FIGURE 7 Regulation of fatty acid oxidation by malonyl CoA. When the mitochondrial oxidation of glucose or fatty acids is prolific, an increase in the acetyl-CoA/CoASH ratio occurs, which is reflected in the cytosol, as carnitine-acetyl translocase (CAT) allows acetyl-carnitine to exit the mitochondria. In the cytoplasm, acetylCoA is reconstituted and becomes a substrate for acetyl-CoA carboxylase(ACC). ACC synthesizes malonylCoA from cytosolic acetyl-CoA, and malonyl-CoA inhibits carnitine palmitoyl transferase 1 (CPT I), preventing the further mitochondrial uptake of palmitoyl-CoA. ACC is under phosphorylation control by 5⬘-AMP-activated protein kinase (AMPK). AMPK activity is increased by AMPK kinase, free AMP, and insulin. As a result, ACC is phosphorylated and inactivated, resulting in a decrease in malonyl-CoA levels and disinhibition of CPT I. Fatty acid uptake and oxidation will increase. Malonyl-CoA levels are reduced by the action of malonylCoA decarboxylase (MCD). Citrate and protein kinase A are important activators of ACC.

In liver, the IC50 of CPT 1 for malonyl-CoA can increase dramatically in various pathologies (97). During these situations, high concentrations of malonylCoA are often required to inhibit fatty acid oxidation. As a result, malonyl-CoA may be less important as a regulator of hepatic fatty acid oxidation. In contrast to liver, however, the sensitivity of heart CPT 1 to malonylCoA inhibition does not change under conditions in which fatty acid oxidation increases (98). Instead, myocardial levels of malonyl-CoA drop (99, 100), resulting in an increase in CPT I activity. Therefore, actual changes in malonyl-CoA levels as opposed to changes in sensitivity of CPT I appear to be the key factor regulating changes in fatty acid oxidation in the heart. The

mechanism of malonyl-CoA production, and malonylCoA degradation, is therefore important. a. Acetyl-Coenzyme A Carboxylase and Malonyl-CoA Production Two isozymic forms of ACC exist. In the heart, a 280-kDa isoform (ACC280) dominates over the hepatic 265-kDa isoform (ACC265) (101). The primary role of this isoform of ACC is to regulate fatty acid oxidation by increasing the production of malonyl-CoA (102). Earlier studies demonstrated that ACC has an important role in ensuring an adequate supply of acetyl-CoA for the TCA cycle and, not surprisingly, is regulated by the acetyl-CoA supply (103). ACC activity is also

32. Myocardial Energy Metabolism

regulated by phosphorylation (104), and modification of ACC by phosphorylation may be a key response to pathological conditions, requiring a change in oxidative energy production. Phosphorylation of ACC leads to a rapid inhibition of enzyme activity. The important regulatory sites on ACC265 have been described, and the kinases responsible for phosphorylation at these sites have been identified (104–106). Although a number of different kinases will phosphorylate ACC265 in vitro, much less is known about the in vivo phosphorylation control of ACC280. However, both protein kinase A and 5⬘-AMP activated protein kinase (AMPK) appear to be important in directly phosphorylating and inactivating cardiac ACC280 and ACC265 (107). b. Role of AMP-Activated Protein Kinase in Phosphorylating ACC AMPK is a key molecular regulator of energy metabolism (108). In general terms, it functions in liver as an ‘‘energy conservation agent’’ by inhibiting anabolic processes when cellular ATP levels are depleted (108, 109). In the heart and in skeletal muscle, however, AMPK acts by upregulating fatty acid oxidation during times of energy demand. Hardie and Carling (109) have therefore referred to AMPK as a ‘‘low fuel warning system’’ that is activated by low ATP levels and subsequent increases in AMP levels. AMPK has been characterized as a heterotrimeric complex with distinct subunits (108, 110). The catalytic subunit of AMPK that predominates in heart (now termed the 2 catalytic subunit) is a 63-kDa protein. An 1 subunit of AMPK has also been characterized, but tissue distribution studies have shown that it is the 2 isoform of AMPK that is expressed predominantly in heart. The two regulatory subunits are responsible for modulating the activity of AMPK in vivo (110). Levels of mRNA for AMPK, as well as AMPK protein expression and activity, are prominent in the myocardium (111, 112). AMPK is itself under phosphorylation control by an AMPK kinase (AMPKK) that phosphorylates and activates AMPK (106, 113). Specific regulators of AMPKK have not yet been characterized, although the cytosolic AMP/ATP ratio appears to be of critical importance to AMPKK activity. Thus, AMP facilitates AMPKK phosphorylation of AMPK and inhibits AMPK dephosphorylation (98). Although AMPKK appears to be part of a kinase cascade regulating ACC activity, no information is presently available as to the characteristics of AMPKK in the heart or whether AMPKK activity is altered following ischemia.

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c. Fate of Malonyl-CoA in the Heart Although the importance of malonyl-CoA in regulating myocardial fatty acid oxidation has been firmly established, little is known of the pathway of malonylCoA degradation in the heart. In liver, malonyl-CoA is a substrate for fatty acid synthase and fatty acid elongation. A similar fate for malonyl-CoA in the heart is unlikely, as fatty acid synthase activity has not been demonstrated and because it is improbable that the energy-requiring process of fatty acid elongation would simultaneously result in the increase of fatty acid oxidation by decreasing malonyl-CoA levels. Another possibility is that malonyl-CoA is decarboxylated by a specific putative enzyme, malonyl-CoA decarboxylase (MCD). Originally demonstrated in the uropygial gland of the goose (114), MCD has been identified and characterized in the myocardium, with levels of malonyl-CoA increasing as MCD activity declines (115). The molecular characterization of mammalian cardiac MCD is at an early stage, but the previously cloned avian enzyme was shown to have two isoforms (114). One of these is expressed in the mitochondria, whereas the other is located in the cytoplasm. Whether a cytoplasmic form of cardiac MCD regulates malonyl-CoA levels and fatty acid oxidation rates in the myocardium remains unresolved. 4. Mitochondrial ␤ Oxidation 웁 oxidation converts acyl-CoA to acetyl CoA by passing the long chain acyl intermediary through the fatty acid oxidation spiral, which removes two-carbon fragments as acetyl-CoA from the carboxyl (-COOH) end of the chain. The enzymes of 웁 oxidation are loosely organized into a multienzyme complex in which the substrate never leaves the complex, except for entering and departing, moving on to the next enzyme in the spiral (116). The basic reactions sequence is illustrated in Fig. 8. Four enzymatic steps are involved in this process: 1. Acyl CoA dehydrogenase removes 2H⫹ atoms, producing an unsaturated acyl-CoA (2-3-trans-enoyl CoA). This step occurs in the presence of flavoprotein (FAD), and the reducing equivalents are transferred directly to the respiratory chain. 2. Enoyl-CoA hydratase reinstates an OH group from water, producing hydroxyacyl CoA (L-3-hydroxyacyl-CoA). 3. L-3-Hydroxyacyl CoA dehydrogenase reduces the OH group to a ketone group, producing 3-ketoacyl CoA and generating NADH from NAD⫹ (required as a coenzyme).

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FIGURE 8 The 웁 oxidation of fatty acids. The sequence commences with the removal of two

hydrogens from acyl-CoA, converting FAD⫹ to FADH2 and forming an 움,웁-unsaturated acyl-CoA. A hydroxyl group is then regained from water to yield 웁-hydroxyacyl-CoA. A dehydrogenase reaction follows, converting NAD⫹ to NADH and the hydroxyl group to a ketone group. Finally, the 웁ketoacyl compound reacts with CoA to cleave two carbons off as acetyl-CoA. Acetyl-CoA will enter the TCA cycle, and the shortened acyl-CoA chain reenters step 1 for a further turn of the spiral.

4. 3-Oxoacyl CoA thiolase then splits the ketoacylCoA at the 2,3 position removing an acetyl-CoA and incorporating another CoA at the COOH⫺ end of the (now shorter) acyl residue. This enters the start of the spiral again at step 1 and the cycle repeats. Two additional auxiliary enzymes are required to achieve the degradation of saturated fatty acids: 3-cis- 2trans enoyl-CoA isomerase and 4-enoyl-CoA reductase. For an in-depth review of the oxidation reactions and enzymatic steps, see Schultz (116). If the supply of fatty acids and oxygen is not limited, the rate of oxidation turnover is primarily dependent on myocardial energy demands. NADH/NAD⫹ and acetylCoA/CoA ratios both decrease in response to an increased energy demand when the cardiac workload increases (117, 118). In the case of acetyl-CoA, the decrease in the mitochondrial acetyl-CoA/CoA ratio releases the suppression of L-3-oxoacyl CoA thiolase activity and also activates hydroxyacyl CoA dehydrogenase, accelerating the pathway. The reverse effect of a reduction in flux through 3-ketoacyl CoA when acetyl CoA levels increase has also been suggested (119), but this is disputed.

Conversely, during deprivation of oxygen, intramitochondrial NADH rises (68), probably because of decreased electron transport. This effect has been suggested to regulate the rate of 웁 oxidation at the hydroxyacyl CoA dehydrogenase step, reducing 웁 oxidation in the myocardial oxygen-deficient heart (120).

C. Critical Features of Fatty Acid Metabolism 1. Fatty acids are the major myocardial fuel of the normal heart, especially in the fasted state, and are able to inhibit the metabolism of glucose. 2. The nonoxidative steps of fatty acid metabolism are ATP consuming, with the CoA activation of acyl residues being a prerequisite to further metabolism. This contrasts with the nonoxidative metabolism of glucose, which generates ATP. 3. Fatty acids that are taken up but not oxidized can form triacylglycerol and myocardial structural lipids, the latter by changes in the degree of saturation and chain length. Generally the heart does not synthesize lipid from glucose or from other nonlipid sources.

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4. CPT I is the key enzyme controlling the flux of long chain fatty acids into the mitochondria. 5. CPT I activity is highly sensitive to inhibition by malonyl-CoA, a three-carbon intermediary formed by the carboxylation of acetyl-CoA. Malonyl-CoA synthesis by acetyl-CoA carboxylase is linked to the mitochondrial acetyl-CoA/CoA ratio. 6. Malonyl-CoA production and disposition are determined by the activity of acetyl-CoA carboxylase and malonyl-CoA decarboxylase, respectively, with the activity of acetyl-CoA carboxylase being linked to the cellular levels of 5⬘-AMP-activated protein kinase.

VI. MITOCHONDRIAL ENERGY METABOLISM A. Citric Acid Cycle Regardless of the form of carbon substrate taken up from the coronary circulation, the final common pathway is the formation of acetyl-CoA and entry into the citric acid cycle (TCA) cycle. In summary, their purpose is the oxidation of acetylCoA, which can be written as CH3UCO CoA ⫹ 3H2O 씮 2CO2 ⫹ 4[2H] ⫹ CoASH (8) Carbon dioxide is a by-product of the reaction, and so the reaction products are the four reducing equivalent pairs produced from hydrolysis of the acetyl intermediary. These reduce the electron-carrying molecules NAD⫹ and FADH, as described later. Apart from this major function, the TCA cycle also acts in the disposition of the products of carbohydrate, fatty acid, and amino acid metabolism. Its intermediaries and their enzymes also provide the means of metabolic regulation (via citrate, and acetyl-CoA), substrate interconversion (via oxaloacetate and 움-ketoglutarate), and the transport of reducing equivalents across the mitochondrial membrane (malate and oxaloacetate). Flux of the TCA cycle is tightly coupled to the capacity for ATP generation, which in turn depends on the availability of oxygen, the cytosolic [ATP]/[ADP][Pi] ratio, and the intramitochondrial [NAD⫹]/[NADH] ratio. A low myocardial demand for ATP (elevated ATP/ ADP ratio) or the nonavailability of oxidized cofactors (NAD⫹ or FAD) is a simple and powerful inhibitor of TCA cycle flux (121). Other steps of special significance apart from oxidation–reduction-coupled reactions are the generation of citrate and the competitive relationship between malate dehydrogenase and citrate synthase for their common substrate, oxaloacetate. Both have similar equilibrium constants Keq for oxaloacetate directed in ‘‘opposite’’ directions. In addition to being

a key intermediary of the TCA cycle, citrate is also a regulator of PFK, and oxaloacetate is a key link to reducing equivalent transport and anaplerosis of TCA intermediaries.

B. Metabolism of NAD and FADH In the process of simplifying fatty acids, glucose, or lactate to reach acetyl-CoA, several dehydrogenation reactions must occur. These remove two hydrogen atoms from the substrate, which are transferred to the carrier molecule nicotinamide adenine dinucleotide (NAD). NAD⫹ is reduced to NADH ⫹ H⫹ (alternatively written as NADH2). The further metabolism of acetylCoA, regardless of its source, takes place in the citrate cycle within the mitochondrial matrix. Further dehydrogenations occur in the citrate cycle producing more NADH. The reduction of flavin adenine dinucleotide (FAD) to FADH2 also occurs, but is of much less quantitative importance. These electron-carrying species, or reducing equivalents, are shuttled through a sequence of transformations called the respiratory chain, or electron transport chain (ETC). The components of the chain are a series of mitochondrial inner membrane proteins and cofactors arranged in a specific sequence. The sequence comprises six proteins: complex I, complex II, coenzyme Q complex III, cytochrome c, and complex IV. The cytochrome complexes are electron-transferring proteins containing iron porphyrin (heme) groups. The iron atoms undergo reversible changes in valency from the ferrous to the ferric form and vice versa. Each of the reactions at complex I, III, and cytochrome oxidase (complex IV) are associated with the ejection of protons from the mitochondrial matrix, with the final transfer of electrons to oxygen, to form H2O. The proton from each of these sites moves back into the mitochondrion via a coupled reaction, with each yielding one ATP.

C. Proton Pumping Mitchell (122) originally proposed the chemiosmotic mechanism for mitochondrial energy conservation based on the concept of a H⫹ electrochemical gradient that might facilitate ATP synthesis. The actual mechanism for ATP manufacture is closely linked with the fate of protons rather than that of electrons. In the respiratory chain, hydrogen atoms are dissociated to H⫹ ⫹ e⫺. With the transfer of electrons in the ETC, protons are pumped across the mitochondrial membrane to yield a transmembrane H⫹ gradient. This H⫹ gradient is the driving force for phosphorylation of ADP. Protons reenter the mitochondrial matrix through a complex of membrane proteins called ATP synthetase,

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or mitochondrial F1F0-ATPase, which is a protein ionophore. Proton movements caused by the transmembrane proton gradient then drive the phosphorylation of ADP. The stoichiometry of mitochondrial oxidative phosphorylation is, however, subject to the phenomenon of ‘‘proton leak,’’ wherein the transmembrane conductance declines, yielding less than the optimal calculated ATP quotient (123). Thus, the mitochondrial oxidation of a single NADH molecule produced by the citrate cycle yields 2.5 molecules of ATP per atom of oxygen reduced (Table I). Expressing ATP production as a molar ratio of the oxygen consumed in its production gives rise to the phosphorylation/oxygen uptake ratio (P/O ratio). In the case of NADH, a ratio of 2.5 is calculated. Other reactions (e.g., pyruvate dehydrogenase) forming NADH2 will also have a P/O ratio of 2.5, but reactions feeding into the ETC at the level of coenzyme Q will have a P/O ratio of 1.5. The first step of the fatty acid oxidation spiral and the succinate dehydrogenase-mediated step in the Kreb’s cycle produce FADH2 , which reacts with coenzyme Q, and therefore has a P/O ratio of 1.5 and not 2.5. The topic is reviewed in more detail elsewhere (124).

D. Cytosolic-Derived NADH Cytosolic NADH is also produced, formed either during glycolysis by GAPDH or with lactate uptake, and its conversion to pyruvate by LDH. When NADH2 is produced in the cytosol, NAD⫹ is depleted and the progression of glycolysis is potentially threatened. Therefore the regeneration of NAD⫹ and the transit of

NADH2 in the mitochondria are vital to allow glycolysis to proceed. When fatty acid oxidation rates are high, relatively little glucose enters the citrate cycle, and the production of lactate allows for NAD⫹ regeneration. However, with a sudden increase in heart work, or during ischemia, glycolysis is accelerated and the flux of pyruvate to acetyl-CoA is increased. NAD⫹ must then be reclaimed. 1. The Malate–Aspartate Shuttle The chief mechanism for the transfer of NADH into the mitochondria is the malate–aspartate shuttle. Malate and oxaloacetate occur both in cytosol and in the mitochondrial space, along with the enzyme malate dehydrogenase, which interconverts these two compounds: malate dehydrogenase

oxaloacetate ⫹ NADH ⫹ H⫹ ⫹ NADH ⫹ H⫹ 씮 malate ⫹ NAD⫹

(9)

During production of NADH2 by glycolysis, oxaloacetate in the cytosol is converted to malate with the reconstitution of NAD⫹ from NADH2 . This process allows glycolysis to proceed by providing NAD⫹ for GAPDH. Malate then passes into the mitochondrial space as part of a complex transport system that ‘‘exports’’ 움-ketoglutarate (125). Once within the mitochondrial space, malate will reform oxaloacetate to enter the TCA cycle. NADH2 also reforms and is accessible to the electron transport chain so that 2.5 ATP are formed for every NADH2 entering. 2. The Glycerophosphate Shuttle An alternate route of disposal of cytosolic NADH in many tissues is entry into mitochondria by way of the 움-glycerophosphate (움GP) shuttle:

TABLE I Comparative Energy Yields of Various Substrates Fully Oxidized, Reflecting Old Convention and Newly Proposed Yields a,b

ATP yield per molecule

Molecule Glucose Lactate Pyruvate Palmitate a

ATP yield per carbon atom

ATP yield per oxygen atom taken up (P/O) ratio

Old

New

Old

New

Old

New

38 18 15 130

32 14.75 12.25 105

6.3 6.0 5.0 8.1

5.2 4.9 4.1 6.7

3.17 3.0 3.00 2.83

2.58 2.46 2.50 2.33

From Verhoeven et al. (112) and Hawley et al. (113). P/O, phosphorylation/oxidation. ATP yield from glucose includes two from glycolysis (old and new). b

움GP dehydrogenase

NADH ⫹ H⫹ ⫹ DHAP ——————씮 NAD⫹ ⫹ 움GP (10) where DHAP is dihydroxyacetone phosphate. The 움GP enters the mitochondria to be oxidized by 움GP oxidase. DHAP then reforms inside the mitochondria, along with FADH2 . The former is transported outward and the latter enters the respiratory chain. It has been difficult to assess accurately the capacity of these systems for the transport of NADH2 and FADH2 in the heart, and the malate–aspartate system may be more important. The chief reason for this conclusion is the relatively low rate of activity of the enzyme 움GP dehydrogenase in heart (126).

32. Myocardial Energy Metabolism

E. Energy Production from Various Substrates When glucose is the source of glycolytic substrate, the glycolytic sequence consumes two ATP and produces four ATP, with a net production of two ATP. When glycogen is the source, an extra ATP is produced. As a result there is an automatic net yield of ATP whenever hexose-phosphates are converted to pyruvate. The major source of energy from either glucose or glycogen, however, lies in the citrate cycle and the conversion of pyruvate to CO2 with the formation of H2O in the ETC. As summarized in Table I, pyruvate dehydrogenation produces 1 molecule of NADH, which will give rise to 2.5 ATP molecules, whereas 9.75 molecules are produced by one turn of acetyl-CoA through the TCA cycle, giving a total of 12.25 ATP for each pyruvate produced and a net yield of 32 ATP from each glucose. In the case of lactate, the dehydrogenation reaction of LDH to form pyruvate produces 1 NADH and eventually 2.5 additional ATP. By comparison, fatty acid activation consumes 2 highenergy phosphates (from 1 ATP) per molecule prior to any energy yield. In the case of palmitate, a 16 carbon chain fatty acid, seven turns of the fatty acid oxidation spiral within the mitochondrion will produce 7 NADH2 (17.5 ATP) and 7 FADH (10.5 ATP). Finally, 8 acetylCoA will produce an additional 78 ATP (9.75 ATP per acetyl-CoA passing through the TCA cycle), with an overall energy yield of 105 ATP per palmitate molecule. Each of the myocardial fuels therefore has a different ATP yield, calculated by molar value, carbon content, or oxygen equivalents consumed per high energy phosphate produced. These relative values are seen in Table I. The highest yield of ATP is from fatty acids such as palmitate, which has the theoretical disadvantage of consuming more oxygen for each mole of ATP produced. Experimentally, a heart metabolizing fatty acids alone would require about 17% more oxygen to produce the same amount of ATP as a heart metabolizing only glucose. The molecular explanation for the relatively lower P/O ratio for fatty acids has already been described.

561

of view of normal energy requirements of the heart, and yet it has special significance for two reasons. First, there may be a subcellular functional ‘‘compartmentalization’’ of glycolytically derived ATP (127). During experimental mild or moderate ischemia, the sustenance of glycolysis in ischemia appears to have beneficial effects, perhaps attributable to the localization of pockets of glycolytic. ATP production, which sustain the activity of certain ATP-requiring enzymes. These are membrane-associated transporters, which are responsible for cellular ion homeostasis, particularly Na⫹K⫹-ATPase (128) and sarcoplasmic reticulum Ca2⫹ATPase (129). Experimental data suggest that sustained glycolysis may help maintain the action potential duration, prevent ischemic contracture, and preserve general membrane integrity (130). Second, and paradoxically, following periods of severe ischemia, increased glycolytic flux with the accumulation of H⫹ ions derived from the anaerobic hydrolysis of ATP can also have adverse consequences. Increased Na⫹ /H⫹ exchange during the reperfusion phase occurs, resulting in an increase in intracellular Na⫹ (131). As a result, the sarcolemmal Na⫹ /Ca2⫹ exchanger is activated, which exchanges intracellular Na⫹ for extracellular Ca2⫹. A high rate of Na⫹ /Ca2⫹ exchange is capable of causing cellular Ca2⫹ overload and cell death (132, 133). The continued production of H⫹ in early reperfusion has also been implicated in the pathophysiology of reperfusion injury (133, 134).

B. Metabolic Regulation under Normal Conditions The myocardium has been termed ‘‘an omnivore’’ because of its eclectic use of a range of energy substrates (121). This term accurately characterizes the ability of the heart to switch from one substrate to an alternate source as and when the need arises. There are nevertheless preferred fuel sources and mechanisms for regulating their relative utilization with the result that fatty acid and glucose metabolism in the heart are linked to one another. Several mechanisms influence the balance of substrate use by the myocardium. 1. Circulating Substrate Levels

VII. INTEGRATION OF MYOCARDIAL ENERGY METABOLISM A. Significance of Glycolytic ATP The amount of ATP generated by glycolysis in the normally oxygenated heart is negligible from the point

Studies in both animals (135) and human subjects (63, 76, 136) have demonstrated that fatty acids, glucose, and lactate are all extracted for oxidation. The use of a particular substrate by the heart is in part dependent on circulating blood levels: in the fasted state, 60–100% of the oxygen consumption is accounted for by fatty acids and 0–20% each by lactate and glucose. In the

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VI. Metabolism and Energetics

isolated working rat heart perfused with high levels of fatty acids, 90% of myocardial ATP produced under aerobic conditions arises from the mitochondrial oxidation of carbohydrates and fatty acids with fatty acids accounting for the majority and carbohydrates playing a secondary role (137, 138). High plasma fatty acid levels (⬎0.8 mM) inhibit both the uptake and the oxidation of glucose and lactate in human myocardium (139, 140), whereas pharmacologically lowering plasma fatty acid levels results in an increase in myocardial glucose and lactate uptake (141, 142). Lactate levels may increase precipitously with vigorous exercise, resulting in an increase in cardiac lactate extraction and oxidation (143). Conversely, the plasma glucose concentration does not determine the amount of glucose extracted or oxidized, if plasma fatty acid levels and insulin are fixed (139, 144). Ketone bodies and amino acids are also potential fuel sources with little role under normal aerobic conditions due to their typically very low circulating levels. 2. Hormonal Regulation Insulin, epinephrine, and norepinephrine are all important regulators of myocardial energy metabolism. Each of these acts through a membrane receptor-linked protein kinase mechanism permitting the intracellular modification of specific enzymes by phosphorylation/ dephosphorylation. They also act by altering substrate supply to the heart. Insulin increases carbohydrate metabolism by stimulating GLUT-4 expression and glucose extraction as described earlier. In the myocardium, insulin stimulates both hexokinase and glycogen synthetase activity and may also act directly on PFK-2 via the PI-3K pathway, increasing F2,6-BP levels and accelerating glycolysis (33). Insulin also inhibits lipolysis in adipocytes and therefore lowers plasma fatty acid levels. Data have demonstrated that insulin also suppresses the expression and activity of 5-AMPK (134), resulting in increased ACC activity, increased malonyl-CoA levels, and decreased fatty acid oxidation. By increasing myocardial contractility, epinephrine and norepinephrine invariably increase energy consumption by the heart. Epinephrine therefore also increases glucose uptake, glycogenolysis, glycolysis, and glucose oxidation (146). Epinephrine also acts systemically to increase circulating fatty acids and therefore fatty acid uptake and oxidation (147). The actions of epinephrine and norepinephrine are mediated through elevated levels of cAMP and protein kinase A, with activation of PFK-2 resulting in elevated levels of F2,6BP, which increase PFK-1 activity (148). Epinephrine is therefore able to reverse the suppression of glycolysis

normally seen in the presence of fatty acids, resulting in a significant increase in H⫹ production from glucose metabolism (149). Epinephrine also increases mitochondrial calcium uptake (150), which activates PDH phosphatase and thereby increases PDH activity, and increases glucose oxidation. Other TCA cycle enzymes (isocitrate dehydrogenase and ketoglutarate dehydrogenase) are also sensitive to intramitochondrial [Ca2⫹]. Long-term thyroid hormone administration is also known to stimulate glucose transport and glycolysis (151). Hypothyroidism results in a reduction in GLUT4 expression and glucose uptake, as well as reduced PFK-1 activity, possibly through a reduction in F2,6-BP levels (152, 153). In addition to these major endocrine hormone effects, glucagon and acetylcholine and several paracrine and cytokine factors known to act locally within the myocardial vascular bed have minor roles in myocardial metabolic regulation. Examples of these not discussed in this chapter include bradykinin, histamine, prostacyclin, tumor necrosis factor, and nitric oxide. 3. Interaction of Fatty Acid and Glucose Metabolism The myocardial requirement for ATP is linked to a complex regulatory mechanism, which balances fatty acid and glucose oxidation rates with these energy demands. This ensures that an adequate supply of intramitochondrial acetyl-CoA is available to support the TCA cycle, without an excess of flux and wasteful oxygen consumption. Classic studies by Randle’s group (154, 155) have described how an increase in fatty acid supply to the heart decreases both glucose oxidation and glycolysis. The mechanisms by which increased glucose oxidation can downregulate fatty acid metabolism have been elucidated more recently (71). Three key enzymes characterized in the heart are important in this process: acetyl-CoA carboxylase, 5⬘AMP-activated protein kinase, and malonyl-CoA decarboxylase. The molecular interactions of these enzymes have been described previously. We have proposed (71) that the supply of acetylCoA to ACC is an important mechanism by which malonyl-CoA synthesis can be increased or decreased to meet the metabolic demands of the heart. Increases in the intramitochondrial acetyl-CoA/CoA ratio due either to decreased metabolic demand (with decreased TCA cycle activity) or to an increase in acetyl-CoA originating from the pyruvate dehydrogenase complex (PDH) will increase the flux of acetyl groups from the mitochondria into the cytosol (Fig. 7). This will stimulate the ACC-mediated synthesis of malonyl-CoA, resulting in a decrease in fatty acid oxidation (157). Thus malonyl-

32. Myocardial Energy Metabolism

CoA links the changes in acetyl-CoA supply arising from lowered metabolic demand or from increasing the utilization of carbohydrate sources to the rate of flux of acetylated long chain fatty acids into the mitochondria. This mechanism explains how increases in glucose oxidation are able to downregulate myocardial fatty acid oxidation and how fatty acid oxidation rates can hence be increased or decreased depending on either carbohydrate availability or the energy demands of the heart. Furthermore, under conditions of decreased ATP production, a small increment in cytosolic AMP levels will result in AMPK activation, downregulation of ACC, and a drop in malonyl-CoA levels with increased fatty acid oxidation and ATP production. In relation to the cellular energy charge, it is clear that this mechanism operates well in advance of any measurable change in this ATP/AMP ratio; thus we must consider the possibility of a localized compartment of AMPK and AMP interaction in the region of the mitochondria.

C. Pathological Alterations in Myocardial Energy Metabolism 1. Diabetes Myocardial glucose transport, glycolysis, and glucose oxidation are all dependent directly or indirectly on insulin and so will all be reduced in the insulin-deficient diabetic state (158). In addition, high levels of circulating fatty acids in the diabetic state contribute to the overall low rates of glucose metabolism. Consequently, the heart becomes almost entirely dependent on fatty acid oxidation to meet its energy requirements, either from exogenous sources or from the turnover of stored triacylglycerol (159, 160). Myocardial AMPK activity in the diabetic heart is increased, ACC activity is significantly reduced, and malonyl CoA levels are low. These effects are reversed in the presence of insulin (161). The mechanical dysfunction of the diabetic heart and its increased sensitivity to ischemia are thought to be closely linked to these changes in energy metabolism (158). 2. Hypertrophy Myocardial hypertrophy constitutes an independent risk factor for decreased function and survival following an ischemic insult, in part due to hypertrophy-induced changes in myocardial metabolism (162, 163). Hypertrophied hearts display a reduction in fatty acid oxidation (164) and an increase in glycolysis (165). Recent work suggests that the mechanism for this shift from fatty acid to glucose-based fuels is a reduction in AMPK expression in the hypertrophied heart, leading to an

563

increase in ACC activity with elevated levels of malonylCoA (166). CPT-1 activity will then be inhibited, reducing fatty acid oxidation. 3. Ischemia Energy metabolism in the ischemic myocardium is principally dependent on the severity of the ischemic insult (167). With the complete interruption of blood flow, there is a rapid decline in high-energy phosphate levels and contractile function declines rapidly. Glycogen is metabolized and lactate accumulates progressively with intracellular acidosis from the anaerobic hydrolysis of ATP. Sarcolemmal integrity then begins to deteriorate and cytosolic calcium overload occurs, resulting in cell death and tissue necrosis. With a 30% reduction in blood flow, a similar rapid decline in contractile function and ATP/phosphocreatine levels occurs, with a brief and rapid increase in lactate output. There is then a slow decline in lactate output over the ensuing 30–90 min (168) and a paradoxical improvement in myocardial ATP levels (169). At this stage, restoration of coronary flow results in a complete recovery of contractile function, and the myocardium has been described as hibernating. ‘‘Hibernating myocardium’’ describes regions supplied by a narrowed coronary artery in which ischemic cells remain viable but contraction is chronically depressed (170). Here blood flow is not low enough to cause progression toward tissue necrosis, but it is low enough to decrease oxidative metabolism and myocardial contractility, persisting from days to months in duration (171). Subnormal perfusion, decreased fatty acid oxidation, and increased glycolysis characterize hibernating myocardium, leading to the term ‘‘metabolismflow mismatch’’ (172, 173). More recent data suggest that fatty acids may still provide the majority of the acetyl-CoA entering the TCA cycle in myocardial hibernation, possibly due to a loss of normal regulatory mechanisms (174). Regardless of the degree of ischemia, there is a reduction in flux through pyruvate, with a reduction in glucose oxidation and a net efflux of lactate. Glycolysis is downregulated at the level of GAPDH by the NAD⫹ /NADH ratio and at PFK by the accumulation of H⫹ ions (39). The decrease in carbohydrate oxidation in mild/moderate ischemia is thought to be due to increased phosphorylation and also end product inhibition of PDH (175, 176). 4. Reperfusion During reperfusion following ischemia, a rapid recovery of mitochondrial electron transport to oxygen re-

564

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plenishes the supply of ATP, allowing for contractile function to recommence. The TCA cycle and myocardial O2 consumption recover rapidly during the early reperfusion of moderate ischemia, but the ratio of cardiac work/MVO2 (cardiac efficiency) decreases. This is due to an alteration in substrate utilization, with high rates of fatty acid oxidation inhibiting glucose oxidation rates and contributing to contractile dysfunction during reperfusion (134). Elevated plasma concentrations of fatty acids probably contribute to these high rates of fatty acid oxidation. ACC activity decreases during reperfusion, with increased AMPK-mediated phosphorylation of ACC being the probable mechanism (100). As a result, levels of malonyl-CoA measured in these hearts are low, with increased fatty acid oxidation and oxygen consumption (103).

VIII. SUMMARY 1. High rates of myocardial energy production are required to maintain the constant demand of the working heart for ATP. 2. A constant supply of oxygen and at least one of the major exogenous substrates—fatty acids, lactate, and glucose—are required for normal metabolic function and for ATP production to continue. 3. Glucose has an inducible transporter, GLUT-4, which is regulated chiefly by insulin, but responds also to the level of myocardial oxygenation and the workload of the myocardium. 4. During hypoxia, tissue levels of ATP and phosphocreatine fall, whereas glucose uptake, glycogen breakdown, and anaerobic glycolysis are accelerated. 5. Glucose is activated to glucose 6-phosphate by hexokinase and can be diverted into either glycogen synthesis or glycolysis. 6. After glucose uptake, the glycolytic enzymes phosphofructokinase (and its allosteric effector, fructose 2,6bisphosphate) and glyceraldehyde-3-phosphate dehydrogenase are the rate-limiting steps of glycolysis. 7. Glycolysis transforms glucose into two three-carbon units (pyruvate), with the net production of ATP and the reducing equivalents (NADH2) without any oxygen requirement. 8. Pyruvate can be oxidized to lactate and released or activated to form acetyl-CoA, which then enters the citrate cycle. 9. Fatty acids provide the major component of myocardial oxidative energy under both conditions of normal oxygen supply and moderate impairment of oxygenation (ischemia).

10. Uptake and transport of fatty acids require fatty acid-binding proteins and intracellular fatty acid transport proteins. 11. Fatty acid activation to acyl-CoA occurs, followed by carnitine-linked translocation into the mitochondrial matrix where acyl-CoA is reformed. 12. Acyl-CoA enters the fatty acid oxidation spiral to form acetyl-CoA units while shedding hydrogen atoms to FAD and NADH, forming FADH and NADH2 . 13. Mitochondrial fatty acid uptake and oxidation are regulated primarily at the level of CPT I by the three-carbon molecule malonyl-CoA. 14. Acetyl-CoA residues are oxidized by the citrate cycle to yield more NADH2 and FADH. NADH2 and FADH are reoxidized by the electron transport chain to yield ATP, with oxygen being the final electron acceptor, forming H2O. 15. ATP provides the energy for contractile function, membrane homeostasis, and cellular functions. Interruption in the supply of substrate and/or oxygen results in significant changes in myocardial energy metabolism and contractile function.

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33 Metabolism and Energetics of Vascular Smooth Muscle CHRISTOPHER D. HARDIN and TARA J. ALLEN

RICHARD J. PAUL

Department of Physiology University of Missouri Columbia, Missouri 65212

Department of Molecular and Cellular Physiology University of Cincinnati College of Medicine Cincinnati, Ohio 45267

I. INTRODUCTION

II. MUSCLE ENERGETICS: SMOOTH MUSCLE VERSUS SKELETAL MUSCLE

The metabolism and energetics of vascular smooth muscle (VSM) contractility have been comprehensively reviewed; we have purposely kept some of the older references to reviews, as a considerable literature may not be found in current retrieval programs (1–13). We will therefore not attempt to be encyclopedic but rather focus on what is widely accepted regarding the energy metabolism of vascular smooth muscle and the demands imposed on that metabolism by contractile activity. Since the appearance of the first edition, there has been substantial growth in the experimental base on which our understanding of smooth muscle behavior is formulated. Much attention has been given to the role of myosin light chain phosphorylation, not only as a mediator of the activation of actin–myosin interaction by calcium, but also in terms of its potential role as a modulator of the mechanical, energetic, and kinetic properties of smooth muscle (12, 14, 15). The body of evidence indicating a functional compartmentation of energy metabolism in smooth muscle has grown considerably (10, 16–18) and has moved from describing the phenomenon to beginning to address the mechanistic basis for this organization of metabolism (19–21). Application of nuclear magnetic resonance (NMR) to smooth muscle studies has provided a means for studying metabolism directly in living preparations. These studies have added many new dimensions to our understanding of smooth muscle mechanochemistry. We have incorporated the results of these studies in this updated review and have focused on several issues that are currently unresolved and topics of some debate.

Heart Physiology and Pathophysiology, Fourth Edition

Amphibian skeletal muscles and vascular smooth muscles represent two extreme modes of how chemical energy stored in the form of the terminal phosphate bond of adenosine triphosphate (ATP) might be provided to supply the motive power needed to drive muscular contraction [see Kushmerick (22) for a schematic summary]. During contraction at 0 ⬚C, amphibian skeletal muscle utilizes ATP approximately 100 times faster than its aerobic metabolism can resynthesize ATP. During a brief isometric tetanus, therefore, the available preformed high-energy phosphate compounds decline rapidly, limiting the ability of the muscle to maintain the developed force. After some period of time, aerobic resynthesis of ATP is activated, and, on a time scale greatly longer than the sustained contractile period itself, the original phosphagen content is restored. To support the brief but intense period of ATP hydrolysis associated with contraction before the onset of aerobic recovery metabolism, skeletal muscles possess a large pool of preformed high-energy phosphate compounds, most notably phosphocreatine (PCr, 15–25 애mol/g fresh muscle). ATP, the substrate used directly by the contractile proteins, is substantially lower (3–5 애mol/g). ADP generated by actomyosin ATPase is immediately rephosphorylated to ATP via the creatine kinase reaction. Vascular smooth muscle operates on the opposite tack. The initiation of contractile activity is associated with virtually no measurable decline in the available ATP ⫹ PCr content (23, 24), as the rates of ATP utilization by the contractile apparatus can be matched by

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aerobic resynthesis. Only if aerobic and glycolytic metabolisms are blocked can a net decline in tissue content of ATP and PCr be noted (25). This then leads to two distinct strategies to study energetics of VSM contraction, both of which are represented in the literature. One method relies on measuring steady-state metabolic rates (i.e., the rate at which ATP is being resynthesized) to estimate the usage of ATP. This method depends on the assumption (verifiable, in most instances) that during the measurement period the intracellular ATP ⫹ PCr pool is constant. Thus ATP utilization and production rates are equal. This method is, of course, subject to the limitation that, on short time scales, such may not be the case. An alternative method is to block all ATP resynthesis (using combinations of oxygen- and substrate-free environments and/or various metabolic poisons) and measure the decline in intracellular ATP ⫹ PCr directly. This method is also subject to inherent limitations. The use of metabolic poisons, in general, raises the issue of how representative these measurements are of the normal tissue [see Daemers Lambert (26), for example]. The most common method for measuring the phosphagen content involves freezeclamping and analysis of tissue extracts. Because one is limited to one data point per tissue, this technique requires statistical comparisons among a large population of tissues. This limitation has been overcome through the use of nondestructive NMR techniques, as each tissue serves as its own control. In addition, 31P NMR provides a myriad of other information relevant to cell metabolism and energetics, such as cytosolic pH and free Mg2⫹. An advantage of this strategy is that it is not dependent on the steady-state assumption and can be applied, in principle, to arbitrarily short time periods. Limiting the time resolution of the method, however, is the extremely low ATPase rate manifest in vascular smooth muscle, even when taken in comparison to the small pool size of preformed high-energy phosphates. Both approaches have been used in the study of smooth muscle energetics, and, for the most part, resulting data have proven complementary so that methodological limitations alone do not seem to play an important role in our understanding of the results. These two energy-provision strategies appear to have evolved to meet the specific physiologic demands placed upon the various muscle types. The VSM role in situ is to maintain blood vessel tone over long periods of time and to adjust tone gradually in response to changing conditions of the cardiovascular system. To do so economically, smooth muscle myosin possesses an extraordinarily low inherent ATPase and yet is capable of developing and maintaining forces quantitatively comparable to or greater than that of skeletal muscles. It has been estimated that only 3 to 5% of the total human

basal metabolism is consumed by the vasculature and only about one-fifth of that is required to maintain circulatory regulation (4). While the total vascular mass is about 10 times greater than that of the heart itself, it consumes less than one-half as much energy in fulfilling its role in distribution of cardiac output. The strategies for energy provision in smooth muscle differ in many ways from those in striated muscle, as the rates of energy turnover in these two muscle types are so dramatically different.

III. CONTENT OF HIGH-ENERGY PHOSPHATES Rather than presenting an exhaustive compilation of data, we will instead focus on the most typical values of various parameters, with the observed ranges indicated when possible. We would like to add the caveat that differences among smooth muscle can be as great as between smooth and striated muscle. Thus, we will try to be cautious in our generalizations, while still attempting to elucidate underlying patterns. Table I summarizes the content of high-energy phosphates found in quiescent mammalian muscles. The most notable feature is simply that vascular smooth muscle has, in general, the lowest content of preformed high-energy stores; in some cases less than one-fiftieth of the preformed energy stores available to skeletal muscles. NMR measurements of the PCr/ATP ratio in smooth muscle generally confirm the measurements of the ratio determined chemically (27, 28). However, GTP and ATP cannot be resolved from each other by NMR, and as much as 12–15% of the total nucleoside triphosphate peak may be GTP, with the remainder being ATP based on measurements in uterus and bladder smooth muscle (27). Because the PCr/ATP ratio determined by NMR generally agrees with that determined by biochemical analysis, virtually all of the ATP appears to be unbound as in skeletal muscle. At room temperature, while generating maximum isometric tension, mammalian skeletal muscles would TABLE I High-Energy Phosphate Content of Resting Mammalian Muscles a Muscle type

ATP

PCr

Total 앑P

Vascular smooth muscle Other smooth muscle Skeletal muscle Cardiac muscle

0.3–1.0 1.0–2.0 3.0–6.0 6.0

0.3–1.0 1.0–3.0 15–30 10

0.6–2.0 2.0–5.0 15–30 16

a All values are 애mol/g wet tissue. These values were compiled from data in Refs. 1–13, 22, 68–71. ATP, adenosine triphosphate; PCr, phosphocreatine; 앑P, total high-energy phosphagen.

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consume their available preformed high-energy phosphates in about 2–3 sec. Smooth muscles, however, would not deplete their supplies substantially for 2–3 min. Using quick-frozen tissues and chemical analysis, no change was found in the ATP ⫹ PCr content of isolated oxygenated bovine carotid arteries after 30 min of maximal activation with potassium chloride, whereas similar experiments with metabolically inhibited iodoacetate-treated arteries led to a complete depletion of high-energy phosphates (29). On a much shorter time scale, Krisanda and Paul (23) did not detect any change in the ATP or PCr content of well-oxygenated hog carotid arteries in times as short as 30 sec following the activation of isometric tension development. NMR data (30, 31) in intact arteries further substantiate the ability of VSM to maintain phosphagen stores for prolonged periods (⬎12 hr) under depolarizing conditions when supplied with substrate. Clearly, in the case of VSM, the aerobic capacity to resynthesize ATP is sufficient to rapidly achieve a balance with the ATPases associated with activation, development, and maintenance of contractile activity.

IV. SUBSTRATE OXIDATION AND RESPIRATION As discussed earlier in vascular smooth muscle, the metabolic demand of the contractile machinery for ATP is matched by its capacity to resynthesize ATP. The biochemical pathways for ATP synthesis appear similar between mammalian skeletal muscle and VSM, as all the enzymes necessary for glycolysis, the citric acid cycle, and the respiratory transport chain are present (32– 34). While VSM has been shown to obtain 앑70% of its ATP production from oxidative metabolism (8), the selection of substrates oxidized by the citric acid cycle has remained controversial. VSM has been shown to oxidize a wide variety of exogenous and endogenous substrates, including glucose (35–38), glycogen (36, 39– 42), fatty acids (37, 39, 43–45), and branched chain amino acids (37, 39). A comprehensive picture of VSM oxidative substrate selection has not been established because many of these studies have examined the oxidation of a substrate by inhibiting the oxidation of alternative substrates. Therefore, while some of these studies have provided examples where a particular substrate can be primarily oxidized and have demonstrated the ability of VSM metabolism to accommodate for altered substrate conditions, they have not determined substrates preferentially oxidized by VSM in vivo. The importance of measuring the oxidation of a substrate in the presence of alternative substrates is illustrated in a study by Chace and Odessey (37). They showed that the oxidation of a given substrate is inhibited more

by a combination of substrates (glucose, palmitate, 웁hydroxybutyrate, and branched chain amino acids) than by any single substrate in that combination. When drawing conclusions regarding substrate metabolism, it is essential to consider the similarity of the experimental substrate conditions to the cocktail of substrates present in vivo. Although there are a variety of substrates present in vivo (including glucose, short and long chain fatty acids, glycogen, endogenous lipids, ketone bodies, and branched chain amino acids), most studies have concentrated on the oxidation of exogenous and endogenous carbohydrates and fatty acids (35–39, 42–46). Because the oxidation of ketone bodies and branched chain amino acids contribute minimally to oxygen consumption (each ⬍5% of total) (37), this section concentrates on the oxidation of exogenous and endogenous carbohydrates and fatty acids by VSM.

A. Carbohydrate Oxidation Early studies found carbohydrates to be the substrate, primarily oxidized by VSM, as indicated by a respiratory quotient of 앑1 (40, 41). This is likely an overestimate of carbohydrate oxidation, as in these studies, glucose was provided at supraphysiological concentrations (11 mM) in the absence of other competing exogenous substrates. That glucose is primarily oxidized is contradicted by data indicating that little glucose (앑5%) is converted to CO2 when radioisotopically labeled glucose was provided (37, 38). This result is supported by VSM’s production of high amounts of lactate, which is derived primarily from glucose under conditions of ample oxygen availability (4, 8, 38). However, the oxidation of glucose in these studies (37, 38, 40, 41) was determined indirectly by measuring the production of radioisotopically labeled CO2 from a single radioisotopically labeled substrate. A novel technique, 13C isotopomer analysis of glutamate, can measure the oxidation of several substrates simultaneously. 13C isotopomer analysis of glutamate quantitates the oxidation of substrates by following the 13C label through the metabolic pathways and then examining its incorporation into glutamate, which is in equilibrium with the TCA cycle intermediate 움-ketoglutarate (47). Based on the 13 C-labeling pattern of glutamate after metabolic incubation, the relative oxidation of several differentially 13 C-labeled substrates can be determined. Using this technique, glucose oxidation was found to account for 앑30–60% of substrate oxidation, despite changes in acetate concentration from 0 to 5 mM in both relaxed and contracted VSM (35). The inability of exogenous acetate, a representative short chain fatty acid, to regulate glucose metabolism is contrary to the classical theories

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of carbohydrate metabolic regulation where elevations in acetyl-CoA inhibit pyruvate dehydrogenase (48) and glucose oxidation (49). This method of regulation can be observed in cardiac muscle where acetate attenuates glucose oxidation by 70% (49). That VSM does not demonstrate this phenomenon could be due to a compartmentation of the enzymes involved in glucose metabolism (50–53) and restricted access of these enzymes to elevated metabolite concentrations (acetyl-CoA) in the cytosol. These data, obtained using 13C isotopomer analysis of glutamate, demonstrate the important role for glucose as an oxidative substrate under highly varied substrate conditions (35, 36). Early data indicated that glycogen oxidation accounts for 20% of oxygen consumption in resting rabbit aorta in substrate-free conditions, which favor maximal glycogen utilization (39). The absence of alternative substrates in this study likely allowed VSM to adapt to the reduced substrate availability and thus utilize the only available substrate. In addition, the activity of glycogen phosphorylase, a key enzyme for glycogenolysis, has been shown to be low in resting aortic VSM (54). It is unlikely that glycogen is a significant oxidative substrate in relaxed VSM, but it may be a larger contributor to substrate oxidation during contraction. This is supported by evidence showing that glycogen phosphorylase activity, a rate-limiting step for glycogenolysis, is well coordinated with contractility (54–56). However, in porcine carotid artery, glycogen breakdown ceases 15–30 min after stimulation (56, 57) with approximately 50% of the initial glycogen content remaining (57). It was concluded that glycogen is utilized as a short-term energy source for tension generation but not tension maintenance (38, 57). It is important to note that in these studies the glycogen contents were lower (2.4 and 2.8 애mol/g) (38, 57) than some of the VSM glycogen contents in vivo (8.9–13.9 애mol/g in cow mesenteric artery) (4, 52). In addition, it has been shown that VSM can accommodate higher glycogen stores (앑7–15 애mol/g glucosyl units of glycogen) than previously thought (52). Because the rate of glycogenolysis increases in proportion to glycogen content (52), it was thought that glycogen might be a larger contributor to substrate oxidation than previously determined (38, 57). To measure the oxidation of glycogen and to determine the role of glycogen in VSM substrate selection, hog carotid arteries with variable glycogen loads were contracted in the presence of glucose (5 mM) and acetate (1 mM) followed by 13C isotopomer analysis of glutamate to measure substrate oxidation (36). These studies showed that as glycogen breakdown increased, it was utilized for both aerobic lactate production (36, 52) and oxidation (36). At the lowest glycogen content examined in this study (앑1 애mol/g), glycogen synthesis rather than glycogen degradation occurred,

showing that glycogen is not a substrate for oxidation at this concentration (36). In fact, even at the highest glycogen content examined (앑11 애mol/g), glycogen accounts for only 앑10% of substrate oxidation and is therefore a minor contributor to substrate oxidation in VSM (36). The increased oxidation of glycogen (앑10%) concomitant with higher glycogen contents resulted in an attenuation of glucose oxidation. However, even at the highest glycogen content examined, glucose oxidation exceeded glycogen oxidation (36). Unlike glucose oxidation, glycolysis (lactate from exogenous glucose) is not regulated by glycogenolysis (36, 52). The independence of glycolytic metabolism from glycogen metabolism suggests a compartmentalization of carbohydrate catabolism in smooth muscle (38, 58) (see later). However, the compartmentation of these two metabolic pathways is not absolute, as glucose oxidation was decreased significantly as glycogen oxidation increased (36). From studies of carbohydrate oxidation it can be concluded that glucose is the primary substrate oxidized by contracting VSM (앑30–60%), whereas glycogen contributes minimally (⬍10%). The continued oxidation of glucose occurs in the presence of increasing concentrations of exogenous acetate (0–5 mM) (35) and glycogen (1–11 애mol/g) (36). While this finding contradicts previous studies depicting both higher (40, 41) and lower (37, 38) percentages of glucose oxidation, this may simply reflect the different substrate conditions among the studies and the indirect nature of the previous measurements of glucose oxidation.

B. Fatty Acid Oxidation Most fatty acids in the plasma are long chain fatty acids, which contain 16 or more carbons (59). The two long chain fatty acids represented most highly in plasma include oleate and palmitate, which comprise approximately 30 and 27% of total serum free fatty acids, respectively (60). In addition to long chain fatty acids, short chain fatty acids found in the plasma include acetate (70 애M), propionate (5 애M), and butyrate (4 애M) (61). Oxidation of the long chain fatty acid palmitate was determined in resting rabbit aortic VSM in the presence of alternative exogenous substrates (glucose, palmitate, 웁-hydroxybutyrate, leucine, isoleucine and glutamine) (37). In this study, palmitate (앑5%) and 웁-hydroxybutyrate (앑8%) were minor and approximately equivalent oxidative substrates (37). Although the oxidation of palmitate increased to 앑17% of total oxygen consumption in the absence of exogenous substrates, it remained a minor contributor to substrate oxidation (39). Because contraction is associated with an increase in energy demand, it is possible that the oxidation of both short and

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long chain fatty acids is increased concomitantly with contractile activation. This is supported by data showing that acetate in contracted hog carotid artery is a large contributor to substrate oxidation, accounting for between 앑30 (36) and 앑50% (45) of oxygen consumption in the presence of glucose. Although evidence suggests that shorter chain fatty acids are oxidized to a greater extent than longer chain fatty acids in relaxed VSM (43, 44), further studies will need to be undertaken to examine the interplay between short and long chain fatty acids for oxidation in contracted VSM. Although the size of the triglyceride pool in VSM is uncertain, it has been shown that VSM has the ability to incorporate a substantial amount of fatty acid into cellular triglyceride pools (62). This demonstrated that VSM has the ability to increase the storage of lipid, which may have ramifications on the utilization of endogenous lipids and substrate selection by VSM. Indirect evidence suggests that endogenous lipids may be the predominant substrate oxidized by resting VSM, as the combined utilization of exogenous substrates (glucose, palmitate, 웁-hydroxybutyrate and branched chain amino acids) accounted for only 30% of oxygen consumption in resting rabbit aorta (37). Because glycogen synthesis rather than degradation occurs at rest, it was concluded that the substrate oxidized primarily by resting VSM is endogenous lipid. This is supported by data showing that endogenous fatty acids account for 앑76% of oxygen consumption in resting rabbit aorta (39). This value was obtained indirectly by measuring the difference between the specific activity of the exogenous fatty acid, palmitate, and the specific activity of the intracellular long chain acyl-carnitine pool. Because acyl-carnitines are the precursors for 웁 oxidation, it was assumed that dilution of the intracellular relative to the extracellular specific activity reflected entry of unlabeled endogenous lipids into the intracellular acyl-carnitine pool for oxidation. It is possible that this assumption overestimated the oxidation of endogenous lipids in resting VSM, as the difference between extracellular and intracellular acyl-carnitine specific activity could be due to a higher turnover of acyl-carnitine derived from exogenous fatty acid (e.g., by preferential oxidation of the pool derived from exogenous sources). If this occurs, dilution of the specific activity by the acyl-carnitine pool from endogenous sources would overestimate the contribution of endogenous lipids to oxidation. Additional evidence suggesting the importance of endogenous lipids for oxidation is based on the maintenance of contractile force and high-energy phosphate levels in the presence of 2-deoxyglucose, an inhibitor of glycolysis (42). It was concluded that since no exogenous substrates were provided and utilization of glycogen was inhibited, an endogenous noncarbohydrate substrate (likely lipid)

maintained the energetic requirements of contraction (42). Because glycogen utilization was inhibited and no exogenous substrates were provided, it is possible that these results reflect adaptation of the VSM to utilize the only substrate available (endogenous lipid). Although several studies have illustrated the oxidation of both endogenous (39, 42) and exogenous (35–37, 43–45) fatty acids, no clear picture of fatty acid oxidation has been established. While shorter chain fatty acids are oxidized to a greater extent than longer chain fatty acids (43, 44), the interplay between exogenous and endogenous fatty acid oxidation is unclear. Although it has been shown that exogenous fatty acids (palmitate and octanoate) decrease the oxidation of endogenous fatty acids in relaxed VSM (39), this phenomenon has not been demonstrated in contracted VSM. Similarly, it is unclear whether the presence of exogenous fatty acids induces a glycogen-sparing effect, as the provision of exogenous fatty acids has been shown to both decrease glycogen breakdown (46, 63) and increase glycogen breakdown (51, 64). Therefore, before conclusions can be made regarding the importance of fatty acids (exogenous and endogenous) as oxidative substrates and their role in altering substrate selection by VSM, the oxidation of fatty acids must be directly measured in the presence of alternative substrates.

C. Summary of VSM Substrate Oxidation Although the overall picture of oxidative substrate metabolism of VSM is far from complete, some initial conclusions can be drawn from the literature. Glucose has been shown to be an important oxidative substrate (앑30–60% of oxidized substrates) for contracted VSM, with glycogen contents spanning the range observed in vivo (1–11 애mol/g) (36) and in contracted and relaxed VSM in the presence of exogenous acetate from 0 to 5 mM (35). Although the importance of carbohydrates as oxidative substrates has been studied thoroughly, the role of fatty acids in oxidative metabolism needs clarification. While many studies have demonstrated the oxidation of exogenous and endogenous lipids under variable substrate conditions, further studies need to be conducted under well-defined substrate conditions to assess the importance of fatty acids as oxidative substrates.

V. RESPIRATORY CAPACITY Mitochondria isolated from vascular smooth muscle do not differ substantially from mitochondria of other mammalian tissues with respect to the P/O ratio, respiratory control ratio, and substrate utilization (65, 66).

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Experiments on intact VSM indicate that the P/O ratio in situ approaches its theoretical maximal value (67). Resting respiratory rates of mammalian smooth and skeletal muscles show relatively similar values. For both red and white skeletal muscles, basal respiratory rates are in the range of 150–300 nmol O2 /min/g tissue (22, 68–70), whereas smooth muscles range from 50 to 200 nmol O2 /min/g tissue (1–7). This suggests that in the absence of an activated actomyosin ATPase and consequent contractile activity, the basal energy cost of housekeeping processes in mammalian muscle types is similar. A major difference arises, however, upon activation of contractile activity. In continuously twitching or tetanized mammalian skeletal muscle, for example, the steady-state O2 consumption rate increases by factors typically 25- to 50-fold (22, 70), as does the recovery O2 consumption rate following tetani of moderate (⬍15 sec) duration (71). In isometrically contracted vascular smooth muscle, the O2 consumption rate increases typically no more than 2-fold (1–7, 24). The much lower

maximum oxygen consumption rate of VSM is, of course, consistent with the differences mentioned previously in inherent actomyosin ATPases, the pool size of preformed high-energy phosphates, and the different energy provision strategies between the two muscle types. A consistent finding in studies of isolated vascular smooth muscle is that increases in the rate of aerobic metabolism are correlated very tightly with the levels of maintained isometric tension under a wide variety of conditions (72–79). This is illustrated in Fig. 1 for studies using strips of hog carotid artery (77) stimulated to develop and maintain varying levels of isometric tension in response to graded increases in the ratio of K⫹ to Na⫹ in the bathing solution. Figure 1A shows continuous recordings of the oxygen tension determined polarographically with an oxygen electrode in the bathing solution. Slopes of the oxygen concentration records are proportional to the rates of oxygen consumption by the arterial segment. It is evident from Fig. 1 that the steady-

FIGURE 1 (A, top) Output of an oxygen electrode in a closed chamber containing a segment of hog carotid artery. The rapid vertical rises in O2 records correspond to changes of the bathing medium that both restored the O2 concentration to approximately the initial value and accomplished the change in ionic composition of the bathing physiologic saline solution. The downward slope of each trace is proportional to the O2 consumption rate of the arterial segment in the chamber [cf. Paul et al. (75)]. (Bottom) The simultaneous record of isometric force produced by the arterial segment in response to the indicated increase in the percentage substitution of Na⫹ by K⫹ in the bathing solution. Na⫹ indicates normal Krebs–Henseleit physiological salt solution. Hist indicates the addition of 10⫺5 M histamine to the already K⫹-depolarized artery segment. (B) Rates of O2 consumption (JO2) are plotted against maintained isometric force (⌬Po) in five hog carotid artery samples in response to varying levels of K⫹ for Na⫹ substitution in the bathing medium, as shown for one artery (open circles). In five other artery samples, maximal activation was maintained by 50% K⫹-substituted saline, but extracellular [Ca2⫹] was varied (after depletion using Ca2⫹-free saline and 0.5 mM EGTA) between 0.1 and 1 mM. Again, JO2 is plotted against the maintained isometric force under these conditions (filled circles). The linear relation between force and JO2 is identical for the two sets of activating conditions. The mean (⫾SD) resting rate of O2 consumption for the 10 artery segments is shown by the bracket near the JO2 axis. The fact that the linear regression through force-dependent JO2 data passes near the resting JO2 value is suggestive that basal JO2 was not altered substantially during isometric contraction.

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state rate of O2 consumption increases progressively as isometric tension increases and returns to the initial resting value when contractile activity ceases. The results of identical experiments with five artery segments are plotted in Fig. 1B (open circles), demonstrating the tight linear correlation between the suprabasal O2 consumption rate and the level of maintained isometric tension. In most cases, the quantitative correlation between isometric tension maintenance and increased O2 consumption rate does not change substantially over long periods of time (up to 12 hr in vitro) nor does not vary significantly with the mode of stimulation of the blood vessel whether ionic, pharmacological, or electrical (1–7, 72–79). Data illustrating this relative invariance are shown in Fig. 1B (filled circles). Additional data beyond that discussed earlier are shown for experiments with hog carotid arteries in which isometric tension was varied by maintaining a constant elevated ratio of K⫹ to Na⫹ in the bathing solution, but varying the extracellular Ca2⫹ concentration (77). Over the range of [Ca2⫹] from 0.1 to 1.0 mM, graded isometric tension responses similar to those shown in Fig. 1A were obtained. The steadystate rates of O2 consumption following this alternative mode of activating graded isometric contractions are shown in Fig. 1B in experiments with five artery samples (filled circles). Clearly, in this case at least, the response of aerobic metabolism to increasing isometric tension maintenance was essentially invariant to the particulars of how the muscle was activated to produce tension. Similar comparative data obtained in studies with bovine mesenteric vein (73–75) display a likewise invariant dependence of steady-state suprabasal O2 metabolism on graded isometric contractions produced by varying the concentration of three pharmacological stimulants: epinephrine, norepinephrine, and histamine. While linearity between O2 consumption and force ordinarily holds, increases in O2 greater than that predicted can be evoked at high forces. This usually requires high levels of stimulation and unphysiologically high levels of Ca2⫹ (80–82). The point of divergence from linearity plays an important role in differentiating between current theories of regulation of contractility as well as interpretation of smooth muscle energetics; this is discussed further in subsequent sections. In comparing various vascular smooth muscles from a variety of species, increases in the O2 consumption rate above basal levels to maintain the maximum level of isometric force are in the range of 50–100 nmol O2 / min/g tissue (1–7). Because of the generally observed close correlation between isometric tension maintenance and suprabasal aerobic metabolism, it is generally believed that ATP hydrolyzed by vascular smooth muscle actomyosin is resynthesized rapidly through primarily oxidative pathways.

VI. REGULATION OF OXIDATIVE PHOSPHORYLATION IN VSM Mechanisms responsible for the control of smooth muscle oxidative phosphorylation that match mitochondrial ATP supply to ATP demand may depend both on the specific smooth muscle and on the metabolic state of a given smooth muscle. 31P NMR has provided a means to monitor the high-energy phosphate content in a single preparation during manipulations that affect the rate of oxidative phosphorylation. Using 31P NMR, PCr was found to decrease substantially during a contraction in the gut smooth muscle taenia coli from guinea pig (83), rabbit (28), and in bladder smooth muscle from rabbit (27). In many gut smooth muscles, the control of oxidative phosphorylation may occur by similar mechanisms as in skeletal muscle, i.e., involving changes in the levels of adenine nucleotide phosphates (e.g., ADP, phosphorylation potential). However, in many VSM preparations, under many conditions, no observable change in PCr occurs during a contraction with an increase in oxygen consumption. In hog carotid artery, no change in total high-energy phosphates (PCr ⫹ ATP), measured biochemically, was observable during a contraction that resulted in a doubling of oxygen consumption (23). In sheep aorta, during a depolarizing contraction with glucose as the substrate, no change in PCr or ATP was observed using 31P NMR, despite a stimulation of oxidative metabolism (24). Hence, in many VSMs, the control of oxidative metabolism appears to occur without a change in the levels of high-energy phosphates and hence without any change in the free [ADP]. Regulation of oxidative metabolism in VSM may occur through an alternative method, which modulates the activity of mitochondrial dehydrogenase enzymes. Mitochondrial dehydrogenase enzymes include pyruvate dehydrogenase, isocitrate dehydrogenase, 움-ketoglutarate dehydrogenase, and succinate dehydrogenase. These enzymes are responsible for the oxidation of substrates, resulting in the transfer of electrons to either NAD(P)⫹ or FAD⫹ producing NAD(P)H or FADH. These reduced electron carriers transport electrons to the electron transport chain where the flow of electrons through the respiratory chain creates an electrochemical energy gradient used to drive ATP synthesis. By regulating the activity of mitochondrial dehydrogenase enzymes, which generate reduced electron carriers, the delivery of NAD(P)H and FADH to the respiratory chain can be regulated and induce changes in oxidative phosphorylation and ATP production in the absence of changes in high-energy phosphate levels. There is considerable amount of evidence illustrating that increases in cytosolic calcium within the physiological range can activate various cellular dehydrogenase

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enzymes in vitro and can stimulate oxidative metabolism in a variety of intact mammalian cells (84). The role of calcium as a stimulator of oxidative metabolism and ATP production in visceral smooth muscle is consistent with the fact that an increase in NADH and FADH precedes contraction in the presence of 0.1 mM calcium (85). Therefore, calcium may serve as a feed-forward signal that primes the production of aerobically produced ATP prior to the increase in energy demand. It has also been shown that in VSM, mitochondrial levels of magnesium rather than calcium are elevated in response to contractile stimulation. It is thus possible that magnesium plays an integral role in regulating oxidative metabolism in VSM in the absence of decreases in highenergy phosphate levels (86). Mitochondrial magnesium has been shown to decrease in response to hormonal signaling in isolated rat liver mitochondria by a cyclic AMP-dependent mechanism involving the adenine nucleotide transporter (87). In addition, changes in magnesium concentration have been shown to regulate the activity of 움-ketoglutarate (88, 89), pyruvate dehydrogenase, and succinate dehydrogenase (88). The ability of magnesium to regulate mitochondrial dehydrogenase enzymes (88, 89) supports a role for magnesium in regulating VSM oxidative phosphorylation, where changes in oxygen consumption occur in the absence of decreases in high-energy phosphates. Therefore, VSM may be able to regulate oxidative metabolism independent of changes in high-energy phosphates by mechanisms involving either magnesium or calcium activation of mitochondrial dehydrogenases. Most measurements of respiratory control in VSM utilize tissues having glucose as the sole exogenous metabolic substrate. However, based on studies in cardiac muscle (90, 91), it is known that the nature of the available substrate may affect the relative role of changes in high-energy phosphates in respiratory control. In sheep aorta, when the glycolytic inhibitor 2-deoxyglucose was substituted for glucose in bathing media, oxygen consumption was stimulated whereas PCr decreased and free [ADP] increased (24). The effect was not directly the result of inhibition of glycolysis, as contractions with no substrate (no glucose) in the bathing solution induced no change in high-energy phosphates, despite the stimulation of oxygen consumption. 2-Deoxyglucose acts as a phosphate sink, thereby driving down PCr and ATP and resulting in an increased free [ADP]. Thus sheep aorta, and perhaps other VSM, can accomplish a control of oxidative phosphorylation by at least two classes of mechanisms, one dependent and the other independent of changes in the levels of high-energy phosphates (calcium and magnesium regulation of mitochondrial dehydrogenase enzymes). The metabolic state of the cell may play a key role in determining which class of mecha-

nism(s) predominates in the control of oxidative phosphorylation.

VII. AEROBIC GLYCOLYSIS In well-oxygenated, resting mammalian skeletal muscles, the steady production of lactic acid is rather small, ranging in the literature from 5 to 80 nmol lactate/min/ g tissue (22, 68, 69, 92, 93), as compared to a resting O2 consumption rate of 150–300 nmol/min/g tissue. In general, the contribution of aerobic glycolysis to total ATP production rate in the resting muscle does not exceed 5%, and the bulk of the glucose or glycogen utilized is metabolized aerobically. In the early phase of skeletal muscular activity, before the onset of recovery metabolism, lactate production may increase substantially. Over longer term tetani, however, or averaged over the full period of recovery metabolism (several minutes), lactate production increases by factors only two to four times over the basal production rate. O2 consumption rates, however, increase by factors typically 20- to 30-fold so that the energetics of both steadystate and/or recovery metabolism in support of mechanical activity in mammalian skeletal muscles is almost entirely oxidative. In vascular smooth muscle, the situation is somewhat different. It has been known for some time that vascular smooth muscles maintain unusually high levels of lactic acid production (40), even in well-oxygenated in vitro preparations. In earlier reports, it was speculated that this reflected some sort of tissue damage. Jlac can be increased three- to fourfold by the inhibition of mitochondrial respiration by hypoxia (Pasteur effect) or CN (94). Similarly, inhibition of the glutamate–malate shuttle has also been reported to increase Jlac . However, modern studies, obtained under conditions suitable to maintain healthy smooth muscle tissue in vitro, consistently report high levels of aerobic glycolysis. Substantial levels of aerobic glycolysis have been reported for a wide variety of smooth muscle types, including uterine, intestinal, and tracheal smooth muscles (72–79, 95–98). For blood vessels with basal O2 consumption rates in the range of 50–200 nmol O2 /min/g, typical resting aerobic lactic acid production rates are in the range of 100–250 nmol lactate/min/g, with a steady-state ratio of 1–2 mol lactate produced per mole O2 consumed being most common. In terms of ATP production, this is a highly inefficient system. If glucose were the sole substrate, it can be calculated that this molar ratio of 1–2 means that 75 to 85% of the glucose equivalents utilized are metabolized only to lactic acid. The 15 to 25% of the remaining glucose, if oxidized completely, nonetheless would provide the bulk of the total ATP

33. Vascular Smooth Muscle

production (70 to 80%) due to the much higher ATP production per glucose molecule of oxidative metabolism.

VIII. ORGANIZATION OF CARBOHYDRATE METABOLISM IN VSM As just described, vascular smooth muscle manifests a high degree of lactic acid production, even in welloxygenated in vitro preparations. On the surface, this inefficiency of carbohydrate metabolism might be viewed as a metabolic defect. However, a growing body of evidence leads to the conclusion that oxidative metabolism and glycolytic metabolism may operate independently and, at times, support different classes of cell functions. Thus the apparent inefficiency of smooth muscle lactate production may really be a consequence of a compartmentalized metabolism–function coupling. This section briefly summarizes some of the results that have helped define the roles for glycolysis and oxidative metabolism in smooth muscle.

A. Glycolysis and the Support of Ion Pumps Some of the first studies demonstrating separate regulation and possible separate roles for glycolysis and oxidative phosphorylation were those of Paul et al. (99), who found that the addition of K⫹ in hog coronary arteries stimulates aerobic glycolysis (i.e., at constant external Na⫹). In contrast, ouabain and K⫹-free or Na⫹free bathing medium depressed aerobic glycolysis. Ouabain, moreover, increases isometric force in these vessels, which is correlated with an increase in JO2 concomitant with the decrease in Jlac . However, there are experimental conditions under which force and JO2 decrease, whereas Jlac and the Na⫹ –K⫹ pump are elevated. For example, in porcine coronary arteries (55), isoproterenol relaxes a KCl-induced contracture, and JO2 shows a parallel decrease, whereas Jlac , and presumably the Na⫹ –K⫹ pump [100], remain elevated. Similarly, readmission of K⫹ to tissues incubated in K⫹-free medium stimulates aerobic glycolysis while decreasing force and JO2 in porcine vessels (101), presumably due to the stimulation of Na⫹ –K⫹ transport. On this basis, it was then proposed that the energy production of aerobic glycolysis is somehow specifically coupled to the Na⫹ –K⫹ transport system of vascular smooth muscles. A coupling between glycolysis and the Na⫹ –K⫹ pump based on similar data has also been suggested for rat aorta (102). In further support of this hypothesis, Campbell and Paul (103) have shown in porcine carotid artery over a wide range of K⫹ transport rates that glycolytic ATP production varied approximately stoichiometrically with K⫹

579

transport. These results indicate that glycolytic support of the Na⫹ –K⫹ pump may be stoichiometric over much of the physiological range. Indeed, this may be a more general feature of muscle metabolism, as it has been demonstrated in a rat sepsis model that hindlimb skeletal muscle exhibited high aerobic lactate production, which was partially inhibited by ouabain (104). In hog carotid arteries, Peterson (77) observed a similar dissociation of lactic acid production from the energy cost of isometric tension maintenance with high K⫹ activation. With maintained activation via high K⫹ depolarization (50% K⫹ for Na⫹ substitution), varying the level of isometric tension development by altering the concentration of extracellular Ca2⫹ exerted no consistent effect on aerobic glycolysis, which simply remained at or near the resting level. Adding histamine to the already K⫹-depolarized artery segment, however, led to a sharp increase in aerobic glycolysis (78) to nearly the same value as that reported for histamine activation alone. During progressive removal and replacement of extracellular Ca2⫹ in arteries stimulated supramaximally with high K⫹ plus added histamine, the level of aerobic glycolysis correlated linearly with the Ca2⫹-activated stable isometric force. On this basis, it was suggested that the energy production of aerobic glycolysis may be coupled to intracellular or plasma membrane Ca2⫹ pumps that are responsible for the sequestration and homeostasis of intracellular [Ca2⫹]. This was proposed, at least in part, as one well-known action of the H2 receptor for histamine is predominantly vasodilatory, suggesting that histamine can activate intracellular Ca2⫹ sequestration or transmembrane extrusion (78). A similar coupling between energy requirements of the Ca2⫹ pump and glycolysis has been proposed by Lundholm and colleagues (6). Using cyclopiazonic acid to inhibit the sarcoplasmic reticulum Ca2⫹-ATPase, Jlac in mouse aorta was inhibited with no effect on JO2 , supporting a coupling of this ion pump to local glycolysis as well (105). The proposed coupling between glycolytically produced ATP and Ca2⫹ pump activity has been investigated in some detail. Because glycolysis was presumed to support membrane ATP-requiring processes and because glycolytic enzymes had been shown to be associated with the plasma membrane in other tissues (106– 110), glycolytic support of membrane ATP-requiring processes should be demonstrable in an isolated plasma membrane preparation. In an isolated plasma membrane preparation from pig stomach smooth muscle, glycolytic substrates and cofactors were sufficient to support Ca2⫹ pump activity as measured by Ca2⫹ uptake of inside-out membrane vesicles (111). These results demonstrate both that the glycolytic enzyme pathway

580

VI. Metabolism and Energetics

is associated with the plasma membrane and that the membrane-associated glycolysis could support one of the membrane ion pumps. In the plasmalemmal vesicle preparation, glycolysis could support the Ca2⫹ pump independent of the total [ATP] measured in the vesicle suspension (112). In addition, at low total [ATP], glycolysis supported a greater extent of Ca2⫹ uptake than ATP added exogenously at matched ATP production rates. Therefore, the smooth muscle Ca2⫹ pump preferentially utilizes glycolytically produced ATP. Conclusions from these studies appear to extrapolate to the intact smooth muscle. In a porcine carotid artery preparation with glycogen stores depleted, the concentration of norepinephrine required for half-maximal contraction was decreased when glucose was removed from the bathing media (113). In addition, after intracellular stores of calcium were depleted by precontractions in the absence of extracellular calcium, readmission of 2.5 mM calcium to the medium resulted in a higher tension transient in the absence of glucose compared to in the presence of glucose. Therefore, glycolytically produced ATP appears to be necessary for normal calcium homeostasis based on experiments using isolated and intact smooth muscle systems. The two proposals that glycolysis may preferentially support the Na⫹ –K⫹ pump and that glycolysis may preferentially support the Ca2⫹ pump may not be mutually exclusive. Indeed, both proposals may be reconciled with the view that glycolytic ATP production may fuel multiple ATP-requiring processes localized at the plasma membrane. For example, in the plasma membrane vesicle preparation from smooth muscle, ATP produced by other plasma membrane-associated kinases was able to support Ca2⫹ pump function comparably to membrane-associated glycolysis (112). Both pyruvate kinase and creatine kinase fueled the Ca2⫹ pump independent of changes in the [ATP] in suspension and to a similar extent as the membrane-associated glycolytic enzyme system. Therefore, all of the membraneassociated kinases preferentially support the Ca2⫹ pump compared to ATP exogenously provided. A specific coupling between each kinase and the Ca2⫹ pump is unlikely. Rather, all membrane-associated kinases may contribute to a pool of ATP that fuels membrane-associated ATP-requiring processes in a preferential manner compared to ATP produced away from the membrane.

IX. COMPARTMENTATION OF GLYCOLYSIS Correlations between oxidative metabolism and isometric force and between aerobic glycolysis and the energy requirements of membrane ion pumps underlie the hypothesis that energy metabolism is functionally

compartmentalized. The ability of oxidative metabolism and aerobic glycolysis to vary independently has been demonstrated for a variety of other smooth muscle preparations (98, 114–117) and suggests that this functional compartmentation of energy metabolism may reflect a biochemical compartmentation of enzyme cascades. Glycolytic enzymes are localized to the plasma membrane in smooth muscle, and the glycolytically produced ATP preferentially supports membrane-associated ATPases such as ion pumps. ATP produced by oxidative phosphorylation appears to support contractile function. A colocalization of ATP supply with ATP demand may underlie both the preferential nature of locally produced ATP in supporting local ATPases and the separate regulation and roles of the two ATP-producing systems of glycolysis and oxidative phosphorylation. Although glucose utilization (glycolysis) may be functionally compartmentalized from oxidative metabolism, under many conditions, glycogen metabolism (glycogenolysis) appears to correlate with contractile activity and hence oxidative metabolism (57). Indeed, glycogen phosphorylase activity is well correlated with contractility (54, 55). A consequence of these observations may be that the pathways for glucose metabolism and for glycogen metabolism may be separate under many conditions. Work by Lynch and Paul (38) demonstrated that when glucose was labeled uniformly with 14 C, the lactate produced had almost exactly the same specific activity, despite a decline in the total tissue glycogen content. If the glycogen were unlabeled, then a dilution of the specific activity in the lactate would be expected if the pathways for glucose utilization and for glycogen utilization mixed freely. These experiments suggested that there may be separate pools of glycolytic and glycogenolytic intermediates in smooth muscle. Further studies demonstrated two pools of one glycolytic intermediate, glucose-6-phosphate (118). Work using 13C NMR provides further insights into the nature of the organization and compartmentation of glycolysis and related pathways in the cytoplasm of vascular smooth muscle. Although 13C NMR is not as sensitive as conventional radioisotope techniques, it has the ability to provide information about label position within a molecule, labeling at adjacent positions, and does not require the separation of small molecules. When 1H NMR is used in conjunction with 13C NMR, the fractional enrichment of 13C at a given position with a molecule can be observed directly. Therefore, NMR spectroscopy has proven to be a valuable tool in studying smooth muscle energetics and the organization of metabolism in VSM. Hardin and Kushmerick (119) used 13 C NMR to directly assess the compartmentation of glycolysis and glycogenolysis in VSM. Glycogen stores were repleted with the 13C label at one position within

33. Vascular Smooth Muscle

the glucosyl units of glycogen, and then glucose labeled at a different position was provided during a subsequent depolarizing contraction. A simultaneous yet separable flux of glycolysis and glycogenolysis was observed with a disproportionate fraction of lactate produced from glycolysis. The compartmentation of glycolysis and glycogenolysis may be surprising since these pathways share 9 common enzymatic steps and 10 common intermediates from glucose-6-phosphate to pyruvate. Because of the shape of VSM cells, which are often approximately 100 애m long and 7 애m wide, diffusion distances should be rather short (less than 4 애m from the center to the surface). Thus it may be expected that the small intermediates of glycolysis and glycogenolysis should mix in the cytoplasm. Although the pathways of glycolysis and glycogenolysis are chemically identical for 9 enzymatic steps, they may be physically nonidentical if the two pathways have distinct and separate localizations within the cell. Enzymes for these pathways have been localized to the plasma membrane (111, 112) and the contractile apparatus [120] of smooth muscle. In a wide variety of other cells, these enzymes have shown various cytoplasmic localizations, such as to microtubules (121), cytoskeletal actin (122), and subdomains of the plasma membrane, such as the caveolae (123) as examples. However, a spatial localization of the pathway intermediates, and not just the enzymes, is necessary for the compartmentation of these pathways. Hardin and Finder (19) investigated whether substrate channeling could mechanistically account for the compartmentation of glycolysis and glycogenolysis. Substrate channeling has been investigated in a number of other systems using a variety of techniques (124–126). Substrate channeling involves the direct transfer of an intermediate from one enzyme to the next in the sequence without release into the cytoplasmic fluid. Therefore, if there were spatially distinct glycolytic cascades, one for glycolysis and one for gluconeogenesis, and if substrate channeling existed within the pathways, the intermediates of the pathways would not mix. Hardin and Finder (19) used suspensions of enzymatically dispersed hog carotid artery cells permeabilized with dextran sulfate to measure whether glycolytic intermediates were released from the permeabilized cells. Using [1-13C]glucose, permeabilized cells produced approximately 40% of the amount of lactate as intact cells, yet no glycolytic intermediates were observed. If substrate channeling did not occur, then the intermediates would have diffused from the permeabilized cells, been diluted 80-fold, and lactate production would not have occurred and glycolytic intermediates in the bathing media would have been observed. Because the permeabilized cells produced significant lactate and no glycolytic intermediates were

581

found in the bathing media, it was concluded that substrate channeling occurs in hog carotid artery cells. Therefore, 13C NMR has begun to elucidate some of the mechanistic basis for the compartmentation of carbohydrate metabolism in VSM. Because 13C NMR provides a direct observation of all soluble products of metabolism of 13C-labeled compounds, this technique has elucidated new characteristics of VSM carbohydrate metabolism. When the glycolytic intermediate fructose 1,6-bisphosphate (FBP) was labeled at the first and sixth carbon, it was observed that [13C]FBP was converted largely to [3-13C]lactate under hypoxic conditions (127). However, when [113 C]FBP was provided to hog carotid artery under welloxygenated conditions, [1-13C]glucose production was observed (128). These studies reveal two novel aspects of VSM metabolism. First, although it is highly charged at physiological pH due to the two phosphate groups, FBP enters the VSM cell readily and is available for metabolism. Finder and Hardin (129) proposed that FBP and 3-phosphoglycerate may enter VSM cells by transport on a plasmalemmal dicarboxylate transporter. Second, these studies reveal that VSM is capable of carrying out a portion of the gluconeogenic pathway and therefore must have the key gluconeogenic enzymes glucose-6-phosphatase and fructose 1,6-bisphosphatase. Although the function of gluconeogenesis in VSM is not clear, it has provided a useful tool to study one aspect of the compartmentation of carbohydrate metabolism in VSM. Further investigation of gluconeogenesis in VSM has revealed that there is no measurable mixing of the intermediates of glycolysis and a portion of gluconeogenesis (128). Therefore, there may be three different components of carbohydrate metabolism that are compartmented, glycolysis, gluconeogenesis, and glycogenolysis. Lloyd and Hardin (20, 21) have used pig cerebral microvessels (PCMV) as a convenient biological system for the study of the structural basis of the compartmentation of glycolysis and gluconeogenesis in VSM. Disruption of microtubules by vinblastine or stabilization of microtubules with taxol resulted in a decrease in glycolytic rate while the rate of gluconeogenesis remained unchanged (20). Therefore, the decrease in glycolysis occurred whether the microtubules were stabilized or whether the microtubules were disassembled, suggesting that the assembly of microtubles did not have a role in glycolytic regulation. It is likely that taxol and vinblastine bind to separate domains of the tubulin protein, both of which are responsible for glycolytic enzyme binding. Therefore, although these agents have opposite effects on microtubular integrity, they have similar effects on displacing the active glycolytic enzyme from the microtubules. Indeed, consistent with this hy-

582

VI. Metabolism and Energetics

pothesis was that the effects of taxol and vinblastine were additive, suggesting that their inhibition of glycolysis was not due to the disruption or stabilization of microtubular structure per se, but rather due to displacement of active glycolytic enzymes from different sites on tubulin molecules. Further work using 웁-escin permeabilized PCMV also demonstrated that substrate channeling of both glycolysis and gluconeogenesis occurred, as flux of both pathways continued (21). However, disruption of the plasma membrane with 웁-escin did alter the accessibility of FBP to glycolytic enzymes, thus allowing lactate production from FBP under welloxygenated conditions. Therefore, the different plasma membrane domains may play an important role in sorting metabolic pathways. Based on these data and the observed coupling between the Na⫹-K⫹ pump and Jlac , we suggested a model (Fig. 2) in which membrane-bound glycolytic enzymes in close opposition to the Na⫹, K⫺ATPase formed one compartment, whereas glycogenolysis and the respiratory machinery were associated more closely with contractile filaments. Indeed, glycolytic enzymes have been localized to the contractile apparatus in smooth muscle (120). The lack of mixing of the intermediates of glycolysis and glycogenolysis may be the result of the two pathways having different locations in the smooth muscle cell. An important feature of the organization of metabolism in vascular smooth muscle is that the compartmentation of ATP and of intermediates of carbohydrate

metabolism is not absolute. When intact artery preparations are contracted while oxidative metabolism is completely inhibited by NaCN, lactate is produced from both glucose and glycogen (119). This result is consistent with a mixing of glycolytic intermediates at least one step in the reaction sequence when the rate of mitochondrial utilization of pyruvate (derived from glycogen) was inhibited. The compartmentation of ATP pools also does not appear to be absolute. When the Na⫹-K⫹ pump rate of intact hog carotid arteries was progressively stimulated above a typical physiological rate, glycolytically produced ATP ceased being the sole ATP source for the Na⫹-K⫹ pump. At high pump rates, oxidatively produced ATP progressively accounted for a greater share of the ATP provision (103). These observations fit well into a model in which compartmentation of small metabolites may be the result of a local balance between production and consumption. That is, for example, with a close juxtaposition of ATP-producing and -consuming enzymes, the local reaction rate may normally dominate the rate of diffusion away from the ATPase. However, when a mismatch exists between local production and consumption, diffusion away from the production locus will become more pronounced. Hence the compartmentation observable in smooth muscle does not appear to rely on strict diffusion limitations, rather it depends on a local balance of reaction and diffusion rates. This reaction/diffusion model for the coupling of colocalized producing and consuming enzymes in smooth muscle may be a reflection of a more general feature

FIGURE 2 Model of smooth muscle indicating the proposed compartmentation of metabolism and function. Energy-requiring processes associated with membrane function, such as the Na, K, or Ca pump, are supplied with ATP by a membrane-associated glycolytic cascade. Glucose is the primary substrate for this pathway with lactate being the major end product. Evidence suggests a similar colocalization of membrane ion pumps, and glycolytic enzymes may also be found for internal membrane structures as the sarcoplasmic reticulum (SR). In contrast, energy requirements for actin–myosin interaction appear to be supported primarily by mitochondrial oxidative phosphorylation. Free fatty acids (FFA) and glucose appear to be major substrates, but a separate glycolytic enzyme cascade, utilizing glycogen but not glucose, also plays a role in this compartment.

33. Vascular Smooth Muscle

of cell energetics. Studies on striated muscle indicate a coupling of the Na⫹ –K⫹ pump to aerobic glycolysis (92, 93, 103). Plasma membrane-localized ATP provided by glycolysis has also been implicated in the gating of ion channels both in cardiac muscle (130) and in smooth muscle (131).

X. CHEMICAL ENERGY UTILIZATION AND CONTRACTILE ACTIVITY Resting vascular smooth muscle consumes ATP at a rate typically 0.5–1.0 애mol ATP/min/g tissue. The bulk of this ATP requirement, typically 70 to 90%, is met from oxidative metabolism and 10 to 30% from aerobic glycolysis (4). Upon maximal activation at muscle lengths near optimal for isometric force development, the total steady-state ATP utilization rate increases by a factor of 2 or so in isolated VSM preparations. The difference between the initial resting metabolic rate and the maximally activated metabolic rate reflects the sum of all energy-consuming processes activated in parallel with or consequent to the mechanical activation. An underlying assumption in this method of measuring contractile energetics is, of course, that the energy requirements of basal processes remain more or less constant during the period of mechanical activity. Most evidence, albeit indirect, indicates this to be the case for pharmacological or ionic methods of stimulation (75–78) (Fig. 1). The increased ATP utilization rate on mechanical activation can be conceptualized, at least, as occurring in three separate parts: (1) actomyosin ATPase in support of mechanical activity, (2) ATP-dependent processes that play some role in initiating and maintaining the activation processes underlying mechanical activity, and (3) all other ATPase activated through the particular means of stimulation chosen. In more recent studies of smooth muscle energetics, some effort has been made to sort out the quantitative subdivision of the total increase in ATPase into these three categories. In many cases, the third category (which is roughly equivalent to a stimulus artifact) appears to make only a small to negligible contribution to the overall increase in tissue metabolism (75, 76, 78) (cf. Fig. 3). The first category is approximated most frequently by tension-dependent metabolism. This is measured by determining how suprabasal energy metabolism changes as the actomyosin interaction (i.e., force development) is varied. During such measurements, the level of stimulation (i.e., in essence, the variable components of categories 2 and 3) is held fixed. If, at constant supramaximal stimulation, for example, the muscle is lengthened or shortened to lengths where tension development is abolished, the

583

tension-dependent ATPase by definition goes to zero and the remaining suprabasal metabolism is called tension-independent metabolism. This division assumed that changes in length per se do not affect the level of stimulation or their energy dependence. There is some evidence that length can influence activation parameters (132). An example of energy partition from measurements of O2 consumption performed with a single segment of bovine mesenteric vein (133) is shown in Fig. 3, which additionally illustrates the invariance of such measurements to the particular pharmacological agonist used. Upon supramaximal activation with epinephrine (10⫺6 M) at the optimal length for force generation (L ⫽ 2.45 cm for this sample), the O2 consumption rate increased by 110 nmol min/g over the initial resting O2 consumption rate of about 140 nmol O2 /min/g. Progressive stepwise shortening of the muscle from this length with supramaximal histamine as a stimulant caused a decline in isometric tension maintenance as expected from the force–length relationship. This in turn led to a linearly correlated decline in the suprabasal O2 consumption rate (upper line). When the muscle had freely shortened to L ⫽ 0.8 cm so that no isometric tension was evident, the O2 consumption rate remained elevated over the basal rate by about 25 nmol O2 /min/g. This value, tension-independent metabolism, is about 20% of the total suprabasal ATPase at maximum isometric tension and was not dependent on whether epinephrine or histamine was used to affect the stimulation. Simultaneous measurements of suprabasal aerobic glycolysis gave similar values for the tension-independent component of aerobic glycolysis in bovine mesenteric vein (74, 134). More recent studies show that tension-independent JO2 can be elevated significantly by supramaximal stimulation, resulting in high levels of myosin light chain phosphorylation MLC-Pi . This aspect is discussed in Section XI. Figure 3 also provides an opportunity to demonstrate the economy of circulatory regulation by vascular smooth muscle. Suppose the vessel segment of Fig. 3 was at a vessel radius equivalent to the segment length L ⫽ 2.45 cm and partially activated so as to maintain that vessel caliber against a blood pressure equivalent to a wall tension of 0.36 kgwt/cm2. Maintaining the pressure in the vessel constant, but maximally activating the vascular smooth muscle would, for the particular sample of Fig. 3, cause the vessel to shorten to a length approximately L ⫽ 1.30 cm (as indicated by the vertical arrow). For the cylindrical blood vessel, this length change is equivalent to a reduction in caliber of about 45%. Using Pouiselle’s law to compute the change in flow resistance of the vessel, this amounts to a 12-fold increase in flow resistance. In terms of energy cost, however, the increased energy metabolism necessary to sup-

584

VI. Metabolism and Energetics

FIGURE 3 Determinants of vascular smooth muscle energy metabolism under steady-state isometric conditions. (A) The dependence of active isometric force (solid line, ⌬Po) and suprabasal rate of oxygen consumption (broken line, JO2) as a function of muscle length (L/Lo , where Lo is the length of the muscle with zero passive force). Data represent the average fitted values for 55 bovine mesenteric veins [adapted from Ref. (2)]. (B) The dependence of the rate of energy metabolism on active isometric force, in which force is varied by changing muscle length at constant, maximal simulation. Changes under these conditions are taken to reflect geometric changes in the number of crossbridges due to sliding filaments. Thus the intercept at zero force defines a tension-independent component, often ascribed to ‘‘activation energetics’’ (see text). (C) Dependence of the rate of energy metabolism on active isometric force, in which force is varied by changing the level of stimulation at the optimum length for force development. The difference in slopes (a and b) of the relationships between metabolism and force in between B and C is attributed to the different levels of activation at the same force. The broken line represents this difference and can be ascribed to the energy utilization associated with tensionindependent, activation processes.

585

33. Vascular Smooth Muscle

port the muscular activity for this circulatory regulation is less than 15% of the resting basal metabolism. Apparently, the energy cost of regulating peripheral circulation is very low. The tension-independent metabolism discussed earlier reflects terms in both categories 2 and 3, as well as the possibility of some residual activated ATP hydrolysis by actomyosin, which nonetheless makes no contribution to tension development. Such a situation could arise, for example, through the generation of internally opposing forces that result in no net external force. That such an ‘‘internal load’’ might be the case is indicated by studies of vascular smooth muscle mechanics (135). Huxley and Simmons (136) proposed that muscle stiffness is a direct measure of the number of actin–myosin crossbridges formed at any instant during mechanical activity. A comparison of resting stiffness (due to inert structural components) with the stiffness found at the extremes of length where active tension development is abolished could indicate the extent to which actin and myosin still interact. In studies of this sort with several arterial preparations, Pfitzer and Peterson (137) found that during the rise of isometric tension following stimulation, arterial wall stiffness increased in direct proportion to the isometric force developed. The same linear proportionality also applied between stable isometric force and stiffness when isometric force was varied by varying the extracellular [Ca2⫹] in the high K⫹ bathing medium (138). These observations suggest that isometric tension and actomyosin interaction in VSM are in-

deed directly related. In hog carotid artery segments maximally activated and at their freely shortened length so that no isometric tension was measurable, stiffness was elevated by some 10 to 15% above the purely passive stiffness, and this component required Ca2⫹ in the bathing medium (J. Peterson, unpublished observation). This is indicative that at least a fraction of the tensionindependent ATPase could be attributable to an actomyosin ATPase. Measurements of steady-state suprabasal energy metabolism and estimates of the actomyosin ATPase of vascular smooth muscles, determined as tension-dependent ATPase, are presented in Table II. Comparisons of the contractile ATPase of the intact preparation determined in this way with that of the ATPase of purified isolated VSM actomyosin, while approximate at best, are in reasonable agreement (4, 139, 140). From studies of these sorts, two principal conclusions arise. (a) Despite the ability to develop forces comparable to or even greater than skeletal and cardiac muscles, the in situactivated ATPase of vascular smooth muscle actomyosin is extremely small. (b) The investment of energy in processes necessary to maintain the activation of contractile activity is substantial, on the order of 0.05 to 0.20 of the total suprabasal energy requirement of at maximal tension development. Under conditions designed to maximize myosin light chain phosphorylation using K⫹ depolarization plus histamine in a Tris-buffered solution, activation energy can be a large fraction (0.4–0.5) of the total energy (96) (see further discussion

TABLE II Suprabasal Energy Metabolism of Isometrically Contracted Vascular Smooth Muscle a

Preparation Bovine mesenteric vein Hog carotid artery

Bovine mesenteric artery Rat aorta Bovine carotid artery a

(1) Total suprabasal metabolism

(2) Tensiondependent metabolism

(3) Tensionindependent metabolsim

nmol ATP/(minⴱg) mN/mm2

nmol ATP/(minⴱg) mN/mm2

nmol ATP (minⴱg)

(4) Tensionindependent ⴜ total suprabasal

16.4 6.8 8.1 4.0 5.1 4.1 12.2 6.6 19.7 8.2

12.8 4.3 4.6

200 270 230

0.06 0.20 0.14

1.6 11.2

71–340 110

0.09 to 0.41 0.05

Stimulus

Ref.

Epi, NE, Hist Hist K⫹ K⫹ K⫹ ⫹ Hist K⫹ to K⫹ ⫹ Hist ⫹ Tris Epi K⫹ K⫹ Electrical

73–75 76 76 77 75 96 6 79 29

In column (1), total suprabasal metabolism has been divided by the observed isometric tension with the mode of stimulation indicated. Column (2) is that part of the total suprabasal metabolism that vanishes when isometric tension is reduced by changing length, but with maintained stimulation, divided by the magnitude of isometric force decrease. Column (2) is taken as an estimate of crossbridge (actomyosin) ATPase activity. Column (3) is the steady-state suprabasal metabolism remaining after force is abolished but with maintained stimulation; this is used as an estimate of activation energy utilization. Column (4) is the fractional cost of activation, i.e., column (3) divided by column (1), calculated using an average force of 200 mN/mm2. Epi, epinephrine; NE, norepineprhine; Hist, histamine; Tris, Tris buffer substituted for Na.

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FIGURE 4 A schematic model of the smooth muscle myosin crossbridge interaction with actin filaments. The transition from state 1 to state 2 represents the Ca2⫹ –calmodulin-dependent phosphorylation/dephosphorylation activation mechanism proposed for smooth muscle regulation. The cycle of interaction (states 2–5) hydrolyzes one ATP molecule and generates one quantum of tension with each pass. Only the angulated attached myosin crossbridge (state 4), however, generates isometric force. The inherent speed of the cycle is much slower in smooth than in striated muscle, with the ATP-dependent dissociation step (states 4 and 5) apparently being the slowest. The dashed lines indicate formation of a proposed dephosphorylated actomyosin crossbridge (150, 151), which then dissociates only very slowly and may be responsible for the very high holding economy of vascular smooth muscle.

in Section XIII.) It is interesting to note that this normal percentage holds for both smooth and skeletal muscle, which indicates that in absolute terms, the energy cost of activation or tension-independent processes is considerably higher in striated muscle than in smooth muscle. The extraordinary economy of tension maintenance in vascular smooth muscle and the detailed nature of the activation processes mentioned earlier are areas of intense interest in VSM physiology. An additional discussion of activation energetics is given in a subsequent section. The very low tension cost (or, alternatively, the high holding economy) of vascular smooth muscle appears to reside primarily in the molecular properties of the actomyosin itself, although structural and geometric factors may also play some part (139–143). Barany (144) first described an inherent correlation between the ATPase activities of various myosins and the contractile velocities of the muscles from which the myosins originated. Vascular smooth muscle appears to be the slowest of all mammalian muscles in terms of its shortening velocity (143), and this mechanical property appears to be a direct reflection of how slowly the actomyosin crossbridges hydrolyze ATP in going through their repetitive cycle of interaction with actin filaments. From comparisons of the actomyosin content of vascular smooth muscles and the observed rates of tension-dependent metabolism, it has been estimated that the VSM

myosin crossbridge goes through its cycle of interaction with actin filaments in about 0.75–1.5 sec (140, 142, 143). This is an extraordinarily slow rate of interaction when compared to skeletal muscle crossbridges, which are estimated to cycle at rates more like 100–150 times per second. If the smooth muscle myosin crossbridge spends a large fraction of this very long cycle time in contact with the actin filament in the force-generating configuration, then the high holding economy of the tissue is not difficult to appreciate. A schematic representation of this model of the actomyosin crossbridge cycle is shown in Fig. 4. The steady-state rate of a single myosin crossbridge is the sum of the time required to make one full pass through states 2, 3, 4, 5, and back to state 2. State 4, however, generates isometric tension so that the average tension maintained by a large number of crossbridges interacting asynchronously is proportional to the time spent in state 4 as a fraction of the total cycle time. Using purified myosins from four different types of vertebrate muscles (including gizzard smooth muscle), Marston and Taylor (145) reported that the time required for ATP to dissociate the smooth muscle crossbridge from actin (states 4 and 5 in Fig. 4) is by far the slowest step. However, though rate limiting, this step was not nearly slow enough to fully account for the very high holding economy of smooth muscle relative to other muscle

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types. Precisely what features of smooth muscle myosin are responsible for these inherent differences in the enzymatic activities and kinetics of otherwise apparently quite similar molecules is currently not understood.

XI. HOLDING ECONOMY AND ACTIVATION ENERGETICS A picture of the actomyosin crossbridge cycle in which the overall cycle time of the crossbridge determines the actomyosin ATPase, whereas the fraction of the cycle time spent in the tension-generating state determines the force developed, yields, in a relatively direct way, the holding economy of the system. In this very simplified model, however, any factors (biochemical, mechanical, or otherwise) that alter the fraction of the cycle time that the crossbridge spends in the tensiongenerating state could alter the observed holding economy of the tissue (cf. Fig. 4). Murphy and colleagues (146–154) reported such a phenomenon in vascular smooth muscle that they have termed the ‘‘latch state.’’ They determined the shortening velocity of hog carotid artery as a function of time during the development and maintenance of isometric tension. The maximum rate at which the muscle is capable of shortening, which is often taken as a direct index of the speed of the individual crossbridge cycle rate, became progressively slower over a period of 5–20 min

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following the activation of mechanical activity. Driska et al. (148) and Aksoy et al. (149) have found a similar decrease in myosin light chain phosphorylation with the increased duration of stimulation, despite the near constant maintenance of isometric force. This temporal correlation is suggestive of the possibility that myosin phosphorylation, in addition to its postulated role as a regulator of the smooth muscle actomyosin interaction [cf. Hartshorne (155) for a review], acts as a modulator of the speed of the smooth muscle actomyosin crossbridge cycle. In their model, phosphorylated myosin crossbridges are dissociated from actin filaments much more rapidly than dephosphorylated myosin crossbridges. This model is illustrated schematically by the dashed lines in Fig. 4. If this is the case, one would expect the holding economy of highly phosphorylated VSM myosin to be less than that of active but dephosphorylated VSM myosin. Available data on hog carotid artery support this hypothesis. Krisanda and Paul (156) measured both suprabasal JO2 and unloaded shortening velocity at various points during the development and maintenance of isometric force. As shown in Fig. 5, both parameters displayed similar, although not superimpossible, biphasic responses. Each of these parameters can be used to estimate the crossbridge cycle rate, and their decline with time of stimulation is similar to that of myosin light chain phosphorylation. The holding economy, as well as velocity in these experiments, differed from their

FIGURE 5 The time course of rate of suprabasal ATP utilization (JATP , open circles), active isometric force (F/Fmax , solid line), and unloaded shortening velocity (Vus , filled circles) in hog carotid artery following stimulation by increasing media KCl by 50 mM. Adapted from Paul (2) and Krisanda and Paul (156), with permission. Bars indicate ⫾SEM. Note that while isometric force increases monotonically to a steady state, both Vus and JATP decrease from the maximum that occurs early in the contraction. Both [Ca2⫹]i and myosin light chain phosphorylation show similar biphasic time courses.

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maximum value by two- to threefold. Thus, even if extrapolated to account for the maximum possible differences in myosin phosphorylation, this mechanism is unlikely to account for the 100- to 2000-fold difference in holding economy between striated and smooth muscle. It is of interest to note that Butler and Siegman (5) also reported that the rate of phosphagen breakdown and velocity in rabbit taenia coli also showed a similar dependence on the duration of stimulation. However, they concluded that this behavior was unlikely to be dependent on myosin light chain phosphorylation. While myosin phosphorylation, JATP , and Vmax do show similar time courses, the question is whether these relations are correlative or causal. An underlying assumption is that this transient in JATP is related to actin– myosin interaction and not a transient in energy utilization ascribable to activation processes. Such processes would differ from that underlying the steady-state tension-independent metabolism, as described in Section X. This energy utilization is a durable component, persisting throughout contractile activity and apparently associated more closely with the maintenance of the activated state of the muscle than with the actual actomyosin ATPase that generates contractile activity. Most frequently, these energy costs are ascribed to processes such as ATP-dependent Ca2⫹ translocation, other altered energy-dependent ion fluxes, protein phosphorylation–dephosphorylation cycles, and other miscellaneous processes (4). It has now been observed repeatedly using a variety of methodologies in vascular smooth muscles (2, 6, 29, 156) and other smooth muscles (5), as well as fast mammalian skeletal muscles (22), that the initiation and development of isometric tension are energetically far more costly than the steady-state maintenance of isometric tension (even when including the previously mentioned steady-state activation energy). It is possible that this excess energy utilization is related to some form of intrinsic internal work performed by the actomyosin systems in stretching internal elastic elements during the development of isometric force, a term that might be essentially similar in all muscle types. For both taenia coli (157) and hog carotid artery (156), this has proven unlikely. Energy utilization has been measured during the redevelopment of isometric force following a fast shortening step that discharged all maintained isometric force. In both cases, the redevelopment of isometric force required no measurable excess energy utilization beyond the observed force maintenance state. This argues that only the activation of tension development from the resting state requires excess high-energy phosphate utilization. Evidence supporting the assumption that this transient excess in JATP is related to actin– myosin interaction was provided by Krisanda and Paul

(156). They showed that this transient was reduced significantly when the muscle was activated at lengths at which little active force was developed. Whereas isometric force increases monotonically to a maintained maximal value after stimulation, many processes decline from maximal values during the steady phase of force maintenance. These include JATP , Vmax , myosin phosphorylation, as we have seen phosphorylase a activity (158, 159), and, importantly, intracellular Ca2⫹ concentration (158–160). An understanding of the role of Ca2⫹ is clearly central. At high extracellular Ca2⫹ concentrations (5–7.5 애M), both Vmax and energy utilization are increased to a greater degree than isometric force (8, 161). Under these nonphysiological conditions, the linear relation between force and JO2 was not invariant, and the tension cost increased at these high Ca2⫹ levels. The implication of these studies was that crossbridge number and cycle rate could be regulated independently. A variable and regulatable holding economy in vascular smooth muscle was reported by Peterson (78). He showed that the addition of histamine to hog carotid arteries already stimulated with high K⫹ leads to an approximate 25% increase in the tension cost of stable isometric force, even when compared at identical levels of isometric tension. Data from Aksoy et al. (149) indicate that in the steady phase of isometric contraction, myosin light chain phosphorylation with high K⫹ as the activator is only about 18% of the total myosin. With histamine as the activator, however, the more or less stable level of myosin phosphorylation is around 45%. If the ‘‘latch’’ model is correct, these extra, more rapidly cycling crossbridges and/or the futile cycle of myosin light chain phosphorylation/dephosphorylation (96) due to histamine activation could be the source of the excess tension cost in Peterson’s experiments. While the change in Vmax correlated well with myosin phosphorylation in porcine carotid artery (149), changes in energy utilization and Vmax at high Ca2⫹ were not correlated with myosin phosphorylation in guinea pig taenia coli (5). What is certain is that crossbridge number and cycle rate can be modulated independently in both striated and smooth muscle. Thus the simple notion that muscle stiffness, ATPase, and force development are all simply more or less equivalent measures of the number of activated myosin crossbridges is no longer tenable.

XII. ENERGETICS OF WORK-PRODUCING CONTRACTIONS: EFFICIENCY ATP utilization under conditions in which the muscle is producing work is of particular interest to those con-

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cerned with muscle energetics. Under conditions of maximum work production, the rate of ATP breakdown is greater in skeletal muscle by up to threefold over the isometric rate (22). The increase in energy utilization in work-producing contractions over that observed under isometric conditions is often referred to as the Fenn effect. Similar studies on taenia coli (162) also show that ATP breakdown increases by as much as 2.7 times over the isometric rate when actively shortening at loads less than isometric. In vascular tissue, however, the rate of ATP estimated from measurements of O2 consumption was not significantly different from the isometric rate when allowed to contract against a load (72) or in isovelocity contractions (163). Chemical measurements of phosphagen breakdown during contractions under conditions of maximal power output in hog carotid artery (164) indicate an increase in JATP of about threefold over the isometric rate. The efficiency of muscle during work-producing contractions, i.e., the work produced per unit free energy change, is an energetic parameter closely tied to our understanding of the mechanism of mechanochemical transduction. Although the tension cost in smooth muscle is substantially less than that of skeletal muscle, this does not appear to be the case in terms of efficiency. Butler et al. (161) reported that for working contractions in rabbit taenia coli, about 4 kJ of work was produced per mole of high-energy phosphagen breakdown. Krisanda and Paul (164) reported similar findings for hog carotid artery. Estimates of the free energy of ATP hydrolysis range from 35 to 50 kJ/mol, which yield an efficiency of about 10% for work production in smooth muscle. This is about four- to fivefold lower than in skeletal muscle (22). In both of these studies, a Fenn effect, as in skeletal muscle, was reported. The effects of calcium on the efficiency of hog carotid artery were reported (165) for contractions in which work was performed after the attainment of a steady state. At a low calcium concentration (0.15 mM), unloaded shortening velocity and isometric force were lower than that observed at 2.5 mM, as reported previously. No Fenn effect was found at the low calcium concentration. However, and perhaps more important, little change in the efficiency was seen in comparison to the contraction at 2.5 mM calcium or when compared to contractions elicited by both KCl and histamine, conditions designed to maximize intracellular calcium levels. It is of interest, in terms of mechanisms of regulation, to know whether the lower velocities and force at low calcium were caused by an ‘‘internal load’’ (135) or were related to a direct effect of calcium on the crossbridge cycle rate. A constant internal load would be expected to decrease the efficiency of work-producing contractions.

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These results thus suggest that the decrease in velocity observed at low extracellular concentrations is not likely to be due to the presence of such an ‘‘internal load.’’ This would appear to rule out models in which slowly or noncycling dephosphorylated ‘‘latch’’ bridges were thought to pose such a load. Butler et al. (157) also presented data that indicated that under conditions designed to optimize the effect of an internal load, no change in efficiency was detected in rabbit taenia coli. Thus it is likely that the low efficiency of smooth muscle and the dependence of unloaded shortening velocity on calcium, potentially mediated through myosin light chain phosphorylation, are not due to the presence of an internal load. Clearly, more work needs to be done before the role of these effectors on smooth muscle efficiency can be stated with certainty. This is an interesting example of how muscle energetics can play a unique role in distinguishing between various theories of the regulation of smooth muscle contractility.

XIII. ENERGETIC CONSEQUENCES AND TESTS OF ‘‘LATCH’’ MODELS The concept of a modulable crossbridge cycle rate was embodied in terms of a kinetic model proposed by Driska (166) and Hai and Murphy (154). These fourstate models permitted quantitative tests of the validity of this type of latch model. In these models, phosphorylation of myosin light chains (MLC-Pi) is necessary for actin–myosin interaction. A novel aspect is that dephosphorylation of an attached crossbridge was proposed to decrease the rate of detachment, leading to the formation of a ‘‘latch’’ bridge. This model can fit available data on the time courses of MLC-Pi and isometric force (154), as well as predicting a near-linear dependence of velocity on MLC-Pi (153), an observation consistent with some, but not all (5), studies of smooth muscle mechanics. The high economy of tension maintenance and relatively lower efficiency of smooth muscle, described in the previous sections, could also be explained by these kinetic models. The high economy was partly ascribable to the lower cycling of crossbridges in the latch state, whereas the relatively lower efficiency had a rather different and controversial explanation (151). Hai and Murphy’s (152) model predicted that ATPase associated with the ‘‘futile’’ cycle of myosin light chain phosphorylation/dephosphorylation was the major (85%) fraction of ATP utilization by smooth muscle during contractile activity. Consequently, the work per total ATP hydrolysis might be expected to be lower than skeletal muscle (152). This latter consequence of their model conflicted with the more traditional view of smooth muscle energetics

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(discussed earlier) in which most (앑80%) of the contractile energy utilization was assigned to crossbridge ATPase. This assignment is based on the dependence of energy utilization on muscle length and is subject to the limitations discussed earlier. In particular, if myosin light chain phosphorylation/dephosphorylation ATP utilization is a type of activation energy, is it dependent on length? However, studies of heat production in skinned fibers (167, 168) would suggest that this futile cycle accounts for less than 15% of the total energy usage. A more readily testable prediction of this model was the dependence of rate contractile ATP utilization as a function of the steady-state level of isometric force generated. The kinetic model of Hai and Murphy (154) was tested by Paul (169, 170), and the predicted relations were highly nonlinear and not compatible with data shown in Figs. 1 and 3. Using other kinetic constants, Paul reported that several different models could fit the force and MLC-Pi time course data, including those with a lower fraction of ATP utilization attributed to myosin light chain phosphorylation/dephosphorylation. However, all were quite nonlinear in terms of the relation between ATP utilization and force. A model with a high crossbridge attachment/detachment rate ratio (Fig. 4), without invoking latch, could fit both sets of data. Hai and Murphy (150) combined the original fourstate model with the Huxley model (171), which incorporates crossbridge position and permits explicit velocity dependencies to be predicted. In this model, about 50% of total ATP utilization was attributable to myosin light chain phosphorylation/dephosphorylation. Although the relationship between ATP utilization and force is curvilinear, there is a region in which ATP utilization is quasi-linear with force. In this model, ATP utilization would be approximately proportional to MLC-Pi. As force is maximized with MLC-Pi at about 30%, it is possible to significantly increase ATP utilization at nearly constant levels of force by increasing MLC-Pi. This could explain the observed nonlinearities at high levels of Ca2⫹ (80–82) discussed earlier. Wingard et al. (95, 96) tested this hypothesis by measuring JO2 , force, and MLC-Pi. They isolated tension-dependent and tension-independent components of JO2 utilizing the force–length relation. It is assumed that the loss of force at long muscle lengths is a simple consequence of a reduction in overlap of thick and thin filaments. At the steady-state levels of MLC-Pi associated with KCl stimulation (앑25%), the magnitude of the tension-independent JO2 was similar to that reported previously and constituted 앑20% of the suprabasal JO2 as discussed in Section XI. However, when MLC-Pi was increased to

50% using a combination of histamine, KCl, and Tris buffer, tension-independent JO2 was similarly increased. These results indicate that a significant fraction of tension-independent JO2 may be associated with myosin light chain phosphorylation/dephosphorylation cycles. Moreover, during the initial rise in tension in intact smooth muscle, the level of MLC-Pi is high, suggesting that the contribution of this futile cycle to the energy cost of contraction during this phase may account for the initially high-energy cost associated with tension development.

XIV. SUMMARY VSM is well designed for its function of slow, sustained contractions for the maintenance of blood pressure and the regulation of flow. The high efficiency of contraction and the high degree of organization of metabolism achieve a tight and specific coupling of metabolism to cell function. Although qualitatively variable among different vascular smooth muscles, the general strategies of energy provision and contractile mechanisms outlined earlier may be applicable to all smooth muscle.

Acknowledgments The authors express their indebtedness to Dr. John W. Peterson for his major contributions to previous editions of this chapter.

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34 Adrenergic Receptors in the Cardiovascular System JON W. LOMASNEY

LEE F. ALLEN

Department of Pathology Northwestern University Medical School Chicago, Illinois 60611

Department of Early Clinical Research Pfizer, Inc. Groton, Connecticut 06340

I. INTRODUCTION

monal regulation of critical cellular processes. The physiological role of ARs in the cardiovascular system is then described in part by examining the in vivo modification of adrenergic signaling pathways via the genetic manipulation of mice. Finally, because it has been estimated that approximately 60–70% of all pharmaceuticals target G-protein-coupled receptors, and many of these target ARs specifically, the role of adrenergic signaling in the pathophysiology of human cardiovascular disease and the implications for therapeutic intervention will be reviewed.

Receptors for the primary adrenal medullary hormone and central neurotransmitter, epinephrine (adrenaline), and the primary sympathetic neurotransmitter, norepinephrine (noradrenaline), mediate various key cellular functions that play a direct role in the control of blood pressure, myocardial contractile rate (chronotropy), myocardial force (inotropy), myocardial relaxation (Iusitropism), airway reactivity, and lipolysis. These receptors, termed adrenergic receptors (ARs), are part of a larger superfamily of integral transmembrane proteins that mediate their effects through interaction with specific guanine nucleotide regulatory proteins (G-proteins); these G-proteins in turn link receptor activation to various other downstream effector molecules. A majority of hormones, neurotransmitters, and autocrine and paracrine factors elicit their physiologic effects by binding to such G-protein-coupled receptors. There are literally thousands of different receptors and at least hundreds of different ligands that can bind to these receptors, thereby regulating important cellular processes. The primary purpose of the receptor is to discriminate among the various extracellular stimuli. The presence of G-protein-coupled receptors in evolutionarily divergent organisms such as slime mold, yeast, and human and the high degree of conservation in their primary structure indicate the critical role of these receptors and their signal transduction pathways in biology. This chapter first discusses the molecular characterization of ARs in order to provide the reader with some insight into the molecular events involved in the hor-

Heart Physiology and Pathophysiology, Fourth Edition

II. MOLECULAR CHARACTERIZATION A. Pharmacology Pharmacological subdivision of the ARs into distinct groups was first proposed by Ahlquist in 1948. 움 and 웁 receptors were distinguished based on differences in the rank order of potency of six sympathomimetic amines for adrenergic responses in various tissues. In the late 1960s and early 1970s, additional groups comparing agonist and antagonist rank order of potencies further concluded that there were in fact two subtypes of each of these two receptors: 웁-ARs included 웁1- and 웁2-ARs and 움-ARs included 움1- and 움2-ARs (Dubocovich and Langer, 1974; Berthelsen and Pettinger, 1977). For many years, the identification and classification of receptors remained primarily dependent on their pharmacologic characteristics. Initially, Ahlquist and others examined the ability of various endogenous hormones and later other naturally occurring or synthetic compounds to

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regulate various physiological processes. A compound that elicited a physiological response was termed an agonist, whereas a compound that blocked the agonistinduced response was termed an antagonist. With the development of techniques to radioactively label agonists and antagonists, investigators were then in a position to actually determine the binding of different compounds to specific receptors and quantitate the density of these specific receptors in individual tissues. In addition, a ligand that bound to a specific receptor with high affinity and specificity could then be used to help purify these very rare integral membrane proteins to homogeneity. This ability to purify receptors subsequently allowed for their more extensive biochemical characterization. The advent of molecular biology during the mid 1980s led to the eventual isolation by the early 1990s of nine distinct genes that encode for the nine individual subtypes of ARs (Fig. 1). This information gave investigators the first look at the primary structure of these proteins and provided an essential tool for their further characterization. Because the primary structure was distinctive and invariant among different laboratories, some investigators suggested using structure as the primary characteristic upon which to classify receptors. Given that structure in biology determines function, this made intuitive sense. This conserved primary structure predicts that these receptors, and in fact all G-proteincoupled receptors, share a similar topological motif consisting of seven hydrophobic segments that span the lipid bilayer. The importance of receptor conformation in binding ligands and coupling to downstream signal transduction elements is discussed later. The nine individual subtypes of ARs belong to three distinct receptor families: (1) 움1-, (2) 움2-, and (3) 웁ARs. Each receptor has its own unique structural, biochemical, pharmacolgical, and physiological properties. After careful consideration by the International Union of Pharmacology (IUPHAR), the present nomenclature

FIGURE 1 Classification of the adrenergic receptor family. Nine distinct gene products are divided into three major families; 움1, 움2, and 웁.

integrates operational, structural, and signal transduction characteristics to arrive at a classification of ARs that includes the following subtypes: 움1a, 움1b, 움1d, 움2a, 움2b, 움2c, 웁1, 웁2, and 웁3 (Girdlestone, 1998). While various reports have suggested the presence of additional AR subtypes, other explanations may account for the discrepancies in receptor characterization, which have perhaps erroneously led to the suggestion that the AR family is larger than its nine current members (Link et al., 1992). Species differences and or differences in tissue-specific G-protein/effector coupling may underlie these observed variations in receptor characteristics.

B. Signaling The classical paradigm of signal transduction through membrane receptors involves agonist binding to its receptor, which in turn induces a conformational change in the tertiary structure. This conformational change increases the affinity of the receptor–ligand complex for its specific regulatory proteins, thereby activating signal transduction pathways. For ARs, heterotrimeric G-proteins serve as the regulatory proteins that link receptor activation to downstream effector molecules. These heterotrimeric G-proteins, as their name implies, consist of three subunits: (1) an 움 subunit (Mr 39–46 kDa), (2) a 웁 subunit (Mr 37 kDa), and (3) a 웂 subunit (Mr 8 kDa). To date, 20 different G움 subunits, 5 웁 subunits, and 10 웂 subunits have been isolated, which conceptually makes a very large number of distinct heterotrimeric G-protein combinations possible. The G움 subunits have themselves been subdivided into four different families: (1) Gs (움s and 움olf), (2) Gi (움g, 움il, 움i2, 움i3, 움o1, 움o2, 움t1, 움t2, and 움z), (3) Gq, (움q, 움11, 움14, 움15, and 움16), and (4) G12 (움12 and 움13). The 움 subunit of heterotrimeric G-proteins primarily determines the specificity of interaction between the Gprotein and a particular receptor. This specificity results from the intimate physical interaction of the G-protein with particular intracellular domains of the receptor, primarily the third intracellular loop and carboxy terminus. Similarly, the site of interaction on G움 subunits also appears to reside in the carboxy terminus part of the protein, specifically in the terminal 12 (⫹/⫺) amino acids. The intricacy of this interaction is further highlighted by the fact that as few as one or two key amino acids in these regions of the receptor or G-protein can determine the specificity of receptor–G-protein binding and that changes in those key residues can switch the specificity of receptor–G-protein coupling (Cotecchia et al., 1992; Hamm, 1998). The G움 subunit can also bind guanine nucleotides and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP) via its intrinsic GTPase activity.

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When GDP is bound to the heterotrimeric G-protein, the G-protein is inactive and downstream signal transduction is blocked. The agonist-occupied form of a receptor, however, promotes the exchange of GTP for GDP and hence activates the heterotrimeric G-protein and its associated signal transduction pathway. Although evidence is relatively sparse, it is presumed that the GTP-loaded G움 subunit then dissociates from the closely associated 웁웂 subunits. Despite clear evidence of subunit dissociation, both the G움 and the 웁웂 subunits have been shown to be able to independently regulate downstream effectors. The intrinsic GTPase activity of the G움 subunit also plays another very important role in turning off signal transduction and the resultant physiologic response. In recent years it has been appreciated that ARs can also interact with G-proteins and activate intracellular signaling pathways in the absence of an agonist; a phenomenon sometimes described as precoupling. Responses generated by this agonist-independent process are almost certainly not robust with physiologic levels of receptor expression, but may play an important role in determining resting or basal tone. Precoupling has been explained by an elegant model put forth by Lefkowitz and colleagues (1993) in which the receptor is hypothesized to exist in a continuum of states ranging from inactive to active. In the absence of an agonist, the majority of receptors are in the inactive conformational state and are unable to couple productively with heterotrimeric G-proteins. However, a small population of receptors exist spontaneously in an active conformation and are capable of coupling productively to G-proteins and stimulating a response even in the absence of an agonist. The role of an agonist in this model, therefore, is to shift the equilibrium between these two conformations of receptors toward the active state. This concept of a population of spontaneously active receptors has been validated further in a transgenic model system in which the overexpression of wild-type 웁2-ARs resulted in baseline cardiac parameters, i.e., heart rate and contractility and adenylate cyclase activity, that paralleled the level of agonist stimulation in control animals (Milano et al., 1994; Bond et al., 1995). Thus, by overexpressing the wild-type receptor to superphysiologic levels, the subpopulation of spontaneously active receptors become predominant physiologically (see later). Several mutations of ARs have also been described, which by themselves shift this equilibrium toward the active state by mimicking the conformational changes induced by agonists. Such receptors have been described for 움1, 움2, and 웁-ARs and have been referred to as constitutively activated receptors (Cotecchia et al., 1990; Samama et al., 1993). Such mutant receptors or receptor overexpression may play a role in the development of human

disease and have been very useful tools in assessing AR function in mice (see later). 움1-ARs can activate the effector phospholipase C via interaction with the Gq class of G움 subunits. This in turn leads to the hydrolysis of membrane inositol phospholipids and the generation of second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) (Fig. 2). IP3 is a potent stimulant of intracellular calcium concentration by causing the release of calcium from the endoplasmic reticulum. DAG activates isoforms of protein kinase C (PKC), which in turn leads to the phosphorylation and hence regulation of cellular targets. 움1-ARs have also been shown to couple to three other effector pathways, including phospholipase A2 (PLA2), phospholipase D (PLD), and calcium channels (Perez et al., 1993). 움2-ARs are primarily linked to the inhibition of adenylate cyclase activity via coupling to the Gi family of G-proteins. Adenylate cyclase generates the second messenger cAMP. Like 움1-ARs, 움2-ARs also couple to multiple downstream effectors, including, in some cases, the stimulation of PLC. All three 웁-ARs primarily couple via Gs to the stimulation of adenylate cyclase increasing the level of cAMP, which in turn leads to the phosphorylation of proteins such as phospholamban, calcium channels, and contractile proteins via cAMP-dependent protein kinase (PKA). Evidence suggests that 웁2 and 웁3-ARs may, under some conditions, also couple to Gi subunits to inhibit adenylate cyclase. This paradoxical coupling may occur in pathologic conditions such as congestive heart failure (CHF). In the cardiovascular system, 움1-ARs are an important regulator of vascular tone and hence blood pressure. They may also have effects on atrial conduction and play a role in the development of cardiac arrythmias. Some 움1-ARs antagonists can have beneficial effects on plasma liporoteins. 움2-ARs are known to participate in the control of blood pressure via central and peripheral actions and also regulate platelet function. 웁-ARs are involved in the control of heart rate (chronotropy), cardiac contractility (inotropy), and smooth muscle relaxation (especially in the lung).

C. Desensitization Agonist interaction with its receptor results in the initiation of two opposing physiologic processes: (1) receptor activation and (2) receptor desensitization (Fig. 3). Desensitization is a general regulatory process in which the intensity of a response wanes over time despite the continued presence of the stimulus. Because many pharmacologic agents targeted to the ARs show diminished effectiveness over time, i.e., tachyphylaxis,

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FIGURE 2 Signal transduction pathways of adrenergic receptors. The three major families of adrenergic receptors (ADR) couple to different guanine nucleotide regulatory proteins (G-proteins), allowing for differential effects. 움1-ARs stimulate phopspholipase C via the Gq family of G-proteins and 웁-ARs stimulate adenylyl cyclase via Gs, whereas 움2-ARs inhibit adenylyl cyclase via Gi. H, agonist; IP3, inositol trisphosphate; DAG, diacylglycerol.

mechanisms that dampen receptor responsiveness and turn off signal transduction are of significant clinical interest. Receptor desensitization is composed of several distinct, but interrelated processes that include receptor: (1) uncoupling, (2) sequestration, and (3) downregulation. Uncoupling is characterized by the loss of G-protein activation by agonist-occupied receptors. This uncoupling typically occurs rapidly (within seconds to minutes) and is a consequence of phosphorylation of the receptor by specific regulatory kinases. 웁-ARs can be phosphorylated by PKA and G-protein-coupled receptor kinases (GRK), leading to the uncoupling of the receptor to Gs. Because PKA can be activated as a result of 웁-AR stimulation, PKA-mediated desensitization can, in some cases, be part of a negative feedback loop, a process termed homologous desensitization. Homologous desensitization refers to the process of receptor-specific uncoupling, i.e., receptors that are being uncoupled are the same ones binding agonist. While PKA can participate in this form of desensitization, it appears that GRKs play a more important role, as this class of unique kinases has greater specificity for the agonist-

occupied form of the receptor. Although the specificity of the six GRKs for different receptors is largely unknown, at least two isoforms, types 2 and 5, are highly expressed in cardiac tissue. Phosphorylated receptors interact with sytosolic proteins, termed 웁 arrestins, which bind to the receptor and block the receptor’s ability to interact with G-proteins. PKA can also be stimulated by agonists binding to receptors other than ARs; therefore, PKA is also part of the mechanism mediating heterologous desensitization. Heterologous desensitization refers to the process uncoupling of receptors other than the receptor to which the agonist is bound. Sequestration is typified by the loss of receptor protein from the surface of a cell. The receptor becomes internalized and is therefore unable to bind most hormones as they are typically water soluble and unable to traverse the plasma membrane passively. Lipophilic radioligands that can traverse the plasma membrane have been shown to bind to these internalized receptors, confirming the presence of sequestered receptors. These sequestered receptors remain functional and can be recycled back to the plasma membrane. In some systems,

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both PKA and GRKs have been found to influence sequestration, although receptor kinases are usually not required for sequestration to occur. Sequestration may also serve as a mechanism by which function is restored to desensitized receptors by facilitating their transport to endosomes where they undergo dephosphorylation. The complete disappearance of receptors from the cell is indicative of the process of downregulation. Downregulation is typically a much slower process than uncoupling and accounts for a larger percentage of receptor desensitization after a prolonged exposure to an agonist. A number of different molecular processes can lead to downregulation. These include receptor degradation by proteolysis and decreased receptor synthesis due to decreases in mRNA synthesis and/or mRNA half-life.

III. ANIMAL STUDIES

FIGURE 3 Adrenergic receptor activation/inactivation. Agonist binding to the 웁2-adrenergic receptor initiates two concurrent processes, one of which results in effector activation, whereas the other results in inactivation. The latter process is referred to as desensitization or tachyphalaxis and is defined as the waning of a response over time. A family of receptor-specific kinases, termed G-protein receptor kinases (GRKs), play an important role in mediating receptor desensitization. The 웁-adrenergic receptor kinase (웁ARK) is an example of a GRK that phosphorylates the agonist-occupied form of 웁-AR, promoting the interaction with another regulatory protein, 웁-arrestin, resulting in uncoupling from the G-protein. AC, adenylyl cyclase; EPI, epinephrine; 움, 웁, and 웂, G-protein subunits.

The ability to manipulate the in vivo expression of ARs in mice has greatly expanded our understanding of the role of specific ARs in mediating the physiological effects of catecholamines. Through both overexpression (transgenic) and loss-of-function (knockout) animal models, the role of ARs in cardiac hypertrophy and the regulation of peripheral vascular tone, blood pressure, and cardiac chronotropy and inotropy have been defined further. In part because adult cardiomyocytes cannot proliferate, hypertrophy is a common adaptive response in the failing heart. Biochemical features of the hypertrophic response include an increase in contractile proteins and the reexpression of embryonic genes, e.g., those coding for atrial naturietic factor (ANF). Cellular signals that lead to cardiac hypertrophy and eventual cardiac failure are now beginning to be elucidated. Many studies point to a common final pathway for cardiac hypertrophy that involves the activation of PLC isoforms. Studies using cultured neonatal myocytes have shown that agonists that stimulate PLC, such as phenylephrine, angiotensin II, endothelin, and prostaglandins, induce cellular hypertrophy (Shubeita et al., 1990; Knowlton et al., 1993; Sadoshima et al., 1993; Adams et al., 1996). In neonatal rat ventricular myocytes, 움1-AR agonists lead to hypertrophy through a signaling cascade that involves Ras and the mitogen-activated protein kinase-kinase (MEKK) and c-Jun NH2-terminal kinase (JNK). (Ramirez et al., 1997) More direct evidence for PLC-induced cardiac hypertrophy comes from several transgenic studies. One such study expressed a constitutively active 움1b-AR in mice using the cardiac-specific murine 움 myosin heavy chain promoter (Milano et al., 1994). 움1b-

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ARs are linked to the stimulation of PLC by the guanine nucleotide regulatory protein, G 움q. These mice demonstrated cardiac-specific 움1b-AR expression with a resultant activation of PLC (a 78% increase in diacylglycerol compared to controls). They also developed cardiac hypertrophy as assessed by an increased overall heart weight (20%), increased myocyte cross-sectional area (62%), and induction of ANF expression (fourfold). Importantly, there was no increase in blood pressure in these transgenic animals, excluding increased afterload as the mechanism for the cardiac hypertrophy. In a second study by D’Angelo et al. (1997), transgenic mice were created that express the G-protein 움 subunit G움q. These transgenic mice also developed cardiac hypertrophy and, in some cases, exhibited biventricular failure. The severity of the phenotype appeared to correlated with the gene dosage of G움q. Echocardiography and in vivo cardiac hemodynamic studies revealed impaired intrinsic contractility manifested as decreased fractional shortening (19% vs 41%), dP/dt max, a negative force frequency response, an altered Starling curve, and blunted contractile responses to adrenergic inotropes (D’Angelo et al., 1997). Taken together, these studies suggest that PLC-mediated signal transduction may be responsible for both compensated and decompensated human cardiac hypertrophy. In the first year of medical school, students are taught that mean arterial pressure (MAP) is the result of cardiac output (CO) ⫻ total peripheral resistance (TPR). ARs are the primary modulators of both CO and TPR. While the distribution of ARs varies among different blood vessels in the body, and indeed the distribution even varies according to the caliber of an individual vessel, 움1-ARs are commonly expressed in the media of large, medium, and small arteries. Because 움1-ARs are coupled primarily to the stimulation of PLC that leads to the mobilization of intracellular calcium, an important regulator of vascular smooth muscle, 움1-ARs have been an important target for therapeutics designed to reduce TPR and hence blood pressure. Indeed, 움1AR antagonists such as prazosin have long been used successfully in the treatment of essential hypertension. The critical role of 움1b-ARs in mediating vascular smooth muscle tone has been confirmed in mouse models in which the 움1b-AR was disrupted or knocked out. (Cavalli et al., 1997). Although there was no change in the basal blood pressure of these 움1b-AR null mice, the hypertensive response to 움1-AR agonists such as phenylephrine or norepinephrine was significantly blunted. The locus ceruleus is an important neural center for the control of central sympathetic tone. Axons of the more than 20,000 cell bodies of this locus are filled with norepinephrine. Presynaptic 움2-ARs play an important

role in regulating the release of NE from this site. The 움2-AR partial agonist clonidine has long been used to treat essential hypertension, presumably by acting at the locus ceruleus to lower central sympathetic tone. Receptor localization studies performed in the rat brain by in situ hybridization techniques have demonstrated the expression of 움2a-ARs, exclusive of the other two 움2-AR subtypes (Scheinin et al., 1994). Subsequent characterization of an 움2a-AR null mouse model indicated that the 움2a-AR is the major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves (Macmillan et al., 1996; Altman et al., 1999). The hypotensive effect of 움2 agonists was completely absent in 움2a-deficient mice. In contrast, 움2band 움2c-AR null mice had principally normal cardiovascular phenotypes. The mammalian heart predominantly expresses 웁1ARs, which comprise approximately 75–85% of all the 웁-ARs present in the myocardium (Brodde 1993). 웁1ARs are coupled efficiently to G-proteins in the heart, and therefore the 웁1AR subtype is sometimes referred to as the cardioselective 웁-AR. Despite this designation, 웁2-ARs can also contribute to the catecholamine-induced stimulation of inotropy and chronotropy. Proof of this hypothesis has come from an examination of transgenic mice overexpressing 웁2-ARs in their ventricles using the cardiac-specific 움-myosin heavy chain promoter. These mice express almost 200-fold more 웁-ARs than wild-type mice. Atrial tension, heart rate, and left ventricular dP/dT were all elevated in the basal state, as was basal adenylate cyclase activity (Milano et al., 1994). Interestingly, responsiveness to the 웁-AR agonist isoproterenol was blunted in these transgenic mice, presumably because there were sufficient 웁2-ARs present in the active conformation in the absence of agonist to maximally stimulate 웁-AR signaling pathways in control animals. Characterization of this mouse model demonstrated the importance of 웁-AR expression in regulating myocardial function. In some pathological states, such as CHF, 웁-AR expression (especially that of 웁1-ARs) is reduced, as are 웁-AR responses. It is not presently known whether a decreased 웁-AR function is causative of CHF or an adaptive response of the failing heart. Decreased 웁-AR responsiveness could have a protective effect on the heart by providing a mechanism for energy conservation. This is especially critical in CHF where the heart already has decreased myocardial stores of high-energy phosphates, such as creatine phosphate. In fact, 웁-AR antagonists have been shown to improve left ventricular function and survival in patients with CHF (Bristow, 1997; Doughty et al., 1997; Doughty and Sharpe, 1997). The dilated and failing heart performs stroke work inefficiently. From the law of Laplace, left ventricular dilation increases the tension and hence the

34. Adrenergic Receptors

amount of work that occurs during contraction. Hence, for any given intraventricular pressure, the tension in a typical dilated heart can be twice as normal. Additionally, because the dilated heart undergoes minimal wall shortening. this increased pressure is not reduced significantly at end systole (앑10%) in stark contrast to the normal heart where ventricular wall tension is reduced by 40% at end systole (Katz 1992). Disruption of 웁1-ARs in mice has been shown to have deleterious effects on survival, with 90% of 웁1AR null mice dying between embryonic days 10.5 and 18.5; increased survival was demonstrated in outbred strains of mice. In surviving mice homozygous for 웁1AR disruption, 웁-agonists completely failed to stimulate heart rate or contractility, even though 웁2-ARs were expressed in the hearts of these animals. This demonstrates the important selectivity of 웁1-ARs for modulating cardiac inotropy and contractility (Rohrer et al., 1996). The failure of 웁2-ARs to regulate cardiac function is somewhat surprising given the dramatic phenotype of animals overexpressing 웁2-ARs and points to the difficulties in interpreting physiological results from animals in which genes are either overexpressed or disrupted. Overexpression of receptors may result in their aberrant coupling to signal transduction pathways to which they normally are not linked because of the increased concentration of receptor protein. Interactions between receptors and G-proteins are driven, like all other interactions between two molecules, by concentration. By increasing the concentration of one of the interacting proteins, it is possible to drive what is normally an unfavorable interaction. Likewise, mice can adapt to gene disruptions in a variety of ways, e.g., by activating redundant or alternate pathways or by modifying the complex network of signal transduction pathways. Indeed, 웁2-AR signaling in 웁1-AR null mice is not normal. There is a reduction in both the 웁2-AR number and the specificity of receptor–G-protein coupling, with the remaining 웁2-ARs coupled aberrantly to Gi (inhibiting adenylate cyclase) instead of their normal coupling to Gs (which would have resulted in the stimulation of cAMP). Study of 웁2-AR knock-out and 웁1/웁2-AR double knock-out animal models seems to confirm the cardioselective effect of 웁1-AR in the mouse heart. Targeted disruption of the 웁2-AR gene leads to mice in which the resting heart rate, the blood pressure, and the chronotropic response to the 웁-AR agonist isoproterenol are normal (Chruscinski et al., 1999). Hypotensive responses to 웁 agonists were also blunted, indicating a role of 웁2-AR in smooth muscle relaxation. As would be expected, 웁-AR agonists were unable to stimulate the heart rate or vascular relaxation in 웁1/웁2 knockout animals (Rohrer et al., 1999). These studies clearly dem-

605

onstrate the importance of 웁1- and 웁2-ARs in the regulation of myocardial function. As has been discussed previously, regulatory kinases such as PKA and GRKs play an important role in AR function. Transgenic mice have been generated that overexpress the two predominant cardiac isoforms of GRK in a cardiac-specific manner: GRK2 and GRK5. Homogenates from the hearts of transgenic mice overexpressing GRK2 and GRK5 showed increased mRNA for GRKs and evidence of an increase in kinase activity as assessed by in vitro phosphorylaton on rhodopsin (Koch et al., 1995; Rockman et al., 1996). Adenylate cyclase activity at baseline and after stimulation with 웁AR agonists was reduced. 웁-AR agonist stimulation of inotropy and chronotropy was diminished. In contrast, transgenic mice that overexpress a peptide inhibitor of GRK2 showed enhanced contractility at rest and normal responsiveness to 웁-AR agonists. It has been demonstrated that the GRK inhibitor can even restore normal 웁-AR responses to transgenic mice overexpressing GRK2 (Akhter et al., 1999).

IV. CLINICAL RELEVANCE OF ADRENERGIC SIGNALING IN THE CARDIOVASCULAR SYSTEM Alterations of the 웁-AR signaling system have been well documented in human CHF. These alterations include a downregulation of 웁-AR (앑50%), desensitization of the remaining 웁-AR, elevation of GRK levels (approximately threefold), and an elevation of basal catecholamine levels (Bristow et al., 1982; Ungerer et al., 1994). These processes lead to an overall blunting of 웁-AR agonist stimulation. Activation of the sympathetic nervous system is a characteristic feature of CHF. Plasma norepinephrine and renin activity are increased and serve as a prognostic factor (Cohn et al., 1984). Elevated catecholamines almost certainly must contribute to the altered 웁-AR signaling by virtue of their ability to desensitize the receptors. The ability of NE and EPI to activate GRKs may play a paramount role in the desensitization. Characterization of mice that lack endogenous NE and EPI supports the role of elevated catecholamines, causing desensitization in human CHF. Homologous disruption of the enzyme 웁-hydroxylase, an enzyme needed to convert dopamine to NE, leads to mice that lack the ability to generate NE or EPI. These mice demonstrated an increase in cardiac contractility, coupled with a decrease in both the level and the activity of GRK2 (Cho et al., 1999). Because the genetic manipulation of 웁-AR signaling in mice has dramatic effects on cardiac function, one could propose manipulating 웁-AR signaling in humans to alleviate some of the symptomatology of CHF. One

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of the more common methods to modulate gene expression in vivo is to infect cells using adenoviral-mediated gene transfer. This method has several drawbacks, primary among them the development of an immune response directed against the virus. The immune response not only can lead to a significant myocarditis, but it also tends to lessen the time during which the transgene is expressed. Despite these drawbacks, the adenovirus has been used successfully to transfer genes to the myocardium, and advances in vector design have reduced the immune response. The recombinant adeno-associated virus (rAAV) is one such advanced vector. rAAV differs from the traditional replication-defective adenovirus (RDAd), as it does not encode viral proteins and hence does not incite a robust immune response. Svensson et al. (1999) reported using rAAV to express the reporter gene 웁-galactosidase (웁-gal) in the myocardium of mice after both intramyocardial and intracoronary injection. Hearts examined at 2, 4, and 8 weeks after direct intramuscular injection demonstrated expression of the 웁-gal in a large number of cardiomyocyates. Little 웁-gal expression was detected 2 weeks after intracoronary injection, although 50% of cardiomyocytes were found to be expressing 웁-gal at 4 and 8 weeks postinjection. Several groups have addressed the ability of various adenoviral-mediated gene transfer constructs to regulate 웁-AR signaling. Drazner et al. (1997) have used a RDAd vector to infect cultured adult rabbit ventricular myocytes with the 웁2-AR and an inhibitor of GRK 2 and 3. Cardiomyocytes infected with the 웁2-AR virus expressed 앑20-fold more 웁2-AR than normal. Both 웁2AR and GRK inhibitor-infected cells had a significantly increased cAMP in response to 웁 agonist stimulation. 웁 agonist-induced desensitization was inhibited in cells infected with the GRK inhibitor. This study suggested that, at least in vitro, viral-mediated perturbation of 웁AR signaling was effective. The same group took this a step further by demonstrating the enhancement of cardiac function after the in vivo delivery of 웁2-AR via an intracoronry injection of a 웁2-AR adenovirus (adeno-웁2) (Maurice et al., 1999). Injection of adeno웁2 via a catheter into the coronary arteries of rabbits resulted in a 5- to 10-fold increase in the expression of 웁2-AR in the heart. At 7 and 21 days postinjection, contractility (dP/dT max) was enhanced at rest and after stimulation with the 웁-AR agonist isoproterenol. The authors proposed that replacement of lost 웁-AR in patients with CHF may represent a novel inotropic therapy. While some groups are proposing increasing inotropic responses in patients via manipulations of the 웁-AR signaling system, such as increasing 웁-ARs or inhibiting GRKs, it seems clear from a large number of random-

ized, double-blind, placebo-controlled clinical trails that patients with CHF actually benefit from the opposite: from treatment with 웁 blockers, which should serve, at least in the short term, as an impediment to inotropic responses in the heart (Doughty and Sharpe, 1997; Packer, 1998). As has been mentioned previously, patients with CHF exist in a hypersympathetic state. This prolonged activation of the sympathetic nervous system may have adverse effects on patients with CHF. It appears that these effects are mediated by 웁1, 웁2, and 움1ARs. 움1 and 웁1-AR play a role in stimulating cardiomyocyte growth, leading to cardiac hypertrophy. Cardiac hypertrophy places undue oxidative stress on the heart and is an independent risk factor for cardiac arrythmias. 움1-AR are important regulators of vascular tone. Hypersympathetic states can lead to 움1-AR activation and an increase in peripheral vascular resistance, leading to an increase in cardiac afterload with resultant oxidative stress and cardiac hypertrophy. Patients with CHF have altered ventricular morphologies due to ventricular dilatation, hypertrophy, fibrosis, or a combination of these. The altered morphology makes the patient with CHF more prone to arrythmias. The arrythmogenic potential is increased further by elevated levels of circulating catecholamines. 웁2-AR appears to be the most important subtype for mediating this proarrythmic effect. Thirdgeneration 웁 blockers, such as Carvedilol, which block the effects of 웁1, 웁2, and 움1-ARs, seem to offer the most therapeutic benefit to patients with CHF (Packer et al., 1996). Several clinical trials have examined the effect of Carvedilol on morbidity and mortality in patients with CHF. All the trials have demonstrated the ability of Carvedilol to limit the progression of CHF (26–49% reduction of the risk of disease progression) and to dramatically decrease (up to 73%) the all-cause mortality rate over the 15- to 24-month treatment course (Packer, 1998).

V. SUMMARY Adrenergic receptors mediate important cardiovascular effects, including regulation of blood pressure, myocardial contractile rate (chronotropy), myocardial force (inotropy), and myocardial relaxation (lusitropism). Hormone or agonist binding to members of the three families of adrenergic receptor 움1, 움2, and 웁 initiates a cascade of molecular events, which includes coupling of the receptor with a heterotrimeric guanine nucleotide regulatory protein (G-protein) and subsequent interaction of the G-protein with various effector molecules. This signaling pathway is itself regulated dynamically, resulting in a dampening of agonist responses over time; a process referred to as desensitization. A family

34. Adrenergic Receptors

of G-protein receptor kinases play an important role in uncoupling the receptor from the G-protein, resulting in desensitization. The generation of mice that overexpress (transgenic) or disrupt (knockout) components of the AR signaling pathway has expanded our ability to probe the exact roles of the nine different ARs in mediating the cardiovascular effects of catecholamines. It appears that 움1AR plays an important role in stimulating cardiac hypertrophy and in regulating vascular smooth muscle tone. 움2-AR primarily regulates central sympathetic tone. 웁1AR primarily regulates cardiac chronotropy and inotropy, although there may be some contribution from 웁2 as well. Congestive heart failure is characterized by increased amounts of circulating catecholamines and by increased sympathetic tone, yet patients also have blunted responses to catecholamines. While gene therapy using adenovirus has been successful in increasing contractility and inotropy at rest and in response to 웁-agonist rabbits, it is unclear whether this novel approach to boost intropy may benefit patients with CHF, as longterm treatment of these patients with 웁 blockers decreases morbidity and mortality.

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Packer, M. (1998). Beta-adrenergic blockade in chronic heart failure: principles, progress, and practice. Prog. Cardiovasc. Dis. 48(1 Suppl 1), 39–52. Packer, M. (1998). Do beta-blockers prolong survival in chronic heart failure? A review of the experimental and clinical evidence. Eur. Heart J. 19(Suppl. B), B40–B46. Packer, M., Colucci, W. S., et al. (1996). Double-blind, placebo-controlled study of the effects of carvedilol in patients with moderate to severe heart failure: The PRECISE Trial. Circulation 94(11), 2793–2799. Perez, D. M., De Young, M. B., et al. (1993). Coupling of expressed 움1B⫺ and 움1D⫺ adrenergic receptors to multiple signaling pathways is both G protein and cell type specific. Mol. Pharmacol. 44, 784–795. Ramirez, M. T., Sah, V. P., et al. (1997). The MEKK-JNK pathway is stimulated by alpha1-adrenergic receptor and ras activation and is associated with in vitro and in vivo cardiac hypertrophy. J. Biol. Chem. 272, 14057–14061. Rockman, H. A., Choi, D. J., et al. (1996). Receptor-specific in vivo desensitization by the G protein- coupled receptor kinase-5 in transgenic mice. Proc. Natl. Acad. Sci. USA 93, 9954–9959. Rohrer, D. K., Chruscinski, A., et al. (1999). Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors. J. Biol. Chem. 274(24), 16701–16708.

Rohrer, D. K., Desai, K. H., et al. (1996). Targeted disruption of the mouse beta1-adrenergic receptor gene: Developmental and cardiovascular effects. Proc. Natl. Acad. Sci. USA 93(14), 7375– 7380. Sadoshima, J., Xu, Y., et al. (1993). Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 75, 977–984. Samama, P., Cotecchia, S., et al. (1993). A mutation-induced activated state of the beta 2-adrenergic receptor: Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636. Scheinin, M., Lomasney, J. W., et al. (1994). Distribution of alpha 2adrenergic receptor subtype gene expression in rat brain. Brain Res. 133–149. Shubeita, H. E., McDonough, P. M., et al. (1990). Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes: A paracrine mechanism for myocardial cell hypertrophy. J. Biol. Chem. 265, 20555– 20562. Svensson, E. C., Marshall, D. J., et al. (1999). Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors. Circulation 99(2), 201–205. Ungerer, M., Parruti, G., et al. (1994). Expression of beta-arrestins and beta-adrenergic receptor kinases in the failing human heart. Circ. Res. 74(2), 206–213.

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35 Cardiac Action of Angiotensin II MASAO ENDOH Department of Pharmacology Yamagata University School of Medicine Yamagata 990-9585, Japan

I. INTRODUCTION

sation of cardiac hypertrophy (12, 13). Ang II receptors exist in cardiac tissue and on cell membranes of various types of cells, including myocardial cells, sympathetic nerve endings, fibroblasts, coronary artery smooth muscle, and endothelial cells (14–17). Two subtypes of Ang II receptors (AT1 and AT2) have been cloned (18–22). AT1 and AT2 receptors are polypeptides containing approximately 360 amino acids with a sequence homology of only 30%. The gene for the AT1 receptor is located on chromosome 3, whereas the gene for the AT2 receptor is on chromosome X (23, 24). Most cardiovascular regulation, including the positive inotropic effect, vasoconstriction, aldosterone and vasopressin secretion, gene expression and protein synthesis leading to cardiac hypertrophy, postmyocardial infarction ventricular fibrosis, and vascular remodeling, is mediated by the activation of AT1 receptors (25–29). The distribution of Ang II receptor subtypes shows a wide range of species- and tissue (atrial or ventricular cardiomyocytes and cardiac fibroblasts)-related variation. In adult rat and rabbit ventricular myocardium, approximately 50–70% of the specific binding is the AT1 subtype, whereas the remaining fraction is the AT2 subtype (30–33). Bovine ventricular myocardium contains approximately 70% AT1 and 30% AT2 receptors, whereas human ventricular myocardium has predominantly AT2 receptors (34); in human atria, 70% are AT2 receptors (35). AT1 receptors have been classified further into the AT1A subtype, which distributes preferentially in liver, kidney, heart, and vascular smooth muscle, and the AT1B subtype, which distributes in adrenal cortex and

Various types of receptor are distributed on the surface membrane of myocardial cells, which are activated by neurotransmitters, neuropeptides, autacoids, and cytokines to lead to the subsequent functional and metabolic adaptation that has to occur in response to physiological and pathophysiological stimuli to the heart. Among these regulatory mechanisms, the tissue (local) renin–angiotensin system (RAS) plays an extremely important role in the pathophysiological modulation of cardiovascular disorders, such as ischemic heart disease, cardiac hypertrophy, heart failure, and hypertension (1–5). RAS may be required for the physiological growth of fetal cells and neonatal cells, including cardiac myocytes as well (6, 7). Angiotensin II (Ang II), i.e., the biologically active principal effector converted from Ang I by angiotensin-converting enzyme (ACE) and chymase (namely in large mammalian species, including human) (8, 9), activates specific Ang II receptors on the myocardial cell membrane. A genetic epidemiologic approach reveals that deletion polymorphism in the gene for ACE is a potent risk factor for coronary heart disease (10) and that AT1 receptor gene polymorphism is associated with left ventricular (LV) hypertrophy (but not with hypertension) (11). Ang II receptors belong to a family of seven transmembrane GTP-binding (G)-protein-coupled receptors. Ang II shares Gq/11 class of G-proteins with endogenous regulatory substances, including endothelin-1, norepinephrine, and prostaglandin F2움 , that have been implicated in the development and ultimate decompen-

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hypothalamus (36–38). It has been shown that AT1A and AT1B subtype mRNAs were differentially regulated in spontaneously hypertensive rat (39). Intracellular signal transduction processes triggered by the activation of AT1 receptors involve divergent pathways, such as acceleration of phosphoinositide hydrolysis through the activation of phospholipase (PL) C웁 by Gq/11 and PLC웂 by tyrosine phosphorylation (40, 41), and activation of various enzymes, including tyrosine kinase (40–43), PLA2 and PLD, mitogen-activated protein kinase (MAPK) (44), S6 kinase, and Jak/STAT (Janus kinase/signal transducers and activators of transcription) (45), which lead to (1) immediate alteration of activity of ion channels and ion exchangers, and intracellular alkalinization; and (2) long-term regulation of gene expression and protein synthesis, which is responsible for cardiac hypertrophy and vascular and cardiac remodeling (46–49). Ang II also affects cyclic AMPmediated signal transduction and decreases cyclic AMP accumulation induced by isoproterenol through the pertussis toxin-sensitive Gi protein in neonatal rat cardiac myocytes (50, 51). Although the role of the AT2 receptor in cellular signal transduction had not been clear (52), accumulating evidence has now revealed the role of AT2 receptors in the regulation of cardiovascular cells, including growth inhibition, proapoptosis, cell differentiation, a decrease in cellular matrix in the heart, inhibition of cell proliferation (53, 54), nitric oxide (NO) production, and a decrease in the cardiac chronotropic effect (55, 56). The importance of AT2 receptor-mediated signal transduction by antagonizing the AT1-mediated response has been revealed (57). Cardiac-specific overexpression of the AT2 receptor causes attenuation of the AT1-mediated pressor and chronotropic effect in transgenic mice (58). Activation of the AT2 receptor elicits the inhibitory regulation of STAT activation by negative cross talk with the stimulation of AT1 receptors and interferon (IFN)-웂, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) in vascular smooth muscle cells (59). Experimental evidence suggesting the potential importance of cross talk of AT1 and AT2 receptors that may play a key role in the pathophysiological regulation of cardiovascular diseases, such as cardiac remodeling after myocardial infarction (60), is described later in the following section. AT3 receptors that respond specifically to Ang III to stimulate cGMP formation in differentiated neuronal cells (61) and AT4 receptors that bind specifically Ang IV with high affinity in mammalian myocardium (62) and that respond to Ang IV in cultured endothelial cells (63) and the blood–brain barrier (64) have also been reported, but they have not yet been cloned and it is

unknown whether they play any role in cardiac regulation. In this chapter, characteristics of the signal transduction processes triggered by the activation of Ang II receptors, namely AT1 receptors, that result in (1) an immediate response of ion channel and ion exchange activities leading to cardiac inotropic and chronotropic regulation and (2) a long-term response that plays a crucial role in adaptation, such as cardiac hypertrophy and pathogenesis of congestive heart failure by modulation of phenotype expression and protein synthesis will be reviewed based on pieces of experimental evidence that support or contradict the potential role of the process in cardiac regulation.

II. IMMEDIATE CARDIAC RESPONSE TO ANGIOTENSIN II In addition to the well-documented vasoconstrictor effect of Ang II, Ang II also has specific cardiac effects, such as positive inotropic, chronotropic, and arrhythmogenic effects in avian hearts (65, 66) and in mammalian hearts, including those of the cat (67, 68), rabbit, calf (69), neonatal rat (70, 71), hamster, and human (72–74). The receptor-mediated acceleration of the hydrolysis of phosphoinositide and the resultant production of inositol 1,4,5-trisphosphate (IP3), which releases Ca2⫹ from the intracellular stores, and diacylglycerol (DAG), which activates protein kinase C (PKC), play a key role in the pathway for signal transduction to lead to functional regulation. A number of endogenous transmitters and hormones, including Ang II, endothelin isopeptides (mainly endothelin-1), and catecholamines, stimulate the hydrolysis of phosphoinositide and produce a positive inotropic effect in the heart of most species. The role of this process in the cardiac contractile regulation is still controversial. Nevertheless, accumulating evidence implies that the acceleration of phosphoinositide hydrolysis by extracellular first messengers of endogenous regulators, such as Ang II, endothelin, and 움-adrenoceptor agonists, is coupled to multiple regulatory processes in the heart, which include the operation of various types of ion channel, such as Ca2⫹, K⫹, Na⫹, and Cl⫺ channels, ion transport systems that include Na⫹ –H⫹ exchange and Na⫹ –Ca2⫹ exchange, and the responsiveness to Ca2⫹ ions of contractile proteins (75–84).

A. Characteristics of the Positive Inotropic Effect of Angiotensin II Besides producing pronounced vasoconstriction, Ang II elicits a positive inotropic effect and a positive

35. Cardiac Action of Angiotensin II

chronotropic effect on the heart. The positive inotropic effect of Ang II (85), as well as that of endothelin isopeptides (86, 87) and 움-adrenoceptor agonists (83, 88) in the isolated rabbit papillary muscle, is consistently associated with a concentration-dependent negative lusitropic effect. Changes associated with isometric contractions are in strong contrast to those produced by the stimulation of 웁-adrenoceptors and are essentially similar to those induced by novel cardiotonic agents that act by an increase in myofibrillar sensitivity to Ca2⫹ ions and are termed ‘‘Ca2⫹ sensitizers’’ (89). Thus, Ang II has a positive inotropic effect, the predominant mechanism of which involves an increase in the Ca2⫹ sensitivity of myofilaments that is associated with intracellular alkalinization and a moderate or little increase in the amplitude of [Ca2⫹]i (90–93) (Fig. 1). Endothelin, as well as 움-adrenoceptor agonists, has also been shown to increase myofibrillar Ca2⫹ sensitivity in mammalian ventricular myocardium, including rabbit (88, 89), ferret (94), and rat (95, 96). Thus, an increase in Ca2⫹ sensitivity of myofilaments appears to be mediated by a common regulatory process that is generally triggered by the receptor agonists that stimulate the hydrolysis of phosphoinositide in cardiac muscle. The mechanism responsible for the increase in myofibrillar responsiveness to Ca2⫹ ions caused by these receptor agonists, however, remains still equivocal and controversial. It is postulated that three signal transduction processes may potentially contribute to the regulation: (1) an intracellular alkalinization induced by activation of the Na⫹ –H⫹ exchange; (2) the phosphorylation of contractile proteins, namely troponin subunits; and (3) a decrease in intracellular inorganic phosphate (Pi). Pieces of evidence accumulated so far support or contradict the role of intracellular alkalinization, as will be discussed in detail in the section on Na⫹ –H⫹ exchange. As concerned with the phosphorylation of contractile proteins, it has long been known that PKC phosphorylates both troponin I and troponin T in vitro (97, 98). Because the PKC-induced phosphorylation of cardiac troponin I and troponin T is associated with an inhibition of the Ca2⫹-stimulated actomyosin Mg-ATPase activity (99), it has been proposed that such phosphorylation might be related to the negative inotropic effect induced by externally applied phorbol esters that activate PKC in cardiac muscles from various experimental animals, including chick and mammals (100–105). Edes and Kranias (106) failed to detect the receptor-mediated phosphorylation of troponin subunits in the perfused guinea pig heart in vivo. However, Talosi and Kranias (107) demonstrated that cardiac 움-adrenoceptor stimulation increased the phosphorylation of myocardial sar-

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FIGURE 1 Effects of Ang II on Ca2⫹ transients and cell shortening in a rabbit ventricular cardiomyocyte. (A) Effects of Ang II (0.1 애M) on the indo-1 fluorescence ratio (upper tracings) and cell shortening (lower tracings) in an isolated rabbit ventricular myocyte. The myocyte loaded with indo-1/AM was stimulated electrically at 0.5 Hz in the cell chamber perfused with the Krebs–Henseleit solution (1 ml/min) equilibrated with 95% O2 and 5% CO2 at room temperature (25–27⬚C). Ang II increased the cell shortening in association with a moderate increase in the amplitude of the indo-1 ratio. (B) Indo-1 fluorescence ratio–cell shortening trajectory in the absence (control) and in the presence of Ang II (0.1 애M). The trajectory was shifted upward compared with the shift induced by the elevation of [Ca2⫹]o , an indication that Ang II increases the myofibrillar sensitivity to Ca2⫹ ions. Five successive contractions in the absence (lower left trajectory) or in the presence of Ang II (upper right trajectory) were superimposed.

colemmal and cytosolic proteins by activation of PKC. It is unknown whether Ang II increases the phosphorylation state of cardiac contractile proteins. It is noteworthy that phorbol esters at low concentrations have a positive inotropic effect on the cardiac muscle of certain species (108–111). In cultured neonatal rat cardiomyocytes the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) mimicked the effects of Ang II to increase the phosphorylation state of a discrete set of proteins, with apparent molecular masses of 32 and 83 kDa, and to increase L-type Ca2⫹ channel activity (102). Clearly, the physiological significance of the PKC-

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induced phosphorylation of contractile proteins requires further clarification in future studies. It has been shown that Ang II is capable of decreasing total intracellular Pi in the heart via two mechanisms: (i) by inhibiting the uptake of Pi into the myocyte and (ii) by increasing the efflux of Pi out of the cell in adult rat ventricular myocytes (112). Because it has been well documented that Pi decreases myofibrillar sensitivity to Ca2⫹ ions in myocardial cells, the Ang II-induced decrease in intracellular Pi might increase the Ca2⫹ sensitivity of myofibrils, but significance of this regulatory mechanism remains unexplored for future studies.

B. Species-Related Differences in the Positive Inotropic Effect of Angiotensin II One characteristic feature of the regulation of cardiac contractile function induced by membrane receptor agonists that stimulate the hydrolysis of phosphoinositide is a wide range of species-dependent variations even among mammals (89). It is controversial whether Ang II elicits a positive inotropic effect in human ventricular myocardium. Moravec and co-workers (73) have reported that Ang II increased the contractile force in human ventricular myocardium, which was less pronounced than in atrial muscle and attenuated in failing compared with normal myocardium. Holubarsch and co-workers (113, 114), however, could not detect a positive inotropic effect in human LV muscle isolated from patients with idiopathic dilated cardiomyopathy, mitral valve stenosis and incompetence, or in normal ventricular muscle and in right ventricle from infants undergoing reconstructive heart surgery for tetralogy of Fallot. It has been reported that Ang II does not elicit a positive inotropic effect in dog, ferret, guinea pig, and adult rat ventricular myocardium (33, 115). In guinea pig atrial and ventricular myocardium, there were dense Ang II-binding sites and Ang II-activated phosphoinositide hydrolysis in these tissues, but it did not elicit a positive inotropic effect in this species (66, 116). There is a report, however, that Ang II elicits a positive inotropic effect on guinea pig right and left atria through the activation of AT1 receptors (117). In cultured neonatal rat ventricular myocytes, Ang II produced a transient rise in [Ca2⫹]i followed by a significant reduction of the amplitude of Ca2⫹ transients (71). In atria isolated from 2-month-old rats, Ang II produced a 17–19% increase in contractile force that was abolished by the AT1 receptor antagonist losartan (118). It has been reported in isolated perfused rat hearts, however, that Ang II decreased diastolic and systolic [Ca2⫹]i and the amplitude of Ca2⫹ transients, thereby depressing cardiac function (119). These observations indicate that various factors, including differences in the stage of development, species, cardiac

regions, and experimental conditions, may play an important role in the detection of inotropic effects of Ang II. 1. Factors Affecting the Variation of Angiotensin IIInduced Inotropic Effects The inotropic effects of Ang II resemble those of 움1agonists and endothelin in so far as the induction of a positive inotropic effect by Ang II is markedly dependent on a variety of modulatory factors, which include location (atrial or ventricular), species, presence or absence of endothelium (120), and conditions of loading to cardiac muscle (121). Rabbit cardiac muscle contains receptors with high affinity for Ang II (15, 122) and responds consistently to Ang II with a pronounced positive inotropic effect (33, 66, 85, 123). It has been reported that Ang II had no significant inotropic effect on isolated cardiac myocyte preparations from guinea pig ventricle, normal and infarcted rat ventricle, and human ventricle and atrium (124), whereas other studies on multicellular preparations of human atrium (but not in ventricular muscle) elicited a definite positive inotropic effect (72, 74, 113, 114, 125). These findings indicate that the inotropic effect of Ang II in single cardiomyocytes may be different from those in multicellular preparations and that Ang II produces a more consistent positive inotropic effect on atrial than ventricular myocardium in most species, including rats (126, 127) and humans (113, 114). Furthermore, Ang II produced a prominent and concentration-dependent positive chronotropic and inotropic effect in blood-perfused dog heart preparations (128) and the pithed rat (129), which implies that the blood-perfused state is favorable for inducing the positive inotropic effect of Ang II. While endothelial cells affect the response of multicellular preparations, it has been shown in isolated cat papillary muscles that the positive inotropic response to Ang II did not necessitate the presence of an intact endocardial endothelium (120). Li and co-workers (121) showed that the inotropic effect of Ang II is dependent on the extent of the preload that determines the sarcomere length in rat ventricular muscles: Ang II administration at Lmax produced a 12% depression of developed tension in papillary muscle from noninfarcted ventricles and a 37% decrease in the viable myocardium of infarcted rats; at 85 and 92.5% Lmax , in contrast, Ang II elicited a positive inotropic effect. In LV cardiac myocytes isolated from pacing-induced heart failure dog, Ang II produced a negative inotropic effect, whereas in myocytes before congestive heart failure, Ang II produced a slight positive inotropic effect, an indication that the inotropic response to Ang II may

35. Cardiac Action of Angiotensin II

be modified by factors that are altered in the course of development of heart failure (130). The regulation of cardiac function mediated by Ang II and endothelin is altered depending on the age of animals. The inotropic effect of Ang II and endothelin that is positive inotropic in the neonate is converted to a negative inotropic effect in adult mice, which implies developmental changes in regulation (131). The number of Ang II- and endothelin-specific binding sites in rat hearts decreases with age (132, 133). Touyz and coworkers (115) showed that Ang II and endothelin-1 increased Ca2⫹ transients in neonatal rat atrial and ventricular myocytes, whereas the effect of Ang II on Ca2⫹ transients disappeared in adult myocytes. In contrast, it has been reported that endothelin-1 (10 nM) elicits a negative inotropic effect, acidification of cytoplasm, and a decrease in Ca2⫹ transients in chick embryonic and rat neonatal cardiomyocytes, effects that contrast strongly with those on the corresponding adult cardiomyocytes (134).

C. Chronotropic Effects of Angiotensin II In cardiac tissue, density of the specific binding of Ang II is higher in the conduction system, including sinoatrial node and atrioventricular nodes, than in ventricular muscle (15, 116, 135). Ang II administered intravenously produces either positive or negative chronotropic effects, mainly through neurohumoral reflex pathways (136, 137). In the absence of a neurohumoral reflex, Ang II elicits a positive chronotropic effect on the heart preparation perfused with circulating blood in the dog (128) and in the pithed rat (129, 138). In cultured neonatal rat heart myocytes, the spontaneous beating frequency of multicellular networks was increased by Ang II, which is not dependent on cAMP or inositol trisphosphate levels, but may involve sustained phosphoinositide hydrolysis (139). Ang II increased the amplitude of Ca2⫹ transients significantly, as well as the frequency of Ca2⫹ spikes in cultured rat neonatal atrial and ventricular cells, but the response to Ang II disappeared in adult myocytes, an indication that the Ca2⫹ response to Ang II may vary with cardiac development (115). In depolarized rabbit right atria, Ang II restored spontaneous mechanical and electrical activity, with the frequency of action potential discharge being concentration dependent (66). Ang II and Ang III elicited a positive chronotropic effect through the activation of AT1 receptors in rat isolated atria (138). However, the heart rate measured in awake rats after heart transplantation was not affected by Ang II infusion (140). In isolated human atrial trabeculae, Ang I and Ang II increased the rate of diastolic depolarization and spontaneous discharges significantly, which were inhibited by captopril and saralasin, respectively, an indica-

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tion that cardiac tissue contains ACE and converts Ang I to the biologically active Ang II rapidly (74). In wild mice, Ang II elicited a concentration-dependent positive chronotropic effect, which was markedly suppressed in transgenic mice with a cardiac-specific overexpression of AT2 receptors (58). These findings imply that AT2 receptors exert biological actions partly through antagonizing the facilitatory effect induced by the stimulation of AT1 receptors. In rabbit sinoatrial cells, Ang II elicited a negative chronotropic effect, probably through attenuation of the ICa current by the inhibition of PKA (141).

D. Phosphoinositide Hydrolysis and Inotropic Effects Induced by Angiotensin II A comparison of the effects mediated by the various types of receptor that result in the stimulation of phosphoinositide hydrolysis will help get an insight into the role of phosphoinositide hydrolysis in cardiac contractile regulation in greater detail, provided that the acceleration of hydrolysis of phosphoinositide plays a crucial role in the signal transduction for contractile function. Therefore, the following issues will be examined: (1) similarities and dissimilarities of regulation of contractile function in association with changes in Ca2⫹ transients induced by Ang II, endothelin, and 움-adrenoceptor agonists (phenylephrine and methoxamine); (2) the influence of available pharmacological tools, including the selective inhibitors of Na⫹ –H⫹ exchange [ethylisopropylamirolide (EIPA)], of Na⫹ –Ca2⫹ exchange (KBR1743) (142), of L-type Ca2⫹ channels (verapamil), of protein kinase C (staurosporine, NA 0345, H-7, calphostin C, and chelerythrine), and the activators of PKC, such as phorbol 12,13-dibutyrate (PDBu), phorbol 12myristate 13-acetate (PMA, the same as TPA), and 1oleyl-2-acetyl-sn-glycerol (OAG) on the receptor-mediated positive inotropic effect in intact cardiac muscle preparations. Ang II and endothelin isopeptides contribute to the pathophysiological modulation of cardiovascular disorders, whereas 움-adrenoceptor stimulation is responsible for physiological regulation (27, 78, 143, 144). The subcellular mechanism, through which the cardiac cells achieve segregation of the signal of physiological and pathological interventions via common and different intracellular pathway, is still unknown. 1. Association of Phosphoinositide Hydrolysis with Positive Inotropic Effect In rabbit ventricular slices that had been prelabeled with myo-[3H]inositol, Ang II as well as 움-adrenoceptor agonists (epinephrine, methoxamine, and phenylephrine) and endothelin isopeptides (endothelin-1 and en-

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dothelin-3) produced a concentration-dependent accumulation of [3H]IP1, [3H]IP2, and [3H]IP3 (80, 84). Accumulation of [3H]IP1 increased continuously during the period of investigation for 60 min, whereas that of [3H]IP2 or [3H]IP3 was in most cases transient, and the levels of these latter products returned to basal values at 30 min after the administration of the 움-adrenoceptor agonists, when the positive inotropic effect of these agonists had reached a steady level (145–147, 149). An excellent correlation between the positive inotropic effect and the hydrolysis of phosphoinositide (when accumulation of [3H]IP1 is used as an indicator of the hydrolysis) induced by Ang II, by 움-adrenoceptor agonists, and by endothelin-1 was found in rabbit ventricular muscle (85, 147). Saralasin, an Ang II receptor partial agonist, and losartan, a selective antagonist of AT1 receptors, but not PD 123319, a selective antagonist of AT2 receptors, antagonized the Ang II-induced positive inotropic effect and the hydrolysis of phosphoinositide (85). In the avian heart, the Ang II-induced positive inotropic effect correlates well with the hydrolysis of phosphoinositide (148). Dissociation of the Ang II-induced acceleration of the hydrolysis of phosphoinositide from cardiac contractile function has also been observed frequently under various experimental conditions and in different species of animals. For example, while the hydrolysis of phosphoinositide is clearly accelerated by Ang II in guinea pig cardiac muscle, no positive inotropic effect is apparent (116). Because the stimulation of hydrolysis of phosphoinositide is coupled to divergent signaling processes that lead to a positive and/or negative inotropic effect, the extent of coupling to respective pathways might be different depending on the species of experimental animals (78–80, 84). While IP3 has been shown to cause Ca2⫹ release from intracellular Ca2⫹ stores by activation of IP3 receptors under certain experimental conditions in cardiomyocytes, the density of IP3 receptors in myocardial cells is much lower than that of ryanodine receptors (150, 151). Furthermore, in myocardial cells, IP3 receptors are localized most densely in the intercalated disk (152). Taken together, it appears that the receptor-mediated activation of PKC in myocardial cells may play a more important role than IP3 in the regulation of E-C coupling (89).

E. PKC Activation and the Positive Inotropic Effect of Angiotensin II Activation of PKC resulting from receptor stimulation has been implicated to play an important role in the functional and genetic regulation of biological systems, including the cardiovascular system (153). It has been shown that endothelin-1 and 움-adrenoceptor agonists

produce activation and translocation of PKC from cytosolic to membrane fractions of myocardial cells. There are little data concerning the activation of PKC in relation to the positive inotropic effect of Ang II, whereas there are abundant pieces of evidence that show that PKC activation plays a crucial role in the long-term regulation of gene expression that is responsible for cardiac hypertrophy and phenotype alteration, as will be discussed in the next section. This section reviews the effects of PKC inhibitors and PKC activators on the positive inotropic effect of Ang II, mainly in intact rabbit ventricular myocardium. 1. Effects of PKC Inhibitors on Intact Cardiac Muscle Staurosporine, NA 0345, H-7, calphostin C, and chelerythrine, over a certain range of concentrations that did not affect the 웁-adrenoceptor-mediated effect, had selective inhibitory actions on the Ang II-induced positive inotropic effect, but the extent of the selective inhibition was only 20–30% of the total response at the maximum (154, 155). Interestingly, the positive inotropic effect of endothelin isopeptides and 움-adrenoceptor agonists was affected by PKC inhibitors in a similar manner (155). When these inhibitors were used at higher concentrations, the 웁-mediated positive inotropic effect was also suppressed, indicating that these inhibitors have nonspecific inhibitory action at the concentration slightly higher than the concentration that they show the selective action on the PKC-mediated effect in intact cardiac muscle (154). It is difficult, therefore, to delineate unequivocally the extent of the contribution of PKC to the positive inotropic effect of Ang II. Experimental evidence implies that the activation of PKC might require some additional processes for achievement of the full contractile regulation. The positive inotropic effect and an increase in [Ca2⫹]i evoked by Ang II in rat cardiac myocytes were inhibited effectively by PKC inhibitors, staurosporine and NPC15437 (156), whereas the effect of Ang II in rabbit papillary muscle was rather resistant to PKC inhibitors (155). In contrast to the previous findings that support the involvement of PKC in the positive inotropic effect of Ang II, endothelin, and 움-adrenoceptor agonists, the activation of PKC has been reported not to be involved in the positive inotropic effect (157) or to be responsible for the negative inotropic effect in the rat ventricular myocardium (158). 2. Effects of PKC Activators on Intact Cardiac Muscle In contrast to our expectation that phorbol esters, such as PDBu and TPA (PMA), and OAG that activate

35. Cardiac Action of Angiotensin II

PKC in vitro may mimic the effect of receptor activation that stimulates the hydrolysis of phosphoinositide, they had a selective inhibitory action on the positive inotropic effect of Ang II, which was more pronounced than the inhibitors of PKC, in the rabbit papillary muscle. The positive inotropic effects of Ang II, endothelin, and 움-adrenoceptor agonists are consistently inhibited by tumor-promoting phorbol esters and OAG (155). In rabbit papillary muscle, PDBu at 3 ⫻ 10⫺8 and 10⫺7 M did not affect the positive inotropic effects of isoproterenol or Bay k 8644, but it abolished the positive inotropic effect of Ang II (33, 85–87,109) and markedly decreased the positive inotropic effect of phenylephrine (109, 159) and endothelin-1 (87, 109). Because the acceleration of phosphoinositide hydrolysis induced by these receptor agonists is similarly inhibited by PDBu (86, 87, 109), it is postulated that, in the rabbit ventricular muscle, PDBu administered externally might have a site of action that differs from that of an endogenously generated PKC activator, DAG. Provided that the receptor-mediated activation of PKC is involved in inhibitory regulation, it would be a cause of tachyphylaxis, but it has been noted that Ang II caused only a limited extent of tachyphylaxis (a decrease in the maximum response by approximately 10%) upon its repeated determination of the concentration– response curve in rabbit papillary muscle (146). Neither 움-adrenoceptor agonists nor endothelin isopeptides caused tachyphylaxis in rabbit papillary muscle (145– 147). It is postulated that PDBu might cause uncoupling of the activation of the receptor and PLC웁, probably via an effect on the GTP-binding protein, Gq/11 , or by a direct effect on PLC웁 (109). Supporting the postulate just given, it has also been shown in cultured vascular smooth muscle cells that phorbol ester and OAG inhibit Ang II-induced activation of PLC웁 (160). It should be noted that the effects of PKC-activating phorbol esters show a wide range of species-dependent variation in mammalian cardiac muscle. For example, whereas PDBu has an inhibitory effect on the receptormediated process in the rabbit ventricular muscle, it has been reported to enhance the positive inotropic effect of 움-adrenoceptor agonists in the LV muscle of the rat (161). In most species of animals, including humans, PKC-activating phorbol esters elicit a negative inotropic effect (89, 157). It is noteworthy that the intracellular application by photorelease of caged DAG in rat cardiomyocytes produced a transient positive inotropic effect preceding the long-lasting negative inotropic effect (162). Therefore, despite accumulating pieces of experimental evidence that implicate the close relationship between the hydrolysis of phosphoinositide and the positive inotropic effect induced by Ang II, as well as endothelin and 움-adrenoceptor agonists, the role of activation of PKC in the regulation of

615

cardiac contractility has not yet been established unequivocally.

F. Effects of Angiotensin II on Ion Channels and Ion Transport System Various modes of regulation of ion channels and ion transport systems, triggered by the activation of Ang II receptors, have been demonstrated in cardiac myocytes of several animal species. These modes of regulation include an increase in the influx of Ca2⫹ ions through L-type Ca2⫹ channels (78, 80, 81), activation of Na⫹ channels (163), inhibition or activation of various types of K⫹ channel (76, 78, 80, 81), activation of Cl⫺ channels (164), and the activation Na⫹ –H⫹ exchange (143, 165– 167) and of Na⫹ –Ca2⫹ exchange (168), which might be responsible for the agonist-induced changes in action potential and the mobilization of Ca2⫹ ions by direct and indirect mechanisms. 1. Effects on Action Potentials In single ventricular myocytes from the rabbit, the effects of Ang II and endothelin-l on the duration of action potential are biphasic, namely a long-lasting prolongation is preceded by a transient abbreviation (80). In contrast, phenylephrine prolongs the action potential in a monophasic manner, which is consistent with the effect on the action potential in a multicellular preparation that is prolonged in parallel to the development of the positive inotropic effect (169). 2. Effects on L-Type Ca2ⴙ Channels The cellular mechanism of the Ang II-induced positive inotropic effect may involve facilitation of the opening of L-type Ca2⫹ channels because the slow inward current (Isi) is increased by Ang II (66, 69). The positive inotropic effects of Ang II in depolarized cardiac preparations, such as rabbit atria and papillary muscles and embryonic chick heart, were blocked by Mn2⫹ (1 mM) and D600 (10⫺7 g/ml), indicating that L-type Ca2⫹ channels may be involved in the signal transduction process triggered by Ang II (66). The results that the positive inotropic effect of Ang II was more susceptible to Ca2⫹ antagonists than the 웁-adrenoceptor-mediated effect (146) are consistent with the findings just given. In isolated rabbit ventricular cardiomyocytes using cell-attached and whole cell patch clamp current-recording techniques, Ang II increased the open-state probability of the Ca2⫹ channel and the L-type Ca2⫹ current, which is considered to be mediated by a Na⫹ –H⫹ antiporter. Therefore, it has been proposed that the increase in the L-type Ca2⫹ channel current induced by Ang II may be secondary to an increase

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in pHi and [Ca2⫹]i due to the activation of Na⫹ –H⫹ exchange (170). In cultured neonatal rat heart myocytes, Ang II increased both steady-state and transient components of the Ca2⫹ current, which is not dependent on cAMP or inositol trisphosphate levels, but may involve sustained phosphoinositide hydrolysis (139). Facilitation by Ang II of the activity of the L-type Ca2⫹ channel may be partly mediated by the activation of PKC because in cultured neonatal rat myocytes the phorbol ester TPA mimicked the effects of Ang II to increase the phosphorylation state of a discrete set of proteins, with apparent molecular masses of 32 and 83 kDa, and to increase the rate of spontaneous beating and L-type Ca2⫹ channel activity (102). 3. Effects on Naⴙ Channels Ang II affects the activity of fast Na⫹ channels in cardiac myocytes. In isolated ventricular myocytes of guinea pig with cell-attached patches, the maximal probability of the Na⫹ channel being opened was increased by Ang II in a concentration range between 0.05 and 1 애M, but was decreased at higher concentrations; the effect of Ang II was not due to an increase in mean open time of the channel but was associated with a delayed inactivation (171). In neonatal rat ventricular myocytes, Ang II applied outside the patch increased the frequency of opening of Na⫹ channels, but in these cells, rates of inactivation of single-channel Na⫹ currents within the patch were also increased (163). As concerned with the subcellular mechanism of these actions of Ang II on Na⫹ channel activity, Moorman and coworkers (163) proposed the crucial role of PKC activation because the phorbol ester TPA mimicked the effects of Ang II. However, Benz and co-workers (172) investigated that PKC-activating OAG diminished reconstructed peak INa to 67 ⫾ 4.9% of the control, whereas Ang II increased the current to 137 ⫾ 17.5% at 3 애M and 176 ⫾ 42% at 30 애M. Thus, although the involvement of PKC activation in Ang II-induced Na⫹ channel activation is still controversial in detail, it is supposed that the effects of Ang II on cardiac Na⫹ currents may predispose toward arrhythmia observed frequently during the course of exacerbation of congestive heart failure. 4. Effects on Kⴙ Channels In rabbit ventricular myocytes, the inwardly rectifying K⫹ current (IK1) is suppressed by the stimulation of 움1-adrenoceptors (173). A PKC activating phorbol ester phorbol 12,13-didecanoate also activates ATP-sensitive K⫹ current in human and rabbit ventricular myocytes

(174). The change in the current induced by Ang II is quite different from that induced by 움1 stimulation and therefore Ang II may activate a current that is different from the IK1 (80). Currents through cardiac K⫹ channels, including transient outward current (Ito), have been shown to be suppressed by the stimulation of 움1-adrenoceptors in rabbit atrial myocytes (175) and in rat ventricular myocytes (176–178). Ang II-induced regulation of K⫹ channels in neuronal system is supposed to play a crucial role in the increase in the neuronal firing rate. Coexpression of K⫹ channel (Kv2.2), which is sensitive to quinine, tetraethylammonium, and 4-aminopyridine, cloned from neuronal cocultures of rat hypothalamus and brain stem and AT1 receptors in Xenopus oocytes demonstrated an Ang IIinduced inhibition of Kv2.2 current (179). 5. Effects on Clⴚ Channels There are pieces of evidence that Ang II stimulates Cl⫺ channels in cardiac cells and noncardiac cells (Fig. 2). In rabbit ventricular cardiomyocytes, it has been shown that the current activated by Ang II is the Cl⫺ current (164, 180), which may involve the activation of PKC as a subcellular mechanism (181–184). These findings in myocardial cells are consistent with those in mesangial cells that Ang II stimulates Cl⫺ channels (185). In these cells, however, the effect of Ang II may be due to tyrosine phosphorylation and the subsequent stimulation of PLC웂 (185). 6. Effects on Naⴙ –Hⴙ Exchange (Regulation of pHi) Experimental evidence indicates that the activity of Na⫹ –H⫹ exchange in cardiac cell membrane is stimulated by Ang II and thereby pHi is elevated in intact cardiomyocytes (92, 186). In adult rabbit ventricular myocytes, Ang II increased steady-state pHi , which was blocked by inhibitors of Na⫹ –H⫹ exchange, amiloride (1 mM) and EIPA (10 애M) (92). Ang II also increased the rate of pHi recovery from intracellular acidosis at a pHi value of above approximately 6.9 (92). Because the positive inotropic effect of Ang II was inhibited markedly by amiloride (1 mM), it was postulated that the positive inotropic effect of Ang II depends, in part, on the stimulation of Na⫹ –H⫹ exchange (92). The cause–effect relationship between pHi and cardiac contraction induced by Ang II is not straightforward. It has been reported that the time course of changes in pHi and the increase in tension developed do not support the cause and effect relationship of these responses induced by Ang II: an increase in tension developed occurred preceding the increase in pHi in

35. Cardiac Action of Angiotensin II

617

FIGURE 2 Effects of Ang II on membrane currents (Cl⫺ current) detected in the absence of [K⫹]o by means of a whole cell voltage clamp method in a rabbit ventricular cardiomyocyte. External K⫹ was omitted, and internal K⫹ was replaced by Cs⫹. (A) The time course of the change in current during superfusion with Ang II (0.1 애M). C, control. Ramp pulses were applied every 15 sec, and a series of square pulses were applied at 10-min intervals. The holding potential was -50 mV. Approximately 40 min was required for the current to reach a maximum steady state. (B) Control currents obtained with 200-msec square pulses with 20-mV steps from a holding potential of ⫺50 mV. (C) Currents recorded with the same protocol but after 40 min of superfusion with Ang II (0.1 애M). (D) The Ang II-induced current, obtained by subtracting the current shown in B from that shown in C. (E) Isochronal I–V curves determined 100 msec after the onset of the square pulses for the currents shown in B (䊉) and C (䊊). (F) I–V relationship for the Ang II-induced current shown in D. (G) I–V curves for the net Ang II-induced current, recorded at 10-min intervals, as indicated by arrows in A. The control current at time 0 was subtracted from each current. Reproduced from Morita et al. (164), with permission.

feline papillary muscle (187). In rabbit ventricular myocytes, the time course of increases in contraction and pHi was more consistent with each other (188). In the study of Skolnick and co-workers (188), however, the increase in contraction was completely dissociated from the alteration of pHi in myocytes isolated from chronically infarcted LV muscle: Ang II increased contraction in association with a decrease in pHi . The authors speculate that Ang II stimulates two pathways involved in the regulation of pHi: (1) activation of Na⫹ –H⫹ exchange and an increase in pHi and (2) facilitation of metabolic acid production and a decrease in pHi . The latter may predominate in myocytes isolated from chronically infarcted ventricles, which leads to a decrease in pHi , whereas the amplitude of Ca2⫹ transients may be increased via Na⫹ –Ca2⫹ exchange due to intracellular Na⫹ accumulation resulting from the Ang IIinduced activation of Na⫹ –H⫹ exchange (188). Involvement of Na⫹ –Ca2⫹ exchange in the positive inotropic effect of Ang II has been supported by findings that KB-R1743, the inhibitor of Na⫹ –Ca2⫹ exchange, abol-

ished the effects of Ang II on indo-1-loaded rabbit ventricular myocytes (189, 190). It has been shown that the activation of Na⫹ –H⫹ exchange plays an important role in stretch-induced gene expression: the stretch-induced activation of Raf-1 kinase and MAPKs followed by an increase in protein synthesis partly due to the enhanced secretion of Ang II and endothelin-1 was markedly attenuated by the exchange inhibitor, HOE 694 (191). While activation of Na⫹ –H⫹ exchange by Ang II is dependent on Na⫹ concentrations, Ang II also stimulates the cardiac Na⫹-independent anion (Cl⫺-HCO3⫺) exchanger by a PKC-dependent regulatory pathway linked to AT1 receptors, which masks the Ang II-induced alkalinization in a HCO3⫺-dependent manner in feline ventricular myocardium (192). During inhibition of Na⫹ –H⫹ exchange, the application of Ang II decreased steady-state pHi , which may be ascribed to activation of the Na⫹-independent anion (Cl⫺-HCO3⫺) exchanger (192) or stimulation of metabolic acid production (92).

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In cultured neonatal rat ventricular myocytes, Ang II also leads to selective activation of the Na⫹ –HCO3⫺ symport and alkalinization through the activation of AT2 receptors and subsequent fatty acid production (193).

III. LONG-TERM CARDIAC RESPONSE TO ANGIOTENSIN II Ang II plays a crucial role in the induction of cardiac hypertrophy, pathophysiological modulation of congestive heart failure, and cardiac remodeling after myocardial infarction through the regulation of phenotype expression and synthesis of cardiac functional proteins and proliferation of cardiac fibroblasts by the activation of AT1 receptors. Modulation of these genetic processes by means of ACE inhibitors and selective AT1 receptor antagonists constitutes an important pharmacological therapy of these cardiovascular disorders.

A. Pathophysiological Interventions and Angiotensin II Mechanical stretch of cardiomyocytes by pressure and volume overload to the heart plays a critical role in determining cardiac muscle mass and its phenotype. Stretch of myocardial cells induces the pathophysiological alterations of cell function and gene expression, including cardiac hypertrophy (194–198). The expression of Na⫹ –H⫹ exchange in myocardial cells is increased in pathophysiological situations such as spontaneous hypertensive rat (199) and cardiac hypertrophy (200). Activation of Na⫹ –H⫹ exchange is postulated to be involved in molecular mechanisms of cardiac hypertrophy and vascular smooth muscle remodeling and intracellular alkalinization in myocardial cells (201), which may be partly mediated by Ang II and endothelin-1 released by myocardial cells in response to stretch of the cells. Cell stretch rapidly activates a cascade of multiple second messenger pathways, including PLC, PKC, tyrosine kinases, PLD, PLA2 , MAPKs, p21ras, Raf-1 kinases, S6 kinases (pp90RSK), and P450 pathways, which may be largely mediated by endogenous regulatory peptides, such as Ang II, endothelin, and cytokines (196, 202, 203). Ang II can directly stimulate amino acid incorporation into proteins of cultured neonatal rat cardiomyocytes and fibroblasts via activation of AT1 receptors (204, 205). Ang II also induces hypertrophy in cultured embryonic chick myocytes (206) and in normal and postmyocardial infarction adult rat ventricular myocytes in vitro (207). In adult feline (quiescent) cardiomyocytes in long-term culture, Ang II increased protein synthesis rates and protein content per cell (208). Acute infusion

of Ang II in isolated rat heart results in an increased rate of protein synthesis (209). In addition, chronic Ang II infusion (e.g., for 7 and 14 days) increased LV mass indexed to body weight compared with control rats, which was independent of cardiac afterload and prevented by losartan (140, 210). Thus, blockade of the AT1 receptor by specific inhibitors prevents partially (but not completely) cardiac myocyte growth and increased cardiac mass produced by the application of a load that alters either stress or strain to cardiac pump function (194, 211). In cultured neonatal rat cardiac myocytes the stretchinduced activation of Raf-1 kinase and MAPKs, followed by an increase in protein synthesis partly due to the enhanced secretion of Ang II and endothelin-1, is likely to be mediated by the activation of Na⫹ –H⫹ exchange, which was markedly attenuated by the exchange inhibitor, HOE 694 (212). In perfused adult rat heart, dynamic (but not static) stretch activates the early response gene, c-fos, probably through the endogenous (tissue) RAS and activation of PKC␧ (213). Overexpression of c-myc and c-fos was associated with the markedly enhanced expression of Ang II receptor mRNA and its receptor protein in myocytes isolated 2–3 days after myocardial infarction in adult rats (214). 1. Downstream of PKC Activation PKC activation is coupled to at least two separate signaling pathways, one including MAPK and another including PI3-kinase and p70s6k as key steps. Activation of the first pathway leads to the reexpression of fetal genes, activation of the second pathway to a general activation of protein synthesis, and cellular growth. In neonatal cardiomyocytes, mechanical stretch causes growth by an activation of an autocrine mechanism, including Ang II and endothelin, which, however, does not operate in a similar manner in adult cardiomyocytes (215). Activation of PLC and PKC may be essential for Ang II-induced c-fos gene expression (202). 2. Observations in AT1A Receptor Knockout (KO) Mice In AT1A KO mice the pressure overload induced cardiac hypertrophy to an identical extent as wild-type (WT) mice (216). In cultured cardiac myocytes of AT1A KO mice, extracellular signal-regulated protein kinases (ERKs) were strongly activated by stretch in KO cardiomyocytes as well as WT myocytes; activation of another member of the MAPK family, p38MAPK, and expression of the c-fos gene were also induced by stretching cardiomyocytes of both types of mice (217). Interestingly, downregulation of PKC inhibited the stretch-induced

35. Cardiac Action of Angiotensin II

619

ERK activation of WT cardiomyocytes, whereas a broad-spectrum tyrosine kinase inhibitor (genistein) and selective inhibitors of the EGF receptor (tyrphostin, AG478, and B42) suppressed the stretch-induced activation of ERKs in KO cardiomyocytes. Furthermore, the EGF receptor was phosphorylated at tyrosine residues by stretching KO cardiomyocytes, an indication that mechanical stretch could evoke hypertrophic responses in cardiomyocytes that lack the AT1A signaling pathway, probably through tyrosine kinase activation. The authors postulate that the AT1A signaling pathway may inhibit the tyrosine–ERK pathway by suppressing the production of some factors that activate tyrosine kinases or by inhibiting some intracellular molecules that are involved in tyrosine kinases signaling (217).

liferation, and subsequent replacement fibrosis in adult rat hearts in vivo (227). While Ang II induces hypertrophy and necrosis of cardiac myocytes and hyperplasia of cardiac fibroblast (228, 229), Ang II-induced signal transduction pathways differ prominently among cell types: in cardiac myocytes, activation of Gq/11 and PKC is important, whereas in cardiac fibroblasts, Ang II activates ERKs through a pathway including the G웁웂 subunit of the Gi protein (which is PTX sensitive), tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, SOS, Ras, and Raf-1 kinase (230). The regional increase in AT1 receptor density in the infarcted region of myocardium after left coronary ligation in rats was associated with fibroblast infiltration and collagen deposition (231).

B. Apoptosis and Angiotensin II

1. Regulation of Angiotensin II Receptor Expression

Ang II also activates the process that leads to programmed myocyte cell death (apoptosis). p53 activates apoptosis in various cell systems, upregulates tissue RAS, and decreases the Bcl-2/Bax protein ratio in the cell (218, 219). Ang II thus generated locally in cardiomyocytes may trigger a mechanism involving PKC-mediated increases in cytosolic Ca2⫹, which results in the stimulation of Ca2⫹-dependent endogenous endonuclease, internucleosomal DNA fragmentation, and apoptosis (220, 221). Thus Ang II generated in myocyte (tissue) RAS may play a crucial role in triggering cell death (apoptosis) and promotes myocyte hypertrophy by a mechanism involving PKC-mediated increases in cytosolic calcium (195, 221–223).

Ang II receptor mRNA and subtype expression are regulated developmentally and by pathophysiological stimuli (232). The expression and function of AT1 and AT2 receptors are differentially regulated in failing human hearts. AT2 receptors upregulated mainly in fibroblasts present in interstitial regions of failing human hearts exert an inhibitory effect on Ang II-induced mitogenic signals, whereas AT1 receptors in atrial and LV tissues were downregulated, and AT1 receptor-mediated functional and biochemical responsiveness was decreased in human failing hearts (233). The downregulation of AT1 receptors was produced in hypertrophied rat LV muscle induced by aortic banding (234). An increase in the amplitude of cell contraction, associated with intracellular alkalinization and a slight increase in peak systolic [Ca2⫹]i that was induced by Ang II (10 nM) in age-matched controls, was abolished in hypertrophied adult ventricular myocytes from ascending aortic banded rats (235). These observations in the animal model are essentially consistent with the findings in failing human ventricle that AT1 receptors were downregulated (236–238), whereas AT2 receptors were unchanged in failing human ventricle (237). In the rat global ischemia model, Ang II receptors are upregulated and Ang II is supposed to act as a compensatory mechanism, ameliorating myocyte contractility in an attempt to sustain ventricular pump function (239). However, accumulating evidence implies that Ang II elicits adverse effects on cardiac function in ischemic heart diseases. For example, Ang II exerts a direct adverse effects on LV diastolic relaxation during low-flow ischemia and recovery in red blood cell-perfused isovolumic rabbit hearts (240). In an isolated guinea pig ventricular free wall model of simulated ischemia and reperfusion, saralasin, a partial agonist of Ang II receptors, exerted antiarrhythmic effects in myocar-

C. Secretion of Atrial Natriuretic Peptide (ANP) by Angiotensin II It has been well documented in congestive heart failure patients that the blood level of ANP is elevated depending on the severity of the symptoms. It has been shown experimentally that Ang II contributes to this phenomenon, inducing ANP secretion from cardiomyocytes directly via a PKC-dependent autocrine pathway that involves cyclooxygenase products and myocardial prostanoid receptors in isolated rabbit (224) and in cultured, spontaneously beating, neonatal rat cardiomyocytes (225). ANP may play a protective role against LV hypertrophy in the rat (226).

D. Differential Myocyte and Cardiac Fibroblast Response to Angiotensin II Ang II arising from either endogenous or exogenous sources is associated with the appearance of abnormal sarcolemmal permeability, myocytolysis, fibroblast pro-

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dial reperfusion by antagonizing the action of endogenous Ang II through a mechanism independent of circulatory and central actions (241). It has been shown that overexpression of the AT1A receptor transgene in the mouse myocardium produces a lethal phenotype: offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first week after birth, an indication that the direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction (242). In AT1A receptor KO mice, Ang II may be critically involved in the induction of ventricular arrhythmias (243). However, AT2 receptor KO mutant mice developed normally, but had an impaired drinking response to water deprivation as well as spontaneous movements; they showed an increased vasopressor response to injection of Ang II (244). Tissue RAS in the heart is upregulated in experimental heart failure (245, 246), and AT1 receptors are upregulated in hypertrophied ventricular myocytes isolated from spontaneous hypertensive rats or two-kidney oneclip renovascular hypertensive rats (232), postinfarction rats (247), and volume-overload rats (248). The regression of cardiac hypertrophy by the normalization of elevated blood pressure completely reversed the increased levels of AT1A receptor mRNA and the receptor density to the control level (232). In the human heart, the ventricular AT1 receptor is downregulated after orthotopic heart transplantation, which may partially reflect a loss of autonomic nerves and thus altered nervous control of the heart (249). In the volume-overload rat hypertrophy model, AT1 receptors on fibroblasts showed a significant downregulation, whereas the Bmax of AT1 receptors and Ca2⫹ sensitivity to Ang II were increased significantly in hypertrophied cardiomyocytes, suggesting that Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload (250). In an identical volumeoverload rat model, AT2 receptors were upregulated, which has been postulated to play an important role in heart enlargement induced by volume overload (251). However, pieces of evidence are accumulating that AT2 receptors play a beneficial role in protecting heart against hypertrophy and cardiac remodeling. For example, in failing myopathic hamster hearts, AT2 receptors are reexpressed by cardiac fibroblasts and exert an antiAT1 receptor action on the progression of interstitial fibrosis during cardiac remodeling by inhibiting both fibrillar collagen metabolism and growth of cardiac fibroblasts (252). It has been shown that expression of the AT2 receptor gene is downregulated by Ang II through a process

involving the PKC–Ca2⫹ pathway in PC12 cells (253). However, in cultured neonatal rat cardiomyocytes, Ang II secreted from stretched myocytes downregulated AT1 and AT2 receptor mRNA levels, whereas stretching of myocytes upregulated the expression of Ang II receptor subtypes (both AT1 and AT2 receptors) transcriptionally and posttranscriptionally through mechanisms involving stretch-activated tyrosine kinases (253).

E. Cross Talk of Renin–Angiotensin and Other Regulatory Systems There is cross talk between the RAS and the sympathetic nervous system when both systems are activated in congestive heart failure. In addition to a facilitatory action of Ang II to release norepinephrine, cross talk between AT1 receptors and 움1-adrenoceptors has also been reported: Ang II selectively downregulates 움1aadrenoceptor mRNA and its corresponding receptor protein and 움1a-adrenoceptor-mediated expression of the immediate early gene c-fos in neonatal rat cardiac myocytes (254). Ang II also interacts with 웁-adrenoceptor-mediated signaling: endogenous Ang II has a major function in maintaining isoproterenol-induced cardiac hypertrophy but does not mediate its induction in the rat (255). As another potential cross talk pathway, it has been shown that insulin-like growth factor-1 negatively influences the myocyte RAS through the upregulation of Mdm2 and its binding to p53 (256). Catecholamines released by Ang II from cardiac sympathetic neuron in vivo may contribute to Ang II-induced myocardial damage, and the acute nature of this damage is associated with a downregulation of 웁1-adrenoceptors (257).

F. Therapeutic Relevance of ACE Inhibitors and AT1 Receptor Antagonists In in vivo studies, ACE inhibitors or AT1 receptor antagonists attenuate ventricular hypertrophy in chronic pressure overload models (211, 258). However, cardiac growth occurs in response to pressure overload even when ACE activity is inhibited in rats (259). Furthermore, it has been reported that the stretch- and Ang II-induced increase in c-fos mRNA in cultured adult and neonatal cardiomyocytes was inhibited by the Ang II receptor antagonist [Sar1,Ile8]Ang II, whereas the increase in Na⫹ –Ca2⫹ exchanger mRNA levels and protein synthesis in response to these interventions were not blocked by the Ang II receptor antagonist (260). Downregulation of expression of the gene coding for SERCA (SR Ca2⫹-ATPase)-2 observed in heart of the cardiomyopathic Syrian hamster (strain Bio 53-58) was prevented by treatment with the ACE inhibitor perindopril (261). In contrast, minoxidil-induced cardiac hyper-

35. Cardiac Action of Angiotensin II

trophy in rats was prevented by losartan but not by enalapril (262). In pressure overload-induced hypertrophy in rats, Ang II does not appear to play a major role, as ACE inhibitors (enalapril and ramipril) were ineffective and losartan was only partially effective in reducing the hypertrophy (263). While these findings indicate that not all phenotype expressions in response to load or stress of cardiomyocytes involve Ang II, ACE inhibitors prolonged the survival of rats with experimental chronic heart failure (264). ACE inhibitors elicit beneficial hemodynamic effects on patients with heart failure (265) and are rare therapeutic agents able to produce a significant reduction of morbidity and mortality of patients with congestive heart failure (266, 267). ACE inhibitors may be of value not only in preventing the progression of heart failure, but also in reversing endothelial dysfunction and preventing the development of atherosclerosis and its consequences, such as myocardial infarction (268). As the subcellular mechanisms of the antihypertrophy and cardioprotective effects of ACE inhibitors, in addition to the decreased Ang II production, an accumulation of bradykinin due to the inhibition of endopeptidase II and the attendant production of NO induced by constitutive NO synthase may contribute in an autocrine/paracrine fashion (269, 270). ACE inhibitors may augment ischemic preconditioning, probably through B2 kinin receptor activation (271). Blunted baroreceptor reflex function in conscious spontaneous hypertensive rats is mediated by AT1 receptors, and NO contributes to the antihypertensive effects of losartan and lisinopril (an ACE inhibitor) (272). In rat heart failure model after occlusion of the left main coronary artery, orally administered captopril or losartan improved but did not normalize cardiac pump performance (273–275). In the rat myocardial infarction model, the inhibition of cardiomyocyte hypertrophy by ACE inhibition (lisinopril) and AT1 receptor blockade (ZD7155) is ascribed to a reduced generation/receptor blockade of Ang II, respectively, whereas the reduction of myocardial collagen accumulation by these agents may involve an increased kinin level because the latter effect is blocked by a B2 kinin receptor antagonist (icatibant) (276). In spontaneous hypertensive rats with LV hypertrophy and adverse structural remodeling of the cardiac interstitium, an ACE inhibitor, lisinopril, reversed fibrous tissue accumulation and medial thickening of intramyocardial coronary arteries and restored myocardial stiffness and the coronary vascular reserve to normal (277). In a postischemic working heart, an Ang II antagonist (L-158-338) alone resulted in a degree of improvement in functional recovery comparable to that observed with ACE inhibitor (lisinopril) treatment (278).

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In human hearts, it appears that bradykinin and AT2 receptors are crucial for the therapeutic differences of ACE inhibitors and AT1 receptor antagonists, respectively (279). This is partly because in large mammals, including humans, a considerable fraction of Ang II production is mediated by chymase. In the dog heart in vivo, conversion of Ang I in interstitial space to Ang II was reduced by captopril by 73%, by chymostatin, a chymase inhibitor, by 43%, and abolished by a combination of both inhibitors (280). In human gastroepiploic arteries isolated from surgically resected stomachs, Ang I-induced vasoconstriction was inhibited only by 30% with lisinopril and was abolished by a combination of lisinopril and chymostatin, an indication that chymase may play a crucial role in the production of Ang II (approximately 70%) in human vasculature (281). Experimental evidence indicates that selective AT1 receptor antagonists provide end organ protection by blocking Ang II effects via the AT1 receptor, leaving the AT2 receptor unopposed. Consequently, these agents may reduce the morbidity and mortality that result from myocardial infarction and other situations resulting from structural alterations in the heart, kidney, and vascular smooth muscle (282). In an acute experimental study in isolated working rat hearts, however, the recovery of mechanical function after ischemia– reperfusion was facilitated by the AT2 antagonist (cardioprotective), whereas the AT1 antagonist did not (283). Thus, the regulation induced via AT1 and AT2 receptors in the course of cardiovascular diseases appears to be of extreme complex. The AT2 receptor is expressed abundantly and widely in fetal tissues, but is downregulated and present only at lower levels in adult tissues. The AT2 receptor, however, is reexpressed/upregulated in various pathophysiological conditions, such as cardiac hypertrophy, myocardial infarction, and neointimal lesions after vascular injury (57, 234, 284, 285). Activation of AT2 receptors has been shown to result in attenuation of neointimal hyperplasia in injured rat carotid artery (57) and antiproliferation in cultured coronary endothelial cells (54). Furthermore, the myocardial AT2 receptor has been shown to be increased in experimental myocardial infarction (285) and in the hypertrophied heart (234) and to be associated with fibrous tissue in human atria (286). Apoptosis is enhanced significantly in ischemic/reperfusion rabbit cardiomyocytes (287) and in hypertensive mouse heart (288). It is therefore hypothesized that apoptosis participates in cardiac remodeling observed clinically after myocardial infarction and that the AT2 receptor may contribute to this process (60). It is further postulated that the regression of cardiac hypertrophy in response to AT1 receptor antagonist therapy may be in part due to the apoptotic action of AT2 receptor activation by Ang II, the concentration

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VII. Signaling Systems

of which is elevated in biophase after administration of the AT1 receptor antagonist. In a murine model of dilated cardiomyopathy, losartan appears to be less effective and less potent than captopril (289). However, in a study of elderly heart failure patients, treatment with losartan was associated with an unexpected lower mortality than that found with captopril (290). Thus, it has not yet been settled whether ACE inhibitors, AT1 antagonists, or a combination of both are more effective than the other in the clinical setting. Namely, the clinical experience with AT1 receptor antagonists is much less than those with ACE inhibitors and therefore the issue requires further clinical study supported by animal studies carried out under appropriate experimental conditions to establish the effectiveness of these selective antagonists on human cardiovascular disorders.

IV. SUMMARY AT1 receptors are coupled to activation of PLC웁 via the Gq/11 protein and of PLC웂 via tyrosine kinase to lead to stimulation of the hydrolysis of phosphoinositide and to the resultant production of IP3 and DAG. Ang II exerts an immediate and long-term regulation of cardiac function and gene expression, thereby playing a crucial role in the pathogenesis of cardiovascular adaptation and in the development of maladaptation and dysfunction, such as cardiac hypertrophy, congestive heart failure, and postinfarction cardiac remodeling. The positive inotropic effect of Ang II is associated with a prolongation of duration of contraction and a retardation of relaxation. These characteristic alterations of contractility are due to the modulation of intracellular Ca2⫹ signaling by Ang II, i.e., a moderate increase in the amplitude of intracellular Ca2⫹ transients and an increase in myofibrillar sensitivity for Ca2⫹ ions. Most probable intracellular mediators of myocardial contractile regulation are the products of hydrolysis of phosphoinositide, although the pieces of experimental evidence supporting the role of phosphoinositide hydrolysis in cardiac contractile regulation are still equivocal. In various tissues, including myocardial cells, IP3 releases Ca2⫹ ions from intracellular stores via the activation of IP3 receptors under certain experimental conditions; however, clear evidence that the IP3-induced Ca2⫹ release contributes to the regulation of cardiac twitch contraction is lacking. PKC activated by endogenously generated DAG stimulates Na⫹ –H⫹ exchange and thereby produces intracellular accumulation of Na⫹ ions and intracellular alkalinization. The former may contribute to an increase in the amplitude of Ca2⫹ transients through Na⫹ –Ca2⫹

exchange and the latter to an increase in Ca2⫹ sensitivity of contractile proteins. PKC inhibitors, such as staurosporine, NA 0345, H-7, calphostin C, and chelerythrine, consistently and selectively inhibited the positive inotropic effect of Ang II at concentrations that produce no effect on the positive inotropic effect of isoproterenol and Bay k 8644 in rabbit ventricular muscle. In contrast, PKC activators, such as PDBu and TPA, and OAG did not mimic the Ang II-induced regulation of myocardial contractility, but inhibited selectively the effect of Ang II on the contractile force and phosphoinositide hydrolysis. These results suggest that externally applied PKC activators act through pathways different from endogenous DAG in intact myocardial cells. Regulation induced by the products of phosphoinositide hydrolysis shows a wide range of species-related variations. In addition, Ang II released by stretching cardiac myocytes plays a crucial role in the induction of cardiac hypertrophy, pathophysiological modulation of congestive heart failure, and cardiac remodeling after myocardial infarction through the regulation of phenotype expression and synthesis of cardiac functional proteins and proliferation of cardiac fibroblasts by activation of AT1 receptors. AT2 receptor activation may exert effects primarily by antagonizing action-induced via AT1 receptors. Thus Ang II is a key endogenous peptide in determining a number of pathophysiological alterations. Modulation of the Ang II signals responsible for genetic expression by means of ACE inhibitors and selective AT1 receptor antagonists constitutes an important pharmacological therapy of these cardiovascular disorders.

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278. Werrmann, J. G., and Cohen, S. M. (1994). Comparison of effects of angiotensin-converting enzyme inhibition with those of angiotensin II receptor antagonism on functional and metabolic recovery in postischemic working rat heart as studied by [31P] nuclear magnetic resonance. J. Cardiovasc. Pharmacol. 24, 573–586. 279. Regitz-Zagrosek, V., Fielitz, J., and Fleck, E. (1998). Myocardial angiotensin receptors in human hearts. Basic Res. Cardiol. 93, 37–342. 280. Wei, C. C., Meng, Q. C., Palmer, R., Hageman, G. R., Durand, J., Bradley, W. E., Farrell, D. M., Hankes, G. H., Oparil, S., and Dell’Italia, L. J. (1999). Evidence for angiotensin-converting enzyme- and chymase-mediated angiotensin II formation in the dog heart in vivo. Circulation 99, 2583–2589. 281. Takai, S., Shiota, N., Jin, D., and Miyazaki, M. (1998). Functional role of chymase in angiotensin II formation in human vascular tissue. J. Cardiovasc. Pharmacol. 32, 826–833. 282. Unger, T., Culman, J., and Gohlke, P. (1998). Angiotensin II receptor blockade and end-organ protection: Pharmacological rationale and evidence. J. Hypertension. (Suppl.) 16, S3–S9. 283. Ford, W. R., Clanachan, A. S., and Jugdutt, B. I. (1996). Opposite effects of angiotensin AT1 and AT2 receptor antagonists on recovery of mechanical function after ischemia-reperfusion in isolated working rat heart. Circulation 94, 3087–3089. 284. Rogg, H., de Gasparo, M., Graedel, E., Stulz, P., Burkart, F., Eberhard, M., and Erne, P. (1996). Angiotensin II-receptor subtypes in human atria and evidence for alterations in patients with cardiac dysfunction. Eur. Heart J. 17, 1112–1120. 285. Nio, Y., Matsubara, H., Murasawa, S., Kanasaki, M., and Inada, M. (1995). Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction. J. Clin. Invest 95, 46–54. 286. Brink, M., Erne, P., de Gasparo, M., Rogg, H., Schmid, A., Stulz, P., and Bullock, G. (1996). Localization of the angiotensin II receptor subtypes in the human atrium. J. Mol. Cell. Cardiol. 28, 1789–1799. 287. Gottlieb, R. A., Burleson, K. O., Kloner, R. A., Babior, B. M., and Engler, R. L. (1994). Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J. Clin. Invest 94, 1621–1628. 288. Hamet, P., Richard, L., Dam, T. V., Teiger, E., Orlov, S. N., Gaboury, L., Gossard, F., and Tremblay, J. (1995). Apoptosis in target organs of hypertension. Hypertension 26, 642–648. 289. Kanda, T., Araki, M., Nakano, M., Imai, S., Suzuki, T., Murata, K., and Kobayashi, I. (1995). Chronic effect of losartan in a murine model of dilated cardiomyopathy: Comparison with captopril. J. Pharmacol. Exp. Ther. 273, 955–958. 290. Pitt, B., Segal, R., Martinez, F. A., Meurers, G., Cowley, A. J., Thomas, I., Deedwania, P. C., Ney, D. E., Snavely, D. B., and Chang, P. I. (1997). Randomised trial of losartan versus captopril in patients over 65 with heart failure (Evaluation of Losartan in the Elderly Study, ELITE). Lancet 349, 747–752.

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36 ATP and Adenosine Signal Transductions AMIR PELLEG

GUY VASSORT

JOHN A. AUCHAMPACH

Departments of Medicine and Pharmacology MCP Hahnemann University Philadelphia, Pennsylvania 19102

INSERM U-390 Physiopathologie Cardiovasculaire FR-34095 Montpellier, France

Department of Medicine University of Louisville, Kentucky 40292

I. INTRODUCTION

topics (Abbracchio and Burnstock, 1998; Belardinelli and Pelleg, 1995; Pelleg and Belardinelli, 1998; Burnstock et al., 1998).

The purine nucleoside adenosine and its related purine nucleotide, adenosine 5⬘-triphosphate (ATP), are two biological compounds found in every cell of the human body where they play a pivotal role in cellular metabolism and energetics. In 1929, Drury and SzentGyorgyi showed for the first time that extracellular ATP and adenosine exert pronounced effects on the mammalian heart, including a negative chronotropic effect on the sinoatrial (SA) node, a negative dromotropic effect on the atrioventricular (AV) node, and a lusitropic effect on the coronary vasculature. These effects were independent of the role of adenosine and ATP in intracellular metabolism. Since then it has been established that adenosine and ATP exert a wide spectrum of effects in various tissues and organs other than the heart and are now considered as endogenous physiologic regulators. Burnstock (1978) hypothesized that specific receptors, which he called purinoceptors, different from cholinergic and adrenergic receptors, mediate the actions of adenosine and ATP. He classified purinoceptors into two groups: P1, the adenosine receptors, and P2, the ATP and other adenine nucleotides receptors. Four different P1 receptors and 20 different P2 receptors have been cloned heretofore. This chapter summarizes the cardiovascular effects of extracellular adenosine and ATP, the signal transduction pathways that mediate these effects, and the potential role of adenosine and ATP in cardiovascular physiology and pathophysiology. The reader is referred to several extensive reviews and books for a more detailed discussion of specific relevant

Heart Physiology and Pathophysiology, Fourth Edition

II. EXTRACELLULAR ATP AND THE CARDIOVASCULAR SYSTEM A. P2 Receptors 1. Structure and Classification The P2 receptors designate cell surface receptors that are activated by ATP and adenine nucleotides. These receptors are divided into two families (Burnstock and Kennedy, 1985; North and Barnard, 1997): ionotropic receptors, P2X, and metabotropic receptors, P2Y, whose only common characteristic is a binding site for ATP. The former family consists of ligand-activated ion channels and the latter family consists of a G-proteincoupled receptors (North Barnard, 1997; Ralevic Burnstock, 1998; Kunapuli Daniel, 1998). The signal transduction pathways of P2X receptors are membrane delimited, whereas those of P2Y receptors involve intracellular second messengers. In each family, several receptors have been identified (P2X1–7 and P2Y1,2,4,6,11 are known in the mammalian cardiovascular system) and the DNA sequences that encode their polypeptide structure have been cloned. The polypeptides of P2X receptors share an overall sequence identity of up to 50% and their size ranges from 388 to 595 amino acids. They consist of two transmembrane domains connected by a relatively long, cysteine-rich, extracellular loop and relatively short intra-

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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VII. Signaling Systems

cellular N- and C-terminals. There is no significant amino acid sequence homology between any of the P2X receptor proteins and other ligand-gated ion channels. ATP-gated ion channels are multimeric complexes; whereas transfected cells expressing a single cDNA clone most often produced functional channels, P2X receptor–channel complexes are probably heteromeric, as was initially shown for P2X2/3 , because multiple P2X mRNAs are present in various cell types. P2Y receptors, like other G-protein-coupled receptors, consist of seven hydrophobic transmembrane domains connected by three extracellular and three intracellular loops, an intracellular C-terminal, and an extracellular N-terminal. Agonist binding to P2Y receptors leads to the activation of a wide range of signal transduction pathways involving various intracellular second messengers. 2. P2 Receptors: Pharmacology The study of P2 receptor pharmacology has been hampered by the lack of selective and specific agonists and antagonists for each receptor subtype. Early on, investigators relied mostly on the rank order of agonist potencies in exerting a specific effect in their efforts to identify the type of receptor that mediates this effect. The following agonists, analogs and related compounds of ATP, have been commonly used: ADP, UTP, UDP, 움,웁-methylene ATP (움,웁mATP), 웁,웂-methylene ATP (웁,웂mATP), 2-methylthioATP (2mSATP), 5⬘-O-(3thio)-ATP (ATP웂S), 웁,웂-imido ATP, 2⬘,3⬘-O-(4-benzoyl)-benzoyl-ATP (Bz-ATP), and 2-Cl-ATP. The following antagonists have been used: reactive blue 2 (RB2) and other related dyes, suramin, and pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonate (PPADS). These compounds not only are nonselective, but all inhibit ectonucleotidases to different degrees. In general, P2X receptors exhibit the following agonist potency rank order: 움,웁mATP⬎웁,웂mATP⬎ATP ⫽ 2mSATP, whereas P2Y receptors are characterized by the rank order of 2mSATP⬎ATP⬎움,웁mATP ⫽ 웁,웂mATP. However, it has been shown that the variable susceptibility of extracellular ATP and its related compounds to degradation by Ca2⫹ /Mg2⫹-dependent ectonucleotidases can directly affect a given agonist potency rank order such that it does not represent the inherent potency of the agonists at the receptor site studied (Tresize et al., 1994). This issue is further complicated by receptor desensitization due to the presence of ATP in the extracellular fluid, impurities of the commercially available adenine nucleotides, and the presence of ectokinase and ectosynthase, which can modulate the level of an agonist at a given P2 receptor site (Dubyak and El-Moatassim, 1993).

a. P2X Ion channels activated by extracellular ATP exhibit inward rectification and are permeable to all small monovalent cations as well as Ca2⫹. Protons, Zn2⫹, and Cu2⫹ can potentiate the current induced by ATP. The mechanism of this potentiation is not fully known. Under physiologic conditions, ATPtotal exists as Mg-ATP2⫺, Ca-ATP2⫺, and ATP4⫺; the latter acts as an agonist at a particular receptor site, i.e., P2X7 (previously called P2Z). In mast cells, macrophages, and thymocytes, the activation of P2X7 by ATP4⫺ leads to cell permeabilization manifested by the increased membrane conductance of cations as well as the permeability of low molecular weight compounds such as ethidium bromide. Thus, P2X7 has been implicated in cytolysis in the immune system. The P2X7 agonist potency order is markedly different from that of other receptors in this family, i.e., BzATPⰇATP⬎2mSATP⬎ATP-웂SⰇADP. The signal transduction pathway linked to this receptor has not been fully determined; however, it has been reported that its activation is associated with pronounced phospholipase D (PLD) activity in murine macrophages. b. P2Y At least two signal transduction pathways have been suggested to be coupled to P2Y receptors, both of which involve the activation of a G-protein. One pathway activates phosphatidyl choline-specific phospholipase C (PLC) and/or PLD, which increase cellular phosphatidic acid and choline but does not affect adenylyl cyclase (AC), whereas the second pathway inhibits AC but has no effect on PLC. The former pathway includes a pertussis toxin (PTX)-insensitive G움q /G11 protein, whereas the latter includes the PTX-sensitive Gi /Go protein. The activation of P2Y receptors results in inositol lipid hydrolysis and/or Ca2⫹ mobilization. This effect is mediated by activation of the mitogen-activated protein (MAP) kinase and tyrosine kinase(s). At least in one preparation, i.e., Chinese hamster ovary cells (CHO), the activation of endogenous P2Y2 receptors stimulates MAP kinase via PTX-insensitive (G움q /G11 mediated) and PTX-sensitive (Gi /Go mediated) pathways, each involving protein kinase C (PKC) to a various degree. A reverse transcription–polymerase chain reaction (RT-PCR) has indicated that multiple P2Y receptor subtypes are expressed in the mammalian heart (i.e., P2Y1 , P2Y2 , P2Y4 , and P2Y6) and that expression changes from the neonate to the adult. A P2Y11 receptor has been described that coupled positively to both PLC and to adenylyl cyclase (see later).

B. Sources of Extracellular ATP ATP is released from ischemic myocytes, activated platelets, nerve terminals as a cotransmitter, inflamma-

36. ATP and Adenosine Signal Transductions

tory cells, erythrocytes, endothelial cells, smooth muscle cells, and exercising muscle cells, as well as electrically driven atrial cells challenged by the cardiotonic agents isoproterenol and forskolin. Strong evidence supports the notion that ATP is a cotransmitter in perivascular sympathetic nerves and that it plays a role in the local regulation of vascular tone as well as in neurotransmitter release via a feedback mechanism. The latter is mediated by adenosine, the product of enzymatic degradation of ATP by ectonucleotidases, some of which can be coreleased with ATP from nerve terminals. Thus, extracellular ATP can be released into the lumen of blood vessels as well as the interstitial space where it activates P2 receptors located on the surface of endothelial and smooth muscle cells and perivascular nerve terminals (Olsson and Pearson, 1990). The net effect of extracellular ATP on vascular tone is dependent on the relative contribution of the action of ATP at each of these three sites. The mechanism by which ATP is transported across the cell membrane is not fully understood. In recent years, the adenine nucleotide-binding cassette (ABC) family of proteins has been identified. These proteins were suggested to be a regulatory component of an ion-channelregulator complex, such as the cystic fibrosis transmembrane conductance regulator (CFTR), which acts as an ATP channel, enabling intracellular ATP to cross the cell membrane and stimulate cell surface receptors. However, whether the CFTR channel is permeable to ATP has been the subject of some controversy (al-Awqati, 1995).

C. Effects of ATP on Vascular Tone Numerous studies have indicated that ATP and related purine nucleotides can either contract or relax blood vessels. Evidence shows that relaxation is mediated by an endothelium-dependent mechanism, whereas contraction is mediated by a direct action on smooth muscle cells. However, at least in the case of the rabbit portal vein, ATP can cause vasodilation by a direct action on smooth muscle cells. Extracellular ATP is rapidly degraded to adenosine, which is a potent vasodilator in different vascular beds. Thus, if the endothelial function is intact, luminal ATP would cause vasodilation due to either its own action on endothelial cells or the action of adenosine on endothelial and smooth muscle cells. In contrast, interstitial ATP can cause either contraction or relaxation of vascular smooth muscle cells. It is now agreed that the vasodilatory action of ATP is mediated by P2Y receptors on endothelial cells and that the ATP-induced contraction of smooth muscle cells is mediated by P2X receptors. However, it has been shown that ATP-induced contraction could be mediated by

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both P2X and P2Y receptors (see later). The mechanism of endothelium-dependent vasodilatory action of ATP is discussed elsewhere in this volume, as well as in previous reviews. The direct effects of ATP’s action on smooth muscle cells are discussed next.

D. P2 Signal Transduction in Smooth Muscle Cells Extracellular ATP evoked a depolarizing transient inward current in smooth muscle cells of the rabbit ear artery. This current resulted from an ATP-activated channel that is cation selective, but one that allows both monovalent and divalent cations to pass across the cell membrane. The channel manifested 3:1 selectivity for Ca2⫹ over Na⫹ at near physiological concentrations and a unitary conductance of 앑5pS in 110 mM Ca2⫹ or Ba2⫹. Its biophysical and pharmacological properties are different from voltage-gated L- or T-type Ca2⫹ channels. The Ca2⫹ influx through this activated channel depolarizes the cell, thereby activating the voltage-dependent channels, which further enhances Ca2⫹ influx. Similar observations were made in cultured rat aortic smooth muscle cells. In the latter preparation, the release of calcium from intracellular stores induced by ATP activated a Cl⫺ current that contributed to the depolarization of the cell membrane as well as a K⫹ current. Similar activation of a Cl⫺ current by ATP was observed in smooth muscle cells of the rat portal vein. The mechanism of the ATP-induced Ca2⫹ influx, inward current, and elevation of intracellular Ca2⫹ could be complex. Ca2⫹ influx could result from the activation of ligand-activated channels, subsequent activation of voltage-dependent channels, release of Ca2⫹ from internal stores [sarcoplasmic reticulum (SR)] via either inositol 1,4,5-trisphosphate (IP3)- or Ca2⫹-sensitive channels, activation of Ca2⫹-dependent channels, or a combination of all of these factors. Thus, both P2X and P2Y receptors could be involved in the modulation of the intracellular Ca2⫹ level ([Ca2⫹]i) by ATP: P2X as the ligand-activated nonselective cation and Ca2⫹ channels and P2Y as mediators of the ATP-induced formation of IP3 and subsequent release of Ca2⫹ from internal stores. In rat aortic smooth muscle strips, evidence was obtained for the P2X- and P2Y-mediated Ca2⫹ influx and P2Y-mediated Ca2⫹ release from internal stores. Similarly, in rat pulmonary artery myocytes, extracellular ATP induced Ca2⫹ influx and [Ca2⫹]i oscillations by activating P2X and P2Y receptors, respectively. In contrast, the P2X agonist, 움,웁-methylene ATP (움,웁mATP), was ineffective in cultured rat aortic smooth muscle cells. Also, in single smooth muscle cells isolated from rat portal vein, ATP released Ca2⫹ from intracellular stores with or without minor involvement of IP3.

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More recently, it has been shown that the ATP-induced contraction of the rat isolated tail artery is mediated by P2X1 receptors as well as by G-protein-coupled P2Y receptors. In cultured human coronary artery smooth muscle cells, extracellular ATP activated an inward current carried by Cl⫺ and an outward current carried by K⫹. Both currents were independent of external Ca2⫹ but dependent on [Ca2⫹]i , and 움,웁-mATP, the P2X agonist, failed to activate these currents. It seems that there is a phenotypic modulation of vascular myocytes in culture associated with changes in the expression and/or function of P2 receptor subtypes. The lack of selective and specific P2 receptor antagonists has resulted in overreliance on the rank order of agonist potency in determining the subtype of P2 receptor responsible for a given action of ATP. However, it has been shown that the rank order of agonist potency at the P2X receptor, which mediates the contraction of rat isolated tail artery, is very different from the potency order for evoking the inward current initiating this contraction. This discrepancy was explained by the relative absence of breakdown of some of the agonists in the single cell preparation used for the electrophysiological study vs the multicellular arterial ring preparation used for the contraction study. It has been shown that both P2X1 and P2X7 expressed in myocytes of the human saphenous vein mediate the contractile effect of extracellular ATP. Because P2X7 mediates the formation of cell membrane pores permeable to large molecules and thereby the cytotoxic effects of ATP, it has been hypothesized that these receptors in the human saphenous vein could be mechanistically involved in smooth cell lysis in the media of varicose veins. However, further studies are required to determine the physiological role of P2X7 signal transduction in vascular smooth muscle cells.

E. ATP and the Vasculature: Concluding Remark It is now recognized that ATP is an important local regulator of vascular tone. ATP is released into the lumen of blood vessels, as well as in the adventitia from endothelial and red blood cells and platelets and perivascular nerves, respectively. It can activate different subtypes of P2 purinoceptors located on the surface of different cell types, including endothelial and smooth muscle cells, as well as sensory nerve endings. In addition, extracellular ATP is degraded by ecto-enzymes to adenosine that can activate P1 receptors, which are also located on these cells. Thus the net effect of extracellular ATP on vascular tone depends on the outcome of several signal transduction pathways triggered by ATP as

well as adenosine, which could be simultaneously operative. Figure 1 is a schematic diagram illustrating the main sites of action of extracellular ATP and adenosine known to mediate the modulatory effects of these compounds on vascular tone.

F. ATP Signal Transduction in Cardiac Myocytes 1. Intracellular Ca2ⴙ In quiescent or stimulated cells, the extracellular application of ATP causes an increase in [Ca2⫹]i . The increase in [Ca2⫹]i results both from the stimulation of Ca2⫹ currents and from a larger Ca2⫹ release from the SR as caffeine and ryanodine markedly reduce it (Danziger et al., 1988). Moreover, the ATP-induced formation of IP3 might have some influence on SR–Ca2⫹ release (see later). Furthermore, ATP directly gates a nonselective cationic channel through which a significant Ca2⫹ influx could also occur. Other hypothesized mechanisms include phosphorylation of an extracellular protein leading to activation of a novel ion channel, as well as ATP-induced acidosis leading to both the depolarizing effect and the increase in [Ca2⫹]i . 2. Cyclic AMP and Cyclic GMP The effects of ATP on second messengers such as cyclic AMP (cAMP), cyclic GMP (cGMP), and IP3 have been studied not only in whole heart tissues, but also, in the most recent studies, in isolated cardiomyocytes. Whether ATP modulates intracellular cAMP has long been controversial. It has been reported that ATP does not affect basal cAMP level in rat ventricular cardiomyocytes, but it facilitates the isoproterenol-induced increase in cAMP. In cardiomyocytes isolated from fetal mice, basal cAMP levels are not changed by ATP, which, however, partially antagonizes the effect of isoproterenol. Stimulation with ATP웂S in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) increases cAMP level twofold; at lower basal levels, a fourfold stimulation was observed. The effect of ATP on cAMP production was poorly potentiated by forskolin and was additive to that of submaximal concentrations of isoproterenol. The ATP-induced activation of adenylyl cyclase is mediated by a 45-kDa Gsprotein, similar to that observed with isoproterenol stimulation. A paracrine effect involving PLA2 activation and the formation of prostaglandins was excluded. Both ATP and isoproterenol increase cAMP in HEK 293 cells expressing type V adenylyl cyclase, whereas cAMP was only increased by 웁-adrenergic stimulation of HEK

36. ATP and Adenosine Signal Transductions

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FIGURE 1 Schematic diagram illustrating the main receptor subtypes for purine and pyrimidine present in most blood vessels. Perivascular nerves in the adventitia release ATP as a contransmitter: ATP released with noradrenaline (NA) and neuropeptide Y (NPY) from sympathetic nerves activates P2X1 purinoceptors of smooth muscle, resulting in vasoconstriction, whereas ATP released with calcitonin gene-related peptide (CGRP) and substance P (SP) from sensory nerves whenever the axon reflex is elicited activates P2Y purinoceptors of smooth muscle cells, resulting in vasodilation. A1-adenosine receptors (i.e., P1 purinoceptors) on nerve terminals of sympathetic and sensory nerves mediate the modulation of transmitter release by adenosine, the product of ATP enzymatic degradation. P2X2/3 purinoceptors on a subpopulation of sensory nerve terminals mediate nociception. A2adenosine receptors on vascular smooth muscle cells mediate vasodilation. ATP and UTP released from endothelial cells during shear stress and hypoxia activate P2Y1 and P2Y2 and P2Y4 receptors, respectively, resulting in the production of nitric oxide and vasodilation. ATP is also released from activated platelets; the latter express ADP-selective purinoceptors (‘‘P2T’’) whereas immune cells of various kinds express P2X7 receptors (P2Z purinoceptors). From Abbracchio and Burnstock (1998), with permission.

expressing type IV and type VI adenylyl cyclases. Thus in rat cardiomyocytes, purinergic and 웁-adrenergic stimulations differentially activate various cyclase isoforms; adenylyl cyclase V is the specific target of purinergic stimulation (Puce´at et al., 1998). In the presence of IBMX, ATP also increases the basal cGMP content of isolated cardiomyocytes. In addition, ATP activates arachidonic acid metabolism in both whole heart and isolated cardiac myocytes. These observations could be related to the ATP-induced in-

crease in cGMP content as arachidonic acid has been reported to activate soluble guanylyl cyclase. 3. Inositol Triphosphate and pH: Role of Tyrosine Kinases ATP accelerates phosphatidylinositol turnover, as assessed by IP3 formation in rat ventricles and isolated fetal mouse cardiomyocytes. This signal transduction pathway is not sensitive to PTX. In rat ventricular cardi-

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omyocytes, ATP activates phospholipase Cg1 , leading to IP3 production by a pathway that involves a tyrosine kinase (Fig. 2). Simultaneously, diacylglycerol (DAG) is produced from phosphoinositide hydrolysis, which should lead to the activation of protein kinase PKC. Direct evidence in favor of an ATP-induced increase in PKC activity had been obtained. ATP triggers redistribution from cytosol to the membrane of both ␧- and 웃PKC, two Ca2⫹-insensitive PKC isoforms expressed in neonatal and adult cardiac cells. PKC also induces the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and the expression of c-fos in neonatal cells, two events known to be mediated by the kinase. These observations are of physiological relevance with regard to the likely specific role of PKC isoforms in cardiac function. Purinergic stimulation activates three major pH-regulating systems: the Na⫹ /H⫹ antiporter, the Na⫹ / HCO3⫺ symporter, and the Cl⫺ /HCO3⫺ exchanger. Intracellular alkalinization is prevented by amiloride derivatives and is attributable in most part to an activation of the Na⫹ /H⫹ antiport. Under experimental acid load, ATP also activates an amiloride-insensitive, HCO3⫺-dependent alkalinizing mechanism. In both cases, the signal transduction pathways have not been clearly established. The most remarkable pH effect of a sudden application of ATP is a large (0.4 pH unit) and transient (1 min) acidosis that requires Cl⫺ ions in the extracellular milieu attributed to the activation of the anionic Cl⫺ /HCO3⫺ exchanger (Fig. 2). The activation of the exchanger is associated with a band 3-like protein phosphorylation on a tyrosine site. Cardiomyocytes express both AE1 and AE3 anion exchanger isoforms: ATP induces tyrosine phosphorylation of AE1 wheares acidosis still occurs in cells in which AE3 expression was blocked. More precisely, ATP activates the tyrosine kinase Fyn and association of both Fyn and FAK with AE1 . Tyrosine

FIGURE 2 Schematic description of the tyrosine kinase transduction pathways suggested to mediate the activation of both the AE1 isoform of the Cl⫺ /HCO3⫺ exchanger and the phospholipase C웂 after stimulation of a P2 purinoceptor. Phosphorylation of the tyrosine kinase FAK allows the docking of another tyrosine kinase of the src family, Fyn, to induce phosphorylation of the exchanger protein. In parallel, this signal transduction cascade activates the PLC웂 isoform to produce IP3 and DAG that activates the PKC.

kinase inhibitors and microinjection of either anti-Cst.1 antibody or recombinant CSK, both of which prevent the activation of Src kinases, significantly depressed the ATP-induced activation of the anion exchanger (Puce´at, 1998). The increases in either cAMP or IP3 have not yet been related to purinoceptor subtypes in cardiac cells. A candidate would be the recently cloned P2Y11 that is coupled to the stimulation of both phosphoinositide and adenylyl cyclase pathways. In other cells (e.g., MDCK cells), ATP increases cAMP preferentially through P2Y2 relative to P2Y1 and P2Y11 . Most P2Y receptors found in cardiac tissues (i.e., P2Y1 , P2Y2 , P2Y4 , and P2Y6) are assumed to be coupled to PLC웁. The purinoreceptor subtype activating the tyrosine kinase cascade is unknown; however, it was shown previously that ATPinduced acidosis requires the presence of Mg2⫹ ions, a characteristic that is not generally observed with presently cloned purinoceptors.

G. ATP Modulates Ionic Currents in Cardiac Myocytes Numerous studies using different experimental models have established the modulatory role of extracellular ATP on transmenbrane ionic currents in cardiac myocytes. These effects of ATP mediated by P2 purinoceptors are summarized in Fig. 3 and discussed in detail later. 1. Nonspecific Cationic Current A rapid, desensitizing inward current activated by ATP in the micromolar range was initially reported in frog atrial cells. A similar current was found in rat and guinea pig cardiac ventricular myocytes. It could not be attributed to a Cl⫺ channel but rather to a nonselective cation channel with a reversal potential near 0 mV, an inwardly rectifying current/voltage relation, and a low unitary conductance. In addition, the external application of ATP at 0.5 mM or above consistently activates a weak, time-independent, weak inward rectifying current in rabbit sinoatrial node cells. The channel is nearly equally permeable to K⫹, Na⫹, or Cs⫹ and only five times less to Tris and N-methyl-D-glucoseamine. Adenosine, ADP, and nonhydrolyzable ATP analogues failed to activate this current (Scamps and Vassort, 1990). The activation of both currents could be due to the stimulation of P2X purinoceptors whose subtype identity remain to be defined. 2. Clⴚ Current Extracellular ATP (5–50 애M) activated an outwardly rectifying, time-dependent Cl⫺ current (ICl) in single

36. ATP and Adenosine Signal Transductions

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FIGURE 3 Schematic presentation of transmembrane ionic currents regulated by extracellular ATP. Gs and Gi/o are G-proteins that mediate stimulation and inhibition of adenylyl cyclase, respectively; IATP , nonselective cationic current; INa , sodium current; ICaT and ICaL , T- and L-type calcium currents, respectively; ICl , chloride current; IKdel , delayed outwardly rectifying potassium current; IK Ach , inward rectifying K⫹ current activated by acetylcholine and adenosine; and IKATP , ATP-sensitive potassium current. IK Ach and IK ATP stimulation imply a Gi/o and a Gs protein, respectively. Pluses and minuses denote stimulatory or inhibitory effects. Inset: After depolarizations induced by ATP.

guinea pig atrial myocytes; ADP, AMP, and adenosine also activate this current. A Cl⫺ current was also activated by extracellular ATP (0.5–100 mM) in single rat ventricular myocytes. This current, blocked by the chloride channel blocker 4,4⬘-diisothiocyanatostilbene-2,2⬘disulfonic acid (DIDS), is not activated by either AMP or adenosine. The differential action of adenosine on ICl , activated by extracellular ATP in atrial and ventricular myocytes, could reflect different Cl⫺ channels in these cells or species variability. Neither the purinoceptor subtype nor the signal transduction pathway mediating the action of ATP on ICl is known. However, it has been reported that genistein at 100 mM, but not daidzein, activates cardiac chloride conductance. This effect is antagonized by Na3VO4 , an inhibitor of phosphotyrosine phosphatase. Comparison of ICl activated by genistein and by forskolin suggested that genistein activates the cAMP-dependent CFTR channel. The physiological importance of ATP enhancement of Cl⫺ current has not been determined. However, the activation of Cl⫺ current whose reversal potential is around ⫺35 mV is potentially arrhythmogenic because such current could depolarize the membrane potential and shorten the action potential plateau. It should also be mentioned that attenuation of isoprenaline-induced Cl⫺ current in guinea pig ventricular myocytes after ATP application has been reported.

3. Naⴙ Current In addition to its effects on ICa , extracellular ATP affects the inward Na⫹ current (INa). Under whole cell patch clamp, extracellular ATP in the micromolar range caused a leftward shift in both activation and availability characteristics of INa in rat single cardiac ventricular myocytes, similar to its effect on ICa . At hyperpolarized potentials, INa could be increased slightly due to the shift in activation, whereas at cell resting and depolarized potentials, INa is decreased because of reduced availability. ATP웂S and 움,웁-mATP exert similar effects, but UTP, 웁,웂-mATP, ADP, and adenosine are without effect. The shifts observed on application of extracellular ATP are not affected by cholera toxin treatment, suggesting that a Gs-protein and cyclic AMP are not involved in this phenomenon. 4. Ca2ⴙ Current Early studies have shown that extracellular ATP and ADP enhance calcium inward current (ICa) and ICadependent phasic tension in muscle bundles isolated from the right atrium of the bullfrog. This effect of ATP was independent of its hydrolysis and was assumed to be mediated by a receptor located at the outer surface of the affected cell membrane. This followed the primary

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VII. Signaling Systems

observation that ATP is able, like isoprenaline, to induce slow action potentials in K⫹-depolarized guinea pig hearts. Extracellular application of micromolar ATP increases the L-type Ca2⫹ current (ICaL) in cells isolated from rat ventricular myocardium (Fig. 4). As with 웁adrenergic stimulation, the increase in current amplitude results mostly from an increase in the probability of opening of single Ca2⫹ channels. ATP웂S exerts a similar effect, but adenosine is much less effective and GTP, UTP, CTP, and ITP are without effect. ATP is unable to alter Ca2⫹ current density after it has been enhanced by cholera toxin. These observations suggest that the P2 purinoceptor leads to an increase in Ca2⫹ current directly following the activation of cholera toxin-sensitive Gs-protein (Scamps and Vassort, 1994). Similarly, ATP increases the transient, low-threshold Ca2⫹ current (ICaT) in frog atrial cells via a pathway that does not involve phosphorylation. In single cells isolated from frog ventricle, ATP (1 애M) increases ICaL by up to twofold; at higher ATP concentrations the increase in ICaL is smaller, and at 100 애M, ATP reduces this current. The ATP-induced increase in Ca2⫹ current is prevented by perturbations that block either signal transduction pathways involving the activation of PLC or its activity. These data were interpreted to suggest that the ATP-induced increase in Ca2⫹ current in frog ventricular myocytes is mediated by P2 purinoceptor and phosphoinositide turnover. In contrast to its positive effect in rat ventricular myocytes and in guinea pig and rabbit atrial myocytes, exracellular ATP inhibits ICaL in a time- and concentration-dependent manner in isolated ferret ventricular myocytes. This effect of ATP is independent of adenosine A1 receptors, but involves ATP binding to P2Y receptor and the activation of PTX-insensitive G-pro-

FIGURE 4 Schematic outline of signal transduction pathways that mediate the action of extracellular ATP on transmembrane Ca2⫹ influx. Activation of P2X purinoceptors induces opening of a nonselective cationic channel and thus influx of Ca2⫹ ions, among other cations, that carry IATP. The L-type Ca2⫹ current, ICaL , is controlled positively by a Gs protein probably directly coupled to the channel protein(s) and negatively by a pertussis toxin-sensitive Gi protein.

tein. Similar observations were obtained in hamster heart cells. An ATP-induced inhibition, involving neither P1 nor P2 purinoceptors, was also reported in guinea pig sinoatrial node cells. Similarly, an ATP inhibitory effect was observed after full activation of ICaL by GTP웂S applied intracellularly. 5. Kⴙ Current A number of K⫹ channels are present in cardiac myocytes that determine, in part, the shape of the action potential and the frequency of spontaneous automatic activity of cardiac pacemakers; extracellular ATP regulates most of these channels. Specifically, extracellular ATP activates two different ionic conductances in bullfrog atrial cells: one transient, nonspecific for cations and depolarizing and the other sustained, probably for K⫹ ions, manifesting an inwardly rectifying current– voltage relation (Friel and Bean, 1988). ATP activates an inwardly rectifying K⫹ channel in calf atrial myocytes. This channel is nearly identical to the one activated by acetylcholine in these cells; conductance of the channel activated by ATP and acetylcholine is 30 and 31 pS, respectively. Also, extracellular ATP and adenosine activate kinetically similar K⫹ channels in atrial myocytes of 1- and 2-day-old rat heart (i.e., single channel conductance and mean open time of 32.0 ⫾ 0.2 pS and 0.5 ⫾ 0.1 msec, respectively, vs 31.3 ⫾ 0.3 pS and 0.9 ⫾ 0.1 msec, respectively). The muscarinic cholinergic receptor and the A1-adenosine receptor are known to be coupled directly to a K⫹ channel (K⫹Ach,Ado channel) via a PTXsensitive, GK-protein. In guinea pig atrial cells, extracellular ATP shortens the action potential. This effect is mediated by a P2 purinoceptor coupled directly to the K⫹Ach,Ado channel through a PTX-sensitive, GK-protein, analogous to the activation of the channel by either acetylcholine or adenosine. In another study on isolated guinea pig atrial myocytes, extracellular ATP (10 애M) transiently activates IK,Ach,Ado; however, when this current was preactivated with either carbachol or adenosine, ATP produced a transient increase followed by a sustained decrease of the current. These data were interpreted as a possible explanation for the biphasic inotropic effect (i.e., a rapid negative followed by a slow positive inotropic effect) of extracellular ATP in the rat atrial preparation. Another inward rectifying K⫹ channel, the intracellular ATP-sensitive channel (K⫹ATP channel), which is inhibited by intracellular ATP, is also regulated by purinergic stimulation (Fig. 5). The K⫹ATP channel consists of a weak inward rectifier subunit, Kir 6.2, plus a member of the ABC superfamily, SUR2. K⫹ATP channel activation during acute ischemia/hypoxia has been shown to exert a protective effect on the heart. Studies in cardiac

36. ATP and Adenosine Signal Transductions

641

phospholipase A2 inhibitor, as well as by inhibition of the cyclic AMP pathway.

H. Other Effects of ATP in Cardiac Cells 1. Glucose Transport Inhibition

FIGURE 5 Schematic description of a signal transduction pathway that has been suggested to mediate activation of the ATP-sensitive K⫹ current by extracellular ATP in cardiac myocytes. A P2Y purinoceptor is linked to adenylyl cyclase (AC isoform V) via the Gs protein. Adenylyl cyclase activation locally reduces the intracellular level of ATP and thereby might lead to activation of the ATP-sensitive K⫹ channel, which is blocked by sulfonylurea (SUR) derivatives. This effect, like that of 웁-adrenergic stimulation, can contribute to the shortening of the action potential and energy saving during local hypoxia.

myocytes in vitro have suggested that the activation of A1-adenosine receptors could result in the activation of K⫹ATP channels. However, at least in the hypoxic guinea pig heart in vivo, endogenous adenosine failed to activate K⫹ATP channels. Studies have shown that extracellular ATP enhances the current flow through this channel (IK,ATP) once it has been partially activated under conditions of metabolic stress (including 100 애M ATP in the intracellular patch pipette solution); the enhancement of IK,ATP by extracellular ATP is inhibited by cholera toxin as well as by inhibition of adenylyl cyclase but it is not sensitive to PKI, an inhibitor of the cAMPdependent PK. Thus, it has been suggested that the mechanism of this effect is the Gs-dependent activation of adenylyl cyclase, which causes increased cAMP production and thereby reduced levels of intracellular ATP (Babenko and Vassort, 1997). Several analogues of ATP, i.e., 움,웁-mATP, 2mSATP, and ATP웂S, exert a similar effect to that of ATP, whereas UTP and ADP have a relatively small effect and AMP and adenosine have no effect. ATP also activates the delayed rectifier K⫹ current (IK), which is activated slowly during the action potential plateau and facilitates repolarization, and whose deactivation also contributes to the depolarization of pacemaker cells. ATP-activated K⫹ currents are also seen in rat ventricular myocytes. First, ATP in the micromolar range increased the inward rectifying current IK,Ach,Ado . In addition, ATP activates a delayed outward K⫹ current that requires the presence of 100 nM intracellular Ca2⫹. The latter effect of ATP is mimicked by the application of arachidonic acid and is blocked by AACOCF3 , a

In addition to modulating plasma membrane conductances for cations and anions, cytosolic Ca2⫹ and H⫹ concentrations, rate of phosphoinositide hydrolysis, and cyclic GMP and AMP production and force of contraction, extracellular ATP markedly inhibits glucose transport. ATP decreases the amount of glucose transporters in the plasma membrane with a concomitant increase in intracellular microsomal membranes. P2X purinoceptors are not involved, as a drastic reduction in extracellular Na⫹ or Ca2⫹ ions does not alter this effect of ATP. The rank order potency of P2 receptor agonists— ATP ⱖ ATP웂S ⱖ 2mSATP ⬎ ADP ⬎ 움,웁-mATP— does not match that of any known P2Y purinoceptor subtype. Inhibition of transmembrane glucose transport was specific as ATP did not inhibit the rate of glycolysis or the rate of pyruvate decarboxylation (Fischer et al., 1997). Nevertheless, the physiological significance of this inhibitory effect of ATP on glucose transport is unclear, although it could be related to the reduced glucose transport in hypertrophied failing heart. 2. Agonist-Induced Internalization of Purinoceptor In the face of persistent stimulation, the response of most receptors fades away. The process of receptor desensitization, one of the many forms of G-proteincoupled receptor regulation, occurs generally via feedback regulation by second messenger-stimulated kinases PKA or PKC that the Gs- and Gq-coupled receptors activate, respectively. This effect was first reported for the 웁2-adrenergic receptor. This phosphorylation is followed by 웁-arrestin binding as a crucial step in the internalization of most heptahelical receptors. Our knowledge of the turnover sequence activation/ desensitization of purinoceptors is rather limited. The tagging of the P2Y2 receptor at its amino terminus with a hemagglutinin epitope sequence revealed that the receptor undergoes agonist-promoted movement to an intracellular compartment. However, this internalization does not establish any functional consequence and is not required for agonist-induced desensitization.

I. Cardiac Physiological Effects of ATP 1. Contractile Effects Most initial reports described negative inotropic effects after applying ATP in mammalian hearts. These

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VII. Signaling Systems

effects were more marked in the atrium and should be attributed to P1 purinoceptor activation after ATP breakdown by ectonucleotidases in the tissue. ATP is a full agonist agent in the frog ventricle, as well as in rat papillary muscle. It also enhances shortening of mammalian ventricular cardiomyocytes. After preventing P1 purinergic stimulation by various antagonists or PTX pretreatment, most ATP analogues and derivatives, as well as UTP, mimick ATP effects with an efficacy and potency order of ATP ⬎ ADP ⬎ AMP Ⰷ adenosine (Mantelli et al., 1993). This is also true in human atria and ventricular tissues. Positive inotropy could be attributable to both an increase in ICa and a facilitation of SR–Ca2⫹ release. Positive inotropy could also result from Ca2⫹ entry during P2X purinoceptor activation. However, in ferret ventricular myocytes, ATP decreases both ICa and force of contraction. 2. Chronotropic and Arrhythmogenic Effects In their seminal work, Drury and Szent-Gyorgyi (1929) reported negative chronotropic and dromotropic effects of purine compounds. Subsequent studies suggested that ATP had dose-dependent effects; small doses produce tachycardia whereas relatively larger doses of ATP slow the heart and induce AVN conduction block. However, these effects could be the result of ATP’s degradation to adenosine and the action of the latter on SA and AV nodes. This was the case during the administration of ATP into the sinus node blood supply (i.e., intracoronary) in dogs, in which model the negative chronotropic action of ATP was attenuated by theophylline, a nonselective adenosine receptor blocker. However, ATP caused cardiac acceleration in 40% of the isolated perfused rabbit hearts. This effect of ATP was not antagonized by either theophylline or PTX pretreatment, but was almost completely blocked by apamin, neomycin, and indomethacin. Based on these data, it was concluded that the positive chronotropic effect of ATP was mediated by P2 purinoceptors coupled to prostaglandins synthesis via a PTX-insensitive pathway involving the stimulation of PLC. It should be noted here that the negative chronotropic action of ATP is independent of prostaglandins. These observations indicate that negative chronotropy is in part due to P1 purinergic activation by ATP or by its degradation product, adenosine. In isolated ventricular myocytes of the guinea pig, ATP alone did not exert any significant electrophysiologic effect. However, when it was applied with drugs known to increase [Ca2⫹]i , ATP facilitated the induction of afterdepolarizations and triggered activity in 앑60% of the cells. In the presence of isoproterenol, ATP increased the amplitude of the transient inward current (Iti), delayed afterdepolarizations

(DADs), and ICa . In the presence of either BayK 8644 or quinidine, ATP further prolonged the action potential duration and also increased the amplitude of early afterdepolarizations (EADs). These findings support the hypothesis that the release of ATP into the extracellular space under pathophysiologic conditions could be arrhythmogenic. It is difficult to anticipate the direct effect of ATP on sinus node automaticity. On the one hand, extracellular ATP activated a time-independent, weakly inwardly rectifying current, which is nonselective for monovalent cations. This current was not activated by ADP, AMP, or adenosine, suggesting that the action of ATP is mediated by a P2 purinoceptor. On the other hand, in contrast to its effect on rat and guinea pig ventricular myocytes, extracellular ATP inhibits, in a concentration-dependent manner, ICa in guinea pig single SA node cells. The rank order of potency of ATP and related compounds in inhibiting ICa in these cells was ATP ⫽ 움,웁mATP Ⰷ 2mSATP ⬎ ATP웂S Ⰷ UTP ⫽ ADP ⬎ AMP ⬎ adenosine. This potency order has not been reported with regard to previously identified P2 purinoceptor subtypes, suggesting the mediation by a novel receptor of ATP’s action. In view of the critical role of ICa in the genesis of the action potential in SA node cells, it was proposed that extracellular ATP may play an important role in the regulation of heart rate. It is even more difficult to extrapolate data obtained in vitro to the human heart in vivo. However, it should be noted that numerous studies in cats, dogs, and human subjects have indicated that, at least in these species, extracellular ATP exerts a negative chronotropic action on cardiac pacemakers, which is mediated in part by the vagus nerve in addition to the action of adenosine. The vagal effect is due to a cardiocardiac depressor reflex elicited by the action of ATP on vagal afferent nerve terminals in the left ventricle, similar to its action on pulmonary vagal afferent terminals. In both organs (i.e., heart and lungs) the triggering of the vagal reflex by ATP is mediated by P2X receptors. The studies just mentioned strongly suggest that extracellular ATP can directly affect ionic currents in SA node cells. However, to what extent this action reflects a physiological role of extracellular ATP in the regulation of SA node automaticity remains to be determined. ATP shows positive chronotropic effects in cultured adult guinea pig myocytes that could be greatly enhanced in the presence of cardiac neurons that also possess P2 purinoreceptors. ATP increased the contractile rate in intrinsic cardiac neurons–myocyte coculture by 40% under control conditions and much more (100%) in the presence of tetrodotoxin. In contrast, ATP induced much smaller effects in noninnervated myocyte cultures (26%). In isolated rat ventricular myocytes, the

36. ATP and Adenosine Signal Transductions

sudden application of ATP at a micromolar concentration range induced cell depolarization and triggered automaticity. A similar phenomenon in rat papillary muscles required the uncaging of ATP by UV-light flash, whereas the application of ATP by changing the bath solution was without significant effect. 3. Hypertrophy Hormones and mechanical stretch can induce cardiac growth. Extracellular ATP constitutes a stimulus sufficient to induce changes in the pattern of expression of immediate-early genes such as c-fos and jun-B that is mediated by a Ca2⫹-dependent pathway in neonatal rat ventricular myocytes. Similarly, extracellular ATP inhibited a norepinephrine-stimulated growth of neonatal rat cardiac fibroblasts and activated c-fos gene expression in these cells. These effects were probably mediated by P2Y recpetors. However, ATP does not induce cell hypertrophy. Such an observation should be compared to the increase in the expression of atrial natriuretic factor (ANF) and myosin light chain-2 (MLC2) genes as well as cell hypertrophy by noradrenaline and angiotensin II. Similarly, it is thought that mitogen-activated protein kinase (MAPK) plays a central role in the regulation of cell growth. Although ATP, like phenylephrine, carbachol, and endothelin, activates the two p42 and p44 isoforms of MAPK, ATP, like carbachol, does not transactivate cardiac-specific promoter/luciferase reporter genes nor causes an increase in ANF expression. Furthermore, these studies suggest that the activation of c-fos, jun-B, PKC, and MAPK are not sufficient by themselves to stimulate hypertrophy and that ATP also activates an inhibitory pathway, as suggested by the fact that it prevents phenylephrine-induced hypertrophy. The purinoceptor subtypes that lead to MAPK activation in cardiac cells are unknown. They could be multiple and complex. Thus, in PC12 cells, different lines could show MAPK activation either by the G-proteincoupled P2Y2 receptor or following Ca2⫹ influx through P2X2 , receptor-activated channels and the involvement of the Ca2⫹-activated tyrosine kinase, PYK2 .

643

both inotropic and chronotropic effects. It is noteworthy that ATP applied abruptly at the same concentration to a cell might lead to its depolarization and anomalous automaticity, a pathological situation encountered during the early phase of ischemia during which ATP is released from the damaged cells. As discussed earlier, it is presently difficult to ascribe a given effect of ATP to a given receptor subtype and signal transduction pathway. The P2X5 receptor was cloned from a rat heart DNA library, with an abundantly expressed mRNA. A hP2X3 clone was also isolated from a heart cDNA library and RNA transcripts detected in human heart by RT-PCR analysis. Using RT-PCR, the expression of P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptor transcripts in whole heart, neonatal cardiac fibroblasts and myocytes, and adult cardiac myocytes was reported. In neonatal rat whole heart, all receptor sequences could be amplified, with P2Y6 being the most abundant. However, using the same procedure in adult rat myocytes, P2Y4 could not be detected, which suggests some changes in purinoceptor expression during development. In a study using human fetal heart, P2X1 , P2X3 , and P2X4 , as well as P2Y2 , P2Y4 , and P2Y6 purinergic receptors, have been identified using degenerated oligonucleotides. Indeed, very little is known about the expression in cardiomyocytes per se. The need of antibodies added to the long need of more specific and well-characterized agonists and antagonists. Moreover, there might be major differences between species as, for example, reexpression of the human and rat P2X4 as well as P2X6 gave different agonist/antagonist profiles. Nevertheless, the diversity in responses to agonists and antagonists produced by functionally expressed receptors are new findings that should help characterize the complex behavior of cardiac tissues under purinergic stimulation and the development of reagents that might prove to have therapeutic value in various cardiac diseases.

III. EXTRACELLULAR ADENOSINE AND THE CARDIOVASCULAR SYSTEM A. P1 Adenosine Receptors

J. ATP and the Heart: Concluding Remarks Adenosine has long been the focus of most studies concerning the effects of purines in the cardiovascular system. Adenosine is a ubiquitous biological compound released into the extracellular space by cells when the oxygen supply does not meet oxygen demand or by the degradation of ATP by ectoenzymes. At much lower concentrations than adenosine, ATP by itself induces quite variable effects on the cardiac tissues that involve

1. Classification and Structure The actions of extracellular adenosine are mediated by binding to specific cell surface receptors termed P1 purinergic receptors (or adenosine receptors). This family of receptors is blocked by methylxanthines such as caffeine and theophylline. All of the P1 receptors identified to date are G-protein-coupled receptors. These receptors are linked to heterotrimeric G-proteins, which dissociate upon adenosine binding, resulting in modula-

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tion of a wide range of intracellular signal transduction pathways. For years, functional and pharmacological evidence accumulated to suggest that multiple subtypes of adenosine receptors exist. Adenosine receptors were found to modulate intracellular levels of cAMP and were initially subdivided into A1 and A2 receptors based on their ability to inhibit or activate adenylyl cyclase, respectively. The existence of two subtypes of A2 receptors, i.e., A2a and A2b , was later proposed based on the identification of two populations of receptors with high and low affinity, respectively, that stimulate adenylyl cyclase in brain tissues. The classification of P1 receptors was validated by the molecular cloning and expression of A1 , A2a , and A2b receptors from multiple species (Luthin et al., 1996; Feoktistov and Biaggioni, 1997; Ralevic and Burnstock, 1998). In addition, a fourth receptor, called the A3 receptor, has been cloned (Linden, 1994). The activation of this receptor results in the inhibition of adenylyl cyclase and reduced levels of intracellular cAMP. The predicted structure of adenosine receptors is similar to that of other G-protein-coupled receptors (such as the P2Y receptors), consisting of seven hydrophobic transmembrane domains connected by three intracellular and three extracellular loops. The carboxyl terminus is assumed to extend into the cell, and the amino terminus is thought to be extracellular. The amino acid sequence identity among the four human receptors is approximately 40%. As would be expected, the sequence identity of the same receptor subtype among species is conserved to a greater degree (70–90%, depending on the receptor subtype). In general, regions with the greatest degree of homology are transmembrane domains and intracellular loops, regions likely to be important in maintaining the overall structure of the receptor, coupling to intracellular signal transduction pathways, and ligand binding. Regions with the least degree of sequence homology include extracellular loops and the carboxyl terminus. Several residues in the intracellular loop regions have the potential to be phosphorylated by protein kinases A and C, which may serve to regulate receptor function. In addition, the carboxy termini of A2a , A2b , and A3 receprs have putative sites for phosphorylation by G-protein-coupled receptor kinases (GRK). It is likely that phosphorylation at these sites is important for rapid receptor desensitization. Evidence in support of this concept includes (1) A1 receptors, which do not have potential GRK phosphorylation sites within the carboxy terminus region and desensitize at a much slower rate (several hours) compared to the other receptor subtypes, i.e., minutes for A3 receptors and 15–20 min for A2a receptors (Palmer and Stiles, 1997); (2) chimeric A3 receptors expressing a carboxy terminus from the A1 receptor desensitize at a slow rate

characteristic of A1 receptors (Palmer et al., 1996); and (3) overexpression of a dominant-negative mutant of Gprotein-coupled receptor kinase 2 results in a profound reduction in the rate of desensitization of A2a and A2b receptors (Mundel et al., 1997). To date, the crystal structure of adenosine receptors has not been attained, hindered by the fact that the receptors are hydrophobic transmembrane proteins. Nevertheless, data compiled from numerous structure–activity relationship studies have led to a model in which ligands are thought to bind within a cleft formed by the transmembrane regions of the receptor and interact with amino acids within transmembrane domains 3, 5, 6, and 7 (Van Rhee and Jacobson, 1996). Human genes encoding A1 , A2a , A2b , and A3 receptors have been characterized and mapped to chromosomes 1, 22, 17, and 1, respectively. In addition, a nonfunctional pseudogene for the A2b receptor has been identified and localized to chromosome 1. All of these genes appear to have one intron interrupting the coding sequence within the second intracellular loop. There is precedence from other G-protein-coupled receptors that alternative splicing can result in the heterogeneity of receptors with different characteristics. Examples include EP3-prostanoid receptors, D2-dopamine receptors, serotonin receptors, and 애-opioid receptors. Because the coding sequence of adenosine receptors contains only one intron, structural variations of adenosine receptor subtypes by conventional means of alternative splicing are not likely. The remote possibility exists, however, that multiple donor–acceptor sites at splice junctions, potentially creating receptors with insertions or deletions (Sajjidi et al., 1996). 2. Signal Transduction Mechanisms A1 and A2a receptors were originally known to inhibit and activate adenylyl cyclase, respectively (Table I). The A2a receptor is still thought to mediate its actions primarily through stimulatory Gs-proteins, which causes increased cAMP levels. However, A1 receptors are now known to be coupled to multiple effectors through pertussis toxin (PTX)-sensitive inhibitory G-proteins. These effectors include phospholipase C (PLC), potassium channels, calcium channels, and phospholipase A2 (Ralevic and Burnstock, 1998). In the heart, A1 receptor stimulation results in the activation of potassium channels. Many of the adenosine-mediated electrophysiological effects are mediated by alterations in potassium conductance and not cAMP production (see later). The transduction pathway activated by the A3 receptor is not fully elucidated. Like A1 receptors, A3 receptors appear to be coupled to inhibitory G-proteins that are sensitive to PTX, which have been demonstrated

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36. ATP and Adenosine Signal Transductions

TABLE I Characteristics of the P1 Adenosine Receptor Effector a

G-protein

Selective agonist

Selective antagonist

A1

Gi/o

앗AC, 앖앗PLC, 앖K⫹ current

CCPA

CVT-124, WRC-0571

A2a

GS

앖AC

CGS-21680

ZM-24135, SCH-58261

A2b

GS , Gq/11

앖AC, 앖PLC, 앖MAPK

None available

Enprofylline

A3

Gi/o

앗AC, 앖PLC

CI-IB-MECA

MRS-1191, MRS-1220, L-249313

a

AC, adenylyl cyclase; MAPK, mitogen-activated protein kinase; PLC, phospholipase C.

in cells expressing recombinant A3 receptors (Linden, 1994). In brain and mast cells, the stimulation of A3 receptors activates PLC, thus, it is likely that A3 receptors are also promiscuous and regulate a variety of signaling pathways. The A2b receptor is a very interesting receptor subtype in terms of its signaling mechanisms. Because its activation stimulates adenylyl cyclase in all tissues in which it is expressed, increases in cAMP undoubtedly are an important component of the signaling mechanism of A2b receptors (Feoktistov and Biaggioni, 1997). In mouse bone marrow-derived mast cells, canine mastocytoma cells, and HMC-1 cells, the A2b receptor also appears to be positively coupled to PLC, and its activation leads to increases in intracellular calcium levels. Regulatory proteins of the Gq family appear to play a role in the coupling of A2b receptors to PLC because the response is still present after pretreatment with PTX. More recently, A2b receptors have also been shown to activate mitogen-activated protein kinases (ERK1/2 , p38, and C-jun kinase) in HEK 293 cells and HMC-1 cells by multiple signaling mechanisms (Gao et al., 1999; Feoktistov and Biaggioni, 1999). These observations raise the interesting possibility that A2a receptors may play important roles in the regulation of cellular growth and differentiation.

the binding affinities of many compounds vary markedly among species. This is not a new concept; however, it has become more prominent with the characterization of the A3 receptor (Linden, 1994). Highly potent and selective agonists and antagonists have been produced for adenosine receptors (Table I). The use of these agents should aid in the understanding of the physiological function of adenosine receptors. Pharmacological characterization of the A2b receptor has progressed more slowly primarily because useful radioligand binding assays for this receptor subtype have only recently been developed. In general, agonists of adenosine receptors are composed of analogues of adenosine with substitutions of various combinations on the C-2 and N 6 position of the adenine radical, or the 5⬘ position of the ribose ring. These substitutions increase the affinity for the individual receptor subtypes and also render the molecules resistant to enzymatic degradation. Prototypical agonists for adenosine receptors include NECA (adenosine-5⬘-N-ethylcarboxamide) and 2-Cl-adenosine, which are nonselective and therefore have relatively equal potency for stimulating all four adenosine receptor subtypes. Traditionally, adenosine receptor antagonists include xanthine derivatives such as the nonselective compounds XAC (xanthine amine congener), theophylline, and 8-SPT (8-p-sulfophenyltheophylline).

3. Pharmacology

a. Adenosine Receptor Agonists

Several reviews have been published that describe in-depth the structure–activity relationship profile for adenosine receptors (Jacobson et al., 1992; Linden, 1994; Luthin et al, 1996; Feoktistov and Biagginoni, 1997; Jacobson et al., 1997). Therefore, this section provides a general description of some of the most useful compounds available for characterizing adenosine receptors and points out new concepts that have been recently emerged. Two general themes are stressed in this discussion. The first is that many compounds are not as selective for a given receptor subtype as originally suggested. This has become apparent now that comparisons are being made among all four receptor subtypes. Second,

A current challenge is the development of selective ligands for A3 receptors. The A3 receptor was discovered only when it was cloned and expressed in 1991. The development of selective agonists for A3 receptors has been hindered by the fact that many potent A3 receptor agonists also exhibit high-affinity binding to A1 receptors (Linden, 1994). One compound that has been identified to be slective for the A3 receptor is IBMECA [N 6-(3-iodobenzyl)-adenosine-5⬘-N-methylcarboxamide]. This compound has substitutions at both the N 6 and the 5⬘ positions of the adenosine molecule, unlike conventional A1 agonists, which have only N 6 substitutions. On the basis of radioligand binding analysis

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with recombinant receptors, the affinity of IB-MECA has been reported to be 1.1 and 54 nM for rat A3 and A1 receptors, respectively. Thus, this compound is approximately 50-fold selective for rat A3 receptors. IBMECA, administered systemically to rats, caused hypotension (secondary to mast cell degranulation) and locomotor depression. These effects were not blocked by A1 or A2a receptor antagonists. It seems, therefore, that IB-MECA can be used to selectively activate A3 receptors. There are two caveats in using IB-MECA to study A3 receptors, however. First, IB–MECA does not appear to be highly selective for A3 receptors in all species. For instance, it is only 앑10–20-fold selective for rabbit A3 receptors. Second, IB-MECA has considerable affinity for A2a receptors (앑50 nM). This has not been fully appreciated as emphasis has been placed on making comparisons between A1 and A3 receptors. In some systems (i.e., coronary vasculature), only a small percentage of A2A receptors need to be occupied to elicit a maximal response due to the high efficacy of its signal transduction pathways. Thus, it is possible that IBMECA may activate A2a receptors at concentrations needed to stimulate A3 receptors. The 2-chloro derivative of IB-MECA (Cl-IB-MECA) has become available. This compound is 2500- and 1400-fold selective for rat A3 receptors versus A1 and A2a receptors, respectively, and therefore should be more useful than IB-MECA. Although it has been difficult to identify agonists that discriminate A3 receptors from A1 receptors, highly selective agonists for A1 receptors are readily available. The two most useful compounds are CPA (N 6-cyclopentyladenosine) and 2-chloro-CPA (CCPA), which are at least 400-fold selective for A1 receptors in all species tested (Luthin et al., 1996). A derivative of NECA, CGS-21680 [2-(4-([2-carboxyethyl]phenyl)ethylamino)- adenosine-5⬘ -N- ethylcarboxamide], is the prototypical agonist selective for A2a receptors (Jacobson et al., 1992). In vivo, CGS21680 causes marked hypotension and reflex tachycardia. CGS-21680 is nearly inactive at A2b receptors and therefore, is useful for discriminating A2a and A2b receptor-mediated responses. Another useful A2a receptor agonist that has been identified is APE [2-(4-[amino]phenylethylamino)-adenosine]. This compound is a highly potent agonist for A2a receptors and can be radioiodinated and used as a high affinity radioligand (Luthin et al., 1996). Although highly potent and selective agonists have been developed for A1 , A2a , and A3 receptors, no selective agonist for A2b receptors has been identified so far. Adenosine itself also has low affinity for the A2b receptor, suggesting that it may only be functionally important during pathophysiological conditions characterized by high extracellular levels of adenosine. NECA

is the most potent A2b receptor agonist; it activates the A2b receptor with an EC50 value of 0.6–2 mM (Fektistov and Biaggioni, 1997). NECA, however, is nonselective and is more potent in activating other adenosine receptors with EC50 values in the low (A1 and A2A receptors) to high (A3 receptors) nanomolar range. A general approach that is effective in characterizing the A2b receptor is to observe the potency order of adenosine agonists. A response in which NECA is effective in the low micromolar range (1–10 애M) and other agonists, such as CGS 21680 and IB-MECA, are less potent is suggestive of mediation by an A2b receptor. b. Adenosine Receptor Antagonists Because expression levels, receptor coupling, and characteristics of the signal transduction mechanisms can influence the effectiveness of agonists, a more reliable approach to study adenosine receptors (or any type of receptor for that matter) is the use of antagonists. Several new selective antagonists have been developed for adenosine receptors. In terms of xanthine molecules, the most useful antagonists have been alkyl substitutions on the 1 and 3 positions (usually propyl or ethyl groups) and a cyclic group on the 8 position. It has been found that cycloalkyl groups at the 8 position increase the affinity of xanthines for the A1 receptor versus other receptor subtypes. One of the most selective xanthine antagonists identified to date for the A1 receptor is CVT124 (S-1,3-dipropyl-8[2-5,6-epoxynorbornyl]-xanthine). This compound, which has high affinity to rat (Ki ⫽ 0.67 nM) and human (Ki ⫽ 0.45 nM) A1 receptors and selectivity of 1:1800 and 1:2400 vs A2a , respectively, is currently being tested as a potential diuretic agent. Another highly selective A1 receptor antagonist is WRC-0571 [C8-(N-methylisopropyl)-amino-N 6-(5⬘-endohydroxy)-endonorbornan-2-yl-9-methyladenine)], a nonxanthine adenosine derivative that displays 60-fold selectivity for the human A1 versus the A2a receptor. WRC-571 has low affinity for the A3 receptor and is the most useful antagonist available for discriminating A1 and A3 receptor-mediated responses (Jin et al., 1997). It is important to point out that CPX (1,3-dipropyl-8cyclopentyl-xanthine), which has long been considered a selective antagonist for A1 receptors, has been shown to be a potent antagonist at A2b receptors (앑40 nM); in fact, its tritiated form has been used to label A2b receptors in binding assays. This compound also has relatively high affinity for the A3 receptor in some species such as the dog (Ki ⫽ 115 nM). Selective antagonists for A2a receptors include KF17837 (7-methyl-8-styryl-caffeine) and CSC [8-(3-chlorostyryl)-caffeinel], both of which are xanthine derivatives with an approximately 20- to 500-fold selectivity for the A1 versus the A2a receptor (Luthin et al., 1996;

36. ATP and Adenosine Signal Transductions

Feoktistov and Biaggioni, 1997). A new generation of nonxanthine antagonists has been developed for the A2a receptor, however, which are more useful than these older compounds. They include ZM-241385 [4-2[7amino-2)-2-furyl-triazolo 兵2,3-a-其-[1,3,5]triazin-5-ylamino)ethyl)phenol]) and SCH-58261 (5-amino-7-(웁-phenylethyl)-2-(8-furyl)pyrazolol[4,3-e]-1,2,4-triazolol[1,5c]pyrimidine). SCH-58261 has been shown to be effective in discriminating A2a vs A2b receptor-mediated responses. Similar problems encountered in developing A3 receptor antagonists were also met in the development of A3-selective agonists. First, it has been difficult to find compounds that are selective for A3 vs A1 receptors. Second, there are marked differences in binding affinities of A3 receptor antagonists. Specifically, the rodent A3 receptor is resistant to blockade by most adenosine receptor antagonists, wherease in other species, such as humans, dogs, and sheep, some antagonists bind to the receptor with high affinity (Linden, 1994). Thus, compounds are available as selective A3 receptor antagonists, but they cannot be used in rodents. Examples of the three most potent A3 receptor antagonists are MRS1191 (a dihydropyridine), MRS-1220 (a triazoloquinazoline derivative), and L 249313 (a triazolonaphthyridine derivative). These nonxanthine compounds are between 500- and 1000-fold selective for A3 vs A1 receptors in several mammalian species (Jacobson et al., 1997). The antagonist pharmacology of the A2b receptor has been characterized only recently. In contrast, with agonists, many antagonists bind to A2b receptors with high affinity. For example XAC, DPX (1,3-diethyl-8phenyl-adenosine), and CPX bind to A2b receptors with affinities of 앑100 nM, but are nonselective. The antiasthmatic drug enprofylline is the most selective A2b receptor antagonist known to date. It binds to human A2b receptors with an affinity of 7 애M, whereas it binds to the A1 receptor with 앑10-fold lower affinity (Feoktistove and Biaggioni, 1997). Thus, this compound is not very potent and is only slightly selective for A2b receptors.

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웁-adrenoceptor activation, and reducing heart rate. Adenosine also delays the development of ischemic injury and stimulates adaptive mechanisms, which protect the heart from additional ischemic insults (see later). Thus, adenosine is a cardioprotective metabolite. Interstitial adenosine is derived primarily from the degradation of adenine nucleotides such as ATP and ADP, which can occur either intracellularly or extracellularly (Fig. 6) (Zimmerman, 1996). Extracellular adenine nucelotides are degraded rapidly by a series of ecto-nucleotidases to form adenosine. As discussed previously in Section III, there are several potential sources of extracellular adenine nucelotides. ATP is released as a neurotransmitter from sympathetic nerve terminals where it is stored in granules with norepinephrine and neuropeptides. In addition, ATP and ADP can be released from circulating blood cells as well as resident leukocytes such as platelets, mast cells, basophils, and macrophages. It has been suggested that cAMP generated intracellularly by adenylyl cyclase can be transported out of cells and degraded to adenosine by an ecto-phosphodiesterase. Intracellularly, cytosolic 5⬘-nucleotidase is involved in the formation of adenosine from AMP, and S-adenosylhomocysteine hydrolase can catalyze the formation of adenosine from S-adenosylhomocysteine, a by-product of transmethylation reactions involving S-adenosylmethionine. The former pathway is the major source of adenosine production during hypoxia, ischemia, or metabolic stress, as AMP levels are increased under these conditions.

B. Sources of Extracellular Adenosine Adenosine is found in the interstitial fluid of all tissues. Under basal conditions, the interstitial adenosine level in the mammalian heart was estimated to be 앑100 nM; however, it increased as much as 100-fold when the oxygen demand exceeded the oxygen supply during ischemia, hypoxia, metabolic inhibition, or exercise. Extracellular adenosine acts to correct the oxygen supply/ demand imbalance by vasodilating the coronary vascular bed, counteracting the positive inotropic effects of

FIGURE 6 Schematic diagram of the metabolic pathways of adenosine and ATP. Extracellular ATP can be degraded by a series of ecto enzymes to adenosine, which can then act on cell surface adenosine receptors. Intracellularly, adenosine is formed from the sequential metabolism of ATP. Adenosine, accumulated in ischemic, hypoxic, or metabolically active cells, can be transported into the interstitial space by a nucleoside transporter. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

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Adenosine can be transported in either direction across the cell membrane by a facilitated transport protein (nucleoside transporter). This membrane-associated protein serves a dual role: First, it serves as a rapid mechanism for the removal of adenosine from the interstitial space. Endothelial cells appear to play an important role in this regard as interstitial concentrations of adenosine increase only when relatively large doses of adenosine are administered intraluminally. Second, when intracellular levels of adenosine are high (i.e., during stressful conditions), the transporter serves to extrude adenosine from the cell, thereby enabling it to access cell surface receptors. Inhibitors of the nucleoside transporter, such as dipyridamole, increase interstitial levels of adenosine, supporting the concept that a significant portion of interstitial adenosine originates from sources other than its facilitated transport out of cells. Under normal conditions, adenosine is reincorporated into the nucleotide pool upon phosphorylation by adenosine kinase. The Km of adenosine kinase is two orders of magnitude lower than that of adenosine deaminase, which removes the amino group from its C-6 position to form inosine. Thus, inosine, as well as its downstream metabolic by-products, xanthine, hypoxanthine, and uric acid, accumulates under hypoxic conditions when adenosine kinase is saturated with AMP. Inosine acts as a relatively potent agonist at the A3 adenosine receptor site of rat (but not human). Therefore, it is possible that inosine plays an important pathophysiological role under hypoxic or ischemic conditions in some species (Jin et al., 1997).

C. Effects of Adenosine in Mammalian Heart Adenosine exerts pronounced slowing of heart rate by suppressing cardiac pacemaker activity (i.e, negative chronotropic effect), slowing of atrioventricular nodal conduction (i.e., negative dromotropic effect), and a negative inotropic effect (Drury and Szent-Gyorgi, 1929). Many of the actions of adenosine in cardiac tissues are mediated by the activation of an inward rectifying potassium outward current, termed IkAdoAch , which is activated also by acetylcholine (ACh) (Pellege and Belardinelli, 1993; Belardinelli et al., 1995). In addition, adenosine modulates the kinetics of Ca2⫹ channels, particularly in AV nodal cells, thereby affecting the inward Ca2⫹ current (ICa). The actions of adenosine on IkAdoAch and ICa constitute direct effects in the heart. Adenosine also exerts an indirect, anti-웁-adrenergic effect (Belardinelli et al., 1995). Catecholamines increase the heart rate and force of contraction by increasing the current through L-type calcium channels (ICa,L) and the hyperpolarization-activated inward current If .

The effects on ICa,L and contractility are mediated by increases in cAMP levels through the activation of Gsproteins. Adenosine reduces cAMP levels and the resultant changes in calcium currents in response to 웁-adrenergic stimulation, although it has no effect on these parameters in the absence of agents that increase cAMP. In supraventricular tissues, adenosine exerts both direct and indirect effects, whereas in the ventricular myocardium of all species other than the rat and ferret, adenosine exerts only the indirect anti-웁-adrenergic effect (Belardinelli et al., 1995). In rats and ferrets, adenosine exerts both effects in the ventricular myocardium. It is well established that the cardiac effects of adenosine are mediated by A1 receptors expressed in muscle cells as well as in specialized cells of the conduction system. These receptors are coupled to PTX-sensitive G-proteins, the activation of which affects potassium channel conductance and inhibits adenylyl cyclase. The effect of adenosine on potassium currents is mediated by the 웁웂 subunit of G-proteins, and the inhibition of adenylyl cyclase is mediated by the a subunit of the Gisubunit (Yamada et al., 1998). Studies in chick and rat ventricular myocytes under specific experimental conditions have suggested a potential positive inotropic action of adenosine mediated by A2a receptors. A2a receptors appeared to mediate an increase in calcium influx by a mechanism independent of cAMP. Thus, it has been suggested that A2a receptors expressed in ventricular myocytes may play an underlying role in regulating ventricular contractility. However, because other studies in the guinea pig ventricular myocardium failed to find evidence for the presence of functional A2a receptors, more studies are required before the potential role of this receptor in the ventricular myocardium is fully elucidated.

D. Effects of Adenosine on Ionic Currents 1. SA Node Activation of A1 receptors on SA nodal cells causes hyperpolarization of the membrane potential and reduced the rate of phase 4 depolarization, thereby slowing the pacemaker activity of the SA node. In single isolated SA nodal cells, adenosine has been shown to activate the inward rectifying, outward potassium current IkAdoAch (Belardinelli et al., 1995). In isolated patches from SA nodal cells, adenosine causes the opening of single K⫹ channels with a unitary conductance of 25 ⫾ 1.9 pS. Adenosine also antagonizes the actions of catecholamines in SA nodal cells. Activation of 웁-adrenoceptors leads to increased current through L-type calcium channels (ICa,L) and the hyperpolariza-

36. ATP and Adenosine Signal Transductions

tion activated inward pacemaker current If . Adenosine attenuates the increase in both ICa,L and If caused by 웁-adrenoceptor agonists.

2. Atrial Myocardium Both ‘‘direct’’ and ‘‘indirect’’ actions of adenosine are evident in atrial tissue (Belardinelli et al., 1995). Adenosine receptors simulation directly activates the IKAdoAch (unitary conductance of 45 ⫾ 4 pS); this effect is independent of changes in cAMP. Adenosine inhibits ICa,L only after it has been enhanced by catecholamines or other agents that increase cAMP levels (e.g., forskolin). Thus, this effect of adenosine is caused by the inhibition of adenylyl cyclase and a reduction in the intracellular levels of cAMP. Adenosine has also been shown to inhibit ICa,L in the absence of drugs that stimulate adenylyl cyclase. This effect, which is relatively small, may be related to the inhibition of basal levels of adenylyl cyclase activity or to other unknown mechanisms independent of cAMP. The activation of IKAdoAch by adenosine also results in the shortening of atrial action potential duration and decreased force of contraction of atrial myocytes. The latter effect is related to reduced Ca2⫹ influx due to the shortening of the atrial action potential plateau.

3. AV Node The negative dromotropic action of adenosine is manifested in the prolongation of the PR and AH intervals as well as complete AV nodal conduction block. Adenosine does not modify the HV interval, indicating that it acts on cells proximal to the His bundle. Thus, the dromotropic actions of adenosine are mediated primarily through its actions on the AV node. The AV node is composed to three distinct regions: atrionodal (AN), nodal (N), and nodal-His bundle (NH). Adenosine has been shown to depress action potentials (i.e., decrease the rate and amplitude) of atrionodal and nodal cells, but to have little or no effect on action potential characteristics of cells from the nodal-His region (Belardinelli et al., 1995). Adenosine causes hyperpolarization, shortening of action potential duration, and slowing of recovery of ICaL from inactivation in both isolated single AV nodal and atrial cells, but it prolongs postrepolarization refractoriness in AV nodal cells only. This differential effect of adenosine on atrial and AV nodal refractoriness could not be explained by the effects of adenosine on IKAdoAch , but could be due to its effects on the reactivation of ICaL , which is the predominant inward current in AV nodal but not atrial cells.

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4. Ventricular Myocardium The effect of adenosine on ventricular myocardium is species dependent (Belardinelli et al., 1995). Adenosine reduces action potential duration and twitch contraction from species that exhibit an adenosine-induced IKAdoAch , i.e, the rat and the ferret. In other species, such as guinea pig, rabbit, and bovine, adenosine does not exert any direct effect on ventricular electrophysiology or contractile function. Similarly, adenosine neither shortens the duration of the monophasic action potential nor has a direct negative inotropic effect on human ventricular myocardium. In contrast, adenosine has a much more pronounced, indirect, antiadrenergic effect in ventricular tissue, which is associated with decreased intracellular levels of cAMP. Catecholamines increase ICa,L , the delayed rectifier potassium current (IK), and the chloride current (ICl); all of these effects are inhibited by adenosine. Afterdepolarizations and triggered activity induced by catecholamines, which are ICa,L and ITi (transient inward current) dependent, are either attenuated or abolished by adenosine. In addition, adenosine can terminate catecholamine-dependent ventricular arrhythmias that are thought to be mediated by cAMP. Adenosine does not, however, influence afterdepolarizations associated with calcium overload induced by elevated extracellular calcium concentration, oubain, or the calcium channel opener, BayK 8644. This notwithstanding, adenosine terminates ventricular tachycardia induced by digoxin toxicity, an effect that is probably mediated by its antiadrenergic action. This interpretation is based on the fact that digoxin toxicity is associated with enhanced sympathetic input to the heart, which plays a mechanistic role in the genesis of the ventricular arrhythmias observed in this setting. Adenosine is effective at reducing these arrhythmias via ‘‘indirect’’ actions, i.e., inhibition of adenylyl cyclase.

E. Effects of Adenosine on Vascular Tone Adenosine is a vasodilator in all vascular beds except for the kidney where it produces vasoconstriction. In the heart, adenosine is a particularly potent coronary vasodilator, acting on conductance vessels as well as on resistant vessels. Traditionally, vasodilation by adenosine has been attributed to actions of A2a receptors expressed in vascular smooth muscle cells. This is based on the potency order of agonists to produce vasodilation in certain vascular beds and on binding studies utilizing isolated vessles. Because activation of adenylyl cyclase is the primary (if not only) effect mediated by A2a receptors, an increase in cAMP levels is a likely component of the signal transduction pathway utilized by A2a recep-

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tors in the vasculature. There is evidence, however, that other adenosine receptor subtypes may play a role in regulating vascular tone. For instance, in some vascular beds, NECA is a potent vasodilator, but CGS-21680 has little effect. This agonist potency profile is suggestive of the involvement of A2b receptors and has been observed in guinea pig aorta, dog saphenous vein, dog coronary arteries, and guinea pig aorta. Furthermore, A3 receptors indirectly regulate vascular tone in some species (i.e., rodents and pigs, but not rabbits) by causing perivascular mast cells to degranulate. Thus, it is likely that multiple subtypes of receptors are involved in regulating vascular tone that may vary depending on the species and the particular vascular bed. There are several potential mechanisms by which adenosine may relax blood vessels. Adenosine may increase intracellular levels of cAMP in smooth muscle cells and cause relaxation by reducing calcium-sensitive myosin ATPase activity secondary to phosphorylation of myofilament-associated proteins by protein kinase A. Alternatively, protein kinase A may phosphorylate the ATP-sensitive potassium (KATP) channel, causing membrane potential hyperpolarization and reduced calcium influx. That glibenclamide, a KATP channel blocker, functionally antagonizes the vasodilatory effect of adenosine in the guinea pig coronary circulation suggests that the KATP channel could play a mechanistic role in the vasodilatory effect of adenosine (Daut et al., 1990). These mechanisms indicate that adenosine acts directly on vascular smooth muscle cells probably by activating both A2A and A2B receptors. Evidence shows, however, that adenosine-induced vasorelaxation may involve the endothelium (Olsson, 1996). For instance, adenosine is able to relax vessels when administered into the lumen of blood vessels, although it is unlikely that it reaches the underlying vascular smooth muscle cells due to its rapid metabolism by the endothelium. Because some studies have demonstrated that inhibitors of nitric oxide synthase (NOS) can attenuate vasodilation in response to adenosine, it is possible that adenosine acting on the endothelium may lead to the activation of endothelial NOS (eNOS). The ‘‘NO hypothesis’’ of adenosine-induced vasodilation has been met with substantial criticism, however, primarily because NOS inhibitors generally produce vasoconstriction, which in itself could reduce the vasoactivity of adenosine. Furthermore, it has been shown that coronary flow responses to adenosine are not diminished in eNOS⫺ / ⫺ knockout mice (G’decke et al., 1998). This experiment strongly argues against an obligatory role of eNOS in the vasoactivity of adenosine, at least in the mouse coronary bed. Other potential mechanisms for endothelial actions of adenosine include effects on

myoendothelial gap junctions; cAMP formed in response to adenosine has been shown to open gap junctions between oocytes and follicle cells. It is possible, therefore, that increased cAMP levels in response to adenosine in endothelial cells may enhance electrical and chemical coupling between these cells and smooth muscles cells, leading to vessel relaxation. In summary, mechanisms of the vascular effects of adenosine are still poorly understood. Adenosine-induced vasorelaxation is likely to involve multiple subtypes of receptors and mechanisms, depending on the species, the particular vascular bed, or the site of action, i.e., endothelium vs smooth muscle cells. Figure 7 is a schematic outline of the potential mechanisms of adenosine’s regulation of vascular tone.

F. Other Effects of Adenosine Several other actions of adenosine have been observed in the cardiovascular system. Adenosine has been shown to increase glucose uptake in the heart by itself or in conjunction with insulin. Exogenous administration of adenosine activates chemoreceptors on nerve terminals and increases sympathetic tone, resulting in increased respiration, hypertension, tachycardia, and increased catecholamine release. These excitatory actions of adenosine are observed in patients in whom adenosine is given systemically for the treatment of arrythmias. Adenosine also acts on intramyocardial sensory fibers, the activation of which can elicit reflexes resulting in the perception of pain. Pain experienced during angina or acute myocardial ischemia may by caused by adenosine acting in this manner. It has only recently become apparent that adenosine may also regulate gene expression (Feoktistov and Biaggioni, 1997). In human mast cells, adenosine acting via A2b receptors increases the expression of interleukin8, an important proinflammatory cytokine involved in inflammation and asthma. Adenosine modulates the expression of the inducible isoform of NOS in cultured neonatal cardiac myocytes, endothelial cells, and vascular smooth muscle cells. Similarly, adenosine increases the synthesis of the angiogenic substance vascular endothelial growth factor. Other studies suggest that adenosine inhibits smooth muscle cell growth, but increases the proliferation of vascular endothelial cells. These opposing actions of adenosine are mediated by A2a and A2b receptors. Whereas most studies of the cardiovascular effects of adenosine have focused on its acute actions on vascular tone and cardiac function, these actions of adenosine suggest that it may also have longterm effects in the regulation of inflammation and cell growth.

36. ATP and Adenosine Signal Transductions

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FIGURE 7 Schematic diagram illustrating potential mechanisms by which adenosine affects vascular tone. Adenosine can act directly on A2a and/or A2b receptors expressed in smooth muscle cells, producing vasodilation by increasing cAMP levels. Adenosine can also act on A2a or A2b receptors expressed on endothelial cells, causing the release of nitric oxide or the activation of myoendothelial gap junctions. In some species, adenosine’s activation of A3 receptors can cause perivascular mast cells to degranulate and release mediators, which can produce vasoconstriction or vasodilation. Mast cells mediators such as leukotrienes, thromboxane, and histamine cause local microvascular constriction. Circulating histamine causes nitric oxide release from the endothelium and thereby systemic vasodilation and hypotension.

G. Cardiovascular Physiology of Adenosine 1. Role as a Local Regulator of Coronary Blood Flow The fact that adenosine is linked to ATP metabolism and is a potent coronary vasodilator suggests that it may be an important physiological regulator of blood flow in the heart. Indeed, this appears to be true in some coronary flow responses, but not in others (Olsson and Pearson, 1990). For instance, after years of study it has been concluded that adenosine plays no role in coronary autoregulation, i.e., the phenomenon whereby blood flow is directly related to oxygen consumption. The ‘‘adenosine hypothesis’’ of autoregulation predicts that localized interstitial adenosine concentrations increase proportionally to increases in oxygen consumption, causing increased blood flow to the metabolically deprived region. Evidence against a role of adenosine in this response includes the observation that the intracoronary administration of the adenosine deaminase inhibitor to attenuate the metabolism of adenosine does not influence autoregulatory responses to changes in perfusion pressure. In addition, adenosine deaminase does not change perfusion pressure distal to a critical stenosis. However, reactive hyperemia is a response in which adenosine plays a contributory role. Adenosine deaminase and adenosine receptor antagonists blunt reactive

hyperemina, although they do not completely abolish it. It appears, therefore, that other metabolic factors (i.e., potassium, CO2), as well as myogenic mechanisms, work in concert with adenosine to produce the hyperemic response. 2. Adenosine and Cardioprotection Adenosine modulates the response of the heart to myocardial ischemia, i.e., the most severe form of oxygen supply–demand imbalance (Ely and Berne, 1992). This has become apparent in experimental animal studies in which the blockade of endogenously produced adenosine with receptor antagonists worsens ischemic injury, whereas exogenous adenosine delays the development of ischemic injury. Specifically, adenosine protects against reversible injury induced by brief periods of ischemia (myocardial stunning) as well as irreversible injury caused by prolonged ischemia (myocardial infarction). Preliminary studies in humans have shown that the administration of adenosine is beneficial in patients during coronary angioplasty, coronary artery bypass surgery, and acute myocardial infarction (Auchampach and Bolli, 1999). The mechanism by which adenosine protects against ischemic injury is unclear. It is effective in isolated, buffer-perfused heart models as well as in models of

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hypoxic injury using isolated cardiomyocytes, thus adenosine does not act solely by altering hemodynamics or increasing collateral blood flow. The major current hypothesis is that adenosine acts directly on A1 adenosine receptors in cardiomyocytes to preserve energy stores. This is accomplished by increasing the current through ATP-sensitive potassium (IKATP) channels expressed either in the sarcolemmal membrane and/or in mitochondria. Activation of cell surface KATP channels is thought to reduce calcium influx indirectly by shortening the action potential duration, thereby decreasing calciumdependent energy-consuming reactions. Increased current through mitochondrial KATP channels may protect against ischemic injury by reducing calcium accumulation in mitochondria or by inhibiting mitochondrial swelling. Because apoptosis is highly regulated by mitochondrial proteins, another possibility is that altering mitochondrial potassium conductance may lead to a decrease in apoptosis. A major focus of current investigations is to determine the relative importance of the two isoforms of the KATP channel in adenosine-induced cardioprotection.

The elucidation of the mechanisms of ischemic preconditioning has uncovered a new physiological role of adenosine (Fig. 8). Ischemic preconditioning is an adaptive phenomenon in which brief ischemia causes the heart to become tolerant to subsequent ischemic episodes (Cohen and Downey, 1995). There are two phases of preconditioning: an early phase that provides immediate protection but only lasts for 앑1 hr and a late phase that becomes apparent 12–24 hr later and lasts for several days. The early phase involves acute changes in metabolism. The time course and duration of the late phase are consistent with a mechanism involving the induction and synthesis of cardioprotective proteins. Both early and late preconditioning are triggered by adenosine that is produced during the transient ischemic episode. This is supported by experimental studies in which adenosine receptor antagonists block the induction of both early and late preconditioning. Furthermore, the exogenous administration of adenosine receptor agonists induces both phases of preconditioning. Strong pharmacological evidence suggests that adenosine induces early and late preconditioning by triggering

FIGURE 8 Schematic diagram of the potential mechanisms by which adenosine provides protection from ischemic injury. Activation of adenosine receptors leads to an enhanced function of sarcolemmal and mitochondrial KATP channels, resulting in the preservation of energy stores. Adenosine receptors are linked to potassium channels, probably via protein kinases. Stimulation of adenosine receptors and activation of signaling pathways involving protein kinases also lead to the activation of transcription factors and the synthesis of cardioprotective proteins. Effects on KATP channels provide immediate cardioprotection (early preconditioning), whereas increased gene expression leads to delayed, longlasting cardioprotection (late preconditioning). Proteins whose synthesis might be induced include antioxidant enzymes, heat shock proteins, and nitric oxide synthase. PTX, pertussis toxin; PLC/D, phospholipase C and D; AC, adenylyl cyclase; KATP , ATP-sensitive potassium channel; SUR, sulfonylurea receptor; APD, action potential duration; mitoKATP , mitochondrial KATP channel.

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a signal transduction cascade involving protein kinase C and MAP kinases. Activation of kinases is thought to alter the sensitivity of KATP channels causing the early preconditioning effect and also to activate transcription factors leading to the induction and synthesis of cardioprotective proteins providing the late preconditioning response. Thus, studies of preconditioning demonstrate that adenosine not only provides protection during acute ischemia, but also acts to trigger adaptive mechanisms to help protect the heart from subsequent ischemic episodes. While it is clear that adenosine plays a major role in preconditioning, it is necessary to point out that other factors, such as opioids, nitric oxide, and oxygen-derived free radicals, have also been implicated as triggers of preconditioning, suggesting that multiple signaling molecules are involved in this adaptive phenomenon, which may act in a cooperative manner. Traditionally, the A1 adenosine receptor has been implicated as the receptor subtype responsible for the cardioprotective actions of adenosine. Evidence shows, however, that the A3 receptor may also be involved (Auchampach and Bolli, 1999). Pharmacological studies have implicated that the activation of A3 receptors reduces infarct size and alleviates myocardial stunning in isolated buffer-perfused rabbit hearts. Furthermore, the A3 receptor agonist IB-MECA has been shown to protect against myocardial stunning and infarction in conscious rabbits. In these studies, IB-MECA was cardioprotective without causing any hemodynamic changes (unlike A1 receptor agonists), suggesting that targeting the A3 receptor may be useful in the clinical setting. It is not clear in these studies, however, whether IBMECA is acting on A3 receptors in cardiomyocytes or on other cell types within the heart (i.e., endothelial cells, mast cells, macrophages). In rats and mice (but not rabbits), A3 receptor agonists produce hypotension secondary to mast cell degranulation. It remains unknown whether a mast cell response occurs in humans.

H. Adenosine and the Cardiovascular System: Concluding Remarks Studies since the late 1920s have demonstrated conclusively that extracellular adenosine can act as a local physiological regulator by activating at least four subtypes of cell surface receptors coupled to different signal transduction pathways. Despite voluminous data accumulated in these studies, the exact roles of extracellular adenosine under physiologic and pathophysiologic conditions have not been fully elucidated. However, sufficient evidence has accumulated to strongly suggest that adenosine in the heart acts as an endogenous cardioprotective metabolite under pathophysiological conditions.

Also, adenosine plays a role as a local regulator of blood flow via its effects on vascular tone. The effects of adenosine in the cardiovacular system constitute a rationale for the use of adenosine receptor agonists and antagonists as therapeutic agents in the clinical setting.

IV. SUMMARY The purine nucleoside, adenosine, and purine nucleotide, ATP, are found in every cell of the human body where they play a critical role in cellular energetics and metabolism. Both compounds are released into the extracellular space under physiologic conditions as well as pathophysiologic conditions characterized by an imbalance between oxygen supply and oxygen demand. Other potential sources of extracellular ATP are UTP, via transphosphorylation, and diadenosine polyphosphates, via degradation. Extracellular ATP is degraded to AMP and adenosine by ectoenzymes. Studies over the past seven decades have demonstrated conclusively that extracellular adenosine acts as a local physiological regulator in the cardiovascular system by interacting with at least four subtypes of cell surface receptors coupled to different signal transduction pathways. Extracellular adenosine exerts negative chronotropic, dromotropic, and inotropic effects and anti-웁-adrenergic action in the heart as well as vasodilatory action on blood vessels. Despite voluminous data accumulated in these studies, the exact roles of extracellular adenosine in many processes have yet to be fully elucidated. For instance, the exact mechanisms and receptor subtypes involved in adenosine-induced vasodilation and cardioprotection remain unclear. The development of adenosine receptor subtype selective agonists and antagonists, as well as genetically modified mice in which specific adenosine receptor subtypes are not expressed, will undoubtedly increase our understanding of the function of extracellular adenosine in the cardiovascular system and hopefully lead to the development of new therapeutic strategies in the clinical setting. Extracellular ATP exerts pronounced effects in the mammalian heart under physiological and pathophysiological conditions. These include positive or negative inotropic and chronotropic effects mediated mainly by P2Y receptors. Under specific experimental conditions, ATP induces acidosis, cell depolarization, and arrhythmias mostly by acting through P2X receptors. Such a variation in activity profile is not generally recognized for other extracellular physiologic regulators acting within the same concentration range. It is noteworthy that most effects of extracellular ATP occur in the micromolar range, a concentration at least a thousandfold

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lower than that required for the intracellular metabolic effects of ATP. ATP could also play a role in various pathophysiological conditions, such as hypertrophy, preconditioning, and apoptosis. A common feature to these P2 receptor-mediated actions is the increase in intracellular calcium ion level ([Ca]i). Because extracellular ATP is degraded to adenine nucleotides and nucleosides, the response of the cardiomyocyte is the integral of the various effects of these multiple compounds generated by the surrounding tissues. Cardiac muscle is considered to be a syncytium. However, the spread of electrical activation can be reduced in the presence of ATP due to the depression of INa as well as cell uncoupling following the increase in [Ca2⫹]i and acidosis. In these circumstances, anomalous electrophysiological activity might occur and propagate due to ATP diffusion and P2X receptor activation, which leads to cell depolarization and contraction, stretching of the neighboring cells, and further ATP release. The cloning of 7 P2X and 13 P2Y receptors in recent years has advanced our knowledge considerably; however, the precise function of these receptors in cardiac myocytes is not clear. Not only is this due to the probable simultaneous expression of multiple receptors in each cardiomyocyte, but also to the fact that both types of P2 receptors can form complex homo- or heterotrimeric receptors whose activity differs with each constitutive oligomer. Furthermore, because phosphorylation of the P2 receptors can modulate trans-cell membrane ionic channel activity, it is difficult to extrapolate the behavior of integrated multiple cellular P2 receptors from the expression of single cloned proteins. However, cloning and expression of these receptor proteins are primary and necessary steps for the development of efficient pharmacological tools and future therapeutic agents.

Bibliography Abbracchio, M. P., and Burnstock, G. (1998). Purinergic signalling: Pathophysiological roles. Jpn. J. Pharmacol. 78, 113–145. al-Awqati, Q. (1995). Regulation of ion channels by ABC transporters that secrete ATP. Science 269, 805–806. Auchampach, J. A., and Bolli, R. (1999). Adenosine receptor subtypes in the heart: Therapeutic opportunities and challenges. Am. J. Physiol. 276, H1113–H1116. Babenko, A., and Vassort, G. (1997). Enhancement of the ATPsensitive K⫹ current by extracellular ATP in rat ventricular myocytes: Involvement of adenylyl cyclase-induced subsarcolemmal ATP depletion. Circ. Res. 80, 589–600. Belardinelli, L., and Pelleg, A. (1995). ‘‘Physiology and Pharmacology of Adenosine and Adenine Nucleotides: From Molecular Biology to Patient Care.’’ Kluwer, Norwell, MA. Belardinelli, L., Shryock, J. C., Song, Y., Wang, D., and Srinivas, M. (1995). Ionic basis of the electrophysiological actions of adenosine on cardiomyocytes. FASEB J. 9, 359–365. Burnstock, G. (1978). A basis for distinguishing two types of purinergic receptors. In ‘‘Cell Membrane Receptors for Drugs and Hor-

mones: A Multidisciplinary Approach’’ (R.W. Straub and L.Bolis, eds.), pp. 107–118, Raven Press, New York. Burnstock, G., Dobson, J. G., Jr., Liang, B. T., and Linden, J. (1998). ‘‘Cardiovascular Biology of Purines.’’ Kluwer, Norwell, MA. Burnstock, G., and Kennedy, C. (1985). Is there a basis for distinguishing two types of P2-purinoceptors? Gen. Pharmacol. 16, 433–440. Cohen, M. V., and Downey, J. M. (1995). Preconditioning during ischemia: Basic mechanisms and potential clinical applications. Cardiol. Rev. 3, 137–149. Danziger, R. S., Raffaeli, S., Moreno-Sanchez, R., Sakai, M., Capogrossi, M. C., Spurgeon, H. A., Hansford, R. G., and Lakatta, E. G. (1988). Extracellular ATP has a potent effect to enhance cytosolic calcium and contractility in single ventricular myocytes. Cell Calcium 9, 193–199. Daut, J., Maier-Rudolph, W., von Beckerath, N., Mehrke, G., Gunther, K., and Goedel-Meinen, L. (1990). Hypoxic vasoilation of coronary arteries is mediated by ATP-sensitive potassium channels. Science 247, 1341–1344. Drury, A. N., and Szent-Gyorgi, A. (1929). The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J. Physiol. (Lond.) 68, 213–237. Dubyak, G. R., and El-Moatassim, C. (1993). Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides, Am. J. Physiol. 265, C577–C606. Ely, S. W., and Berne, R. M. (1992). Protective effects of adenosine in myocardial ischemia. Circulation 85, 893–904. Feoktistov, I., and Biaggioni, I. (1997). Adenosine A2B receptors. Pharmacol. Rev. 49, 381–402. Feoktistov, I., and Biaggioni, I. (1999). Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells. Mol. Pharmacol. 55, 726–734. Fischer, Y., Becker, C., and Loken, C. (1999). Purinergic inhibition of glucose transport in cardiomyocytes, J. Biol. Chem. 274, 755–761. Friel, D. D., and Bean, B. P. (1988). Two ATP-activated conductances in bullfrog atrial cells. J. Gen. Physiol. 91, 1–27. Gao, Z., Chen, T., Weber, M. J., and Linden, J. (1999). A2B adenosine receptors and P2Y2 receptors stimulate mitogen-activated protein kinase in HEK-293 cells: Cross-talk between cAMP and protein kinase C pathways. J. Biol. Chem. 274, 5972–5980. G’decke, A., Decking, U. K. M., Ding, Z., Hirchenhain, J., Bidmon, H. J., G’decke, S., and Schrader, J. (1998). Coronary hemodynamics in endothelial NO synthase knockout mice. Circ. Res. 82, 186–194. Jacobson, K. A., van Galen, P. J. M., and Williams, M. (1992). Adenosine receptors: Pharmacology, structure-activity relationships, and therapeutic potential. J. Med. Chem. 35, 407–422. Jacobson, K. A., Park, K. S., Jiang, J. L., Kim, Y. C., Olah, M. E., Stiles, G. L, and Ji, X. D. (1997). Pharmacological characterization of novel A3 adenosine receptor-selective antagonists. Neuropharmacology 36, 1157–1165. Jin, X., Shepherd, R. K., Duling, B. A., and Linden, J. (1997). Inosine binds to A3 adenosine receptors and stimulates mast cell degranulation. J. Clin. Invest. 100, 2849–2857. Kunapuli, S. P., and Daniel, J. L. (1998). P2 receptor subtypes in the cardiovascular system. Biochem. J. 336, 513–523. Linden, J. (1994). Cloned adenosine A3 receptors: Pharmacological properties, species differences and receptor functions. Trends Pharmacol. Sci. 15, 298–306. Luthin, D. R., Auchampach, J. A., and Linden, J. (1996). Adenosine receptors. In ‘‘Biomembranes’’ (A. G. Lee, ed.), Vol. 2, pp. 321– 347. JAI Press, Greenwich, CT. Mantelli, L., Amerini, S., Filippi, S., and Ledda, F. (1993). Blockade of adenosine receptors unmasks a stimulatory effect of ATP on cardiac contractility. Br. J. Pharmacol. 109, 1268–1271.

36. ATP and Adenosine Signal Transductions Mundel, S. J., Benovic, J. L., and Kelly, E. (1997). A dominant negative mutant of the G proteincoupled receptor kinase-2 selectively attenuates A2 receptor desensitization. Mol. Pharmacol. 51, 991–998. North, R. A., and Barnard, E. A. (1997). Nucleotide receptors. Curr. Opin. Neurobiol. 7, 346–357. Olsson, R. A. (1996). Adenosine receptors in the cardiovascular system. Drug Dev. Res. 39, 301–307. Olsson, R. A., and Pearson, J. D. (1990). Cardiovascular purinoreceptors. Physiol. Rev. 70, 761–845. Palmer, T. M., Benovic, J. L., and Stiles, G. L. (1996). Molecular basis for subtype-specific desensitization of inhibitory adenosine receptors: Analysis of an A1-A3 chimeric receptor. J. Biol. Chem. 271, 15272–15228. Palmer, T. M., and Stiles, G. L. (1997). Structure-function analysis of inhibitory adenosine receptor regulation. Neuropharmacology 36, 1141–1147. Pelleg, A., and Belardinelli, L. (1993). Cardiac electrophysiology and pharmacology of adenosine: Basic and clinical aspects. Cardiovasc. Res. 327, 754–761. Pelleg, A., and Belardinelli, L. (1998) ‘‘Effects of Extracellular Adenosine and ATP on Cardiac Myocytes.’’ R. G. Landes Company, Austin, TX. Puce´at, M. (1998). Purinergic activation of a tyrosine-kinase-dependent pathway in cardiac cells. Drug Dev. Res. 45, 427–433. Puce´at, M., Bony, C., Jaconi, M., and Vassort, G. (1998). Specific

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activation of adenylyl cyclase V by a purinergic agonist. FEBS Lett. 431, 189–194. Ralevic, V., and Burnstock, G. (1998). Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 415–492. Sajjadi, F. G., Boyle, D. L., Domingo, R. C., and Firestein, G. S. (1996). cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant. FEBS Lett. 382, 125–129. Scamps, F., and Vassort, G. (1990). Mechanism of extracellular ATPinduced depolarization in rat isolated ventricular cardiomyocytes. Pflu¨g, Arch. 417, 309–316. Scamps, F., and Vassort, G. (1994). Pharmacological profile of the ATP-mediated increase in L-type calcium current amplitude and activation of a non-specific cationic current in rat ventricular cells. Br. J. Pharmacol. 113, 982–986. Tresize, D. J., Bell, N. J., Kennedy, I., and Humphrey, P. P. A. (1994). Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: Implications for P2 purinoceptor classification. Br. J. Pharmacol. 113, 463–470. Van Rhee, A. M., and Jacobson, K. A. (1996). Molecular architecture of G protein-coupled receptors. Drug Dev. Res. 37, 1–38. Yamada, M., Inanobe, A., and Kurachi, Y. (1998). G protein regulation of potassium ion channels. Pharmacol. Rev. 50, 723–757. Zimmerman, H. (1996). Extracellular purine metabolism. Drug Dev. Res. 39, 337–352.

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37 Kinase Signaling in the Cardiovascular System JUN-ICHI ABE, CHEN YAN, JAMES SURAPISITCHAT, and BRADFORD C. BERK Cardiovascular Research Center University of Rochester Medical Center Rochester, New York 14642

I. INTRODUCTION

strates and regulators. Finally, induction and activation of phosphatases that inhibit kinases are prominent mechanisms for turning off signal transduction. These general concepts are discussed in greater detail with respect to several growth factors and ROS-mediated signal events.

The functions of individual cells in multicellular organisms are regulated and orchestrated by a vast array of extracellular signals, including growth factors, cytokines, and reactive oxygen species (ROS). This chapter discusses the role of kinases in agonist-mediated signal transduction in vascular smooth muscle cells, endothelial cells and cardiac myocytes. Studies indicate important roles for growth factors, cytokines, and ROS in atherosclerosis and cardiac hypertrophy. Exciting findings show that these extra stimuli regulate intracellular kinases and control cell function and growth. This chapter focuses on how receptors on the cell surface modulate cell and tissue function and how they transmit signals that activate intracellular kinases.

A. Receptor Tyrosine Kinases Growth factor receptors with protein tyrosine kinase (PTK) activity possess structural features that transmit growth signals from the cell exterior to interior. These conserved features include a large, glycosylated extracellular-binding domain, a single hydrophobic transmembrane region, and a cytoplasmic tail that contains a tyrosine kinase catalytic domain. Receptor PTKs exist in at least eight superfamilies, defined by similarities in their extracellular domains (e.g., cysteine-rich regions, immunoglobulin-like sequences), their existence as heteroligomers [hepatocyte growth factor (HGF) and insulin-like growth factor (IGF) receptor subclasses], or the presence of an insert region in their kinase domain [fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) receptor subclasses]. Binding of growth factor activates the receptor by stimulating the phosphorylation of tyrosine residues in the intracellular domain. The precise activation mechanism is family specific, but in all cases a conformational alteration of the receptor’s extracellular domain facilitates receptor– receptor oligomerization (Heldin, 1995). The juxtaposition of the kinase domains of two receptors allows phosphorylation in trans between receptors, often referred to as autophosphorylation. Autophosphorylation serves

II. INITIATION AND CONTROL OF EXTRACELLULAR STIMULI Signal transduction events progress from the cell surface to generate intracellular second messengers. These molecules then transmit the signal to intracellular mediators present in both the cytoplasm and the nucleus. This transition occurs by the integration of multiple extracellular stimuli by shared, convergent kinases. In addition there is amplification of signal transduction events by kinase cascades with activation of multiple downstream kinases by single upstream kinases. Positive and negative cross talk between kinase cascades modulates signal transduction. Also, translocation and subcellular localization of enzymes determine access to sub-

Heart Physiology and Pathophysiology, Fourth Edition

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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VII. Signaling Systems

two important functions: it increases the catalytic activity of the kinase and it provides docking sites for downstream signal transduction molecules. To illustrate these concepts, the PDGF 웁-receptor is described in Fig. 1. Upon tyrosine phosphorylation the tyrosine kinase activity of the oligomerized receptors is increased, resulting in transphosphorylation of neighboring intracellular domains. One site is a conserved tyrosine residue localized inside the kinase domain (Tyr857). Mutation of this residue to a phenylalanine residue lessens kinase activity, suggesting that phosphorylation of Tyr857 is important for activation of the kinase (Fantl et al., 1989). The other phosphorylation sites are spread out over the cytoplasmic part of the receptor; a total of 11 of 15 tyrosine residues in the noncatalytic part of the receptor are phosphorylated (Claesson, 1994).

Receptor tyrosine phosphorylation establishes a confirmation that is capable of recruiting and stabilizing high-affinity protein–protein complexes with cytoplasmic signaling molecules at the plasma membrane. Such interactions are directed by different types of domains, e.g., Src homology domain 2 (SH2) and phosphotyrosine-binding domain (PTB) domains, which recognize phosphorylated tyrosine residues in specific environments (Pawson, 1995). At least 10 different SH2 domain-containing molecules bind to different phosphorylated tyrosine residues in the PDGF 웁-receptor in a specific manner (Fig. 1); the specificity is primarily determined by the character of the three to six amino acid residues downstream of the phosphorylated tyrosine. No PTB domain is known to bind to the receptor. SH2 domain proteins that interact with the PDGF 웁receptor fall into two categories: molecules with enzymatic or other activity and adaptor molecules that connect the receptor with other molecules, as mentioned later.

B. Signal Initiation by Receptors Lacking Intrinsic PTK Activity 1. Heptahelical Receptors

FIGURE 1 Schematic illustration of the interaction between the autophosphorylated PDGF 웁-receptor and downstream signal transduction molecules. Tyrosine residues known to be autophosphorylated are indicated by ‘‘P’’. The specificity of the interaction between SH2 domain molecules and autophosphorylated tyrosine residues is indicated; broken lines for Src, Shc, and PLC-웂 show interactions of lower affinity.

Neurohumoral agonists that dynamically regulate vascular tone have been shown to initiate signal transduction via the seven transmembrane-spanning (hepathelical) G-protein-coupled receptor superfamily. These receptors are typically coupled to heterotrimeric G-proteins and lack intrinsic PTK activity, in contrast to receptors for growth factors such as PDGF, FGF, and epidermal growth factor (EGF). The topography of heptahelical receptors is characterized by amino-terminal extracellular and carboxy-terminal intracellular ‘‘tails,’’ connected by seven transmembrane segments with three intervening extracellular and three intracellular loops. Nonpeptide agents such as 움1-adrenergic agonists, as well as peptides such as endothelin-1 (ET-1), angiotensin II, and 움-thrombin, are capable of inducing hypertrophic phenotypes in cultured cardiomyocytes, generally via pertussis toxin-insensitive G-proteins of the G움q family. Activation of G-protein-coupled receptors elicits a profound change in the transmembrane 움 helices. This changes the conformation of the intracellular loops, which in turn uncovers previously masked G-proteinbinding sites (Altenbach et al., 1996; Bourne, 1997; Wess, 1997). This causes the exchange of GDP for GTP bound to the G-protein 움 subunit and a conformational change in three flexible ‘‘swich regions’’ of G움, activating G움 and causing its dissociation from the 웁웂 heterodimers (Lambright et al., 1994; Sondek et al., 1994). In

37. Kinase Signaling

turn, GTP-bound G-protein 움 subunits or 웁웂 complexes initiate intracellular signaling responses by acting on effector molecules such as adenylyl cyclases, phosphodiesterases, and phospholipases or regulating the activity of ion channels, ion transporters, and a growing number of kinases. As an example of the G-protein-coupled receptor, we discuss the angiotensin AT1 receptor, which is composed of 359 amino acids with seven transmembrane domains arranged as shown in Fig. 2. Interactions among these domains and corresponding intracellular loops are responsible for determing the nature of angiotensin II binding and receptor activation after binding. For example, the angiotensin II-binding site appears to be the result of interactions among several of these domains, including the NH2 terminal extension, Tyr92 in extracellular loop 1, extracellular loop 3 (Asp278 and Asp281), Tyr292, and Tyr225 (Fig. 2). Several transmembrane domains are responsible for G-protein interactions with the receptor, including domains present in the second

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and third intracellular loops. Modeling analysis suggests that an interaction between Asp74 and Tyr292 is critical for angiotensin II binding and activation (Marie et al., 1994), as are the conserved residues Tyr292 and Tyr215 (Hunyady et al., 1995a). In addition, the motif Asp125Arg126-Tyr127 in the amino-terminal region of the second cytoplasmic loop is important in G-protein activation (Ohyama et al., 1992). 2. Cytokine Receptors During inflammation and other forms of stress, vascular smooth muscle cells (VSMC) and cardiomyocytes are exposed to cytokines and interferons. Receptors for these ligands are constitutively associated with members of the Janus kinases (JAK) PTK pathway. Although cytokine and interferon receptors are not receptor tyrosine kinases themselves, their activation mechanisms are so similar to the receptor PTKs that they should be considered in the same context. Ligand binding activates

FIGURE 2 Representation of amino acids and signaling domains in the rat AT1a receptor. Helices were positioned on the basis of modeling of G-protein-coupled receptors. Ser, Thr, and Tyr residues are depicted, as well as residues thought to be involved in specific signal transduction events. Highlighted residues in transmembrane domains may be important in ligand binding even if they are not phosphorylated. The receptor contains putative docking motifs for Stat3 (YXXQ; Y223-Q226; Y312-Q315), Src (YXXI; Y127-I130), SHP-2 (YIXP; Y319-P322), and PI-3 kinase (YFYM; Y54-M57) that may participate in JAK-STAT coupling and activation of other signaling pathways. (Berk et al., 1997)

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VII. Signaling Systems

signal transduction by inducing receptor oligomerization and concomitant JAK activation, probably via a transmembrane signal resulting in the transphosphorylation of neighboring intracellular tails. As is the case for receptor tyrosine kinases, this allows for binding of cytoplasmic signaling molecules, which may be substrates for JAK phosphorylation. For example, cytokines of the LIF/IL-6 family induce activation of the gp130 transmembrane receptor, which does not possess any kinase activity. The functional importance of gp130 hypertrophic signaling in vivo was demonstrated in transgenic mice, which overexpress circulating IL-6 as well as a soluble form of the IL-6 receptor. These animals develop marked cardiac hypertrophy, which is dependent on myocardial expression of gp130. Stimulation of gp130 rapidly activates two classes of signaling kinases: JAKs and nonreceptor kinases (such as BtK, Tec, and Fes).

III. INTRACELLULAR SIGNALING PATHWAYS A. Receptor Phosphorylation in Heptahelical Receptors 1. G-Protein-Coupled Receptor Kinases (GRKs Serine/Threonine Kinases) To respond selectively to new stimuli or changes in magnitude of stimulation, biological systems consistently diminish their responses via a process termed desensitization or adaptation. Desensitization manifests itself at the cellular level in biological processes as diverse as bacterial chemotaxis (Spring and Krebs, 1999), mating responses in yeast (Reneke et al., 1988), light perception in Drosophilia (Dolph et al., 1993), and neurotransmission in mammals (Arriza et al., 1992). Phosphorylation appears to be a critical event in the desensitization of G-protein-coupled receptors such as the 웁2adrenergic receptor (Bouvier et al., 1988; Ferguson et al., 1995; Oppermann et al., 1996). Although these receptors do not possess any intrinsic kinase activity, they can be phosphorylated on serine and threonine residues by several kinases, including GRK (Premont et al., 1995). Agonist activation of the 웁2-adrenergic receptor leads to the activation and membrane recruitment of GRK2 and GRK3 (Bouvier et al., 1988; Ferguson et al., 1995; Hausdorff et al., 1989). The GRKs phosphorylate the activated receptor protein, which promotes the binding of arrestin proteins to the phosphorylated receptor and uncoupling of the receptor from further G-protein activation (Lefkowitz, 1998; Pitcher et al., 1998). In addition, the phosphorylation and binding of arrestin promote the physical sequestration of the inactivated 웁2AR from the cell surface. Thus, the GRK/arrestin pathway

promotes the desensitization and downregulation of agonist-activated heptahelical receptors (Lefkowitz, 1998; Pitcher et al., 1998). Like the G-proteins themselves, GRKs recognize and respond to the activation state of the receptors. First, GRKs ‘‘translocate’’ in response to receptor activation to the plasma membranes where the receptors are found (Lefkowitz, 1998; Pitcher et al., 1998). Second, GRK kinase activity is activated directly by interaction with agonist-activated receptors (Lefkowitz, 1998; Pitcher et al., 1998), but not unactivated receptors. Thus GRKs can be considered as effectors for G-protein-coupled receptors, functioning in a negative feedback manner to regulate the receptors themselves. Paxton et al. (1994) and others (Kai et al., 1994) have shown that the AT1 receptor in vascular smooth muscle is phosphorylated basally and in response to angiotensin II. The majority of phosphorylation in vivo was found on serine residues, and there was a small increase in total phosphoserine content after agonist exposure. GRK phosphorylation domains in G-protein-coupled receptors include the third intracellular loop and the carboxyl-terminal tail (Premont et al., 1995). Although multiple serines may be phosphorylated by GRKs, it appears that only the initial one or two phosphorylations are physiologically relevant. These sites, in a number of receptors, are characterized by a pair of acidic amino acids located amino terminal to the initial site of phosphorylation (Fredericks et al., 1996), such as Asp/Glu-Asp/Glu-X(0-5)-Ser/ Thr. It has been shown in transfected 293 cells that the AT1 receptor can be phosphorylated by GRK2, GRK3, and GRK5 (Oppermann et al., 1996). However, the actual role of GRKs in phosphorylation of the AT1 receptor in relevant tissues such as vascular smooth muscle remains speculative. It has been shown that AT1 receptor sequestration is regulated by a motif in the cytoplasmic tail involving residues carboxyl to Tyr312 (Hunyady et al., 1994) and to Leu314 (Thomas et al., 1995b) (Fig. 2). This region lacks highly acidic amino acids typical of the GRK phosphorylation motif (especially Asp and Glu). However, one region, Asp343-Asn344Met345-Ser346-Ser347-Ser348, has partial homology to the consensus GRK phosphorylation motif. In summary, serine phosphorylation may play a role in AT1 receptor desensitization, although the precise phosphorylation sites remain to be determined. 2. Tyrosine Phosphorylation in Hepthahelical Receptors Studies have suggested that tyrosine residues in the carboxyl tail may also be important in G-protein-coupled receptor sequestration. Specifically, Ferguson et al. (1995) identified a highly conserved tyrosine (Y326) in

37. Kinase Signaling

the motif NPXXY, which is involved in the sequestration of the 웁2-adrenergic receptor, although these investigators did not demonstrate tyrosine phosphorylation at this site. This motif is present in the AT1 receptor at Tyr302 (NPLFY). However, AT1 receptors in which Tyr302 was mutated to Ala or in which triple alanine replacements of Phe301, Tyr302, and Phe304 were performed showed no substantial changes in their internalization kinetics (Hunyady et al., 1994). The AT1 receptor contains tyrosine residues in cytoplasmic loops and the C terminus that could undergo phosphorylation and participate in signal transduction by serving as docking sites for adaptors (Fig. 2). In fact, tyrosine phosphorylation of the AT1 receptor has been shown to occur (Paxton et al., 1994), suggesting a role in receptor-mediated events such as signal transduction. Immunoprecipitation of the AT1 receptor and Western blot analysis using an antiphosphotyrosine antibody showed a small increase in agonist-dependent tyrosine phosphorylation up to 6 min after angiotensin II treatment (Marrero et al., 1995c). Tyr302 appears to be important in signal transduction because two groups (Hunyady et al., 1995b; Laporte et al., 1996) have shown that mutating this residue to Ala or Phe caused alterations in G-protein activation and IP3 production. Tyr319 is of interest because it is part of the motif Tyr-Ile-Pro-Pro, which is analogous to the SH2-binding motifs found within the PDGF receptor (Tyr-Ile-Ile-Pro) and within the EGF receptor (Tyr-Leu-Pro-Pro) (Fantl et al., 1993; Pascal et al., 1994). In the PDGF and EGF receptors, these motifs have been shown to be target sequences for signaling mediators when the tyrosine is phosphorylated. SH2-binding domains contain a critical tyrosine residue that, when phosphorylated, promotes interactions with signaling proteins that contain the SH2 domain. For example, PLC-웂 contains an SH2 domain that interacts with the Tyr-Ile-Ile-Pro and Tyr-Leu-Pro-Pro sequences present in PDGF and EGF receptors (Fantl et al., 1993). Thus, tyrosine phosphorylation of the AT1 receptor may be important in signal transduction events mediated by the binding of SH2 domain-containing proteins.

B. Modular Domains in Intracellular Signaling Specificity in the recruitment and activation of signaling molecules by receptor and nonreceptor PTKs is determined by sequences in both the kinases and the signaling molecules themselves. These protein–protein interactions are the consequence of highly specific interactions between tyrosine-phosphorylated proteins and modular domain-containing signaling adaptors, regulators, and effectors. Significant progress has been made in elucidating the basis for these interactions (Pawson,

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1995; Songyan and Cantley, 1995). For example, SH2 domains, found in many cytosolic signaling molecules, direct the interactions of these proteins with tyrosinephosphorylated receptor PTKs. Other domains, such as Src homology 3 (SH3), pleckstrin homology (PH), and the phosphotyrosine-binding (PTB) domain, localize PTKs and their substrates to specific cell compartments and/or mediate specific interactions between signal mediators. 1. SH2 Domain Of these protein modules, the SH2 domain has been characterized most extensively. Consisting of approximately 100 amino acids, the globular region contains a pocket-like region (Pawson, 1995). SH2 domains bind ligands containing phosphotyrosine residues within a specific sequence (Pawson, 1995). High-affinity binding is provided by the phosphotyrosine residue itself and by residues carboxyl terminal to it, p-YXX⌿ (X: any amino acid or a selected amino acid from subset that correlates with the ␧ determinant—those amino acids within domains which determine ligand predilections. ⌿: hydrophobic amino acid; p-Y: phosphotyrosine) (Songyan and Cantley, 1995). Songyang and Cantley (1995) have utilized degenerate phosphopeptide libraries to identify sequence determinants of SH2 domainbinding specificity. These investigators have postulated that the identify of the amino acids at the ⫹1 and ⫹3 positions on the carboxyl-terminal side of the phosphorylated tyrosine, in combination with the identity of the residues at the fifth position of 웁-sheet D (the notation indicates fifth amino acid in the fourth 웁-strand; D indicates the fourth strand), is sufficient to define functional subclasses of SH2 domains (Songyan and Cantley, 1995). For examples, these sequences may dictate the preference of receptor PTKs for binding to targets such as the p85 regulatory subunit of PI3-kinase or PLC-웂, whereas cytoplasmic PTKs such as Src may bind preferentially to other cytosolic targets. 2. SH3 Domain SH3 domains are composed of approximately 60 amino acid residues and contain a prolin-rich core of 10 amino acids (Cohen et al., 1995). These sequences also mediate binding of molecules that are critical to PTK-based signal transduction. Similar to SH2 domains, SH3 domains regulate protein localization, enzymatic activity, and often participate in the assembly of multicomponent signaling complexes (Schlessinger, 1994; Mayer and Eck, 1995). An example is the adaptor protein Grb2, which links activated receptor PTKs to the Ras regulator known as Sos. SH3 domains may also

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be important in directing the compartmentalization of cytosolic proteins to locations such as the cytoskeleton or the plasma membrane (Malarkey et al., 1995). The minimal sequence requirement for SH3 domain ligands is the PxxP motif (Ren et al., 1993), and structural analysis indicates that the ligands adopt a left-handed polyproline II helix conformation (Yu et al., 1994). The general consensus sequence for SH3 domain ligands is ⌿PX⌿P (Mayer and Eck, 1995). 3. PH Domain PH domains consist of two perpendicular antiparallel 웁 sheets followed by a carboxyl-terminal amphipathic 움 helix. They are found in many types of proteins, including PTKs, substrates for these kinases, effectors such as PLC and GTPases, and cytoskeletal proteins. In vitro studies suggest that PH domains function by tethering small signaling molecules to membranes, thus mediating protein–lipid interactions (see section on PI3-K). However, the specific details of their function in intact systems remain to be determined. 4. Phosphotyrosine-Binding (PTB) Domain In general, PTB domains play a role similar to SH2 domains (Gustafson et al., 1995). Based on ligand recognition, PTB domains fall into two groups: group I represents those PTBs that bind to ligands containing NPXp-

Y (N-asparagine, P-proline) cores where tyrosine is phosphorylated (van der Geer and Pawson, 1995). The PTB domains of Shc and IRS-1 belong to this class and bind ligands with ⌿XNPXp-Y⌿ and ⌿⌿⌿XXNPXp-Y consensus sequences, respectively (van der Geer and Pawson, 1995). Group II PTBs bind to ligands with NPXY cores where tyrosine is not necessarily phosphorylated (Zambrano et al., 1997). A common structural element in the NPXp-Y/NPXY motif is that it forms a type I 웁 turn that is well accommodated by the binding pocket of the PTB domain.

C. G움-Dependent Signals Cells receive many signals through heptahelical transmembrane receptors, which couple heterotrimeric Gproteins. Heterotrimeric G-proteins consist of 움, 웁, and 웂 subunits and regulate the activity of various effector enzymes and ion channels (Neer, 1995). G-proteins are defined by the identity of their 움 subunits. So far, 17 genes encoding G-protein 움 subunits have been detected in mammals, and they appear to be involved in all aspects of mammalian physiology. The 움 subunits of mammalian G-proteins are divided into four subfamilies based on homologies in sequence and function (Simon et al., 1991) (Table I). Most G-proteins couple specifically to certain receptors and effectors; however, individual receptors and effectors can interact with more than one type of G-protein.

TABLE 1 G움 Proteins: Their Receptors and Effectorsa Family G움s

G움i

G움q

G움12

a

Subtype

Expression

Receptors

G움s

Ubiquitous

웁1,2-adrenergic, dopamine, glucagon, A2adenosine, ACTH, LH, MSH, and TSH

G움olf

Olfactory epithelium

Odorant receptors

G움i1 G움i2 G움i3 G움0

Widely distributed Ubiquitous Widely distributed Neuronal, neuroendocrine

움2-adrenergic, thrombin m2,4-Muscarine acetylcholine, vasopressin, somatostatin Muscarinic

G움t1,2 G움g G움z

Retinal codes Taste cells Neuronal, platelets

Opsins Taste receptors Dopaminergic, 5-HT1A

G움q G움11 G움14

Ubiquitous Ubiquitous Kidney, lung, spleen

움1-adrenergic, LPA Chemokine, bradykine Acetylcholine, bradykinin

G움15

Hematopoietic cells

LPA, vasopressin, thromboxane, chemokine, histaminergic

G움12 G움13

Ubiquitous Ubiquitous

Thrombin, thromboxane, LPA Thrombin, thromboxane, bradykinin, LPA

Dhanasekaran et al., 1998; Offermanns et al., 1998.

Effectors Adenyl cyclase 앖 Ca2⫹ channel 앖 Na⫹ channel 앖 Adenyl cyclase 앖 Adenyl cyclase 앗 K⫹ channel 앖 Ca2⫹ channel 앗 K⫹ channel 앖 Ca2⫹ channel 앖 cGMP-phosphodiesterase 앖 cGMP-phosphodiesterase 앖 Adenyl cyclase (?)

Phospholipase C-웁 앖 (웁3⬎웁1⬎⬎웁2:웁4)

GEFs,GDIs,GAPs (?) GEFs,GDIs,GAPs (?)

37. Kinase Signaling

Upon activation by an appropriate signal, the receptor interacts with the G-protein and catalyzes the exchange of ‘‘bound GDP’’ for GTP in the 움 subunit (G움). Subsequently, the GTP-bound 움 subunit and 웁웂 subunits (G웁웂) dissociate from the receptor as well as from each other. The ‘‘active’’ 움 subunit and the ‘‘free’’ 웁웂 subunits initiate cellular responses by altering the activity of intracellular effector molecules. Meanwhile, the intrinsic GTPase activity of the G움 hydrolyzes the bound GTP to GDP, thus deactivating itself. G움-GDP then reassociates with G웁웂, possibly attenuating the 웁웂 effector function as well. The reassociated G움웁웂-GDP heterotrimer is then prepared itself to interact with another receptor molecule (Hepler and Gilman, 1992). The heptahelical receptors for 움1-adrenergic agonists, ET-1, and angiotensin II share many features and couple to pertussis toxin-insensitive heterotrimeric Gproteins of the G움 family. Both G움q and G웁11 have been identified in the heart, and a growing body of evidence supports an important role for the G움q/G움11 pathway for cardiac hypertrophy, as explained later. The major known effect of G움q activation is the stimulation of phospholipase C-웁 (PLC-웁). The activation of PLC-웁 leads to cleavage of phosphatidylinositols generating inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). The DAG generated from these pathways activates PKC, which in turn can stimulate ERK through Raf kinase (Zou et al., 1996). Thus, in cell types such as CHO cells, it has been observed that G움q can activate ERK-mediated proliferative pathways through a PKCdependent but Ras-independent mechanism (Fig. 3). However, in other cell types, such as the rat vascular smooth muscle and NIH/3T3 cells, G움q appears to activate ERK through a novel pathway involving prolinerich tyrosine kinase-2 (Pyk2) and Ras (Lev et al., 1995) (Fig. 3). This pathway appears to be activated by the IP3 generated by the activation of PLC-웁. IP3 increases the intracellular levels of Ca2⫹, presumably through a Ca2⫹ /calmodulin-dependent kinase, which stimulates the activity of Pyk2 to activate Shc through tyrosine phosphorylation. The resulting formation of the Shc– Grb2-SOS complex, stimulates Ras, which leads to the activation of ERK (Lev et al., 1995). Thus, G움q can regulate cell growth and hypertrophy through either a Ras-dependent or Ras-independent, PKC-dependent ERK pathway (Fig. 3). Either of these pathways can couple G움q signaling to nuclear events through the activation of various transcription factors.

D. G웁웂-Dependent Signals The ability of 웁웂 subunits to activate PLC and hence PKC or Ca2⫹-dependent pathways suggests that 웁웂 subunits can activate the ERK pathway through more than

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FIGURE 3 Schematic model of ERK1/2 activation by PLC.

one mechanism (Gutkind, 1998). Because G웁웂 can physically interact with and activate PLC, it is likely that the IP3 derived from the PLC pathway can increase intracellular Ca2⫹, which can activate Pyk2 kinase. Pyk2, in turn, can phosphorylate Shc, leading to the activation of Grb2 and Sos. The resultant activation of Ras leads to the activation of ERK through the Raf-MEK pathway (Della Rocca et al., 1997). G웁웂 activation of PLC can also lead to an increase in DAG and subsequently to activation of PKC. Therefore, at least in some cell types, G웁웂 should be able to activate the ERK pathway through a PKC-dependent but Ras-independent pathway similar to that described for G움q. It has also been reported that the 웁웂 subunit activation of Ras and the subsequent activation of ERK may be dependent on the nonreceptor tyrosine kinase activity of Src as well as PI3-K (Hawes et al., 1996; Luttrell et al., 1996). In cells overexpressing 웁웂 subunits, wortmannin treatment (a PI3-K inhibitor) blocks 웁웂-induced Src phosphorylation as well as ERK activation (Hawes et al., 1996). The molecular basis for the activation of Src by PI3-K is speculated to be phosphatidylinositol3,4,5-triphosphate, one of the products of PI3-K (Hawes et al., 1996). The role of Src in activating Ras through the Shc-Grb2-SOS pathway has been established already

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VII. Signaling Systems

FIGURE 4 Domain structure of c-Src. (Schwartzberg et al., 1998)

(Gutkind, 1998). Hence, the recruitment of different signaling proteins, such as Src, Pyk2, and PI3-K by 웁웂 subunits, appears to be cell type and/or receptor dependent. PLC is a family of at least three related genes: PLC웁, PLC-웂, and PLC-웃 (Rhee and Choi, 1992). Isoforms of PLC-웁 are differentially regulated by G-protein 움 and 웁웂 subunits (Smrcka et al., 1991; Taylor et al., 1991). In contrast, PLC-웃 isoforms are regulated by tyrosine phosphorylation (Kim et al., 1991). Regulation of PLC웃 isoforms is unclear but may involve changes in intracellular calcium. For most G-protein-coupled receptors, it appears that PLC-웁 is critical for the production of IP3 . Two different, but not mutually exclusive, mechanisms may account for IP3 formation by angiotensin II. Similar to G-protein-coupled receptors, such as the 움1-adrenergic receptor, G움q activated by angiotensin II may stimulate PLC-웁. Alternatively, angiotensin II may activate PLC-웂 via tyrosine phosphorylation. This distinction is important because the activation of calcium-dependent tyrosine kinase, such as Pyk2, would be ‘‘downstream’’ from PLC-웁, if tyrosine phosphorylation of PLC-웂 is the primary mechanism by which angiotensin II increases intracellular calcium. Alternatively, if angiotensin II increases calcium by stimulating PLC-웁 (via G움q), then activation of Pyk2 would be an important ‘‘upstream’’ tyrosine kinase event.

IV. OVERVIEW OF TYROSINE KINASE SIGNAL TRANSDUCTION Three major tyrosine kinase-regulated pathways will be discussed in this review on the basis of activation of

specific kinases shown to be regulated by angiotensin II and PDGF in VSMC : Src kinase family, FAK, and Janus Kinases (JAK and TYK) that phosphorylate signal substrates such as Shc, Raf, and PLC-웂. The purpose of these multiple receptor-activated kinases is to provide an integrated series of regulated cellular events. It is unlikely that all these effects occur simultaneously in vivo. Thus, an important area of future research is to determine the physiological variables and tissue-specific factors that determine the relative extent to which these signal transduction pathways are activated.

A. Src Family Kinases The 60-kDa c-Src is the best characterized member of a family of nine cytoplasmic protein tyrosine kinases that participate in growth factor signal transduction. Tissue-specific expression of alternatively spliced gene products yields at least 14 different Src-related kinases (Bolen et al., 1992). Three family members (c-Src, Fyn, and Yes) are expressed ubiquitously and appear to have partially overlapping functions on the basis of studies with transgenic mice (Pascal et al., 1994). Functional domains shared by Src family kinases include an aminoterminal myristoylation sequence for membrane targeting, SH2 and SH3 domains, a kinase domain, a carboxyl-terminal noncatalytic domain, and a unique domain (Fig. 4). These regions participate in a complex tonic inhibition of Src family kinases that can be overcome when cells are exposed to mitogens. One of the residues that appears to be critical for the regulation of c-Src is Tyr527, which is not present in v-Src. Phosphorylation of Tyr527 by C-terminal Src kinase (Csk) inhibits C-Src activity (Klages et al., 1994),

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37. Kinase Signaling

whereas dephosphorylation of this residue appears to be an activating mechanism. Autophosphorylation of Tyr416 in the catalytic domain may be an activating signal. c-Src activity is also inhibited via intramolecular interactions of the carboxy-terminal catalytic domains with both SH2 and SH3 domains (Cooper and Howell, 1993). These SH2 and SH3 domains probably also stimulate c-Src activity through interactions with regulators and downstream/kinase substrates. Functional properties of the amino-terminal unique region have not been well defined. It has been assumed that this domain may be required for specific interactions between particular Src family kinases and downstream targets. Studies of the interaction of Src with and phosphorylation of the NMDA receptor support this view: phosphorylation is dependent on the Src unique region (Yu et al., 1997). Activation of Src during the M phase is accompanied by phosphorylation of Src on certain Ser and Tyr residues, which can lower activation of Src during M phase, but does not eliminate it (Shalloway et al., 1992). Several laboratories, including ours, found that JAK activation is specifically regulated by Fyn, but not Src. Hansen et al. (1997) have shown that Fyn is phosphorylated on Tyr28 in the unique aminoterminal part of the molecule after interaction with the intracellular domain of the PDGF 웁 receptor. Furthermore, activated Fyn undergoes autophosphorylation on Tyr30, Tyr39, and Tyr420. When Fyn mutants (Tyr28, Tyr30, or Tyr39 replaced with phenylalanine) were transfected into NIH/3T3 cells, decreased activation after PDGF stimulation was observed, suggesting the functional importance of tyrosine phosphorylation of this unique domain. Further study of this domain in the context of normal cellular functions of Src will be revealing. Studies from several laboratories (Dhar and Shukla, 1994; Marrero et al., 1995a), including our laboratory (Ishida et al., 1995), suggest that c-Src plays an important role in angiotensin II signal transduction. We demonstrated that angiotensin II stimulation of VSMC was associated with a rapid activation of c-Src (Ishida et al., 1995). Specifically, angiotensin II stimulated a two- to threefold increase in activity of c-Src within 2 min, measured by either autophosphorylation or kinase activity toward enolase (Ishida et al., 1995). Bernstein’s laboratory (Marrero et al., 1995a) studied the functional consequences of inhibiting c-Src activity by electroporating a monoclonal anti-c-Src antibody into cultured VSMCs. They demonstrated significantly greater inhibition of PLC-웂 phosphorylation and IP3 formation by the anti-cSrc antibody compared with purified murine IgG. Data from Izumo’s laboratory (Sadoshima and Izumo, 1996) support the importance of Src family kinases as mediators of angiotensin II function. They showed in cardiac fibroblasts that angiotensin II activated both c-Src and

Fyn (Rodriguez-Linares and Watson, 1994). These findings demonstrate that the activation of Src family kinases is one of the earliest signal events stimulated by angiotensin II and strongly suggest that c-Src is involved in the angiotensin II-stimulated tyrosine phosphorylation of PLC-웂.

B. FAK Because changes in cytoskeletal architecture are related intimately to cell proliferation and differentiation, there has been intense interest in the role of PTKs in mediating these effects of growth factors. It has become clear that focal adhesion complexes, specialized sites of cell adhesion, act as supramolecular structures for the assembly of signal transduction mediators. The best characterized tyrosine kinase localized to focal adhesion complexes is a 125-kDa protein termed FAK. This protein was originally isolated from v-Src-transformed chicken embryo fibrolasts by Kanner et al. (1990). FAK exhibits protein tyrosine kinase activity toward other proteins with which it colocalizeed at these sites, such as paxillin (Burridge et al., 1992). FAK lacks modular domains such as SH2 or SH3 and resembles known protein tyrosine kinases only in its catalytic domain. FAK is autophosphorylated at Tyr397 in resting substrate-attached cells and possesses sites favored for phosphorylation by Src, such as tyrosines 407, 576, and 577. Autophosphorylation of FAK at Tyr397 results in the binding of the SH2 domain of Src and Fyn (Schaller et al., 1994). The interaction between these two tyrosine kinases accelerates Src-mediated FAK phosphorylation. To date, Tyr407, Tyr576/577, Tyr861, and Tyr925 have been identified as the sites on murine FAK that are phosphorylated by Src (Calalb et al., 1995, 1996; Schlaepfer et al., 1994; Schlaepfer and Hunter, 1996). Phosphorylation of Tyr576/577 has been suggested to increae the kinase activity of FAK (Calalb et al., 1995), whereas Tyr925 phosphorylation allows Grb2 binding, which may lead to the activation of Ras and triggers the MAP kinase cascade (Schlaepfer et al., 1994; Schlaepfer and Hunter, 1996). Src family kinases such as Src and Fyn then phosphorylate a number of FAK-associated proteins, including paxillin, tensin, and p130CAS (Nojima et al., 1995). FAK appears to play an important role in events leading to cell proliferation, as suggested by the finding in v-Srctransformed fibroblasts that hyperphosphorylation of FAK occurs in concert with the loss of a requirement for cell attachment for growth (Guan and Shalloway, 1992). Two laboratories have independently reported the cloning of a second FAK family member, denoted Pyk2 (Lev et al., 1995) or cell adhesion kinase-웁 (Sasaki et al., 1995). In cells of neural origin, Pyk2 was found to be activated by G-protein-coupled receptor agonists,

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VII. Signaling Systems

PKC stimulation, or increased cytoplasmic calcium levels (e.g., potassium depolarization or calcium ionophore). Pyk2 has been postulated as a potential link between calcium-dependent signaling pathways and protein tyrosine kinase pathways (Fig. 4). Dikic et al. (1996) reported that activated Pyk2, like FAK, complexes with c-Src and that this interaction is required for Pyk2-mediated ERK activation. As such, Pyk2 is a candidate both to regulate c-Src and to link G-proteincoupled vasoconstrictor receptors with protein tyrosine kinase-mediated contractile, migratory, and growth responses.

C. JAK-STAT Signaling Myocardial hypertrophy is induced by various stimuli in vivo, such as pressure or volume overload. The activation of the cytokine receptor protein gp130 in cardiac myocytes was reported to induce myocardial hypertrophy. Activation of gp130 stimulates two pathways in cardiac myocytes: a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and a MAP kinase pathway. While JAKs associate with gp130 in the unstimulated state, gp130 receptor stimulation promotes kinase activation, and JAKs phosphorylate gp130 tyrosine residues, as well as cytoplasmic proteins called STATs. To date, the activation of STAT3 in hypertrophied myocytes from IL-6 binary transgenic mice provides the best evidence for its involvement in gp130 hypertrophic signaling (Hirota et al., 1995). STAT3 is also subsequently phosphorylated on serine (Boulton et al., 1995), possibly by ERK, with enhancement of its transcriptional activity. Studies indicate that the AT1 receptor shares properties with cytokine receptors, such as the interleukin-2, IFN-웂, and IFN-움 receptors(Berk and Corson, 1997). Similar to these classical cytokine receptors, the AT1 receptor stimulates tyrosine phosphoryla, activates the MAP kinase pathway, and induces c-fos mRNA expression (Duff et al., 1992; Huckle et al., 1992). We have found that angiotensin II rapidly activates JAK2 and TYK2 and stimulates tyrosine phosphorylation of STAT1 in VSMCs (Marrero et al., 1995b), and Baker’s group showed that angiotensin II activates STAT3 in cultured neonatal cardiac fibroblasts (Bhat et al., 1994). These data support the role of angiotensin II as a cytokine similar to IFN-움 and IFN-웂 (Darnell et al., 1994; Ruff et al., 1993). In addition, JAK2 was found to immunoprecipitate with the AT1 receptor, suggesting a role for this kinase in mediating the earliest events activated by angiotensin II (Marrero et al., 1995b). These observations and the demonstration that thrombin also activates JAK2 (Rodriguez-Linares and Watson, 1994) indicate the great extent to which G-protein-coupled receptors

share signal mechanisms with cytokine and growth factor receptors.

D. Mitogen-Activated Protein (MAP) Kinase Pathway MAP kinases are serine and threonine protein kinases that are activated in response to a wide variety of extracellular stimuli and are encoded by a multigene family (Fig. 5) (Blenis, 1993). MAP kinases are activated by phosphorylation on Thr and Tyr residues within a T-X-Y phosphorylation motif, where ‘‘X’’ can be Glu (E), Pro (P), or Gly (G). Three classes of MAP kinases may be defined based on their phosphorylation motifs (TEY, TPY, and TGY), which we will term ERK1/2 and BMK1, c-Jun N-terminal protein kinases (JNK, also called SAPK), and p38, respectively. Activation of the three classes of MAP kinases is characteristic for particular stimuli. For example, growth factors and phorbol myristate acetate (PMA) activate ERK1/2 strongly, but JNK and p38 kinases weakly (Cano et al., 1994). Hyperosmolar stress and TNF-움 are strong stimuli for p38 (Han et al., 1994). In VSMC we have shown that growth factors and angiotensin II are powerful activators of ERK1/2 (Duff et al.,1992). Arachidonic acid and 15HETE (a 15-lipoxygenase product of arachidonic acid) have both been show to activate ERK1/2 in VSMC (Rao et al., 1994). The specificity for MAP kinase activation is determined, in part, by members of the MEK family, which exhibit unique pairing with downstream MAP kinases. For example, MEK1 and MEK2 activate ERK1/2, MKK3 and MKK6 activate p38, and MKK4 and MKK7 activate JNK (Fig.5). Thus, cell- and stimulus-specific events regulate MAP kinase activity. The specificity of activation of MAP kinases by individual stimuli is paralleled by specific substrates for each class (Fig. 5). Common substrates for the MAP kinases are transcription factors that, upon phosphorylation, may be activated and induce changes in gene expression. ERK1/2 phosphorlate the ternary complex factor (TCF)/ElK-1 on sites essential for transactivation (Marais et al.,1993). JNK phosphorylates c-Jun and increases its transcriptional activating potential (Kyriakis et al., 1994). ATF2 is phosphorylated and activated by both JNK and p38 (Gupta et al., 1995; Raingeaud et al., 1995). BMK1 activates MEF2 transcription factors (Kato et al.,1997). 1. Extracellular Signal Regulated Kinases 1 and 2 (ERK1/2) (Abe et al., 2000) The most significant feature that distinguishes MEK1/2 from other dual-specificity kinases is the high level of stringency exhibited by MKKs in phosphorylat-

37. Kinase Signaling

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FIGURE 5 Schema for MAP kinase cascade.

ing their substrates. MEK1/2 phosphorylate Tyr183 and Tyr185 of ERK1/2. Phosphorylation of both of these residues is essential for the activation of ERK1/2. The activated ERKs initiate the nuclear events that regulate cell proliferation. Consistent with this view, it has been observed that the sustained activation of MEK1-ERK1 was essential for the PDGF-stimulated G1 to S phase transition and cell proliferation in various cell types (Cowley et al., 1994). In these cells, expression of dominant-negative MEK1 (Cowley et al., 1994) or the treatment of cells with a specific MEK1/2-inhibitor (PD98059 (Weber et al., 1997) inhibits ERK activity as well as cell growth. Griendling and colleagues found that angiotensin IImediated VSMC hypertrophy was dependent on the generation of ROS, as angiotensin II rapidly stimulated NADH oxidase and hypertrophy was inhibited by catalase, SOD, and inhibition of NADH oxidase (Fukui et al., 1995). Both ERK1/2 and p38 appear necessary for angiotensin II-mediated hypertrophy as inhibition of these MAP kinases with PD98059 and SB205380, respecitvely, led to additive decreases in cell protein synthesis (Bass and Berk, 1995; Ushio-Fukai et al., 1998). 2. c-Jun N-Terminal Kinase JNKs are represented by more than 10 different isozymes encoded by three genes (JNK 1-3) (Kyriakis and Avruch, 1996). Stress stimuli do not alter JNK expression but rather activate existing JNK proteins through phosphorylation by upstream kinases and inactivation

of associated inhibitors. First, JNK activity as a kinase requires its phosphorylation on Thr 183 and Tyr 185 by the upstream kinases MKK4/SEK1 or MKK7 (Toyoshima et al., 1997) (Lin et al., 1995). MKK4 itself undergoes prior phosphorylation by its upstream kinases (which include MEKK1, DLK,MLK,and ASK1) (Dhanasekaran and Premkumar Reddy, 1998). MEKK1 phosphorylation and proteolysis by caspase 3 are alternate pathways involved in MEKK1 regulation and subsequent activation of MKK4 and JNK (Deak et al., 1998). The initial event leading to the activation of MKK4-JNK involves guanine nucleotide exchange in Rac or CDC42. The GTP-bound Rac or CDC42 then stimulates the Ser/Thr kinase of p21-activated kinases (PAKs), a mammalian homologue of yeast STE20 kinase involved in the mating phermone response pathway. JNK activity is inhibited by its associated protein JIP, which contributes to the cytoplasmic retention of this kinase (Dickens et al., 1997). Unlike most kinases, JNK is tightly bound to its substrates, c-Jun, ATF2, Elk-1, and to the nonsubstrate transcription factor JunB (Adler et al.,1992). While the role of the MEK1/2-ERK pathway in cell proliferation and differentiation pathways has been well characterized, the role of MKK4 and JNK in different physiological contexts is largely unknown. Initial observations indicated that JNK activation is essential for the Ras transformation of REF3T3 cells, thus suggesting a mitogenic role for JNK. In contrast, it has been reported that the expression of MEKK1 strongly inhibits cell proliferation in NIH/3T3 cells. The observation that

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VII. Signaling Systems

MKK4 (-/-) homozygous animals die before embryonic day 14 suggests a critical role for MKK4 in embryonic survival (Yang et al., 1997). These apparently contradictory effects of JNK on cell growth and differentiation may depend on the impact from other parallel signaling pathways. Stimulation of VSMC with H2O2 or O2- (generated by LY83583) failed to activate PAK1, the upstream regulator of both p38 and JNK. In contrast, angiotensin II stimulated PAK1 rapidly and stimulated p38 and JNK in VSMC (Schmitz et al., 1998; Ushio-Fukai et al., 1998). These results suggest that additional signals activated by angiotensin and/or the nature of intracellular ROS generation versus extracellular ROS provide critical differences in signal transduction events. 3. p38 Kinase p38 kinase is a mammalian homologue of the yeast osmosensing protein kinase HOG-1. Like JNK, p38 kinase also phosphorylates transcription factors (ATF2, CHOP, and MEF2C) (Clerk et al., 1998; Sugden and Clerk, 1998), increasing their trans-activating activity. In addition, p38 kinase phosphorylates and activates the MAPK-activated protein kinases (MAPKAPs) 2 and 3 (Kumar et al., 1997), which in turn phosphorylate the small heat shock proteins (Hsp25/27). This may modulate the cytoprotective activity of Hsp25/27 (Clifton et al., 1996). However, direct validation of this concept will require analysis in genetically engineered mice. 4. Big MAP Kinase 1 (BMK1/ERK5) A MAP kinase termed big MAP kinase 1 (BMK1) or ERK5 was cloned by two groups (Lee et al., 1995; Zhou et al., 1995). Because the primary structure and molecular mass (앑 110 kDa) differ from ERK1/2 (Lee et al., 1995), the name BMK1 will be used. BMK1 has a TEY sequence in its dual phosphorylation site, like ERK1/2, but has unique carboy1-terminal and loop-12 domains, suggesting that its regulation and function may be different from ERK1/2. The upstream kinase that phosphorylates BMK1 has been identified as MEK5 (Lee et al., 1995; Zhou et al., 1995). However, upstream regulators of MEK5 remain unknown. In our laboratory we have observed that in response to several different VSMC agonists, BMK1 was stimulated to the greatest extent by H2O2 , with a relative potency of H2O2 ⬎⬎⬎ PDGF ⬎ PMA ⫽ TNF움 (Abe et al., 1996). It is noteworthy that while ERK1/2 and JNK are activated by ROS, they are activated to a much greater extent by growth factors and cytokines. Thus, these findings suggest that BMK1 is the first MAP kinase that is specifically redox sensitive.

We found that BMK1 was rapidly and specifically activated by H2O2 , but not by growth factors in VSMC. H2O2 caused a time- and concentration-dependent activation of BMK1, which was calcium and tyrosine kinase dependent, as shown by inhibition with thapsigargin and herbimycin A, respectively (Abe et al., 1996). H2O2 stimulation of BMK1 appeared ubiquitous as shown by increases in BMK1 activity in human skin fibroblasts, human VSMC, and human umbilical vein endothelial cells (Abe et al., 1996). These findings demonstrate that activation of BMK1 is different from ERK1/2, JNK, and p38 and depends on calcium and tyrosine kinases, but not on PKC. To show that BMK1 activation in VSMC is regulated by the redox state, we used ebselen (a selenium-based antioxidant shown previously to enter cultured cells rapidly). VSMC were treated with 30 애M ebselen or vehicle for 30 min and then exposed to 200 애M H2O2 for 20 min. Ebselen completely inhibited H2O2-mediated increases in BMK1 activity. Analysis of the signaling mechanism indicates that MKK5-BMK1 activation results in the phosphorylation of MEF2C, a transcription factor belonging to the myocyte enhancer factor-2 (MEF2) family (Kato et al., 1997). Further studies have indicated that MEF2C is a protein substrate for BMK1 and that MEF2C activation is involved in the expression of several immediate early genes, including c-Jun (Kato et al., 1998). Although, the functional significance of this pathway remains unclear, it is significant that both p38MAPK and ERK2 can also phosphorylate and activate MEF2C. In this context, it is interesting to note that MKK5 and ERK5 are highly expressed in cardiac myocytes (Zhou et al., 1995), whereas the MEF2 family of transcription factors is mainly expressed in skeletal muscle and brain tissue (McDermott et al, 1993). Taken together, these observation may be indicative of the critical role played by MKK5-ERK5 signaling in cardiac physiology (Zhou et al., 1995). An essential role for c-Src in H2O2-mediated BMK1 activation in VSMC is suggested by four experiments (Abe et al., 1997). (1) H2O2 stimulated c-Src activity rapidly in VSMC and fibroblasts (peak at 5 min), which preceded the peak activity of BMK1 (20 min). (2) Specific Src family kinase inhibitors (herbimycin A and CP-118,556) blocked BMK1 activation by H2O2 in a concentration-dependent manner. (3) BMK1 activation in response to H2O2 was completely inhibited in cells derived from mice deficient in c-Src, but not Fyn. Mouse fibroblasts from transgenic animals that lacked Src (Src-/-), Fyn (Fyn-/-), or both (Src-/-&Fyn-/-) were prepared (Soriano et al., 1991; Thomas et al., 1995a). BMK1 activation was decreased only in Src-/- and Src-/-&Fyn-/- cells. (4) BMK1 activity was much greater in v-Src-transformed NIH/3T3 cells than wilde-type

37. Kinase Signaling

cells. These results demonstrate an essential role for c-Src in H2O2-mediated activation of BMK1 and suggest that the redox-sensitive regulation of BMK1 is a new function for c-Src. 5. p90 Ribosome S6-Kinase (p90RSK) We have proposed previously that p90 Ribosomal S6 kinase (p90rsk), a downstream substrate of ERK1/2 (Sturgill et al., 1988), was a physiologically relevant NHE-1 kinase (Phan et al., 1997; Takahashi et al., 1997a). Using two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identified Ser703 as the major serum-stimulated amino acid. Mutation of Ser703 to alanine had no effect on acid-stimulated Na⫹ /H⫹ exchange, but completely prevented the growth factormediated increase in NHE-1 affinity for H⫹. In addition, we showed that p90 ribosomal S6 kinase (p90rsk) is a key NHE-1 kinase, as p90rsk phosphorylates Ser703 stoichiometrically in vitro, and transfection with kinaseinactive p90rsk inhibits serum-induced phosphorylation of Ser703 in transfected 293 cells. These findings establish p90rsk as a serum-stimulated NHE-1 kinase and a mediator of increased Na⫹ /H⫹ exchange in vivo. 6. Phosphatidylinositol 3-Kinase (PI3-K) Another important mitogenic pathway stimulated by receptor PTKs is initiated by the action of PI3-K. Analysis of PDGF 웁-receptor deletion mutants demonstrated that activation of PI3-K is required for mitogenic signaling by the PDGF 웁 receptor. The 85-kD a regulatory subunit of PI3-K binds the PDGF 웁 receptor via its carboyl-terminal SH2 domain (Klippel et al., 1992). Subsequently, the 110-kD a PI3-K effector subunit is recruited to a neighboring region of the 85-kD a regulatory subunit and is activated. This kinase then phosphorylates membrane phosphatidylinositides (PtdIns) at the 3 position of the inositol ring, generating PtdIns3-P, PtdIns(3,4)P2 , and PtdIns(3,4,5)P3 . These phosphorylated species are thought to function as mitogenic second messengers, as no phospholipase has been found that can cleave inositol phosphorylated at the 3 position. However, until recently, the targets of these phosphorylated lipid species were unclear. There is growing evidence that important targets include the serine/threonine kinase designated Akt/PKB. Akt/PKB was discovered by virtue of its homology with PKC and PKA, and was found to be the cellular homologue of the transforming oncogene v-Akt (Bellacosa et al., 1991). Akt/PKB consists of a carboxyl-terminal catalytic domain and an amino-terminal PH domain and is activated rapidly by a variety of mitogenesis. A direct mechanism of Akt/PKB activation by PI3-K that

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involves the binding of PtdIns(3,4,5,)P3 , to the PKB PH domains has been described (Pitcher et al., 1995). Because the PI3-K products, PtdIns(3,4)P2 , and PtdIns(3,4,5,)P3 can regulate the activities of a wide varety of proteins, including PKC isoforms and proteins that regulate the actin organization (e.g., profilin), the consequences of PI3-K activation likely extend beyond Akt.

E. Signaling Pathways and Hypertrophy of Cardiac Myocytes 1. MAP Kinases Endothelin-1, phenylephrine, and PMA are powerful hypertrophic agonists in cultured cardiac myocytes. It was suggested that activation of the ERK cascade by these agonists may mediate the hypertrophic response. Although there is considerable evidence that ERKs participate in myocyte hypertrophy, it is now apparent that ERKs are not the sole mediators of this response. JNK and p38 kinase have been proposed to affect cardiac hypertrophy, but the involvement of these stress-responsive kinases in cardiac hypertrophy may not be simple(Sugden and Clerk, 1998). It has been reported that constitutively activated MKK3 diminishes cell survival, and cotransfection experiments (with p38 kinase) show that this effect is dependent on p38 kinase. The nature of these kinases in hypertrophy requires further investigation. 2. G␣q Family Genes A growing body of evidence supports an important role for G움q pathways in the pathogenesis of cardiac hypertrophy. G움q/G움11 double-deficient embryos show a thinning of the myocardial layer of the heart that is already visible at embryonic day 9.5 and is most likely responsible for embryonic death due to cardiac failure. These data indicate that both G움q and G움11 may be necessary for the proper growth of myocardial cells during embryogenesis (Offermanns et al., 1998). Transgenic expression of a constitutively activated mutant of the 움1-adrenergic receptor, as well as of the wild-type form of G움q in the heart, has been shown to result in cardiac hypertrophy (Offermanns et al., 1998). Thus, G움q and G움11 appear to be involved in physiological as well as pathological growth of cardiac myocytes. Identification of the precise downstream pathways that mediate these various effects will be of interest in the coming years. 3. Calcineurin Increased cytoplasmic Ca2⫹ concentrations activate calcineurin (also called protein phosphatase 2B), lead-

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VII. Signaling Systems

ing to dephosphorylation of the transcription factor NFAT. This allows NFAT to migrate into the nucleus, where it upregulates the transcription of a number of genes, such as GATA-4. Molkentin et al. (1998) reported that myocytes transiently transfected with plasmids encoding calcineurin A(1씮398), NFAT3, and GATA4 showed a dramatically increased expression of a BNP reporter gene. Cyclosporin A or FK-506 inhibited the increases in cell size and myofibrillar assembly of myocytes exposed to Ang II or phenylephrine for 72 hr, as well as the Ang II-induced increase in ANF mRNA expression (Molkentin et al., 1998).

intracellular signal events similar to those resulting from agonist–receptor coupling such as growth factors and cytokines. Flow experiments with cultured endothelial cells have demonstrated that flow stimulates the phosphorylation of multiple cellular proteins. Flow-induced responses are mediated by a complex signaling system in which protein phosphorylations by different protein kinases appear to be critical the next section summarizes current knowledge of the participation of protein tyrosine kinases and MAP kinases in flow-stimulated signaling.

F. Kinase Activation during Myocardial Ischemia and Reperfusion

V. FLOW ACTIVATES PROTEIN TYROSINE KINASES (PTKS)

Sugden’s group has reported that p38 kinases and the downstream MAPKAP2 are activated during ischemia and that their activation is sustained or increased during reperfusion (Clerk et al., 1998). Activation of p38 kinase by cellular stresses is associated with the activation of MAPKAP2 and the phosphorylation and disaggregation of Hsp25/27 (Sugden and Clerk, 1998). The activation of MAPKAP2 is completely inhibited by SB203580, implicating particularly p38움 and/or p38웁 in its activation in the heart. In contrast, JNKs are not activated during global ischemia but are strongly activated during the reperfusion phase. It is probable that phosphorylation and activation of c-Jun/ATF2 by JNKs and p38 kinase are involved in the upregulation of these genes after ischemia and reperfusion.

A. Activation of Receptor Tyrosine Kinases (RTKs) by Flow

G. Kinase Activation under Fluid Shear Stress Mechanical forces acting on vessels due to blood flow can be simply resolved into two principal parts, the perpendicular part representing blood pressure and the parallel part creating a frictional force (shear stress) on the luminal surface of vessel wall (Grabowski and Lam, 1995). Endothelial cell morphology, as well as functional properties, is regulated by shear stress in a time- and force-dependent manner (Davies, 1995). Responses take only seconds, such as a burst in inositol triphosphate generation, an increase in cytosolic calcium, and generation of a membrane potassium current (Takahashi et al., 1997b). Some responses require minutes to hours, such as activation of MAP kinases and tyrosine kinases (Takahashi et al., 1997b). The flow-stimulated responses defined in vitro may account for many in vivo adaptive responses involving changes in gene expression, vessel tone, cell shape, and cytoskeletal rearrangement. Flow stimulates the expression of plateletderived growth factor (PDGF) A and B chains, tissue plasminogen activator, endothelial nitric oxide synthase, and endothelin (Traub and Berk, 1998). Flow stimulates

The role of RTKs in flow-stimulated signaling has not been well characterized. Studies from our laboratory and others suggest that RTKs may be involved in flowdependent signaling. Our work indicates that flow-stimulated NO production is likely dependent on the vascular endothelial growth factor (VEGF) receptor Flk-1, which is tyrosine phosphorylated by flow (Ueba et al., 1999). Suramin, a PTK inhibitor, completely inhibited flow-stimulated ERK2 activity, but not JNK activity, highlighting a role for growth factor receptors in ERK2 activation (Hu et al., 1998b). In A431 cells, flow rapidly induces the tyrosine phosphorylation of EGF receptor as early as 30 sec (Shyy et al., 1999). In vascular smooth muscle cells, physical forces rapidly induce the phosphorylation of PDGF receptor 움 (Hu et al., 1998a). Thus, mechanical stresses may act directly on the cell surface to alter receptor conformation, thereby initiating signaling pathways normally used by growth factors. For example, JNK activation in response to UV irradiation of osmotic stress appears to be mediated by EGF receptor aggregation and internalization (Rosette and Karin, 1996).

B. Activation of Nonreceptor PTKs by Flow The importance of nonreceptor PTKs as intracellular mediators is that they are among the kinases activated most rapidly, and many proteins are tyrosine phosphorylated in flow-stimulated endothelial cells. For example, studies from our laboratory (Takahashi and Berk, 1996) and others (Fleming et al., 1998; Jalali et al., 1998; Li et al., 1997; Shyy et al., 1995) provide evidence that flow stimulates the tyrosine phosphorylation of multiple proteins in endothelial cells within minutes. Furthermore, several nonreceptor RTKs have been shown to be activated by flow. Among them, Src family kinases have

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been identified most often. Many groups have shown that c-Src was stimulated rapidly by flow (Jalali et al., 1998; Shyy et al., 1999; Takahashi and Berk, 1996). For example, an in vitro kinase assay using enolase as the substrate showed that Src kinase activity was increased by flow at the onset of 1 min and peaked at 5–10 min (Shyy et al., 1999). p130 Crk-associated substrate (Cas), a putative Src substrate, was tyrosine phosphorylated within 1 min in response to flow (Takahashi et al., 1999; Okuda et al., 1999). Similarly, Fyn, another member of the Src family, was also stimulated by flow (Shyy et al., 1999). In addition, Src and other herbimycin A-sensitive tyrosine kinases appear to be especially important for the flow-induced activation of MAP kinases. Our studies showed that BMK1 activation by flow was inhibited by herbimycin A but it was independent of Src, suggesting that a tyrosine kinase other than c-Src mediates BMK1 activation by flow (Yan et al., 1999). Another tyrosine kinase, focal adhesion kinase, has been shown to be activated and tyrosine phosphorylated by flow in BAEC (Li et al., 1997). The flow activation of FAK is dependent on actin structure integrity, as it is attenuated by pretreating endothelial cells with cytochalasin B. In addition, FAK activation was found to be necessary for the flow-induced activation of both ERK1/2 and JNK (Li et al., 1997).

VI. FLOW ACTIVATES MAP KINASES (MAPKs) Among MAPK family members, ERK1/2, JNK, p38, and BMK1 have been shown to respond to flow in endothelial cells.

A. Activation of ERK1/2 by Flow ERK1/2 is activated in cultured endothelial cells by a physiological range of shear stress with peaks between 15 and 30 dynes/cm2. The signal transduction events by which ERK1/2 is activated have been characterized, although the exact sequence of events has not been elucidated. The importance for activation of a heterotrimeric G-protein was first demonstrated using the nonhydrolyzable GTP analogue GDP-웂S (Tseng et al., 1995). Jo et al. (1997) further identified that G움i2 is the G-protein isoform mediating ERK1/2 activation by flow because the expression of mutant G움i2 and the antisense G움i2 prevented flow-dependent activation of ERK1/2. The involvement of PKC in flow-induced ERK1/2 activation was also characterized. Inhibition of PKC with staurosporine or downregulation of PKC with phorbol 12,13-dibutyrate completely blocked ERK1/2 activation by flow (Traub et al., 1997; Tseng et al., 1995). However, chelating Ca2⫹ with BAPTA-AM had no effect, suggesting the involvement of a Ca2⫹-independent

PKC isoform (Traub et al., 1997). Using specific antisense PKC oligonucleotides, Traub et al. (1997) found that PKC-␧ is specifically required for the activation of ERK1/2 by flow in HUVEC. Ras is another important mediator for flow-induced activation of ERK1/2. Flowactivated Ras (Li et al., 1996) and the activation of ERK1/2 by flow was blocked in cells overexpressing a dominant-negative Ras (N17Ras) (Jo et al., 1997). In addition, an important role for tyrosine kinase(s) in flow-mediated ERK1/2 activation is suggested by the ability of herbimycin A (Takahashi and Berk, 1996) or genistein (Jo et al., 1997) to inhibit ERK1/2 activation completely.

B. Activation of JNK by Flow The effects of flow on JNK activity are conflicting. In BAEC growth arrested by serum deprivation, these groups reported that JNK was activated by flow, even though the time course for peak JNK activation varied in these reports (Jo et al., 1997; Li et al., 1996; Hu et al., 1998b). In contrast, our group has observed inhibition of JNK in density-arrested BAEC and HUVEC. Flow activated JNK via a signaling pathway different from ERK1/2. Flow-induced JNK activation was partially inhibited by the expression of 웁ARK-ct, which blocks G웁웂, suggesting that 웁 and 웂 subunits of heterotrimeric G-proteins are the upstream regulators (Jo et al., 1997). Expression of a dominant-negative Ras completely prevented flow-dependent activation of JNK, suggesting the importance of Ras (Li et al., 1996). Rho family GTPases, Cdc42 and Rho, have been reported to play important roles in flow-induced JNK activation because the dominant-negative mutant of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated flowinduced JNK activity (Li et al., 1999). In addition, a tyrosine kinase is likely involved because genistein prevented the flow-induced activation of JNK (Jo et al., 1997). The identity of the tyrosine kinase remains to be identified. PI3K웂, a G-protein-dependent form of phosphatidylinositol 3-kinase, is activated by flow (Go et al., 1998). Treatment of BAEC with an inhibitor of PI3K wortmanin inhibited the flow-dependent activation of JNK, but had no effect on ERK1/2 (Go et al., 1998). Further, expression of a kinase-inactive mutant (PI3K웂K99R) in BAEC inhibited the flow-dependent activation of JNK but not ERK1/2 (Go et al., 1998). These results suggest that PI3K웂 selectively regulated the flow-induced JNK pathway. Akt kinase, the downstream target of PI3K, has been shown to be activated by flow with a maximal increase up to six fold after 1 hr of flow exposure (Dimmeler et al., 1998). The stimulation of Akt by flow seemed to be mediated by PI3K. It is not clear whether Akt activation leads to JNK activation induced by flow.

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FIGURE 6 Flow activates p38 in HUVEC (A) and BAEC (B) Western blot analysis showing p38 kinase activity with phospho-p38 antibody.

In contrast, data from our laboratory showed that steady laminar flow (12 dynes/cm2, 10 min) decreased JNK activity compared to static controls in growth-arrested HUVEC (2 days after reaching confluence) (Surapisitchat and Berk, 1999). In addition, flow prevents JNK activation mediated by a physiological stimulus, TNF-움. PD98059, a specific mitogen-activated protein kinase inhibitor, blocked the flow-mediated inhibition of TNF-움 activation of JNK, suggesting that flow inhibits TNF-움-mediated signaling events in HUVEC by a mechanism that is dependent on the activation of the ERK1/2 signaling pathway (Surapisitchat and Berk, 1999). Possible explanations for the discrepancies in these studies may be the culture condition (serum-free medium vs complete medium). For example, results showing that JNK was activated by flow were achieved from BAEC growth arrested by serum deprivation; however, the result showing flow decreased JNK activity was achieved from HUVEC growth arrested by contact inhibition in the 10% serum-containing medium. It is likely that the JNK pathway is inhibited in cells cultured in the serum-containing medium because flow potently activated the ERK1/2 signaling pathway in these cells and the ERK1/2 pathway inhibits JNK activation.

C. Activation of BMK1 by Flow Studies from our laboratory showed that flow is the strongest known stimulus tested for BMK1 activation in both BAEC and HUVEC (Yan et al., 1999). Flow (12 dynes/cm2) activated BMK1 within 10 min with a peak at about 60 min in a force-dependent manner. The signal events linking flow to BMK1 are different from other MAPKs. For example, flow-stimulated BMK1 activation is dependent on Ca2⫹ mobilization from internal Ca2⫹ stores, but not Ca2⫹ influx. The requirement of

tyrosine kinase(s) in BMK1 activation was demonstrated by the complete inhibition by herbimycin A. However, Src is not required for BMK1 activation because the Src-specific inhibitor PP1 did not block BMK1 activation by flow and the expression of dominant-negative Src using an adenovirus vector did not block BMK1 activation either. Using specific inhibitors and activators, it was found that flow-mediated BMK1 activation was not regulated by the intracellular redox state; intracellular NO; protein kinase A, C, or G; calcium/calmodulin-dependent kinase; phosphatidylinositol 3-kinase; or arachidonic acid metablism.

D. Activation of p38 by Flow The effects of flow on p38 activity have not been well characterized. Studies from our laboratory showed that p38 was activated by flow (Fig. 6). Western blotting with specific antibodies against phosphorylated p38 demonstrated a time-dependent stimulation of p38 phosphorylation by flow with a maximal increase up to 10-fold after 1 hr of flow exposure. Activation of p38 by flow was observed in both BAEC and HUVEC. A similar observation was reported for BAEC by Shyy et al. (1999). The signaling pathway leading to p38 activation remains to be determined.

VII. SUMMARY Studies of signal transduction have provided insight into the complexity of interactions among different signaling pathways. Specifically, it has become clear that signal transduction involves an intracellular network that acts to integrate multiple receptors, signal transducers, and second messengers. A superficial analysis of

37. Kinase Signaling

these networks often reveals functional and molecular redundancy with apparent coincidental signaling, extensive signal crosstalk, and frequent signal overlaps. However, a closer look at individual signaling pathways, as described in this chapter indicates that the mechanisms involved in signal integration (between different pathways) are as important as the primary signal transduction pathways. Thus, to understand the meaning of a signaling pathway within the context of a signal network, it is important to define the subsets of proteins and signal transduction mediators responsible for distinct cellular events, such as cell proliferation, apoptosis, hypertrophy, or alterations in mechanical force. This chapter discussed the pathways from cell surface receptor to nucleus. Specifically, we focused on receptors belonging to different superfamilies, including G-protein-coupled receptors, receptor tyrosine kinases, cytokine receptors, cell adhesion receptors, and antigen receptors. Common to all these receptors is signal transduction to the nucleus through sequentially phosphorylating kinases collectively known as a kinase signaling module. The best studied of these kinase modules is the MAP kinase module. Many extracellular stimuli activate MAP kinases, including growth factors, hormones, cytokines, and antigens, as well as physical and chemical stimuli, such as oxidative stress, heat shock, osmotic imbalance, and fluid shear stress. Because many other kinase modules are also activated by these same stimuli, MAP kinases represent a logical starting point in examining the interactions among kinase modules. In addition to interactions among kinase modules, a further level of complexity that remains to be explored in greater detail are interactions among these kinases which regulate the activity of transcription factors. An even more complex level of regulation is the effect of resulting changes in gene expression (such as the induction of MAP kinase phosphatases) that feedback on the signal networks. While on one hand the complexity of signal transduction represents a daunting challenge to understanding the biology of cell function, on the other hand the diversity offers multiple therapeutic targets for pharmacological intervention.

Acknowledgments The authors thank M. Corson and many members of the Berk laboratory, including M. Ishida, T. Ishida, U. Shmit, A. Baas, M. Okuda, H. Ueba, O. Traub, J. Duff, H. Tseng, T. Peterson, M. Takahashi, E. Takahashi, and M. Kusuhara.

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38 Calcium Signaling DEREK TERRAR, STEVAN RAKOVIC, and ANTONY GALIONE Department of Pharmacology Oxford University Oxford OX1 3QT, England

I. INTRODUCTION

mechanical activity. Cytosolic calcium can also regulate communication between muscle cells, mainly via gap junctions (Lamont et al., 1998), influencing the spread of electrical activity through the heart. A further possible role for cytosolic calcium has been suggested in the control of pacemaker activity in the sinoatrial node. Longer term influences of calcium signaling mechanisms may occur in determining gene expression, leading, for example, to changes in protein expression in the failing or hypertrophied heart. The aim of the present chapter is to consider evidence for these various aspects of calcium signaling in the heart.

Calcium ions are unique among intracellular messengers in that they are neither created nor destroyed, but are moved from a place of storage or quiescence into cellular regions where they can trigger a variety of cellular changes by specific interactions with target proteins such as troponin C in cardiac muscle (Clapham, 1995). Cardiac myocytes are typical of excitable cells in which changes in the electrical activity of the plasma membrane lead to an influx of calcium ions through calcium channels, which can be further amplified by a larger calcium release through intracellular calcium channels of the (sarco)endoplasmic reticulum calcium store termed calcium-induced calcium release (CICR) (Fig. 1). Many of the proteins involved in cardiac calcium regulation have been identified, and the combination of ultrastructural (Fig. 2) and functional studies has been important in our understanding of the subtleties of the role of this ion in regulating cardiac function. The intricacies of subcellular calcium patterning have only been appreciated with the employment of confocal microscopy, which has been paralleled by the identification and appreciation of subcellular localizations of the proteins generating these signals (Niggli, 1999). Calcium signaling in the heart involves a variety of interrelated processes. The most obvious role for calcium in the heart is in the regulation of the mechanical activity of cardiac muscle to enable effective pumping of blood. This contraction is controlled by electrical activity to which calcium fluxes contribute, but ionic currents underlying the electrical changes can themselves be modified both by cytosolic calcium and by the

Heart Physiology and Pathophysiology, Fourth Edition

II. EXCITATION–CONTRACTION COUPLING Control of cardiac contractile force is discussed extensively in an excellent monograph by Bers (1991), and recent reviews of cardiac excitation–contraction (EC) coupling have also been published (Niggli, 1999). The basic scheme for excitation–contraction coupling in mammalian ventricular muscle is reviewed in Fig. 1. The cardiac action potential triggers the ‘‘calcium transient,’’ which is a brief elevation of cytosolic calcium from its resting level of approximately 100 nM to a value in excess of 1 애M. The mechanism requires calcium entry across the surface membrane (including that of the transverse tubules delivering the action potential signal rapidly to the center of the cell), which leads to CICR from sarcoplasmic reticulum (SR) stores (Shacklock et al., 1995). The importance of calcium release is to amplify the calcium signal from calcium entry and to ensure that calcium rises rapidly throughout the cell. The extent

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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FIGURE 1 Excitation–contraction coupling in cardiac muscle. Scheme showing calciuminduced calcium release in a cardiac muscle cell. The main pathway for calcium entry is thought to be L-type calcium channels, and a local change in calcium close to the inner mouth of these channels is thought to provoke calcium release from nearby ryanodine receptor/channels located in the terminal cisternae of the sarcoplasmic reticulum. Calcium from these two sources (calcium entry and calcium release) then diffuses to the myofilaments to activate contraction by combining to troponin C. Note that the time course of local calcium changes will also depend on the density of calcium-binding sites that ‘‘buffer’’ calcium so that the rate of change of calcium close to the myofilaments need not be the same as that close to transverse tubules.

of this amplification of the calcium signal by calcium release from the SR may vary with species and experimental conditions, as discussed in more detail later.

A. Calcium Fluxes across the Sarcolemma Evidence in support of the importance of calcium entry providing an important trigger for calcium release and contraction has accumulated since the time of Ringer (1882), who showed that the removal of extracellular calcium rapidly abolishes cardiac muscle contraction, whereas effects of extracellular calcium removal in skeletal muscle are much less and are slower to develop. Direct evidence for CICR from SR stores was obtained in an extensive series of experiments on mechanically skinned cardiac muscle by Fabiato (1983). The magnitude and rate of change of calcium applied by pressure ejection from micropipettes in these skinned fibers appeared to be important for triggering calcium release from the SR under the conditions of the experiments. Early voltage clamp experiments (Reuter, 1967) in multicellular cardiac muscle preparations showed the importance of calcium entry across the surface membrane through voltage-gated calcium channels in controlling contraction. When single cardiac ventricular cells were isolated, it was found that the calcium current was activated by voltage more rapidly than previously

thought, reaching a peak in approximately 3 msec at mammalian body temperature, and triggering contraction within milliseconds (Mitchell et al., 1983). A useful tool for investigating the importance of SR stores in excitation–contraction coupling is the plant poison ryanodine, which was shown by Sutko and colleagues (1997) to interfere with SR calcium release. In single rat ventricular cells, ryanodine suppresses contraction greatly without reducing peak calcium current (Mitchell et al., 1984), reflecting the importance of calcium release from the SR in supporting contraction in this species. In other mammalian ventricular cells, significant residual contraction remains after ryanodine, but the calcium transients and accompanying contractions are relatively slow to develop compared with observations in the same cells before ryanodine is applied (White and Terrar, 1990). It seems therefore that calcium entry through the surface membrane is relatively poor in activating contraction directly, and calcium release from SR stores reinforces the magnitude of the calcium transient and enhances its rate of development. Interesting observations in experiments of Beam and colleagues have highlighted the importance of calcium entry through L-type calcium channels in cardiac muscle and demonstrated differences between the role of such channels in skeletal and cardiac muscle (Tanabe et al., 1990). The molecular structure of L-type calcium chan-

38. Calcium Signaling

nels in these two types of muscle, although broadly similar, shows some differences, especially concerning a particular cytosolic loop in the amino acid chain. Chimeric proteins were constructed in which this loop was manipulated in the two types of channel and studied in dysgenic skeletal muscle fibers that do not themselves express L-type calcium channels. When the cardiac form of the loop was introduced into skeletal muscle L-type channels, the muscles showed cardiac-like EC coupling (with CICR), whereas EC coupling typical of skeletal muscle (showing voltage-gated calcium release from the SR) was seen when the skeletal loop was introduced into cardiac L-type calcium channels. Calcium entry through T-type calcium channels may also lead to contraction, although these currents are

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both smaller and apparently less effective in triggering calcium-induced calcium release compared with currents through L-type calcium channels (Sipido et al., 1998). Under some conditions, calcium entry through sodium–calcium exchange working in ‘‘reverse mode’’ may trigger calcium release from the SR, but again this pathway appears to be less effective than that through L-type channels (Lipp and Bootman, 1998). Under some conditions (e.g., stimulation of 웁-adrenoceptors), calcium may also enter through voltage-gated sodium channels, which are said to show ‘‘slip mode’’ conductance after phosphorylation by protein kinase A (Santana et al., 1998). While this has proved controversial, it seems that for such a mechanism to occur, certain subunits of voltage-gated sodium channels must be

FIGURE 2 Localization of cellular components in cardiac cells. Montage of guinea pig cardiac cells. (A) Sinoatrial node cell and (B) ventricular cell labeled with Di-8-ANEPPS to reveal the surface membrane. Note that the ventricular cell shows extensive invaginations corresponding to the transverse tubules at approximately 2-애m intervals, whereas the sinoatrial node cell shows clear labeling of the surface membrane but appears to lack transverse tubules. (C–E) Ryanodine receptors revealed by immunocytochemistry (ryanodine receptors labeled with mouse monoclonal antibody to the canine cardiac ryanodine receptor, Clone C3-33 from Oncogene Research Products; fluorescence indicated by FITC-conjugated goat antimouse IgG secondary antibody) in individual myocytes from sinoatrial node (C), atrium (D), and ventricle (E) of the guinea pig. Note the appearance of ryanodine receptors in bands with a separation of approximately 2 애m (corresponding to the expected sarcomere spacing). Unpublished observations of L. Rigg, B. M. Heath, Y. Cui, and D. A. Terrar.

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coexpressed to allow the phenomenon to be observed (Nuss et al., 1999).

B. Calcium Release from the Isolated Sarcoplasmic Reticulum The majority of calcium for stimulating contraction comes from the SR. This is a large and extensive structure in which large amounts of calcium are loosely bound to luminal calcium-binding proteins such as calsequestrin (see Bers, 1991). Certain regions, the terminal cisternae, are expanded and express a higher density of calcium release sites. The direct study of calcium release from cardiac SR was first performed by Fabiato (1983) using permeabilized cells. It was initiated by perfusing cells with calcium-containing solutions and hence was termed calcium-induced calcium release. A similar phenomenon had been observed previously in skeletal muscle SR (Endo et al., 1970; Ford and Podolsky, 1970). The study of the molecular nature of the calcium release channel itself, and its eventual isolation, was made possible by the use of the plant alkaloid, ryanodine. Ryanodine was isolated from the South American shrub Ryania speciosa, named after the Scottish botanist who discovered it, John Ryan. It is highly toxic, having

been used as a poison and insecticide. Its pharmacological effects on muscle were puzzling. While it induced contraction of skeletal muscle, it inhibited that of cardiac (Sutko et al., 1997). However, its use paved the way for the study of calcium release mechanisms in isolated SR vesicles by 45Ca flux techniques (Chamberlain et al., 1983, 1984) and, most importantly, as a highaffinity probe leading to the purification of its binding site, which on incorporation into lipid bilayers formed a large calcium channel with many of the pharmacological characteristics of the calcium release channel in SR (McPherson and Campbell, 1993). This protein is now known as the ryanodine receptor (Fig. 3). Three distinct isoforms of ryanodine receptors have now been sequenced and are encoded by three separate genes: ryr1, ryr2, and ryr3. Because the distribution of these isoforms is often cell specific with the type 1 channel and type 2 channel predominant in skeletal and cardiac muscle, respectively, this provides an explanation for the differences in action of ryanodine in these cells. Each comprises four identical subunits, which form a homotetrameric structure. Because each subunit is of the order of 560 kDa, the ryanodine receptor complex is one of the largest protein structures present in cells, with dimensions of 29 ⫻ 29 ⫻ 12 nm. Little wonder then that they

FIGURE 3 Modulation of ryanodine receptor gating. Schematic representation of key factors regulating ryanodine receptor activity in cardiac muscle. Calcium entry via L-type calcium channels (dihydropyridine receptors) in the sarcolemma (SL) increases the local calcium concentration around ryanodine receptors. Calcium may activate ryanodine receptor opening, thus promoting CICR. In addition, many other factors modulate ryanodine receptor gating. These include small molecules such as ATP and cADPR, cytosolic proteins such as sorcin, FK506BPs, calmodulin, and various kinases that are known to phosphorylate ryanodine receptors or the accessory proteins. SR luminal proteins as well as the calcium load in the SR may also regulate ryanodine receptor gating. The structure of the ryanodine receptor is based on reconstructions by Hamilton’s group (Serysheva et al., 1999).

38. Calcium Signaling

can be visualized by electron microscopy and were first noted as the ‘‘foot’’ structures spanning the space between sarcolemma and SR in skeletal muscle cells (Franzini-Armstrong and Protasi, 1997). Purification of the protein has allowed detailed electron microsopy of these giant structures. Three-dimensional reconstructions have now given us a clear picture of these channels (Sharma et al., 1998) and remarkably a molecular description of how the channel in skeletal muscle may be gated by calcium (Serysheva et al., 1999). Molecular evidence for the importance of RYR2 in cardiac function has come from the cardiac abnormalities and lethal consequences observed in RYR2 knockout mice (Takeshima et al., 1998). This is probably a consequence of the disruption of calcium homeostasis required for normal development rather than simply a disruption of excitation–contraction coupling. The enormous RYR complex, around 2000 kDa in size, allows a myriad of factors, including proteins to interact and in turn modulate channel gating by calcium. Important factors include the proteins, calmodulin, FK506-binding proteins, sorcin (Valdivia, 1998), and the low molecular weight nucleotides, ATP and cyclic ADPribose (see Fig. 3), the latter of which is discussed in detail later.

C. Calcium Release from the Sarcoplasmic Reticulum in Cardiac Myocytes One aspect of CICR that has received extensive consideration in recent years is whether calcium entry across the whole surface of a cardiac cell could be thought of as reaching a single ‘‘common pool’’ in the cytoplasm into which the contents of SR stores of calcium were also added by CICR (Stern, 1992; Niggli, 1999). This simple system is expected to be unstable as a small amount of calcium entering the cell, raising the calcium concentration in the common pool, would lead to SR calcium release, which would in turn become amplified in a positive feedback manner to give an allor-nothing calcium transient. More realistic descriptions of the cardiac cell take account of the organization of the ryanodine receptor release sites on the terminal cisternae of the SR, closely associated with L-type calcium channels in the surface membrane. Narrow spaces between the sarcolemma and SR membranes might form microdomains, allowing calcium to rise and fall locally without an all-or-none response involving the entire cell. This would allow greater flexibility in the control of whole cell calcium transients, as a larger number of contributing microdomains of calcium elevation would increase the total amount of cytosolic calcium available for controlling contraction. Several explanations have been advanced to account for graded calcium

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release from SR. These are based on mechanisms proposed to counter the positive feedback inherent in the CICR process. In his pioneering studies, Fabiato (1983) proposed that a calcium-dependent inactivation of calcium release from the SR occurred, as the magnitude of calcium release from stores was crucially dependent on the rate of trigger calcium application to trigger release. More recently, studies based largely on the single channel properties of ryanodine receptors reconstituted into artificial lipid bilayers have led to other proposals. One is the concept of adaptation of the ryanodine receptor. Fill and colleagues observed that calcium activation of ryanodine receptor openings appeared to exhibit adaptation rather than inactivation, as channels remained sensitive to incremental increases in calcium by exhibiting further openings (Velez et al., 1997). This phenomenon has been demonstrated for both rapidly applied calcium by fast perfusion techniques and by photolysis of caged calcium. However, the latter experiments with flash photolysis have been questioned, as others have suggested that the adaptation phenomenon may be associated with artifacts associated with the flash photolysis technique (Lamb, 1997). Perhaps the most important single observation in the field since the publication of the monograph by Bers (1991) is the discovery of ‘‘calcium sparks’’ (Cheng et al., 1993; Niggli, 1999; Wier, 1998). These are localized increases in cytosolic calcium (with dimensions of approximately 2 애m, lasting about 50 msec), representing a fundamental building block of the whole hierarchy of cardiac calcium signals (Fig. 4), and are thought to arise from CICR from SR stores. These experimentally observed events have obvious parallels with the theoretical arguments in the preceding paragraph. The transient rise in calcium, which accompanies the ventricular action potential and which controls contraction, can be thought of as comprising many calcium sparks occurring synchronously, although (as discussed in more detail later) smaller units of increase in cytosolic calcium than sparks may occur in some circumstances (Niggli, 1999). Whether the calcium sparks are ‘‘unitary’’ events or not, important insights have been gained from their study. Sparks occurring spontaneously, e.g., in rat cells (Fig. 5), can be detected readily, but in order to see individual sparks during a whole cell calcium transient it is usually necessary to reduce their probability of occurrence. This can be done in a variety of ways, such as blocking a large fraction of calcium entry with a dihydropyridine antagonist of L-type calcium channels or applying voltage clamp protocols that minimize calcium entry. The activation curve for calcium currents is biphasic, with approximately similar peak currents occurring at ⫺20 and 20 mV; interestingly, the probability of occurrence of calcium sparks is greater for the

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FIGURE 4 Hierarchy of calcium signals in cardiac myocytes. This diagram shows types of activity in the form of changes in cytosolic calcium that can be detected using confocal microscopy in cardiac ventricular cells. In each case, fluo-3 fluorescence was excited by a laser, which repeatedly scanned along a single line positioned over the cell (‘‘line scan images’’ or ‘‘xt’’ mode): position/distance across the cell is shown top to bottom, and time (with repeated scans) is shown from left to right. (A) The myocyte was stimulated to fire an action potential, and calcium was seen to rise synchronously across the cell (calcium transient). (B) There was a spontaneous local increase in calcium (calcium spark). (C) A propagating calcium wave in which calcium rose first at the lower edge of the cell (perhaps triggered by a spark) and traveled progressively across the cell. In this form of display, a wave with faster conduction velocity would show a steeper gradient. Each linescan image has dimensions of 100 애m in a vertical direction and 1336 msec in a horizontal direction.

FIGURE 5 The calcium spark. (A) Line scan image (obtained using confocal microscopy) showing a spark similar to that shown in Fig. 4. (B) The amplitude of fluorescence in the region of the spark is shown on the same time scale. It can be seen that the fluorescence at the peak of the spark was approximately two to three times greater than the background level. The linescan image has dimensions of 100 애m in a vertical direction and 1336 msec in a horizontal direction.

38. Calcium Signaling

current at ⫺20 mV than for that at 20 mV, despite their similar magnitudes. A possible reason for this becomes clear when it is appreciated that the current at ⫺20 mV represents the activity of a small number of calcium channels with large unitary currents arising from the greater driving force for calcium entry compared with 20 mV where the same magnitude peak current reflects more channels, each with a smaller unitary current. It appears that unitary currents at ⫺20 mV are better able to evoke sparks than those at 20 mV, perhaps because the larger unitary currents at ⫺20 mV give rise to a higher local calcium concentration in the vicinity of the ryanodine receptor release sites. Plots of the probability of spark occurrence per unit of calcium current at a series of membrane potentials derived from experiments of this kind are said to reflect the ‘‘gain’’ of calcium-induced calcium release (with a steeper relationship reflecting a higher gain) (Cannell and Soeller, 1999), but the exact relationship between calcium current flowing through calcium channels and the probability of spark occurrence remains the subject of debate. In physical terms, functional groupings of L-type calcium channels and calcium release channels termed couplons have been proposed by correlating the occurrence of morphological structures observed at the electron microscope level with subcellular calcium imaging studies (Franzini-Armstrong et al., 1997). The gain of calcium-induced calcium release calculated in this way from the probability of spark occurrence may be reduced during pathological conditions such as hypertrophy and heart failure, even though the current density of calcium currents is not altered greatly (Gomez et al., 1997). This was interpreted as perhaps reflecting a change in the geometry of the space between L-type calcium channels and ryanodine receptor release channels. In another series of experiments in which hypertrophy was induced by constriction of the abdominal aorta, a decreased gain of calcium-induced calcium release was detected only when extracellular calcium was reduced (McCall et al., 1998).

D. Modulation of Calcium Release by Cyclic Adenosine Diphosphate Ribose In a variety of tissues, calcium release from ryanodine-sensitive intracellular stores is regulated by the recently discovered signaling molecule, cADP-ribose (cADPR) (Lee, 1997). Since the mid-1990s, experiments have indicated a possible role for cADPR in regulating calcium release from the SR in cardiac muscle. Cytosolic application of the competitive antagonist 8-aminocADPR has been shown to reduce the amplitudes of calcium transients and contractions accompanying action potentials (Rakovic et al., 1996). This was interpre-

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ted in terms of antagonism of endogenous cADPR, which is known to be present in cardiac tissues, along with enzymes for its synthesis and degradation. 8-aminocADPR was without effect on L-type calcium currents and did not decrease caffeine-releasable stores of calcium. The effects were, however, prevented by pretreatment of myocytes with ryanodine or thapsigargin. It was therefore suggested that endogenous cADPR might increase the calcium sensitivity of CICR from SR stores and that the effects of cADPR might be antagonized by 8-amino-cADPR. More direct evidence in support of this hypothesis was provided by experiments in which cADPR applied to the cytosol via a patch pipette enhanced calcium transients and contractions (Iino et al., 1997). These actions were again sensitive to drugs that disrupt SR calcium storage, ryanodine and thapsigargin, and were antagonized by 8-amino-cADPR and 8-BrcADPR (another competitive cADPR antagonist). The competitive effects of 8-Br-cADPR could be overcome by increasing the concentration of the agonist, cADPR. Interestingly, effects of both agonists and antagonists were markedly temperature dependent. This may explain some previous difficulties in observing actions of cADPR in cardiac myocytes where experiments were carried out at room temperature and may also provide important clues concerning its mechanism of action on cardiac release channels (see later). Calmodulin has been shown to play a role in the actions of cADPR at RyRs on sea urchin egg microsomes, where its interactions with calcium release channel components are necessary to confer cADPR sensitivity (Lee et al., 1994). Evidence has shown that this may also be the case in cardiac muscle (Cui et al., 1999). FK-binding proteins have been shown to regulate calcium release in cardiac muscle (Marks, 1996; Valdivia, 1998; Xiao et al., 1997), and there have been suggestions of a possible involvement of this substance in actions of cADPR (Noguchi et al., 1997). Effects of cADPR may therefore require the concerted action of several proteins involved in CICR. Interestingly, we have found that photoreleased cADPR causes an enhancement of calcium transients to a similar degree to that observed with patch-applied cADPR, but there is a delay between photorelease and maximal effect of approximately 15 sec (Cui et al., 1999) (Fig. 6). It has been speculated that this delay and the temperature dependence may both reflect the complexity of the pathway mediating the enhancement of the calcium sensitivity of CICR by cADPR. In rat cells, which show calcium spark activity at rest, Cui et al. (1999) found that photorelease of cADPR increases the frequency of sparks (an approximate doubling) without a change in their characteristics (amplitude, rise time, rate of decay, and distance to half ampli-

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FIGURE 6 Photoreleased cADPR potentiates action potential-evoked calcium transients in guinea pig ventricular myocytes. (A) Line scan images (obtained using confocal microscopy) showing an abrupt rise in Fluo-3 fluorescence, representing the calcium transient accompanying an action potential (stimulated by a brief current pulse applied to the cell). (B) Photorelease of cADPR leads to an increase in the magnitude of the calcium transient (shown by brighter fluo-3 fluorescence). (C) A more quantitative representation of the calcium transient shown in A, obtained by averaging the magnitude of fluorescence across the cell. (D) Photorelease of cADPR caused a substantial increase in the magnitude of the calcium transient (averaged fluorescence from B). (E) Calcium transients presented on a compressed time scale, illustrating the time for development of the effect of photoreleased cADPR. Note that the effect of photoreleased cADPR on calcium transients developed slowly, taking more than 10 sec for development of the full effect. Data from Cui et al. (1999), reanalyzed by S. Rakovic. Each linescan image has dimensions of 45 애m in a vertical direction and 1336 msec in a horizontal direction.

38. Calcium Signaling

tude) (Fig. 7). The effects of cADPR to increase whole cell calcium transients, apparently as a consequence of enhancement of the calcium sensitivity of calciuminduced calcium release, might be described as an increase in ‘‘gain’’ of the system. However, this has not yet been tested in experiments of the kind described earlier in which gain is determined from the steepness of the relationship between probability of spark occurrence per unit calcium current and membrane potential. The influence of cADPR on spark frequency has been confirmed in a remarkable study by Lukyanenko and Gyorke (1999), in which they reported calcium spark occurrence and waves in saponin-permeabilized rat ventricular myocytes. Examples of sparks that may be recorded in such preparations are shown in Fig. 8. Using calcium buffers to carefully control calcium in the perfusing solution, it was observed that increasing the calcium concentration enhanced sparking occurrence and, at high enough levels, led to the initiation of propagating calcium waves. Furthermore, cADPR and calmodulin were also found to increase the frequency of calcium spark occurrence. In addition to the effects of cADPR on the stimulated contraction of healthy myocytes, there may be effects under conditions resembling pathophysiology. When spontaneous activity associated with overloading of SR calcium stores was provoked by high doses of isoproterenol or by ouabain, this was suppressed by cytosolic application of 8-amino-cADPR. Types of spontaneous activity found to be suppressed by 8-amino-cADPR include afterdepolarizations, spontaneous action poten-

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tials, transient inward currents under voltage clamp conditions, spontaneous calcium transients, and calcium waves. In experiments using confocal microscopy, 8amino-cADPR applied to the cytosol did not reduce the conduction velocity of calcium waves before the waves were abolished. Taken together, these experiments provide convincing evidence that the proposed action of cADPR to increase the calcium sensitivity of CICR also occurs under arrhythmogenic conditions, presumably predisposing the cell to exhibit calcium oscillations involving the SR, and that these effects are suppressed by the antagonistic effect of 8-aminocADPR (Rakovic et al., 1999). We have also studied possible mechanisms of synthesis of cADPR in microsomal preparations of cardiac ventricular muscle. The synthetic enzyme that normally catalyzes the formation of cADPR from the substrate NAD can also synthesize cGDPR when provided with NGD. Because cGDPR fluoresces at 410 nm when excited by UV light at 300 nm, the activity of the synthetic enzyme ADP-ribosyl cyclase in producing cGDPR can be followed by cuvette-based fluorimetry. We have found that the enzyme appears to be associated with SR vesicles from guinea pig ventricle. Interestingly, enzyme activity is enhanced by exposure to the catalytic subunit of cAMP-dependent protein kinase (Heath et al., 1999), raising the possibility that hormones and neurotransmitters that alter the concentrations of cAMP may regulate the levels of cADPR in intact cells. In addition, there may be direct coupling among 웁-adrenergic receptors, G-proteins, and activation of cADPR-synthesizing

FIGURE 7 Photoreleased cADPR enhances the frequency of calcium spark occurrence in rat ventricular myocytes. (A) Line scan images (obtained using confocal microscopy) showing localized increases in fluorescence, representing calcium sparks. (B) The frequency of occurrence of sparks was enhanced following the photorelease of cADPR. Data from Cui et al. (1999).

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VII. Signaling Systems

FIGURE 8 Local calcium signals in rat permeabilized ventricular myocytes. Line scan images showing spontaneous calcium release events, resembling calcium sparks, observed in two rat ventricular myocytes permeabilized with saponin. Following permeabilization to remove the sarcolemma [based on the method described by Lukyanenko and Gyorke (1999)], cells were perfused with fluo-4 and imaged using a confocal microscope. Such preparations provide a useful means by which membrane-impermeant drugs may be applied directly to a functioning sarcoplasmic reticulum without the need of patch electrodes.

ADP-ribosyl cyclase associated with the sarcolemma (Higashida et al., 1999), which contributes to their positive inotropic actions.

E. Role of Inositol 1,4,5-Trisphosphate Receptors Molecular characterization of the inositol 1,4,5-trisphosphate receptor (IP3R) indicated that, similar to ryanodine receptors, they are large homotetrameric structures and share significant homologies in the amino acid sequences of their proposed channel structures (Patel et al., 1999). Each subunit is around 310 kDa and, similar to RyRs, they have large cytoplasmic domains. Three distinct isoforms have been reported encoded by three distinct genes, as is the case for ryanodine receptors. These structural similarities are mirrored in their functional properties too, particularly their modulation by calcium ions and ATP. It is likely that both classes of calcium release channels have evolved from a common

gene and are thus part of the same gene family. Although IP3Rs bind IP3 selectively (Kd 앑2–100 nM), they can also be activated by calcium and thus exhibit CICR, although IP3 binding is required. Although IP3Rs are prominent in nonexcitable cells, they are also found in excitable cells, with particularly high levels of type 1 receptor found in cerebellar Purkinje neurons. Evidence shows that low levels of IP3Rs are expressed in cardiac tissue (Gorza et al., 1993). For example, the type 2 receptor has been demonstrated in rat ventricular tissue. IP3 is generated from a minor constituent of the lipid bilayer, phosphatidyl inositol 4,5-bisphosphate, by the action of receptor-coupled phospolipase C (Berridge and Irvine, 1989). The other product of the reaction is diacylglycerol, which activates protein kinase C (PKC), which in turn phosphorylates and hence regulates a number of important regulatory proteins. PLC-웂 is activated by many tyrosine kinase-linked receptors, whereas PLC-웁 isoforms are activated by G-proteincoupled receptors. In the heart, the most significant

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38. Calcium Signaling

receptors coupled to IP3 signaling are 움1-adrenergic, endothelin, and thrombin receptors (Scholz et al., 1988). IP3 receptors in the heart respond only weakly to IP3 and release calcium much more slowly, which may preclude them from activating CICR. It is thus thought that they have only a marginal role in normal excitation– contraction coupling compared to their larger relative, the RyRs. However, a number of reports indicate that IP3Rs may play a role in a number of cardiac pathologies. IP3-evoked calcium release mechanisms have been implicated in a number of diseases, such as myocarditis, Chargas’ disease, and transplant rejection. IP3 may be important in disturbances of rhythm and, in the longer term, an important mediator of apoptotic pathways. IP3 has also been demonstrated to induce slow calcium oscillations, generating arrhythmias due to stimulation of Iti, resulting in delayed afterdepolarizations, slowing of repolarization favoring reentry mechanisms, and promotion of cell–cell uncoupling. Thrombin, whose receptors are coupled to IP3 production in myocardia, together with 움1-adrencopter activation, is proarrhythmic under ischemic and reperfusion conditions. The phospholipase C inhibitor U-73122 has been shown to inhibit the effects of thrombin. In the failing heart, there is more reliance on 움1-adrenoceptor rather than 웁1-adrenocopter activation, which may also be involved in the initiation of biochemical responses resulting in cardiac hypertrophy. In addition, myocardial tissue from patients with dilated cardiac myopathies have higher levels of mRNA encoding IP3Rs.

III. INFLUENCE OF CALCIUM DURING PACEMAKER ACTIVITY The possible importance of calcium release from the SR in regulating pacemaker activity of the sino-atrial (SA) node has been raised (Rigg and Terrar, 1996). Application of ryanodine to spontaneously beating atrial preparations slowed the rate of occurrence of action potentials recorded with surface electrodes. A similar reduction of spontaneous activity in these preparations was seen when SR function was suppressed by a different method—application of cyclopiazonic acid to interfere with SR Ca ATPase. Both ryanodine and cyclopiazonic acid also caused similar changes in electrical activity recorded using intracellular electrodes in these preparations. When spontaneous activity was slowed by these agents, the most negative potential shifted to more depolarized levels and there were changes in the slope of the pacemaker depolarization. Similar observations concerning slowing effects of ryanodine and accompanying effects on electrical activity were reported in small

preparations of rabbit SA node. Studies in single cells isolated from rabbit SA node also showed slowing effects of ryanodine on pacemaker activity. Studies of cytosolic calcium using fluorescent probes in toad pacemaker cells have provided direct evidence that the slowing effects of ryanodine are accompanied by the suppression of calcium transients (Ju and Allen, 1999). Preliminary evidence shows similar changes in guinea pig SA node cells (Rigg and Terrar, 1998a). Because 웁-adrenoceptor stimulation increases the calcium released from stores as well as calcium entry across the surface membrane in ventricular cells, it seems possible that 웁-adrenoceptor agonists may also enhance SR calcium release in pacemaker cells. It has been shown that the slowing effect of ryanodine on pacemaker activity is even greater after 웁-adrenoceptor stimulation than in the absence of agonists. Exposure to ryanodine also causes the log(concentration)–response curve for the action of isoproterenol on spontaneous beating to be less steep in atrial preparations (Rigg and Terrar, 1998b; Rigg et al., 2000). It has been shown in toad pacemaker cells that effects of 웁-adrenoceptor stimulation on ryanodine-sensitive calcium transients may contribute to positive chronotropic effects mediated via this pathway (Ju and Allen, 1999). There is rather little information on the density and organization of ryanodine receptors in the SA node. Immunohistochemical evidence using specific antibodies against RyR2 receptors shows that these receptors are indeed present in guinea pig pacemaker cells. These appear to be organized in bands with a 2-애m spacing resembling that of sarcomeres, even though no transverse tubules (which may be labeled with the surface membrane dye, Di-8-ANEPPS; see Lipp et al., 1996) appear to be present in these cells.

IV. SUMMARY This chapter emphasized some of the new and exciting developments in cardiac calcium signaling. The cardiac myocyte is an exquisite machine for generating complex calcium signals. Although the release of calcium via RyRs during excitation–contraction coupling is critically under the tight control of trigger calcium entering via L-type calcium channels, additional biochemical mechanisms appear to be important in modulating this process of CICR. While the release of calcium from the SR is undoubtedly important for control of the mechanical properties of the heart, new evidence suggests that it may also play a crucial role in pacemaking and developmental processes.

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Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E., and Walseth, T. F. (1994). Cyclic ADP ribose activation of the ryanodine receptor is mediated by calmodulin. Nature 370, 307–309. Lipp, P., and Bootman, M. D. (1998). Which cardiac Ca2⫹ channels control E-C coupling? J. Physiol. (Lond.) 508, 331. Lipp, P., Huser, J., Pott, L., and Niggli, E. (1996). Spatially nonuniform Ca2⫹ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture. J. Physiol. (Lond.) 497, 589–597. Lukyanenko, V., and Gyorke, S. (1999). Ca2⫹ sparks and Ca2⫹ waves in saponin-permeabilized rat ventricular myocytes. J. Physiol. (Lond.) 521, 575–585. Marks, A. R. (1996). Cellular functions of immunophilins. Physiol. Rev. 76, 631–649. McCall, E., Ginsburg, K. S., Bassani, R. A., Shannon, T. R., Qi, M., Samarel, A. M., and Bers, D. M. (1998). Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am. J. Physiol. 274, H1348–H1360. McPherson, P. S., and Campbell, K. P. (1993). The ryanodine receptor/ Ca2⫹ release channel. J. Biol. Chem. 268, 13765–13768. Mitchell, M. R., Powell, T., Terrar, D. A., and Twist, V. W. (1983). Characteristics of the second inward current in cells isolated from rat ventricular muscle. Proc. R. Soc. Lond. B Biol. Sci. 219, 447–469. Mitchell, M. R., Powell, T., Terrar, D. A., and Twist, V. W. (1984). Ryanodine prolongs Ca-currents while suppressing contraction in rat ventricular muscle cells. Br. J. Pharmacol. 81, 13–15. Niggli, E. (1999). Localized intracellular calcium signaling in muscle: Calcium sparks and calcium quarks. Annu. Rev. Physiol. 61, 311–335. Noguchi, N., Takasawa, S., Nata, K., Tohgo, A., Kato, I., Ikehata, F., Yonekura, H., and Okamoto, H. (1997). Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2⫹ from islet microsomes. J. Biol. Chem. 272, 3133–3136. Nuss, H. B., Marba´n, E., Balke, C. W., Goldman, L., Aggarwal, R., Shorofsky, S. R., dos Santos Cruz, J., Santana, L. F., Frederick, C. A., Isom, L. L., Malhotra, J. D., Mattei, L. N., Kass, R. S., Xia, J., An, R.-H., and Lederer, W. J. (1999). Whether ‘‘slip-mode conductance’’ occurs. Science 284, 711a. Patel, S., Joseph, S. K., and Thomas, A. P. (1999). Molecular properties of inositol 1,4,5-trisphosphate receptors. Cell Calcium 25, 247–264. Rakovic, S., Cui, Y., Iino, S., Galione, A., Ashamu, G. A., Potter, B. V., and Terrar, D. A. (1999). An antagonist of cADP-ribose inhibits arrhythmogenic oscillations of intracellular Ca2⫹ in heart cells. J. Biol. Chem. 274, 17820–17827. Rakovic, S., Galione, A., Ashamu, G. A., Potter, B. V. L., and Terrar, D. A. (1996). A specific cyclic ADP-ribose antagonist inhibits cardiac excitation–contraction coupling. Curr. Biol. 6, 989–996. Reuter, H. (1967). The dependence of slow inward current in Purkinje fibres on the extracellular calcium-concentration. J. Physiol. (Lond.) 192, 479–492. Rigg, L., and Terrar, D. A. (1996). Possible role of calcium release from the sarcoplasmic reticulum in pacemaking in guinea-pig sinoatrial node. Exp. Physiol. 81, 877–880. Rigg, L., and Terrar, D. A. (1998a). Possible influence of cytosolic calcium on the rate of beating of sino-atrial node cells isolated from guinea-pig heart. Br. J. Pharmacol. 123, 328P. Rigg, L., and Terrar, D. A. (1998b). Influence of ryanodine on the positive chronotropic response to beta-adrenergic stimulation with isoprenaline in guinea-pig sino-atrial node. Br. J. Pharmacol. 123, 327P. Rigg, L., Heath, B. M., Cui, Y., and Terrar, D. A. (2000). Localization and functional significance of ryanodine receptors during beta-adre-

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Stern, M. D. (1992). Theory of excitation-contraction coupling in cardiac muscle. Biophys. J. 63, 497–517. Sutko, J. L., Airey, J. A., Welch, W., and Ruest, L. (1997). The pharmacology of ryanodine and related compounds. Pharmacol. Rev. 49, 53–98. Takeshima, H., Komazaki, S., Hirose, K., Nishi, M., Noda, T., and Iino, M. (1998). Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2. EMBO J. 17, 3309–3316. Tanabe, T., Beam, K. G., Adams, B. A., Nidome, T. and Numa, S. (1990). Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling. Nature 346, 567–569. Valdivia, H. H. (1998). Modulation of intracellular Ca2⫹ levels in the heart by sorcin and FKBP12, two accessory proteins of ryanodine receptors. Trends Pharmacol. Sci. 19, 479–482. Velez, P., Gyorke, S., Escobar, A. L., Vergara, J., and Fill, M. (1997). Adaptation of single card†iac ryanodine receptor channels. Biophys. J. 72, 691–697. Wier, W. G. (1998). Ca2⫹-induced Ca2⫹ release: Physiological experiments on a new level. J. Physiol. (Lond.) 508, 645. White, E., and Terrar, D. A. (1990). The effects of ryanodine and caffeine on Ca-activated current in guinea-pig ventricular myocytes. Br. J. Pharmacol. 101, 399–405. Xiao, R. P., Valdivia, H. H., Bogdanov, K., Valdivia, C., Lakatta, E. G., and Cheng, H. (1997). The immunophilin FK506-binding protein modulates Ca2⫹ release channel closure in rat heart. J. Physiol. (Lond.) 500, 343–354.

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39 Diadenosine Polyphosphate Signaling in the Heart ALEKSANDAR JOVANOVIC and SOFIJA JOVANOVIC

ANDRE TERZIC

Tayside Institute of Child Health Ninewells Hospital and Medical School University of Dundee, Dundee, Scotland

Division of Cardiovascular Diseases Department of Internal Medicine Mayo Clinic and Foundation Rochester, Minnesota 55905

I. INTRODUCTION

et al., 1998a). Using HPLC, micromolar concentrations of Ap5A were detected in guinea pig ventricular tissue extracts (Jovanovic et al., 1998c). In a following study, Ap2A and Ap3A were found, this time, in human ventricular tissue (Luo et al., 1999). In addition, Ap2A and Ap3A were also present in porcine myocardial granules, the content of which is released into the extracellular space (Luo et al., 1999). The concentration of Ap2A and Ap3A in granules was estimated to be in the millimolar range. Taken together, these findings strongly indicate that ApnA are present in heart tissue. In fact, micromolar concentrations in cardiac tissue extracts and millimolar concentrations in the granular fraction suggest that biologically effective concentrations of ApnA can be reached in both intracellular and extracellular milieu.

Diadenosine polyphosphates (ApnA) contain two adenosine moieties linked through ribose 5⬘ carbons to a phosphate group chain, with the number of phosphate groups (n) from 2 to 6 (Fig. 1). Over the last decades, ApnA have been implicated as signaling molecules in the regulation of various cellular functions in both lower and higher organisms. In prokaryotic cells, changes in the concentration of cytosolic ApnA have been reported during stress and associated with inhibition of heat shock and oxidative stress proteins. In mammalian cells, the involvement of ApnA in cell differentiation, apoptosis, tumorigenesis, and cellular response to interferons and cytokine signaling has been documented (Table I). Although both intracellular and extracellular ApnA have been implicated in the regulation of vital cellular functions (Table II; see Baxi and Vishwanatha, 1995; Javanovic et al., 1997; Kisselev et al., 1998; MirasPortugal et al., 1998), only recently has the role for ApnA been considered in the myocardium itself (Jovanovic et al., 1997, 1998c; Kisselev et al., 1998; MirasPortugal et al., 1998; Flores et al., 1999; Lou et al., 1999). The aim of this chapter is to outline what is currently known about the cardiac effects of ApnA and discuss the possible signaling role of these nucleotides in the heart.

III. SYNTHESIS AND METABOLISM OF ApnA The exact pathways responsible for the synthesis and degradation of ApnA in the heart have yet to be determined. In other mammalian cell types, such as the liver and megakaryocytes, the metabolism of ApnA has been established. Synthesis of Ap3A and Ap4A is catalyzed by aminoacyl-tRNA synthases, involving aminoacylAMP formation followed by AMP transfer to ATP or ADP, respectively (reviewed by Yakovanko and Formazyuk, 1993; Fig. 2). It starts with the formation of an enzyme-bound aminoacyl adenylate, which is the usual intermediate in the synthesis of aminoacyl-tRNA. In the absence of tRNA, the fate of the enzyme-bound

II. PRESENCE OF ApnA IN THE HEART In 1998, member of the ApnA family was identified for the very first time in cardiac tissue (Jovanovic

Heart Physiology and Pathophysiology, Fourth Edition

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TABLE I Changes in ApnA Levels in Different Cell Types Following Exposure to Various Factorsa Factor

Cell

ApnA

Effect

Interferon 웁

Human cultured cells

Ap4A

10-fold decrease

Interferons 움 and 웂

Human cultured cells

Ap3A

3- to 5-fold increase

Heat shock

Chicken erythrocytes

Ap4A

10-fold increase

Glucose

Murine pancreatic cells

Ap3A, Ap4A

30- to 70-fold increase

Phorbol ester (TPA)

Human cell line HL60

Ap3A

4- to 5-fold increase

Topoisomerase II inhibitor (VP16)

Human cell line HL60

Ap3A, Ap4A

3-fold decrese and 4-fold increase, respectively

a

From Weinmann-Borch et al. (1984), Bonaventura et al. (1992), Ripoll et al. (1996); and Vartanian et al. (1996, 1997).

FIGURE 1 General structure of ApnA, which are dinucleotides composed of two adenosine moieties linked through ribose 5⬘ carbons to the phosphate group chain. The number of phosphate groups (n) is 2 in Ap2A, 3 in Ap3A, 4 in Ap4A, 5 in Ap5A, and 6 in Ap6A.

TABLE II Effects of ApnA in Target Tissuesa Effect

Target cell or tissue

ApnA member ApnA (n ⫽ 3–6)

Stimulation of DNA synthesis

Renal mesangial cells

Induction of Ca2⫹ oscillations

Hepatocytes

Ap3A, Ap4A

Inhibition of sperm motility

Human spermatozoa

Ap3A, Ap4A

Vasoconstriction and vasodilation

Mesenteric and renal arteries

ApnA (n ⫽ 2–6)

Glycogen phosphorylase activity

Hepatocytes

Ap3A, Ap4A

Activation of ryanodine receptors

Skeletal and cardiac muscle

Ap3A, Ap4A

Prevention of aggregation

Platelets

Ap4A

Increase in intracellular Ca2⫹

Neutrophils

ApnA (n ⫽ 3–6)

Feedback inhibition of excitation

Hippocampus

Ap4A, Ap5A

Stimulation of gluconeogenesis

Proximal tubules

Ap3A, Ap4A

Delay in apoptosis

Neutrophils

Ap5A, Ap6A

a From Chan et al. (1991), Zamecnik et al. (1992), Klishin et al. (1994), Ralevic et al. (1995), Green et al. (1995), Schulze-Lohoff et al. (1995), Holden et al. (1996), Keppens (1996), Gasmi et al. (1996, 1997), Edgecombe et al. (1997), and Delanye et al. (1997).

39. Diadenosine Polyphosphate Signaling

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FIGURE 2 Schematic representation of the synthesis of Ap4A by aminoacyl-tRNA synthetases.

aminoacyl adenylate is determined by the competition of several reactions. The reverse reaction results in the formation of ATP (ADP) and amino acid. Alternatively, aminoacyl adenylate can be attacked by the pyrophosphate moiety of ATP leading to the formation of Ap4A, or hydrolyzed, or alternatively dissociated from the enzyme. In the absence of substrate tRNA, the reaction equilibrium is shifted significantly toward pyrophosphorolysis of the aminoacyl adenylate. Cleavage of pyrophosphate (PPi) formed in the course of amino acid activation by inorganic pyrophosphatase activates the synthesis of Ap4A. In contrast to Ap3A and Ap4A, the mechanism of in vivo Ap5A and Ap6A synthesis is still unknown. It is believed that Ap5A and Ap6A are synthesized by the same enzymes as Ap3A and Ap4A when adenosine tetraphosphate (Ap4) or adenosine pentaphosphate (Ap5) enters into the reaction described in Fig. 2 as one of the subtrates. In higher eukaryotes, Ap3A and Ap4A are hydrolyzed by specific enzymes, Ap3A hydrolase (EC 3.6.1.29) and asymmetrical Ap4A hydrolase (EC 3.6.1.17). Ap3A hydrolase cleaves Ap3A into AMP and ADP, whereas asymmetrical Ap4A hydrolase cleaves Ap4A into AMP and ATP. Ap4A hydrolases are specific for Ap4A, although they can also cleave Ap5A and Ap6A. In addition to specific hydrolases, ApnA may be degraded nonspecifically by a variety of phosphodiesterases and nucleotidases. In contrast to intracellular hydrolases, which are specific for the length of the phosphate chain, the extracellular catabolism by plasma enzymes, as well as by cell-bound ectoenzymes, seems less specific (reviewed by Yakovenko and Formazyuk, 1993; Baxi and Vishwanatha, 1995; Kisselev et al., 1998).

drops from micro- to submicromolar levels (Jovanovic et al., 1998a; Fig. 3). The significance and mechanism of the ischemia-induced decrease in cardiac Ap5A are unknown at present. In other cell types, the stressregulated dynamics of ApnA have been associated with the regulation of proteins containing nucleotide-binding domains. In fact, such proteins are commonly involved in cellular signaling coupled to DNA replication, growth, and cell division (see Baxi and Vishwanatha, 1995; Kisselev et al., 1998; Miras-Portugal et al., 1998). In cardiac cells, a major sensor of metabolic stress is the ATP-sensitive K⫹ (KATP) channel, an ATP-gated ion conductance which opening promotes cellular survival under ischemic conditions (Noma, 1983; Hearse, 1995; Jovanovic et al., 1998a,b, 1999; Gross and Fryer, 1999). The nucleotide-binding domains of the KATP channel complex, along with other nucleotide-binding proteins, may serve as targets for ApnA in the heart (see later). Because ApnA are present in the heart, with levels regulated by metabolic stress, the role for these molecules in the myocardial response to ischemia has been considered (Jovanovic et al., 1998c). In particular, Ap5A has been found to antagonize the opening of ischemiasensitive KATP channels with an EC50 at 13 애M, a value close to the actual level of Ap5A present in the myocar-

IV. REGULATION OF ApnA LEVELS IN THE HEART As ApnA have been discovered only recently in the heart (Jovanovic et al., 1998c; Luo et al., 1999), their regulation still remains largely unknown. In noncardiac tissues, numerous factors, mostly associated with stress conditions, can change cellular levels of ApnA (Table I). In the heart, global 10-min-long ischemia produces a significant decrease in ventricular Ap5A, which

FIGURE 3 Concentrations of Ap5A, estimated by HPLC, in guinea pig ventricular tissue extracts obtained from normoxic hearts and hearts exposed to a 10-min-long global ischemia (n ⫽ 4–6 hearts). Vertical bars represent mean ⫾ SEM. Modified from Jovanovic et al. (1998c).

696

VII. Signaling Systems

In the heart, it has also been reported that specific binding sites for ApnA may be present (Walker et al., 1993; reviewed by Flores et al., 1999). Specifically, a specific binding site for Ap4A has been identified on the surface of isolated cardiomyocytes. ATP, ADP, AMP, and adenosine at micromolar concentrations do not bind to this receptor, but Ap5A and Ap6A may act as competitive agonists. Ap4A binding is specific, saturable, and reversible and can be antagonized by a monoclonal antibody raised against the receptor (Walker et al., 1993). However, the signaling cascade coupled with such a putative ApnA receptor in the heart, as well as the effects of activation of this receptor type, remains elusive. FIGURE 4 Concentration–response curve of Ap5A versus KATP

channel activity. Data points represent mean ⫾ SEM (n ⫽ 3–6). The patch clamp technique was employed on isolated ventricular cardiomyocytes in the inside-out configuration. Holding potential: ⫺60 mV. Modified from Jovanovic et al. (1998c).

dium (7 애M; Jovanovic et al., 1998c). Thus, concentrations of Ap5A, found in normal hearts, could maintain a low probability of KATP channel opening, whereas concentrations of Ap5A found in hearts following ischemia are associated with a higher probability of channel opening (Fig. 4). Indeed, Ap5A fluctuates during ischemic stress within a range of concentrations that correspond closely to the EC50 that defines the action of this ApnA on the probability of KATP channel opening. Therefore, Ap5A may serve as an ischemia-sensing regulator of cardiac KATP channels. In this regard, further investigation is required to determine whether changes in ApnA and opening of cardiac KATP channels are closely associated and whether other consequences of ApnA fluctuations may also occur in the ischemic myocardium.

V. DOES A SPECIFIC RECEPTOR FOR ApnA EXIST IN THE HEART? Some of the biological effects of ApnA have been attributed to their interaction with cell surface receptors. ApnA can bind and activate P2X and P2Y receptor subtypes to which ATP can also bind (reviewed by Ralevic and Burnstock, 1998). the existence of a specific receptor for ApnA is still not definitely accepted, although it has been proposed (Hoyle, 1990). In noncardiac tissue, it has been proposed that there are two distinct classes of ApnA receptors called P2YAp4A (also known as P2D ; Pintor et al., 1993) and P4 (Miras-Portugal et al., 1998). It has been suggested that the putative P2D receptor is coupled to G-proteins and protein kinase C. However, the P4 receptor seems to be coupled to Ca2⫹ channels.

VI. CARDIAC EFFECTS OF ApnA A number of ApnA effects have been reported in the heart (Table III). These effects may be due to the action of uncleaved ApnA molecules or to mononucleotides/adenosine yielded from ApnA (Fig. 5). ApnA themselves, as well as different products of ApnA degradation, may bind to numerous receptors and nucleotide-binding proteins, including P1 and P2 purinoceptors, a putative ApnA receptor, nucleotidebinding enzymes, and ion channels.

A. Negative Inotropic and Chronotropic Effect of ApnA In 1996, ApnA (n ⫽ 2–6) were found to induce a direct negative inotropic and chronotropic effect, as well as inhibit an electrical field stimulation (EFS)-induced positive inotropic response in a cardiac preparation (Rubino and Burnstock, 1996; Vahlensieck et al., 1996). These effects were concentration dependent, achieved at micromolar levels of ApnA. Such cardiac effects of ApnA were inhibited by the adenosine A1 receptor antagonist. It is thus likely that ApnA inhibit neurotransmission and regulate the force and rate of cardiac contraction via prejunctional and postjunctional adenosine A1 receptors. It is not yet established, however, whether these effects of ApnA are due to a direct agonistic action on A1 receptors or mediated by adenosine yielded from ApnA. So far, there is no evidence that ApnA directly binds and activates adenosine receptors. Moreover, the whole cell configuration of the patch clamp technique revealed that ApnA do not activate I(KACh) current in atrial myocytes, a potassium ion conductance that serves as an effector in the signal transduction pathway of A1 receptors in supraventricular cardiac myocytes (Brandts et al., 1998). In contrast, data suggest that adenosine itself, probably originated by hydrolysis from ApnA,

697

39. Diadenosine Polyphosphate Signaling

TABLE III Cardiac Effects of ApnAa Effect

ApnA tested

Negative inotropic and chronotropic effects

ApnA (n ⫽ 2–6)

Positive inotropic effect

Ap4A

Inhibition of 웁 receptor-stimulated Ca2⫹ current in ventricular cardiomyocytes

Ap2A, Ap3A, Ap6A

Activation of ATP-regulated K⫹ current in atrial cardiomyocytes

ApnA (n ⫽ 4–6)

Inhibition of muscarinic K⫹ current in atrial cardiomyocytes

ApnA (n ⫽ 4–6)

⫹

ApnA (n ⫽ 4–6)

Inhibition of ATP-sensitive K channels in ventricular cardiomyocytes

a From Jovanovic and Terzic (1995, 1996), Rubino and Burnstock (1996), Jovanovic et al. (1996a), Vahlensieck et al. (1996, 1998), Brandts et al. (1998), Stavrou et al. (1998), and Luo et al. (1999).

may mediate ApnA action on the heart. Specifically, it has been shown that inhibitors of adenosine uptake potentiate the ApnA-induced negative inotropic and chronotropic effects in isolated perfused heart and that only a fraction of the initially applied ApnA is detected in the effluent of the perfused heart (Brendts et al., 1998).

B. Positive Inotropic Effect of Ap4A It has been reported that Ap4A, as opposed to other members of the ApnA family tested so far, induces a positive inotropic effect in the human myocardium (Vahlensieck et al., 1999). This action of Ap4A is not due to the release of catecholamines, adenosine, and/ or activation of adrenoceptors. The Ap4A-induced positive inotropic effect can be blocked by the P2 purinoceptor antagonist suramin, suggesting that the P2 purinoceptor subtype(s) may mediate this effect of Ap4A. It remains to be determined what is the exact subtype of the P2 purinoceptor involved in the Ap4A action and

whether this is a direct effect of Ap4A or an effect of products, possibly mononucleotides, yielded from Ap4A.

C. Electrophysiological Effects of ApnA It has been demonstrated that ApnA, when applied from the extracellular or intracellular side of the sarcolemma, can regulate certain ion channels. In particular, ApnA inhibit I(KACh) in atrial cardiomyocytes, activate the ATP-regulated K⫹ current (I(KATP)) in atrial cardiomyocytes, block the 웁-receptor-activated Ca2⫹ current in ventricular cardiomyocytes, and inhibit ATPsensitive K⫹ (KATP) channels in ventricular cardiomyocytes.

D. Inhibition of I(KACh) ApnA inhibit acetylcholine-, adenosine-, or GTP-웂S-induced activation of I(KACh) in atrial cardiomyocytes (Brendts et al., 1998). ApnA induce this effect regardless

FIGURE 5 Possible modes of ApnA action in the heart. Mode 1: acting as uncleaved molecules on target proteins specific for ApnA (e.g., putative ApnA receptor). Mode 2: acting as uncleaved molecules on target proteins nonspecific for ApnA (e.g., different P2 receptor types). Mode 3: cleaved and yield mononucleotides and adenosine that target specific and nonspecific protein targets (e.g., P2 and A1 receptors).

698

VII. Signaling Systems

of whether they are applied from the extracellular or intracellular side of the sarcolemma. It has been interpreted that this ApnA effect may be due to an intracellular action of ApnA. However, because there is no evidence that ApnA can cross intact cell membranes or can be taken up by cardiomyocytes (Walker et al., 1993), such a possibility awaits to be examined further.

E. Activation of I(KATP) When applied on an atrial cardiomyocyte from the extracelullar side of the sarcolemma, ApnA induce I(KATP) current (Brandts et al., 1998). This effect of ApnA has been also ascribed to an intracellular action of these compounds. Specifically, it has been proposed that ApnA may be internalized into cardiomyocytes and then hydrolyzed to yield AMP. In turn, AMP is a substrate for the adenylate kinase reaction: AMP ⫹ ATP } 2ADP. Through this reaction, AMP increases the concentration of ADP, a known activator of KATP channels (Elvir-Mairena et al., 1996). This, in turn, would lead to the activation of KATP channels and induction of I(KATP) current. The main challenge to this theory is the mechanism of ApnA uptake into cardiomyocytes.

F. Inhibition of 웁-Adrenoceptor-Activated Ca2⫹ Current In isolated cardiomyocytes, ApnA do not affect the amplitude of inward Ca2⫹ current per se, but block isoprenaline-induced Ca2⫹ current. This strongly resembles the electrophysiological action of adenosine (Vahlensieck et al., 1999; Luo et al., 1999). In fact, this effect of ApnA is sensitive to antagonists of adenosine A1 receptors. Because there is lack of evidence that ApnA themselves are able to bind and activate adenosine A1 receptors, it is conceivable that adenosine yielded from ApnA is responsible for such an electrophysiological effect (Brandts et al., 1998).

G. Inhibition of Ventricular KATP Channels The ventricular KATP channel is the ion conductance presumed to link the metabolic state of the cell with its membrane excitability (Noma, 1983; reviewed by Aguilar-Bryan and Bryan, 1999). This ion channel complex is characterized by its selectivity for K⫹ and nucleotide-binding properties (reviewed by Seino, 1999). That ApnA act as inhibitory ligands of KATP

FIGURE 6 Diadenosine tetra (A)-, penta (B)-, and hexaphosphate (C) (ApnA, n ⫽ 4–6)-

induced inhibition of ATP-sensitive K⫹ channels in ventricular cardiac cells. The patch clamp technique was employed on isolated cardiomyocytes in the inside-out configuration. Holding potential: ⫺60 mV. Dotted lines correspond to the zero-current level. Modified from Jovanovic and Terzic (1995, 1996) and Jovanovic et al. (1996a).

39. Diadenosine Polyphosphate Signaling

TABLE IV Summary of C50 (애M ) and Hill Coefficient (n) Values Defining the Concentration-Dependent Inhibition of Ventricular KATP Channels by Diadenosine Polyphosphates (ApnA, n ⫽ 3–6)a IC50

n

Ap3A

NDb

NDb

Ap4A

17

1.2

Ap5A

16

1.6

Ap6A

14

1.1

a Data from Jovanovic and Terzic (1995, 1996) and Jovanovic et al. (1996a). b Not determined.

channels has first been described for Ap6A applied on membrane patches excised from ventricular cardiomyocytes (Jovanovic and Terzic, 1995; Fig. 6C). Further investigation, using the inside-out configuration of the patch clamp technique, indicated that in addition to Ap6A, micromolar concentrations of other members

699

of ApnA, namely Ap4A and Ap5A, also inhibit KATP channel opening in a reversible manner (Jovanovic and Terzic, 1996; Jovanovic et al., 1996a) (Fig. 6 and Table I). The site of action of ApnA is most likely intracellular, as these molecules are poorly membrane permeable and so far there is no evidence for fast ApnA uptake in ventricular cardiomyocytes (Walker et al., 1993; see also Yakovenko and Formazuyk, 1993). The effect of ApnA occurs in the absence of GTP, ruling out the possibility for the involvement of a GTP-binding protein in the transduction of the inhibitory effect of ApnA on KATP channels. The concentration dependence and saturable nature of the action of ApnA on KATP channel opening suggest the existence of specific binding sites for ApnA within KATP channel subunits or a closely associated protein (Jovanovic et al., 1997). Regardless of the length of the phosphate chain, the potency of various ApnA, e.g., Ap4A, Ap5A, and Ap6A, to inhibit KATP channels is within a close micromolar range (Table IV). This is in contrast with a variable affinity displayed by members of the ApnA family in binding to other nucleotide-binding proteins, such as adenosine kinase, adenylate kinase, terminal deoxynucleotidil transferase, and carbamyl phosphate syn-

FIGURE 7 Diadenosine polyphosphates, Ap4A and Ap5A, do not inhibit spontaneous KATP channel openings in the presence of the nucleotide-diphosphate UDP, but inhibit UDP-evoked KATP channel openings, after ‘‘run-down’’ of spontaneous channel activity. Average NP0 (where N represents the number of channels and Po the probability for channels to be open) of spontaneously (A) or UDP-evoked (B) KATP channel openings in the absence (1) and presence (2) of Ap4A or Ap5A. Stars indicate statistical significance of differences between two means (P ⬍ 0.01; n ⫽ 4–8).

700

VII. Signaling Systems

thase (reviewed by Yakovenko and Formazuyk, 1993; Baxi and Vishwanatha, 1995). Despite their different selectivity toward other nucleotide-binding proteins and their different affinities for the ATP-binding site located on these proteins, it appears that the property of ApnA to inhibit KATP channels is common to all ApnA tested so far. The outcome of the interaction between ApnA and KATP channels can be set by the operative condition of KATP channels (Jovanovic et al., 1996b). KATP channels can operate under two operative conditions: operative condition 1, when KATP channel activity is spontaneous and not enhanced by nucleoside diphosphates versus operative condition 2, when after the decline of spontaneous channel activity (‘‘run-down’’), nucleoside–diphosphate induce the channel to reopen (Terzic et al., 1994). The effect of Ap4A and Ap5A on KATP channels under different operative conditions was tested in membrane patches excised from ventricular cardiac cells (Jovanovic et al., 1996b). Under operative condition one, the application of nucleoside–diphosphates prevented the Ap4A- and Ap5A-induced inhibition of spontaneous KATP channel activity (Fig. 7). In contrast, under operative condition 2, nucleoside diphosphateinduced KATP channel openings were inhibited by Ap4A or Ap5A (Fig. 7). Thus, the outcome of the interaction between an ApnA and nucleoside diphosphates on KATP channel opening is not constant but may change with the operative condition of the channel. However, the mechanism responsible for the switch in the responsive behavior of KATP channels toward ApnA is still unknown.

VII. COMPARISON OF ApnA WITH MONONUCLEOTIDES AND ADENOSINE ApnA are structurally related to ATP and there are strong parallels between their cardiac actions. The greater stability of ApnA compared with mononucleotides and adenosine has significant implications with respect to their extracellular and intracellular actions. ATP appears to be degraded much more rapidly than ApnA by nucleotidases and ectohydrolases (Hoyle et al., 1996). Therefore, despite a lower tissue concentration. ApnA could be at least as effective as ATP. ATP and ApnA inhibit opening of KATP channels with a similar potency (Table II). As described previously for ApnA, the outcome of interaction between ATP and KATP channels is also governed by the operative condition of the channel and the presence of nucleoside diphosphates (Terzic et al., 1994). However, in contrast to ATP, which serves not only as a ligand to close the KATP channel, but also maintains channel activity (see Terzic et al., 1995). ApnA do not promote a sustained channel

TABLE V Comparison of Effects of ATP and Diadenosine Polyphosphates (ApnA, n ⫽ 4–6) on Ventricular ATP-Sensitive K⫹ (KATP ) Channelsa ATP Channel inhibition

ApnA

Yes

Yes

24–30 애M

14–17 애M

1.7–1.8

1.1–1.6

Operative condition dependent

Yes

Yes

Maintenance of channel activity

Yes

No

IC50 Hill coefficient

a Data from Terzic et al. (1994), Jovanovic and Terzic (1995, 1996), and Jovanovic et al. (1996a).

activity (see Jovanovic et al., 1997). Maintenance of channel activity by ATP can be observed after removal of this nucleotide and is the exclusive property of the Mg2⫹-bound form of ATP (MgATP), not shared by nonhydrolyzable analogues of ATP. Because both Mg2⫹ and ATP hydrolysis are necessary for kinase activity, maintenance of KATP channel activity may relate to an enzyme-dependent action of ATP, such as phosphorylation. When KATP channels are in ‘‘run-down,’’ treatment of membrane patches with MgATP reactivates spontaneous channel activity (see Terzic et al., 1995). In contrast, ApnA do not reverse channel activity after ‘‘rundown’’ (see Jovanovic et al., 1997). Thus, ApnA possess a ‘‘pure’’ inhibitory property on KATP channel opening, without having the ability to maintain channel activity. In this regard, the action of ApnA on KATP channels mostly resembles that observed with nonhydrolyzable analogues of ATP (Table V).

VIII. SUMMARY In prokaryotic cells and some noncardiac tissue, ApnA are signal molecules proposed to act during stress conditions. In the heart, there is evidence in favor of a signaling role for ApnA. Specifically, this class of ‘‘uncommon’’ nucleotides is (1) present in the heart; (2) senses the metabolic state of the myocardium; and (3) induces significant cardiac effects, including the regulation of contraction and excitation, as well as the regulation of cardioprotective mechanisms. Therefore, it seems that ApnA harbor properties of signaling molecules in the heart. However, future studies are required to fully understand and establish the precise role of ApnA in the myocardium. Such studies may thus also provide novel insight into the regulatory mechanism of cardiac function and cardiac response under metabolic stress.

39. Diadenosine Polyphosphate Signaling

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VII. Signaling Systems

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phosphate (Ap4A) in human and animal cardiac preparations. J. Pharmacol. Exp. Ther. 288, 805–813. Vahlensieck, U., Boknik, P., Knapp, J., Linck, B., Muller, F. U., Neumann, J., Herzig, S., Schluter, H., Zidek, W., Deng, M. C., Scheld, H. H., and Schmitz, W. (1996). Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (Ap6A) via A1-adenosine receptor. Br. J. Pharmacol 119, 835–844. Weinmann-Dorch, C., Hedl, A., Grummt, I., Albet, W., Ferdinand, F. J., Friis, R. R., Pierron, G., Moll, W., and Grummt, F. (1984). Drastic rise of intracellular adenosine(5⬘)tetraphospho(5⬘)adenosine correlates with onset of DNA synthesis in eukaryotic cells. Eur. J. Biochem. 138, 179–185. Vartanian, A., Narovlyansky, A., Amchenkova, A., Turpaev, K., and Kisselev, L. L. (1996). Interferons induce accumulation of diadenosine triphosphate (Ap3A) in human cultured cells. FEBS Lett. 381, 32–34. Vartanian, A., Prudovsky, I., Suzuki, H., Dal Pra, I., and Kisselev, L. L. (1997). Opposite effect of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures. FEBS Lett. 415, 160–162. Walker, J., Lewis, T., Pivorun, E. P., and Hilderman, R. H. (1993). Activation of the mouse heart adenosine 5⬘,5⬙⬘-P1,P4-tetraphosphate receptor. Biochemistry 32, 1264–1269. Yakovenko, I. N., and Formazuyk, V. E. (1993). Diadenosine oligophosphates: Metabolic pathways and role in regulation of the functional activity of cells. Biokhimiya 58, 3–24. Zamecnik, P. C., Kim, B., Gao, M., Taylor, G., and Blackburn, G. M. (1992). Analogues of diadenosine 5⬘,5⬙⬘-P1,P4-tetraphosphate (Ap4A) as potential anti-platelet-aggregation agents. Proc. Natl. Acad. Sci. USA 89, 2370–2373.

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40 Cardiac Development and Regulation of Cardiac Transcription FRE´DE´RIC CHARRON*,† AND MONA NEMER*,†,‡ *Laboratory of Cardiac Growth and Differentiation, Montreal Clinical Research Institute; † Department of Medicine, Division of Experimental Medicine, McGill University; and ‡ Department of Pharmacology, University of Montreal, Montreal, Quebec, Canada H2W 1R7

I. INTRODUCTION

paradigm in tissue-specific and pathological control of gene expression, will be presented. Finally, we will discuss the emerging role of cardiac transcription factors in two different types of cardiac pathologies, namely hypertrophic cardiomyopathies and congenital heart diseases.

Cardiovascular malformations are the largest cause of human birth defects. The susceptibility of the heart to congenital malformations reflects the complexity of the morphogenetic events involved in its development. Despite the fact that cardiac malformations have been characterized extensively at the anatomical level, the genetic bases for these abnormalities remain largely unknown. However, remarkable progress has been achieved in the identification of genes involved in heart development. It is very likely that the elucidation of the role of these genes could provide a better understanding of the causes of cardiac abnormalities and help prevent or correct congenital heart malformations. This chapter reviews the various stages of cardiac development along with genes known to be involved in these events. A particular emphasis will be given to genes coding for transcription factors, given the crucial role that these proteins play in the regulation of tissuespecific gene expression and in the control of cell fate determination. Our discussion will integrate and summarize results obtained in various vertebrate model species. Due to space limitations, we will focus primarily on the development of the three layers of the heart, namely the myocardium, endocardium, and the pericardium, together with the generation of the cardiac septa and valves—the development of the cardiac outflow tract and conduction system is beyond the scope of this chapter and we refer the interested reader to two extensive reviews (Creazzo et al., 1998; Moorman et al., 1998). In addition, the combinatorial mechanism underlying cardiac gene regulation, which is emerging as a

Heart Physiology and Pathophysiology, Fourth Edition

II. CARDIAC DEVELOPMENT A. Overview of Cardiac Development Cardiomyocyte precursors originate from mesodermal tissue and initially migrate to generate a straight heart tube composed of an outer myocardial layer and an inner endocardial layer, which are separated by an extracellular matrix-rich layer, the cardiac jelly. This heart tube, already patterned into ventricular and atrial domains, will then loop and undergo complex morphogenetic changes to give rise to the mature multichambered heart. The following sections discuss in detail the morphological and molecular events underlying these processes.

B. Formation of the Heart Field During gastrulation, in vertebrates, cardiac precursor cells from the epiblast invaginate through the primitive streak and migrate anterolaterally to form the anterior lateral plate mesoderm (Fig. 1). This pair of bilaterally symmetrical regions of the embryonic mesoderm, which contains the cardiac precursor cells, is termed the heart field, whereas the two independent bilateral regions are

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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FIGURE 1 Migration of cardiac precursors and generation of the straight heart tube. During gastrulation, cells invaginate through the primitive streak (A) and migrate anterolaterally to form the anterior lateral plate mesoderm (B). The cardiac primordia then fuse at the midline (C) to form the linear heart tube (D). Note that the relative anteroposterior positions of the cardiac precursors in the primitive streak (A) are retained in the heart field (B and C) and in the heart tube (D). hdpc, human days postcoitum; mdpc, mouse days postcoitum; An, anterior; P, posterior; PS, primitive streak; CT, conotruncus; V, ventricle; A, atrium; SV, sinus venosus.

40. Cardiac Development and Regulation

referred to as the cardiac primordia. Subsequently to its formation, the lateral plate mesoderm splits into two layers: the somatic mesoderm, which is composed of skeletal muscle progenitors, and the splanchnic mesoderm, which includes the cardiac precursors for the three layers of the heart tube, namely the myocardium, endocardium, and pericardium (Fig. 2). The commitment of these precursor cells to a cardiac fate results from inductive interactions during gastrulation. A major source of inductive signals is the anterior lateral endoderm, which is in contact with, and appears to migrate along, cardiac precursors. Indeed, endoderm ablation experiments have shown that this embryonic layer is required for heart formation. Moreover, the anterior lateral endoderm is able to induce cardiac differentiation of noncardiac mesoderm explants, suggesting that the anterior endoderm secretes factors that initiate the cardiogenic program in the adjacent mesoderm (Harvey, 1996). Candidates for endodermalderived, cardiac-inducing signals are the fibroblast

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growth factor 4 (FGF4) and the transforming growth factor (TGF)-웁–family member bone morphogenic protein 2 (BMP2), which are both expressed in the anterior endoderm. In the current model, both BMP2 and FGF4 would be required; BMP2 would specify mesodermal cells to the cardiac lineage whereas FGF4 would promote the proliferation and survival of these specified cells (Lough et al., 1996; Schultheiss et al., 1997).

C. Transcriptional Regulation in the Heart Field 1. The NK2 Family Mechanisms by which FGF4 and BMP2 induce cardiogenesis remain unknown. However, studies using the fruit fly Drosophila have begun to reveal genetic pathways controlling cardiogenesis and suggest that these molecular events are highly conserved across species. In Drosophila, the dorsal vessel, a primitive

FIGURE 2 Heart tube formation as seen in transverse sections. As the embryo is folding ventrally, the splanchnic mesoderm (A) differentiates into the primitive epicardium, endocardium, and myocardium (B). Ventral folding leads to the fusion of cardiac tubes (C) into a single tube (D) and also generates the primitive gut.

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heart-like structure contracting rhythmically and pumping hemolymph through an open circulatory system, is analogous to the straight heart tube of the vertebrate embryos. Formation of the dorsal vessel requires the homeodomain transcription factor tinman, which is expressed in the early mesoderm and later in the dorsal vessel (Harvey, 1996). tinman is thought to specify the formation of the dorsal mesoderm, the tissue from which cardiomyocyte precursors originate. Screens for vertebrate tinman homologues led to the identification of the homeodomain transcription factor Nkx2-5 (Table I). Nkx2-5 binds to T(C/T)AAGTG sequences and activates the transcription of cardiac genes, such as atrial natriuretic factor (ANF) and cardiac 움-actin. Nkx2-5 is expressed in the lateral plate mesoderm and is one of the earliest markers of heart field induction. In mice homozygous for a null mutation in Nkx2-5, the heart tube forms but cardiomyocytes do not fully differentiate, as indicated by the downregulation of many cardiac genes, such as genes coding for cardiac contractile protein [ventricular myosin light chain 2 (MLC2V)], cardiac hormones [ANF, B-type natriuretic peptide (BNP)], and cardiac transcription factors (MEF2C, eHAND, and N-myc) (Table II) (Tanaka et al., 1999). However, in contrast to the tinman mutant in drosophila, cardiac mesoderm and cardiomyocytes are still specified. The less drastic effect of the Nkx2-5 mutation in mice compared to the tinman mutation in flies could reflect partial

compensation by other tinman vertebrate homologues, namely Nkx2-3, Nkx2-7, and Nkx2-8, which are expressed in the cardiogenic mesoderm. Indeed, it was shown that the expression of dominant repressor mutants, which can interfere with all NK2 proteins, completely blocks myocardial gene expression and heart formation (Grow and Krieg, 1998; Fu et al., 1998). The exact role of Nkx2-5 or other NK2 proteins in the heart is still unclear. Detailed analysis of a null mutation of Nkx2-5 in mice, as well as an analysis of chimeric mice generated from Nkx2-5 null embryonic stem (ES) cells, suggests that Nkx2-5 is likely required for later stages of myocyte differentiation, such as spatial or asymmetric regionalization (Tanaka et al., 1999). Whether other NK2 proteins are able to substitute or compensate for earlier functions of Nkx2-5 in cardiomyocyte recruitment and/or commitment is presently unknown. Gain-of-function studies in xenopus and manipulations of cardiac induction in chick embryos have shown that (i) Nkx2-5 is able to recruit additional ‘‘permissive’’ cells to the cardiac lineage, thus enlarging the heart field, and (ii) together with transcription factor GATA-4, Nkx2-5 is the nuclear effector of cardiac inductive signals (Durocher and Nemer, 1998). The bulk of these data suggests that Nkx2-5 acts in concert with other factors present in precardiomyocytes to alter the expression of a subset of precardiac cells and affect later stages of differentiation. One such Nkx2-5 collaborator

TABLE I Transcription Factor Families Involved in Cardiovascular Development Family

DNA-binding domain

Binding site

Family members

NK2

Homeodomain

T(C/T)AAGTG

Nkx2–5 Nkx2–3 Nkx2–7 Nkx2–8

Cardiac progenitors Cardiomyocytes

GATA

C4 zinc finger

(A/T)GATA(A/G)

GATA-4

Cardiac progenitors Cardiomyocytes Cardiac progenitors Endocardial cells Cardiac progenitors Cardiomyocytes Vascular smooth muscle cells

GATA-5 GATA-6

HAND

bHLH

CANNTG

dHAND

eHAND

Cardiovascular expression

Cardiac progenitors Cardiomyocytes (right ventricle) Neural crest cells Cardiac progenitors Cardiomyocytes (left ventricle) Neural crest cells

MEF2

MADS

(T/C)TA(A/T)4TA(A/G)

MEF2A MEF2B MEF2C MEF2D

Cardiac progenitors Cardiomyocytes Vascular smooth muscle cells Endothelial cells

NF-AT

Rel-homology domain

GGAAAAT

NF-AT3 NF-ATc

Cardiomyocytes Endocardial cells

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TABLE II Loss of Function Phenotypes and Putative Roles of Cardiac Transcription Factors Target genesa

Gene

Loss of function phenotype

Nkx2–5

Arrest in cardiac development after looping

ANF, BNP, MLC2V, MEF2C, eHAND, N-myc, Msx2, CARP

Cardiomyocyte differentiation Heart tube regionalization

GATA-4

Cardia bifida (no fusion of the cardiac primordia)

ANF, BNP, 움-MHC, 웁-MHC, cTnl, PDGFR웁

Cardiomyocyte differentiation Cardiac primordia fusion Maintenance of the cardiac phenotype Hypertrophic response

GATA-6

Extraembryonic endoderm defects

GATA-4, ANF, BNP, 움-MHC, 웁-MHC, cTnI, PDGFR웁

Extraembryonic endoderm differentiation Cardiomyocyte progenitor proliferation

MEF2C

Arrest in cardiac development at the looping stage (absence of the right ventricle) Vascular and endocardial defects

ANF, Cardiac 움-actin, 움-MHC, MLC1A, dHAND, angiopoietin-1, VEGF

Cardiomyocyte differentiation (late stage) Endocard development

dHAND

Arrest in cardiac development at the looping stage (absence of the right ventricle) Neural crest defects

Ufd1

Heart tube regionalization Regulation of gene(s) involved in neural crest development

eHAND

Placentation defects Arrest in cardiac development at the looping stage

?

?

NF-ATc

Valve formation and heart septation defects

?

?

a

Putative roles

Target genes listed are those identified by loss-of-function studies.

is GATA-4, which interacts physically with Nkx2-5 and cooperatively enhances transcription of Nkx2-5 target genes (see later). 2. The GATA Family Members of the GATA family of transcription factors are zinc finger proteins that bind specifically to (A/T)GATA(A/G) DNA sequences (Charron and Nemer, 1999). The founding member of this family, GATA-1, as well as GATA-2 and GATA-3, is largely restricted to the hematopoietic lineage, and targeted disruption of their genes has revealed an essential nonredundant function for each of these factors in hematopoiesis. Analysis of cardiac-specific promoters led to the cloning of an additional member of the GATA family, GATA-4, whose expression is mainly restricted to the heart and gonads (Gre´pin et al., 1994). GATA-4 can be detected in the bilateral cardiac primordia and, together with Nkx2-5, constitutes the earliest markers of heart field induction. Later, GATA-4 transcripts and proteins are detected throughout the myocardium and endocardium and persist at all stages of heart development. Transfection studies in noncardiac cells established that GATA-4 is a potent transactivator of numerous cardiac promoters. Moreover, ectopic expression of GATA-4

in vivo was shown to activate the transcription of cardiac contractile genes (Jiang and Evans, 1996). These properties are consistent with a key role for GATA-4 in cardiac transcription. The first evidence for a role of GATA-4 in heart differentiation came from loss-of-function studies in the pluripotent P19 embryonic carcinoma cell line, which provides a model of inducible cardiac differentiation. P19 cells expressing GATA-4 antisense transcripts were unable to achieve terminal cardiac differentiation and massive apoptosis of precardiac cells was observed; conversely, overexpression of GATA-4 in P19 cells markedly potentiated cardiomyocyte differentiation (Gre´pin et al., 1997). Loss-of-function studies in postnatal cardiomyocytes revealed that GATA-4 is essential for the expression of many cardiac genes and maintenance of the cardiac phenotype (Charron et al., 1999). Consistent with an important role for GATA-4 in the heart, mice homozygous for a null mutation in the GATA-4 gene are not viable due to the inability of the bilateral cardiac primordia to fuse and form the heart tube (Kuo et al., 1997; Molkentin et al., 1997). Nevertheless, cardiomyocytes expressing differentiation markers are detectable in these mice, raising the possibility that other factors, including other members of the GATA family, may compensate, at least in part, for GATA-4 deficiency

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during early cardiac development. Two other GATA factors, GATA-5 and GATA-6, which are expressed predominantly in the heart and gut, have been identified by low stringency hybridization to a cDNA probe corresponding to the mouse GATA-1 DNA-binding domain. GATA-4, GATA-5, and GATA-6 are homologous in their amino acid sequence, particularly in the DNAbinding domain, and form a distinct subclass of GATA factors. Within the heart, the three GATA factors are regulated differentially throughout development, with GATA-4 being the predominant transcript in cardiomyocytes at all stages. GATA-6 is also expressed in the precardiac mesoderm and is later found in myocardial and also in vascular smooth muscle cells. GATA-5 transcripts are largely restricted to endocardial cells. Interestingly, the expression of GATA-6 is highly upregulated in GATA-4-deficient mice. Two lines of evidence indicate that both GATA-5 and GATA-6 can indeed substitute for some of the GATA-4 functions in the heart. First, all three factors are potent activators of GATA-dependent cardiac promoters (Durocher et al., 1997), suggesting that, if required, they could compensate for each other with respect to the transcription of several cardiac genes. Second, ectopic expression of GATA-4, GATA-5, or GATA-6 was equally efficient at activating the transcription of cardiac contractile genes in vivo (Jiang and Evans, 1996). However, other data suggest that each GATA factor fulfills a unique role(s) in heart development. Injection of GATA-6, but not GATA-4, mRNA in xenopus embryos resulted in enlargement of the cardiogenic field and suggested that GATA-6 might regulate the proliferation of cardiac progenitor cells (Gove et al., 1997). Unfortunately, inactivation of the GATA-6 gene revealed a requirement for GATA-6 in the formation of the extraembryonic endoderm and resulted in very early embryonic lethality, precluding analysis of GATA-6 function in heart development (Morrisey et al., 1998). However, loss-offunction studies in an in vitro culture model revealed an essential role for GATA-5 in endocardial differentiation that could not be compensated by GATA-4 or GATA6 (Nemer et al., unpublished data). Taken together, these results are consistent with critical roles for GATA factors in the differentiation and proliferation of cardiogenic cells.

D. Formation of the Heart Tube Commitment of splanchnic mesoderm cells to the cardiogenic lineage is followed by their bilateral migration to form the two cardiac primordia, which in turn will migrate and fuse at the ventral midline to form the primitive cardiac tube (Fig. 2). The molecular mechanisms underlying these very early morphogenetic events

are not well understood, although they are linked temporally to the ventral closure of the embryo. Saturation mutagenesis in zebrafish has yielded mutant animals in which the cardiac primordia do not fuse (a condition known as cardia bifida), but the genetic loci responsible for these cardiac defects are still unidentified. The only gene known to be required for fusion of the cardiac primordia is GATA-4, as in mice homozygous for a null mutation in GATA-4, the two cardiac primordia do not migrate ventrally and fail to form the cardiac tube (Kuo et al., 1997; Molkentin et al., 1997). Experiments using chimeric expression of GATA-4 in the endoderm showed that GATA-4 is necessary and sufficient to rescue the cardia bifida phenotype, suggesting that GATA-4 plays an essential role in the early cross talk between endodermal and mesodermal layers, which is critical for proper cardiac development.

E. Heart Tube Regionalization and Looping As it is forming, the heart tube begins to contract. The first contractions are peristaltic and subsequently become sequential. Along with the functional maturation of the heart, specialization of the contractile protein machinery occurs and chamber-specific contractile genes are detected well before morphological evidence of chamber demarcation. The beating heart tube is organized with an anteroposterior polarity, where the anterior region will become the outflow region of the mature heart and the posterior region will become the inflow region (Fig. 3). Thus, specific segments of the heart tube are already fated to become, from anterior to posterior, the aortic sac, the conotruncus, the right ventricle, the left ventricle, the atria, and the sinus venosus of the mature heart. Mechanisms controlling heart tube regionalization are poorly understood, and the timing of atrial and ventricular specification is presently controversial; although fate-mapping studies suggest that atrial and ventricular lineages are specified and separated during gastrulation, work in chicken and zebrafish suggest that the fate of cardiac progenitors can be altered—by retinoic acid treatment, for example—within a specific window of time, raising the possibility that lineage commitment occurs later on (Stainier et al., 1993; Yutzey and Bader, 1995). A homeobox gene, Irx4, has been identified and shown to be expressed only in ventricular myocytes at all stages, making it the only cardiac gene with chamber-specific expression throughout development. Gain and loss of function in chick embryos suggest that Irx4 serves to impose a ventricular phenotype over a default atrial pathway (Bao et al., 1999). As it grows, the linear heart tube undergoes rightward looping, which will ultimately bring the already fated regions of the linear heart tube into their

40. Cardiac Development and Regulation

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FIGURE 3 Heart tube looping and cardiac morphogenesis. The beating straight heart tube (A) undergoes rightward looping (B and C). By 25 hdpc (D), the atrium and the ventricle are morphologically different and the ventricle is beginning to thicken by growth of the wall and addition of trabeculae. The cardiac jelly separates the myocardium from the endocardium and becomes thicker in the outflow tract and the AV regions, where cushions and valves will form. From the AV cushions and the interventricular and interatrial septa, the atrium and the ventricle divide into right and left chambers (E). (A–D) The developing heart from the left side and (E) a frontal view.

mature relative positions. At this stage, the atrial and ventricular chambers start becoming morphologically identifiable. Subsequent growth and maturation of individual chambers result in the mature heart. Which extrinsic or intrinsic factor drives cardiac looping remains to be elucidated. The process clearly depends on a number of concurrent events, which include proper lineage differentiation and differential myocyte proliferation as well as asymmetric signaling. Laterality signals governing asymmetric heart looping probably originate from the asymmetric process of egg fertilization (Harvey, 1998). A few cell divisions later, the TGF-웁 family member Vg1 seems to coordinate the elaboration of the left/right axis signaling pathway. According to the current model, Vg1 would upregulate the expression of a member of the hedgehog family, Sonic hedgehog (Shh), specifically on the left side of the embryo. Via its receptor, Patched (Ptc), Shh

would induce the expression of the TGF-웁 family members nodal and lefty-2, which are expressed in the caudal region of the forming heart tube, where the first cardiac asymmetries are seen. The homeodomain transcription factor Pitx2 lies downstream of nodal and lefty-2 and is expressed along the left side of the forming heart tube, with expression persisting in the left side of the atria and ventricles during looping. Remarkably, expression of Pitx2 in right-sided cardiac primordia induces bilaterally symmetrical hearts. These results suggest that the asymmetric signaling cascade regulating heart morphogenesis converges on Pitx2, the currently most downstream gene involved in the laterality pathway. The timing of heart looping also coincides with initiation of the embryonic circulation, which can also interfere with rotational movements directly or as a result of alterations in myocyte growth and/or proliferation.

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Gene inactivation studies have linked two other families of transcription factors to looping phenotypes. 1. The HAND Family eHAND and dHAND are two transcription factors of the basic helix-loop-helix (bHLH) family expressed in the precardiac mesoderm (Srivastava, 1999). Later, dHAND is expressed throughout the straight heart tube and becomes restricted predominantly to the future right ventricle during looping. In contrast, eHAND expression is restricted to the anterior and posterior regions of the heart tube, which are fated to become the conotruncus and left ventricle, respectively. Finally, in the mature heart, dHAND and eHAND display complementary expression in the right and left ventricles, respectively. Inactivation of the dHAND gene in mice results in embryonic lethality at the looping stage. Interestingly, these mice fail to develop the segment of the heart tube that will form the right ventricle, consistent with the predominant expression of dHAND in this segment. Inactivation of the eHAND gene results in early embryonic lethality due to placentation defects, making analysis of its role in cardiac morphogenesis ambiguous. Nevertheless, chimeric analysis (that rescue the placentation defect) suggests that, like dHAND, eHAND is also required to ensure proper cardiac looping, but not cardiomyocyte differentiation. However, it is not clear whether these looping defects are due to an intrinsic effect of the HAND genes on cardiomyocytes or are due to defects of other structures essential for proper cardiac development, such as the neural crest, which is defective in dHAND null mice. 2. The MEF2 Family A second family of transcription factors has also been linked to heart looping. Myocyte enhancer binding factor 2 (MEF2) family members, MEF2A, MEF2B, MEF2C, and MEF2D, belong to the MADS [MCM1, agamous, deficiens, and serum response factor (SRF)] box family of transcription factors (Black and Olson, 1998) and are enriched in striated myocytes. MEF2 factors activate transcription through a conserved AT-rich motif found in the promoter of many skeletal and cardiac muscle genes. MEF2C and MEF2B are the earliest members expressed in the heart field, where their transcripts are detected shortly after GATA-4 and Nkx2-5. Later, MEF2A and MEF2D transcripts are also found in the developing heart. In Drosophila, the single MEF2 gene is not required for the initial specification of cardiac cells as the dorsal vessel forms normally in MEF2 mu-

tants. However, terminal muscle differentiation is not achieved, as evidenced by the lack of contractile gene expression in both cardiac and skeletal myocytes. This phenotype, together with the finding that the MEF2 gene is a downstream transcriptional target of tinman, indicates that MEF2 controls late stages of cardiomyocyte differentiation. Consistent with the just-described conclusion, null mutation of the MEF2C gene in mice results in developmental arrest at the looping stage (Black and Olson, 1998). These mice exhibit complex vascular malformations that impair blood circulation and embryonic growth. Also notable are defects in endocardial development likely due to the reduced myocardial expression of angiopoietin-1 and vascular endothelial growth factor (VEGF) (Bi et al., 1999). Other cardiac differentiation markers, including cardiac 움-actin, 움-MHC, ANF, and myosin light-chain 1A (MLC1A), were also downregulated, indicating that MEF2C is essential for the transcription of a subset of myocyte genes and that proper myocyte differentiation is essential for endocardial development. Again, it is not clear whether the arrest at the looping stage is due to a direct effect of the MEF2C mutation on cardiomyocytes or due to defects in vascular development.

F. Endocardium Development, Valve Formation, and Heart Septation The endocardium, a cellular layer lining the interior of the heart, has a developmental origin distinct from the vascular endothelium. During development, endocardial progenitors are localized at the periphery of the cardiogenic field and they become surrounded by the two myocardial layers of the linear tube as the two cardiac primordia fuse (Fig. 2). The cellular and molecular mechanisms of endocardial differentiation are unclear, and the transcription factors involved in this process are largely undefined. In the heart, two transcription factors, NFATc and GATA-5, are restricted to endocardial cells. As described later, gene inactivation studies have implicated NFATc in valve formation, a specialized function of the endocard during development. The role of GATA-5 in endocardial cells is not established yet; however, GATA-5 preferentially transactivates the promoter of endocard-specific genes, such as endothelin-1 (Nemer et al., 1999). Given the role of other GATA factors in lineage differentiation, it is tempting to speculate on a putative regulatory role for GATA-5 in endocardial cell differentiation. The endocardium is essential for the generation of heart valves and membranous septa. Cardiac cushions, which consist of localized swellings in the cardiac jelly,

40. Cardiac Development and Regulation

are the primordia for the valves and septa. During development, endocardial cells undergo an epithelial to mesenchymal transformation while migrating to the cushions and proliferating there. Migration of the endocardial cells occurs in response to secreted molecules. Many evidence support a role for the TGF-웁 family members TGF-웁1, TGF-웁2, and TGF-웁3 in this process. Consistent with a role for the TGF-웁 signaling pathway in the migration of endocardial cells to cushions, it was shown that the type III TGF-웁 receptor is essential for endocardial cell transformation and migration (Brown et al., 1999). Atrioventricular (AV) canal cushions, which appear as a constriction between ventricular and atrial chambers of the looping heart, expand and fuse in the midline to partition the AV canal into right and left sides. The orifices will ultimately become the tricuspid and mitral valves that connect the septated atrial and ventricular chambers. The formation of the outflow tract valves contains many similarities to that of the AV canal formation; however, in this case, they arise from outflow tract cushions and further develop into the aortic and pulmonary valves. Interatrial and interventricular septa, which divide the atria and the ventricle into right and left chambers, have multiple origins. A cardiac muscle wall is first elaborated between the left and the right sides of the atria and ventricles. Then, portions of interatrial and interventricular septa arise from growth of the endocardial cushions toward the anterior and posterior directions of the looped heart, respectively. Many transcription factors have been shown to be involved in septation and myocardial growth. Mice homozygous for a null mutation in genes encoding the retinoic acid X receptor 움 (RXR움), N-myc, TEF1, Wilms tumor (WT1), and neurofibromatosis (NF1) display ventricular septal defects and ventricular wall hypoplasia (Rossant, 1996). However, in most cases, the precise role of these transcription factors in cardiac development and their transcriptional targets remain to be determined.

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NF-AT family members bind DNA rather weakly on their own and require interaction with other transcription factors for high DNA-binding affinity. Cyclosporin, a calcineurin inhibitor, prevents nuclear translocation of NF-AT proteins and thus transcriptional activation by these factors. During early development, NF-ATc expression is restricted to the endocardium, with higher expression in the endocardium of the AV canal and the outflow tract (Nolan, 1998). Interestingly, inactivation of the NF-ATc gene in mice completely prevents formation of the pulmonary and aortic valves. Moreover, the interventricular septum and the tricuspid and mitral valves are defective in these mice. Remarkably, these defects correlate with regions that express higher levels of NF-ATc. Given that another member of the NF-AT family, NF-AT3, is known to interact with the GATA family members GATA-4, GATA-5, and GATA-6 (see later), it is tempting to speculate that the only two known endocardium-specific transcription factors, NF-ATc and GATA-5, may cooperate in endocardial differentiation and/or valve formation.

G. Pericardium Development The pericardium is the outer layer of the heart and contributes to form the vascular and connective tissues within the heart. The pericardium originates from a population of cells near the sinus venosus that migrates in a posteroanterior fashion to cover the developing heart and penetrates into the ventricular chamber walls to form the vessels of the coronary arteries. The epicardial 움4-integrin and the myocardial VCAM-1 cell adhesion molecules are both required for adhesion of the pericardium to the myocardium (Rossant, 1996). Transcription factors expressed and/or involved in the differentiation of the pericardium remain unknown. Analysis of the promoter regulatory elements specifying the epicardial expression of the 움4-integrin gene could give insights into the mechanisms involved in epicardial cell development.

1. The NF-AT Family NF-ATc, a member of the NF-AT (nuclear factor of activated T cells) transcription factor family, has been shown to play a crucial role in valvular and septal development. NF-AT proteins belong to the Rel family of transcription factors, which also includes NF-␬B (Rao et al., 1997). These factors are constitutively localized to the cytoplasm, but upon Ca2⫹-dependent activation of the phosphatase calcineurin, NF-AT proteins are dephosphorylated and shuttled to the nucleus where they bind DNA and modulate gene transcription. However,

III. REGULATION OF CARDIAC GENE EXPRESSION: COMBINATORIAL MECHANISMS A. Combinatorial Interaction between GATA-4 and Nkx2-5 The establishment and maintenance of the cardiac phenotype require the activation of cardiac-specific genes in a tightly regulated temporal and spatial manner. An emerging theme in the tissue-specific transcrip-

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tional regulation of gene expression is that this process is governed by the combinatorial action of cell-restricted as well as ubiquitous transcriptional regulators (Charron and Nemer, 1999). As stated previously, studies in P19 cells indicated that overexpression of GATA-4 potentiates cardiogenesis only in ‘‘permissive’’ cellular environments, suggesting that GATA-4 action requires a cofactor. In many species, the expression pattern of GATA-4 overlaps with that of Nkx2-5 in the heartforming region. Interestingly, ectopic expression of Nkx2-5 results in enhanced myocyte recruitment, but is not sufficient to initiate cardiac gene expression or differentiation, suggesting that Nkx2-5 acts in concert with other transcription factors to specify the cardiac phenotype. The fact that GATA-4 and Nkx2-5, two of the earliest markers of precardiac cells, are essential for heart formation and that overexpression of either alone cannot initiate cardiogenesis, yet enhances recruitment and/or differentiation of committed precursors, raised the possibility that these proteins may be mutual cofactors. Because ANF was the only transcriptional target for both GATA-4 and Nkx2-5, it provided a useful tool to investigate potential functional cooperation between GATA-4 and Nkx2-5. Indeed, it was shown that GATA-4 and Nkx2-5 are mutual cofactors, as coexpression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter (Durocher et al., 1997). This molecular interaction may provide cooperative cross talk between two pathways that are critical for the early events of cardiogenesis.

B. The GATA-4/Nkx2-5 Interaction: A Mediator of BMP Signaling Because GATA-4 and Nkx2-5 are the earliest markers of myocardial cell fate, the identification of the upstream regulators of GATA-4 and Nkx2-5 in the precardiac mesoderm should give important insights on the nature of the inducers of cardiac fate. Interestingly, BMP2, which is able to specify mesodermal cells to the cardiac lineage, also induces Nkx2-5 and GATA-4 expression in anterior lateral mesoderm (Schultheiss et al., 1997). Thus, it is possible that Nkx2-5 and GATA-4 are downstream effectors of BMP2 and that the functional interaction between these two factors would be required for cardiomyocyte differentiation. This hypothesis is consistent with the gain-of-function studies that demonstrated that neither GATA-4 nor Nkx2-5 could alone initiate cardiogenesis, although either protein could potentiate it in committed cells. This functional interaction, which potentiates the transcriptional activities of both proteins, would be especially important at low concentrations of GATA-4 and

Nkx2-5, a situation that likely occurs in the early moments of cardiac cell fate induction.

C. Heterotypic Interactions between GATA-4 and GATA-6 and the Maintenance of Cardiac Gene Expression in the Postnatal Heart Because mice lacking GATA-4 or GATA-6 die prior to formation of the primitive heart tube, they are not useful for assessing the role of GATA-4 and GATA-6 in postnatal heart development. Thus, an adenovirusmediated antisense strategy that specifically inhibits GATA-4 or GATA-6 protein production in cardiomyocytes was developed and used to assess the role of these factors in postnatal cardiomyocytes (Charron et al., 1999). Results indicate that several endogenous cardiac genes, including ANF, BNP, cardiac troponin I (cTnI), 움-myosin heavy chain (움-MHC), 웁-myosin heavy chain (웁-MHC), and platelet-derived growth factor receptor 웁 (PDGFR웁), are downregulated in cardiomyocytes lacking either GATA-4 or GATA-6, suggesting that these genes are transcriptional targets for both GATA-4 and GATA-6. Interestingly, the promoter of all these genes contains at least one GATA element, suggesting that they are direct downstream targets for GATA-4 and GATA-6 in postnatal cardiomyocytes. Moreover, this approach revealed that a subset of genes is targeted by both GATA-4 and GATA-6; remarkably, removal of both GATA proteins had the same effect as removing either one by itself, suggesting that GATA-4 and GATA-6 might be part of the same active transcription complex. Indeed, experiments using ANF and BNP promoters revealed that GATA-4 and GATA-6 form a heterotypic complex that binds a single GATA element and synergistically activates the transcription of cardiac promoters. Taken together, these results suggest that GATA-4 and GATA-6 can act in concert to regulate the expression of cardiac genes.

IV. TRANSCRIPTION FACTORS INVOLVED IN HYPERTROPHIC CARDIAC GROWTH A. The Calcineurin/NF-AT Pathway Soon after birth, cardiomyocytes lose their ability to proliferate and respond to growth stimulation by increasing their size, but not their number, a process known as cardiac hypertrophy. This process is characterized by the reinduction of a fetal genetic program, where many genes that where downregulated postnatally are reinduced. In vitro, many hypertrophic stimuli, such as endothelin-1, angiotensin II, the 움-adrenergic agonist

40. Cardiac Development and Regulation

phenylephrine, stretch, and others, are able to reinduce the fetal genetic program in isolated cardiomyocytes. However, the nuclear events that they trigger to genetically reprogram the heart and establish cardiomyocyte hypertrophy are still unclear. Pharmacologic manipulations have implicated many signal transduction pathways in cardiomyocyte hypertrophy, however, the transcriptional downstream effectors of most of them have not yet been identified clearly (for more details, see Chapter 58). One exception to this is the calcineurin/ NF-AT signaling pathway, where it was shown that NF-AT3, a downstream effector of calcineurin (discussed earlier), is able to interact and cooperate with GATA-4 to activate cardiac gene expression in a Ca2⫹-dependent manner (Walsh, 1999). Moreover, overexpression of an activated calcineurin or its effector NF-AT3 has been shown to induce cardiac hypertrophy in transgenic mice. Of note, in this model, cardiac hypertrophy induced by activated calcineurin was inhibited by cyclosporin. Cyclosporin was also shown to prevent the development of cardiac hypertrophy in transgenic mice harboring cardiac-specific overexpression of a variety of mutant sarcomeric proteins. However, cyclosporin is not able to inhibit the induction of hypertrophy in conventional genetic (spontaneously hypertensive rats; SHR) and hemodynamic overload (aortic banding) rodent models of cardiac hypertrophy. These results raise the question of whether the calcineurin/NF-AT signaling pathway is involved in only a subset of cardiac hypertrophy.

B. GATA Factors and Hypertrophic Development In addition to its functional and physical interaction with the transcription factor NF-AT3, which mediates calcineurin-dependent cardiac hypertrophy, other lines of evidence are consistent with a role for GATA factors in cardiac hypertrophy. For example, using direct injection of DNA into the myocardium, three groups have found that GATA elements present on the promoters of the angiotensin type 1A receptor (AT1AR), 웁-MHC, and BNP genes are required for the activation of these promoters in response to pressure or volume overload (Herzig et al., 1997; Hasegawa et al., 1997; Marttila et al., 2000). Moreover, analysis of genetic and experimental models of hypertrophy, SHR and one kidney one clip rat models, revealed increased GATA-4 transcripts in association with ventricular hypertrophy (Nemer et al., unpublished data). Thus, GATA-4 may play a role in the genetic reprogramming of the hypertrophied heart either through increased expression of the protein or through recruitment of other cofactors, such as NF-AT3.

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V. TRANSCRIPTION FACTORS AND CONGENITAL HEART DISEASES (CHD) The most common types of CHD are those related to incomplete septation of the atria, ventricles, or AV canal. Mutations in the genes coding for two transcription factors, Tbx5 and Nkx2-5, have been identified in families with a high incidence of atrial or ventricular septal defects. Dominant mutations of the Tbx5 gene, a T-box transcription factor, cause the Holt–Oram syndrome, which is characterized by a developmental disorder affecting the heart and the upper limbs (Li et al., 1997; Basson et al., 1997). The most frequent cardiac abnormalities found in these patients are atrial and/or ventricular septal defects and conduction defects. These abnormalities correlate with the expression of Tbx5 in the heart and limbs during development. Most of the mutations identified in the Tbx5 gene result in premature stop codons, preventing synthesis of the fulllength protein. Dominant mutations in the Nkx2-5 gene were shown to be linked to septal defects and AV conduction abnormalities in four different families (Schott et al., 1998). The three mutations identified are predicted to affect DNA binding and two of them result in truncated proteins. AV canal, atrial, and septal defects are also present in 60% of patients with deletions or inverted duplications of chromosome 8p23.1. Haploinsufficiency of the GATA-4 gene has been reported in most patients with CHD associated with 8p23.1 monosomy (Pehlivan et al., 1999). The dHAND transcription factor may also be involved indirectly in a different type of CHD, the DiGeorge syndrome. The most common cardiac defects presented by DiGeorge syndrome patients are conotruncal defects (persistent truncus arteriosus) and interruption of the aortic arch, which are both due to neural crest defects. More than 80% of affected individuals have microdeletions of chromosome 22q11, suggesting that one or more genes regulating neural crest cells may map to this region. Accordingly, many efforts have been directed at identifying the gene(s) localized in that region that could be responsible for the DiGeorge syndrome, without success. However, based on the idea that dHAND is required for the survival of cells of neural crest-derived branchial and aortic arch arteries, but does not itself map to 22q11, it was hypothesized that dHAND could regulate the expression of a gene involved in the DiGeorge syndrome and mapping to chromosome 22q11. Indeed, a screen for dHAND targets led to the cloning of the Ufd1 gene, a mouse homologue of a yeast gene involved in the degradation of ubiquitinated proteins and which maps to 22q11

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(Yamagishi et al., 1999). Interestingly, Ufd1 is expressed in virtually all tissues affected in the DiGeorge syndrome, making it a good gene candidate for the DiGeorge syndrome.

Acknowledgment The authors acknowledge the dedicated efforts of all members of the Nemer laboratory whose results were cited here.

Bibliography

VI. SUMMARY Remarkable progress has been achieved in the identification of genes involved in heart development. NK2 and GATA transcription factor families, and more specifically Nkx2-5 and GATA-4, appear to play critical roles in heart field generation and cardiomyocyte differentiation. Later, fusion of the two cardiac primordia at the ventral midline to form the heart tube requires GATA-4. Although two transcription factor families, the HAND and the MEF2 families, have been linked to heart tube looping, it is not known whether they exert a direct intrinsic effect on cardiomyocytes or whether the developmental arrest observed at the looping stage in mice deficient for these genes is due to a defect in another tissue essential for proper heart development. However, a member of the homeodomain transcription factor family, Pitx2, has been shown to regulate asymmetric heart morphogenesis directly, although its role in cardiomyocytes remains undefined. Shortly after the initiation of cardiac looping, the cardiac valves and septa, which originate from endocardial cells, begin to form. NF-ATc, a member of the Rel family of transcription factors, which is specifically expressed in endocardial cells, is essential for valve formation and heart septation. However, genes involved in the development of the two other layers of the heart, the endocard and the pericard, are still unknown. The combinatorial regulation of gene expression is clearly emerging as a paradigm for cardiac-specific gene expression under normal and pathological cardiac conditions. The best characterized example of cardiac combinatorial gene regulation is the functional and physical interaction of Nkx2-5 and GATA-4, which could be important in mediating the cardiac-inducing activity of BMP2. In some pathological cardiac conditions, the calcineurin/NF-AT pathway appears to be involved in the regulation of cardiac gene expression; its effects may be exerted via interaction with GATA-4. Other lines of evidence are also consistent with a role for GATA factors in cardiac hypertrophy. Finally, the identification of mutations or haploinsufficiency in the transcription factors Nkx2-5, Tbx5, or GATA-4 in patients with congenital heart defects suggests that further investigation of the role of these transcription factors could benefit our understanding of the causes of cardiac abnormalities greatly.

Bao, Z. Z., Bruneau, B. G., Seidman, J. G., Seidman, C. E., and Cepko, C. L. (1999). Regulation of chamber-specific gene expression in the developing heart by Irx4. Science 283, 1161–1164. Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T. A., LeblancStraceski, J., Renault, B., Kucherlapati, R., Seidman, J. G., and Seidman, C. E. (1997). Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nature Genet. 15, 30–35. Bi, W., Drake, C. J., and Schwarz, J. J. (1999). The transcription factor MEF2C-null mouse exhibits complex vascular malformations and reduced cardiac expression of angiopoietin 1 and VEGF. Dev. Biol. 211, 255–267. Black, B. L., and Olson, E. N. (1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14, 167–196. Brown, C. B., Boyer, A. S., Runyan, R. B., and Barnett, J. V. (1999). Requirement of type III TGF-beta receptor for endocardial cell transformation in the heart. Science 283, 2080–2082. Charron, F., and Nemer, M. (1999). GATA transcription factors and cardiac development. Sem. Cell Dev. Biol. 10, 85–91. Charron, F., Paradis, P., Bronchain, O., Nemer, G., and Nemer, M. (1999). Cooperative interaction between GATA-4 and GATA-6 regulates myocardial gene expression. Mol. Cell. Biol. 19, 4355– 4365. Creazzo, T. L., Godt, R. E., Leatherbury, L., Conway, S. J., and Kirby, M. L. (1998). Role of cardiac neural crest cells in cardiovascular development. Annu. Rev. Physiol. 60, 267–286. Durocher, D., Charron, F., Warren, R., Schwartz, R. J., and Nemer, M. (1997). The cardiac transcription factors Nkx2-5 and GATA4 are mutual cofactors. EMBO J. 16, 5687–5696. Durocher, D., and Nemer, M. (1998). Combinatorial interactions regulating cardiac transcription. Dev. Genet. 22, 250–262. Fu, Y., Yan, W., Mohun, T. J., and Evans, S. M. (1998). Vertebrate tinman homologues XNkx2-3 and XNkx2-5 are required for heart formation in a functionally redundant manner. Development 125, 4439–4449. Gove, C., Walmsley, M., Nijjar, S., Bertwistle, D., Guille, M., Partington, G., Bomford, A., and Patient, R. (1997). Over-expression of GATA-6 in Xenopus embryos blocks differentiation of heart precursors. EMBO J. 16, 355–368. Gre´pin, C., Dagnino, L., Robitaille, L., Haberstroh, L., Antakly, T., and Nemer, M. (1994). A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription. Mol. Cell. Biol. 14, 3115–3129. Gre´pin, C., Nemer, G., and Nemer, M. (1997). Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor. Development 124, 2387–2395. Grow, M. W., and Krieg, P. A. (1998). Tinman function is essential for vertebrate heart development: Elimination of cardiac differentiation by dominant inhibitory mutants of the tinman-related genes, XNkx2-3 and XNkx2-5. Dev. Biol. 204, 187–196. Harvey, R. P. (1996). NK-2 homeobox genes and heart development. Dev. Biol. 178, 203–216. Harvey, R. P. (1998). Links in the left/right axial pathway. Cell 94, 273–276.

40. Cardiac Development and Regulation Hasegawa, K., Lee, S. J., Jobe, S. M., Markham, B. E., and Kitsis, R. N. (1997). cis-Acting sequences that mediate induction of betamyosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction. Circulation 96, 3943–3953. Herzig, T. C., Jobe, S. M., Aoki, H., Molkentin, J. D., Cowley, A. W., Jr., Izumo, S., and Markham, B. E. (1997). Angiotensin II type1a receptor gene expression in the heart: AP- 1 and GATA-4 participate in the response to pressure overload. Proc. Natl. Acad. Sci. USA 94, 7543–7548. Jiang, Y. M., and Evans, T. (1996). The Xenopus GATA-4/5/6 genes are associated with cardiac specification and can regulate cardiacspecific transcription during embryogenesis. Dev. Biol. 174, 258–270. Kuo, C. T., Morrisey, E. E., Anandappa, R., Sigrist, K., Lu, M. M., Parmacek, M. S., Soudais, C., and Leiden, J. M. (1997). GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 11, 1048–1060. Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H., Gebuhr, T., Bullen, P. J., Robson, S. C., Strachan, T., Bonnet, D., Lyonnet, S., Young, I. D., Raeburn, J. A., Buckler, A. J., Law, D. J., and Brook, J. D. (1997). Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nature Genet. 15, 21–29. Lough, J., Barron, M., Brogley, M., Sugi, Y., Bolender, D. L., and Zhu, X. (1996). Combined bmp-2 and fgf-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm. Dev. Biol. 178, 198–202. Marttila, M., Hautala, N., Toth, M., Vuolteenaho, O., Nemer, M., and Ruskoaho, H. (2000). Activation of the B-type natriuretic peptide gene expression in response to hemodynamic stress. Submitted for publication. Molkentin, J. D., Lin, Q., Duncan, S. A., and Olson, E. N. (1997). Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 11, 1061–1072. Moorman, A. F., de Jong, F., Denyn, M. M., and Lamers, W. H. (1998). Development of the cardiac conduction system. Circ. Res. 82, 629–644. Morrisey, E. E., Tang, Z., Sigrist, K., Lu, M. M., Jiang, F., Ip, H. S., and Parmacek, M. S. (1998). GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev. 12, 3579–3590.

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Nemer, G., Qureshi, S. A., Malo, D., and Nemer, M. (1999). Functional analysis and chromosomal mapping of GATA5, a gene encoding a zinc finger DNA-binding protein. Mam. Genome 10, 993–999. Nolan, G. P. (1998). Transcription and the broken heart. Nature 392, 129–130. Pehlivan, T., Pober, B. R., Brueckner, M., Garrett, S., Slaugh, R., Van Rheeden, R., Wilson, D. B., Watson, M. S., and Hing, A. V. (1999). GATA4 haploinsufficiency in patients with interstitial deletion of chromosome region 8p23.1 and congenital heart disease. Am. J. Med. Genet. 83, 201–206. Rao, A., Luo, C., and Hogan, P. G. (1997). Transcription factors of the NFAT family: Regulation and function. Annu. Rev. Immunol. 15, 707–747. Rossant, J. (1996). Mouse mutants and cardiac development: New molecular insights into cardiogenesis. Circ. Res. 78, 349–353. Schott, J. J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E., and Seidman, J. G. (1998). Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108–111. Schultheiss, T. M., Burch, J. B., and Lassar, A. B. (1997). A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11, 451–462. Srivastava, D. (1999). HAND proteins: Molecular mediators of cardiac development and congenital heart disease. Trends Cardiovasc. Med. 9, 11–18. Stainier, D. Y. R., Lee, R. K., and Fishman, M. C. (1993). Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development 119, 31–40. Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, N., and Izumo, S. (1999). The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development 126, 1269–1280. Walsh, R. A. (1999). Calcineurin inhibition as therapy for cardiac hypertrophy and heart failure: Requiescat in pace? Circ. Res. 84, 741–743. Yamagishi, H., Garg, V., Matsuoka, R., Thomas, T., and Srivastava, D. (1999). A molecular pathway revealing a genetic basis for human cardiac and craniofacial defects. Science 283, 1158–1161. Yutzey, K. E., and Bader, D. (1995). Diversification of cardiomyogenic cell lineages during early heart development. Circ. Res. 77, 216–219.

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41 Developmental Changes of Ion Channels HISASHI YOKOSHIKI

NORITSUGU TOHSE

Department of Cardiovascular Medicine Hokkaido University Graduate School of Medicine Sapporo 060-8638, Japan

Department of Physiology Sapporo Medical University School of Medicine Sapporo 060, Japan

I. INTRODUCTION

embryonic/fetal development (Sperelakis and Shigenobu, 1972; Kojima et al., 1990). The value reaches their adult levels at the late stage of embryo/fetus. It becomes almost unchanged after birth. Because [K⫹]i of embryonic chick ventricular cells is already as high as that of adult, low RP is not due to the alteration of equilibrium potential for K⫹ (EK) (Sperelakis and Shigenobu, 1972; Sperelakis and Haddad, 1995). By measuring RP as a function of [K⫹]o, it was demonstrated that PNa /PK decreased during embryonic development. In general, PK in adult hearts is so high (i.e., very low PNa /PK) that the RP is determined by EK. However, in early embryonic hearts, the PK is low (i.e., higher PNa /PK), which accounts for the low RP (Sperelakis and Shigenobu, 1972) (Fig. 1). This is also in agreement with high membrane resistivity in early embryonic hearts. Thus, the increase in RP during development is due to the increase in PK . Spontaneous activity (which is often seen in the early stage of embryo/fetus) is in part ascribed to the low RP. In the early embryonic period, the low RP of the ventricular portion is not stable, but exhibits a spontaneous depolarization, the pacemaker potential (phase 4 diastolic depolarization). The maximum diastolic potential increases (hyperpolarized) and the slope of the pacemaker potential decreases progressively during embryonic development. When the RP has attained the adult level in the late embryonic period, the pacemaker potential disappears. Thus, automaticity of the ventricular cells is lost by the middle embryonic period. Possible factors in the loss of automaticity are the decrease in the PNa /PK ratio and the resultant hyperpolarization. These factors are closely related to the increase in the

During the ontogeny of avian and mammalian hearts, important physiologic, electrophysiologic, pharmacologic, and biochemical alterations occur in concert with ultrastructural changes. For example, dramatic changes in electrophysiological properties occur during development, which affect the physiological functions of hearts. In hearts, a great deal of ion channels determine electrophysiological properties. Therefore, it is important to clarify developmental changes of ion channels/currents. Developmental changes in ion channels include alterations in the types, the number, and the kinetic properties. As a consequence of these changes, resting potential (RP) and action potential (AP) are altered greatly during the developmental stages. For example, RP increases in amplitude during development, and large changes occur in the AP rate of rise, overshoot, and duration. In general, the rate of rise increases markedly, the overshoot increases, and the duration decreases during development. This chapter focuses primarily on the ion channels of cardiomyocytes where most is known about the developmental changes. The reader is referred to a number of review articles that deal with this topic (e.g., Sperelakis and Haddad, 1995; Wetzel and Klitzner, 1996).

II. DEVELOPMENTAL CHANGES A. Action Potential Parameters The transmembrane RP of ventricular cells from chick and rat increases (i.e., hyperpolarizes) during

Heart Physiology and Pathophysiology, Fourth Edition

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inward rectifier K⫹ current (IK(IR)) and the loss of the hyperpolarization-activated inward current (Ih or If) (see later). The maximal rate of depolarization (max dV/dt) increases during embryonic/fetal development with a time course similar to that of RP (Sperelakis and Shigenobu, 1972; Kojima et al., 1990) (Fig. 2). That is, a gradual increase in max dV/dt is observed until around the day of hatching/birth. The AP amplitude also increases during development, as can be indicative of an increase in the rate of rise of the AP. The low max dV/dt is not simply caused by the low RP (which inactivates fast Na⫹ current) because hyperpolarizing RP to ⫺80 mV (i.e., the value for adult heart) is not enough to produce high max dV/dt seen in adult heart. An increase in fast Na⫹ channel density can explain the development of max dV/dt (see later). The large increase in max dV/dt indicates the greater local circuit current, thereby producing faster propagation (see Chapter 6). Therefore, it allows synchronous contraction of the developing heart whose

growth is rapid and prominent. The greater cell size (i.e., diameter) seen during development also promotes faster propagation because propagation velocity is a function of the square root of cell diameter. Change in AP duration (APD) during development is species dependent. In chick embryonic hearts, APD remains essentially unchanged during development (Sperelakis and Shigenobu, 1972). In contrast, APD in rat and mouse ventricular muscles decreases during fetal and postnatal development and reaches the level of adult around 2 weeks after birth (Kojima et al., 1990; Wang et al., 1996; Wang and Duff, 1997) (Fig. 2). In guinea pig and rabbit ventricular cells, APD decreases from late fetus to neonate and increases after birth (Sa´nchez-Chapula et al., 1994; Kato et al., 1996; Haddock et al., 1998). In principle, the major determinant for APD is the balance between inward Ca2⫹ current and outward K⫹ current. For example, the developmental abbreviation of APD seen in rat hearts is largely due to an increase in transient outward current (Ito), with no sub-

FIGURE 1 Developmental changes of resting potential (Em) as a function of external K⫹ ion concentration ([K⫹]o) in embryonic chick hearts. Experimental Em values for each heart (days 3, 5, and 15) were plotted against each [K⫹]o on a logarithmic scale. The solid line is the theoretical curve predicted by the constantfield equation (shown in inset) of resting Em as a function of [K⫹]o for various assumed PNa /PK ratios of 0.001, 0.01, 0.05, 0.1, and 0.2. Calculations were made assuming a [K⫹]i of 150 mM, [Na⫹]i of 30 mM; the sum of [K⫹]o ⫹ [Na⫹]o was held constant at 152 mM (which was the method used to obtain experimental data). Reproduced, with permission, from Sperelakis and Shigenobu (1972).

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FIGURE 2 Developmental changes in action potentials (AP) in rat ventricular muscles. (A) APs at various ages are given. (a–d) APs recorded on postgestation days 12 (a, b), 14 (c), and 18 (d). (e–i) APs recorded on neonatal days 0 (e), 2 (f), 6 (g), 14 (h), and 20 (i). (j) AP recorded in the adult (a: spontaneous AP; and b–j: APs evoked by electrical stimulation at 0.5 Hz). The upper, middle, and lower traces represent zero potential, AP, and dV/dt, respectively. The peak excursion of dV/dt gives Vmax , i.e., max dV/dt. (B) Summary of developmental changes in parameters of APs. Changes in (a) max dV/dt, (b) resting potential, (c) overshoot, (d) AP amplitude, and (e) APD50 (䊉) and APD90 (䊊) are given (AP durations at 50 and 90% repolarization levels, respectively). Each point represents the mean ⫾ SE. Reproduced, with permission, from Kojima et al. (1990).

stantial change in total Ca2⫹ current (ICa), after birth (however, the different time course of postnatal changes in APD and Ito is also reported). In addition, attenuation of the sustained component of fast Na⫹ current contributes to the shortening of APD from fetus to neonate (see later). However, in guinea pig and rabbit hearts, ICa is not fully developed at the time of birth and increases substantially during postnatal development. In addition, recovery of ICa from inactivation is slowed in immature rabbit ventricular cells (see later). Such alterations of

ICa can explain the developmental increase in APD in these animals.

B. (Na–K)-ATPase/Pump The remarkable increase in RP occurs during the ontogeny of embryonic chick hearts, as discussed previously. This is accomplished by a decrease in membrane resistivity due to an increase in PK without changing EK . Because intracellular Na⫹ and K⫹ distribution is

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determined by Na–K pump activity, it is necessary to increase its activity to maintain the large electrochemical gradient for K⫹ against the increase in PK . As expected, Na–K pump activity in heart increases during chick embryonic development (Sperelakis, 1972). In rat ventricles, there is an isoform switch from 움3 to 움2 subunit (both of which are Na–K pump isozymes with high affinity to cardiac glycosides) during postnatal development. In contrast, the low-affinity 움1 subunit remains unchanged (Lucchesi and Sweadner, 1991).

C. Na⫹ Channels The observation of the greatest increase in max dV/ dt and AP amplitude during ontogeny of early embryo/ fetus indicates that inward depolarizing currents are enhanced progressively. Because the max dV/dt is highly dependent on [Na⫹]o, such an inward current is carried through Na⫹ channel (Sperelakis and Shigenobu, 1972). Consistent with this thought, a progressive increase in Na⫹ current (INa) density occurs during embryonic/fetal development (Sada et al., 1995; Davies et al., 1996) (Fig. 3). The density of INa reaches its maximal level (which is the value of adult) at the day around the birth (or hatching). The property of INa also changes, especially during the ontogeny of embryo/fetus. For example, in early embryonic (3-day-old) chick and fetal rat cardiomyocytes, the sustained component of INa (steady-state or no inactivation or ‘‘late’’ Na⫹ current) is present (Josephson and Sperelakis, 1989; Conforti et al., 1993). Thus, the rate of INa inactivation is slowed in immature cardiomyocytes. This unique feature of INa results from multiple reopenings and closings of Na⫹ channels (thereby producing a birst-like behavior), which are evident in a significant number of Na⫹ channels in early

embryonic hearts (Josephson and Sperelakis, 1989) (Fig. 4). Such a birst-like channel behavior is more sensitive to inhibition by tetrodotoxin (TTX) compared to a normal brief opening (observed only during the very early depolarization), probably due to the slow binding and increased affinity of TTX to the open Na⫹ channel (Josephson and Sperelakis, 1989). The sustained component may reflect the ‘‘window current’’ produced by a balance between the activating (m) gate and the inactivating (h) gate. As expected, a positive shift of the steady-state inactivation curve (with the unchanged steady-state activation curve) is observed in immature hearts, thereby producing the greater ‘‘window current’’ (Zhang et al., 1992; Sada et al., 1995). [However, there is a study reporting the negative shift of the steady-state inactivation (h gate) in neonatal rat ventricular cells vs adult.] It is interesting to know that such a developmental change in INa can be mimicked by innervation with sympathetic neurons, especially through the 웁adrenergic/cAMP-dependent mechanism (Zhang et al., 1992). Because the sustained component of INa plays a significant role in fetal rat ventricular cells, a relatively low concentration (1 애M) of TTX shortens APD (without abolishing AP), suggesting that the sustained component of INa contributes to the prolonged APD seen in immature rat hearts (Conforti et al., 1993). As the AP in late embryonic hearts is abolished by TTX, it is generated through a fast, TTX-sensitive Na⫹ channel. However, in early embryonic (ca. 3-day-old) chick hearts, the AP and its max dV/dt were insensitive to TTX (Sperelakis and Shigenobu, 1972; Sperelakis and Haddad, 1995). This TTX-insensitive AP was thought to be evoked by a slow, TTX-insensitive Na⫹ channel (not by a slow Ca2⫹ channel) because it was abolished in Na⫹-free solution with normal Ca2⫹ levels (Sperelakis

FIGURE 3 Fast Na⫹ current in embryonic chick ventricular cells. Current traces recorded in response to different voltage steps in (A) 3- and (B) 17-day-old embryonic chick ventricular cells. Holding potential (HP): ⫺90 mV; pulse rate: 0.5 Hz; temperature: 16–17⬚C. (C) Current–voltage relationships for 3 (䊊)-, 10 (䉭)-, and 17 (䊐)-day-old cells. Peak Na⫹ current values (mean ⫾ SE) were normalized with respect to cell membrane capacitance (pA/pF). Reproduced, with permission, from Sada et al. (1995).

41. Ion Channels

723

FIGURE 4 Single Na⫹ channel currents recorded from an outside-out patch in embryonic chick ventricular cells. The HP was ⫺110 mV and the step potential was ⫺60 mV (applied at 1 Hz). Single channel traces were grouped into those displaying a rapid inactivation following opening (A) and those displaying multiple channel openings or bursts (B). The arrow in the top trace of (A) indicates the time of onset of the voltage clamp step. Dotted lines give the closed, or zero current level. Reproduced, with permission, from Josephson and Sperelakis (1989).

and Shigenobu, 1972). In the later study, TTX reduced max dV/dt even in 4-day-old embryonic chick hearts (Iijima and Pappano, 1979). Therefore, in contrast to earlier findings, it is suggested that the reduced sensitivity to TTX in the early embryonic chick AP may result from the larger contribution of the slow Ca2⫹ channel. Thus, there may be the change from Ca2⫹ to Na⫹ dependence of the AP rise during embryonic development. However, two families of binding sites for TTX are present in rat ventricular cells, i.e., high- and low-affinity sites (Renaud et al., 1983). High-affinity sites are absent in immature hearts, and their number gradually increases to reach a maximum level around 45 days after birth. In addition, mRNA levels of a newly cloned Na⫹ channel (from a mouse AT-1 atrial tumor cell line) in mouse hearts are highest around the day of birth and decrease rapidly thereafter (Felipe et al., 1994). Therefore, it remains undetermined whether a slow, TTXinsensitive Na⫹ channel is present.

D. Ca2⫹ Channels The current density, the opening and closing behavior, and the type of Ca2⫹ channels change during development. In chick embryonic heart (in contrast to the Na⫹ channel), the density of peak ICa is already high (ca. 8 애A/cm2) at 3 days and decreases (to ca. 5 애A/

cm2) at 17 days (Tohse et al., 1992b) (Fig. 5). In rat and mouse hearts, the density of ICa increases during fetal development and reaches its maximal level (i.e., the adult level) around the day of birth (Cohen and Lederer, 1988; Masuda et al., 1995; Davies et al., 1996) (Fig. 5). However, in guinea pig and rabbit hearts, the density at birth is not as high as that for adult (Osaka and Joyner, 1991; Huynh et al., 1992; Kato et al., 1996), i.e., the ICa density gradually increases from fetus to adult (Fig. 5). In addition, the recovery from inactivation is slowed in neonatal rabbit cardiomyocytes, suggesting a relative deficiency of Ca2⫹ channels at birth (Huynh et al., 1992; Wetzel and Klitzner, 1996). The developmental increase in ICa after birth appears to be promoted by innervation with sympathetic neurons, especially through the 움-adrenergic-dependent mechanism (Pignier et al., 1998). No apparent shifts of steady-state activation and inactivation curves are observed during the ontogeny of embryonic chick and postnatal rabbit (Tohse et al., 1992b; Osaka and Joyner, 1991). In contrast, the smaller window current in immature rabbit hearts is also reported (Wetzel and Klitzner, 1996). In rat ventricular cells, the window current is augmented in neonatal vs adult as a result of the positive shift of the steady-state inactivation (f) curve in neonatal cells (Cohen and Lederer, 1988). Moreover, the decay of ICa (elicited at poten-

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VIII. Developmental Changes and Aging

FIGURE 5 Developmental changes of Ca2⫹ current in (A) embryonic chick, (B) fetal/neonatal rat, and (C) fetal/neonatal rabbit cardiomyocytes. (A) Current–voltage curves [normalized as current density (in 애A/cm2) (mean ⫾ SE)] for L-type Ca2⫹ current (ICa(L)) in 3-day (䊉) and 17-day (䊊) embryonic chick heart cells. ICa(L) was elicited by depolarizing steps from a HP of ⫺40 mV. 1.8 mM [Ca2⫹]o, 35⬚C. (B) Ba2⫹ currents through Ltype Ca2⫹ channels (IBa(L)) were elicited by depolarizing steps from a HP of ⫺40 mV (22⬚C). Current–voltage curves [normalized as current density (in pA/pF) (mean ⫾ SE)] are shown for the different developmental stages (from day-12 fetal to day-10 neonatal). (C) Current–voltage curves [normalized as current density (in 애A/cm2) (mean ⫾ SE)] for Ca2⫹ current (ICa) in rabbit cardiomyocytes of the different developmental stages (from day-21 fetus to adult). HP of ⫺80 mV was employed. 10 mM [Ca2⫹]o , 23⬚C. Reproduced in modified form, with permission, from Tohse et al. (1992), Masuda et al. (1995), and Huynh et al. (1992).

tials positive to 0 mV) is slower in neonatal cells vs adult. In the presence of ryanodine (10 애M) [which locks sarcoplasmic reticulum (SR) Ca2⫹ channels open, thereby disturbing Ca2⫹-induced Ca2⫹ release from the SR], the time course of ICa inactivation in adult cells is also slowed, as in neonatal cells. These observations are probably due to the underdeveloped SR and its impaired Ca2⫹-induced Ca2⫹ release in neonatal hearts, thereby diminishing the effect of [Ca2⫹]i-dependent inactivation of ICa (Cohen and Lederer, 1988). Ca2⫹ currents in hearts generally consist of two components: (a) a high threshold slow (L-type) Ca2⫹ current [ICa(L)] and (b) a low threshold early transient (T-type) Ca2⫹ current [ICa(T)]. ICa(L) is dominant and expressed ubiquitously in hearts. The expression pattern of ICa(T) depends on species, tissues, and developmental stages. That is, ICa(T) is relatively abundant in the conduction system, such as sinoatrial nodal cells and Purkinje cells, whereas it is negligible (or absent) in some ventricular cells (e.g., rat, mouse). In immature or developing (growing) cardiomyocytes, the appearance of ICa(T) is relatively high. For example, during the postnatal development of rat atrial myocytes, the density of ICa(T) is well correlated with growth rate (Xu and Best, 1992) (Fig. 6). It appears that an increased expression of ICa(T) is closely related to cellular growth and/or hypertrophy. In addition to L-type and T-type channels, a novel type of Ca2⫹ channel, F type (fetal type), is proposed to be present only during the fetal period of rat ventricles

(Tohse et al., 1992a). The F-type channel current is resistant to the dihydropyridine Ca2⫹ antagonist nifedipine, a blocker of the L-type channel. Both T-type channel blockers (Ni2⫹, tetramethrine) and an N-type blocker (웆-conotoxin) do not inhibit the F-type channel current.

FIGURE 6 T-type and L-type Ca2⫹ current density as a function of postnatal ages. T-type (䊉) and L-type (䊏) Ca2⫹ current density (mean ⫾ SE) in atrial myocytes isolated from female rats. Whole body growth rates (䊊) at different ages are also given. T-type Ca2⫹ current density is correlated linearly with growth rate as shown in the inset. Reproduced, with permission, from Xu and Best (1992).

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41. Ion Channels

probability are higher in 3-day-old compared to 17-dayold embryonic chick cardiomyocytes (Tohse et al., 1992b).

E. Inward Rectifier K⫹ Channel Family The prominent changes of inward rectifier K⫹ current (IK(IR)) occur during the ontogeny of embryonic/fetal hearts with respect to its density, conductance, and open probability. It is most likely that these changes in the IK(IR) channel are responsible for the developmental increases in PK, thereby making RP close to the EK value. The current density of IK(IR) increases greatest during the embryonic/fetal period (Fig. 8) and reaches its quasimaximal level around the day of birth (Huynh et al., 1992; Masuda and Sperelakis, 1993; Xie et al., 1997). From neonate to adult, a further small increase is observed in rabbit ventricular cells (Huynh et al., 1992), whereas some gradual decrease (due to increase in cell

FIGURE 7 Presence of long openings of slow (L-type) Ca2⫹ channels in young embryonic (3-day) chick heart cell. (A) Single channel activity elicited by consecutive command pulses to 0 mV (from a HP of ⫺80 mV) every 2 sec. Sweep-to-sweep variations of the probability of the channel opening (Po) are given in the right-hand column. (B) A histogram of Po data from nine cells (30 sweeps each). Note that many sweeps showed long openings and high Po. Reproduced, with permission, from Tohse and Sperelakis (1990).

The dihydropyridine-sensitive slow L-type Ca2⫹ channel usually exhibits very brief openings and closings. On top of this normal behavior, long(-lasting) openings are often observed in early embryonic chick and fetal rat ventricular cells (Tohse and Sperelakis, 1990; Tohse et al., 1992b; Masuda et al., 1995) (Fig. 7). The long openings are reminiscent of mode-2 behavior, which becomes more frequent in the presence of BAY K 8644, a dihydropyridine Ca2⫹ channel agonist. The incidence of (naturally occurring) long openings decreases during development (Tohse et al., 1992b; Masuda et al., 1995). Thus, the mean open time and open

FIGURE 8 Developmental changes in density of whole cell inward rectifier K⫹ current [IK(IR)] in fetal/neonatal rat ventricular cells. Currents are shown as current densities [pA/pF (A) or 애A/cm2 (B)]. (A) Superimposed traces of IK(IR) from 12-day fetal, 18-day fetal, and 5-day neonatal ventricular cells elicited by test potentials (⫺60 to ⫺120 mV) from a HP of ⫺40 mV. (B) Current–voltage relationships at different developmental stages (from fetal day 12 to neonatal day 10). Reproduced, with permission, from Masuda and Sperelakis (1993).

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VIII. Developmental Changes and Aging

ventricular) tissues. This channel is stimulated by a neurotransmitter acetylcholine (ACh) and is often referred to as the ACh-activated K⫹ (KACh) channel. It is also stimulated by adenosine (Ado). Although different types of receptors [i.e., mucarinic (M2) and adenosine (A1)] are involved, the mechanism of stimulation is mediated through a common GTP-binding protein signal transduction pathway, probably by 웁웂 subunits of the pertussis toxin-sensitive G-protein. When estimating the ontogenic changes of this channel, a complication may exist if the number and property of the receptors, as well as the downsteam signaling mechanisms, are altered during development. In rat atrial cells, the muscarinic K⫹ current induced by ACh (IK,ACh) and that induced by (IK,Ado) exhibits development changes with a slight difference. The absolute amplitude of IK,ACh increases gradually from fetus to adult, whereas that of IK,Ado reaches its maximal level around 10–20 days and decreases later on (Takano and Noma, 1997) (Fig. 10). When evaluating the current density (i.e., the value normalized by cell membrane capacitance), both IK,ACh and

FIGURE 9 Current–voltage relationships for single IK(IR) channels in different developmental stages. (A) Single–channel current recording from typical fetal ventricular cells (days 12 and 18). (B) Current– voltage relationships for single IK(IR) channel in three different developmental stages: fetal days 12 and 18 and neonatal day 5. Reproduced, with permission, from Masuda and Sperelakis (1993).

membrane capacitance) is evident in rat ventricular cells (Xie et al., 1997). An increase in single channel conductance also occurs. For example, in rat ventricular cells, the conductance of the IK(IR) channel is 11 pS in the 12day fetus, 30–31 pS in the neonate (Fig. 9), and 42 pS in the adult under the condition of symmetrical 150 mM K⫹ (i.e., [K⫹]i ⫽ [K⫹]o ⫽ 150 mM) (Masuda and Sperelakis, 1993). There is a parallel increase in open probability in association with the change in conductance. In the later study, the small conductance events of 11 pS seen in fetal hearts is suggested to be sublevels of the 30-pS channel (Xie et al., 1997). However, alteration of the single IK(IR) channel conductance is also observed during the ontogeny of embryonic chick as well as postnatal rabbit hearts (Sperelakis and Haddad, 1995; Wetzel and Klitzner, 1996). Taken together, the increase in the number, unit current amplitude (conductance), and open probability of the IK(IR) channel would account for the development of IK(IR) (i.e., PK), concomitant with a decrease in membrane resistivity. The muscarinic K⫹ channel, a member of an inward rectifier K⫹ channel family, is abundunt in atrial (supra-

FIGURE 10 Developmental changes of muscarinic K⫹ current in rat atrial myocytes. Muscarinic K⫹ current was induced by acetylcholine (IK,ACh) and by adenosine (IK,Ado). (A) Peak amplitudes of IK,ACh (solid bars) and IK,Ado (open bars) measured at ⫺40 mV. The values are expressed as mean ⫾ SE. (In response to agonists, the muscarinic K⫹ current exhibits rapid activation followed by a decay to a quasisteady level, i.e., desensitization. Therefore, the peak amplitudes are given.) (B) Densities of IK,ACh and IK,Ado normalized by cell membrane capacitance. Reproduced, with permission, from Takano and Noma (1997).

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41. Ion Channels

IK,Ado increase from fetus to late neonate (10–20 days) and decrease thereafter. As for the conductance and kinetic (the open time), no apparent differnces are observed between fetus and adult cells. Thus, the distinct subtypes of the KACh channel are not found during development. A small but substantial amount (ca. 1 pA/pF at ⫺50 mV) of IK,ACh is also present in ventricular cells. In contrast to atrial cells, there are no significant differences in the current density among fetal, neonatal, and adult ventricular cells (Xie et al., 1997). The ATP-sensitive K⫹ (KATP) channel, a member of the inward rectifier K⫹ channel family, is expressed ubiquitously in heart. Its distinct properties include inhibition by [ATP]i and sulfonylurea compounds and stimulation by nucleotide diphosphates (NDPs) and K⫹ channel opening drugs. Robust expression of the KATP channel is observed in the embryonic mouse heart (Davies et al., 1996), although its physiological significance requires further investigation. In rabbit ventricular cells, the conductance of the KATP channel is reported to be higher in adults (66 pS) than in neonates (56 pS) (under the condition of symmetrical 150 mM K⫹) (Chen et al., 1992). In rat ventricular cells, the current density of IK,ATP (induced by metabolic inhibition) gradually increases from the fetal period and reaches its maximal level around the day of birth. Its value in the neonatal

period (1–20 days old) stays constant, but that in the adult becomes smaller due to the increase in cell membrane capacitance (Xie et al., 1997). This pattern of developmental change is similar to that in the IK(IR) density of rat ventricular cells. In contrast to rabbit hearts, the conductance (ca. 70 pS) and the opening and closing kinetics are quite similar among fetal, neonatal, and adult rat ventricular cells. However, developmental differences are evident with respect to the density, open probability (Po), and ATP sensitivity of the channel. The density (estimated as the number of the channel per patch) in adult myocytes is about four times higher than that in fetus, but is almost the same as that in neonate myocytes. The Po in absence of ATP (i.e., the maximal Po) is lower in fetal myocytes vs adult myocyte. The rank order of the half-maximal inhibition (IC50) value for ATP is adult ⬎ fetus ⬎ neonate, indicating that the KATP channel in neonatal myocytes is most sensitive to inhibition by ATP (Xie et al., 1997) (Fig. 11). Alteration of both the channel density and the Po would account for the developmental increase in IK,ATP.

F. Transient Outward Current In heart, two types of transient outward current (Ito) are found: (a) Ca2⫹-insensitive Ito (Ito1) and (b) Ca2⫹sensitive Ito (Ito2). Ito1, which carries mainly the K⫹ ion,

FIGURE 11 Developmental changes in the sensitivity of ATP-sensitive K⫹ (KATP) channels to the intracellular ATP concentration ([ATP]i) in rat ventricular cells. (A) Representative channel responses to application of 10 애M ATP in 14-day fetal (14 dF), 1-day neonatal (1 dN), and adult cells. HP was set to ⫺40 mV. Dotted lines indicate closed-current levels. Overlap of IK(IR) channels is observed during an initial one-half in record of the adult cell. (B) [ATP]i-inhibition relationships for KATP channels in 14 dF (䉭), 1 dN (䊊), and adult (䊐) cells. Values of product of number of channels and open probability (NPo) measured at different [ATP]i were normalized to that in ATPfree solution [NPo(ctr)] in individual patches. Each plot is expressed as mean ⫾ SE. Reproduced, with permission, from Xie et al. (1997).

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VIII. Developmental Changes and Aging

is expressed ubiquitously in hearts and is characterized by the inhibition by 4-aminopyridine. Ito2 is thought to actually be a Cl⫺ current, and its prevalence depends on species. Because Ito1 is the major component of total Ito, the term Ito designates the former one, i.e., 4-aminopyridine-sensitive Ito (Ito1) in this chapter. Developmental changes of Ito have been examined extensively. A postnatal increase in Ito is observed consistently in ventricular cells from mouse, rat, and rabbit (Kilborn and Fedida, 1990; Sa´nchez-Chapula et al., 1994; Shimoni et al., 1997; Wang and Duff, 1997). In human atrial cells, a developmental increase also occurs (Crumb, et al., 1995), although it requires further investigation. Ito at the day around birth is very small or essentially absent and gradually increases thereafter (Fig. 12). Such an increase in Ito contributes to the age-related shortening of APD, especially in rat and mouse. As for the recovery from inactivation, it is slower in young human atrial cells (Crumb et al., 1995), as well as neonatal rat ventricular cells, compared to that of adult. In contrast to these species (i.e., human and rat), the faster recovery kinetics of neonatal cells is observed in rabbit ventricles (Sa´nchez-Chapula et al., 1994). In mouse ventricular cells, the recovery kinetics is altered from a monoexponential function in neonate to a biexponential

one in adult (Wang and Duff, 1997). In addition, the rate of Ito inactivation becomes slowed significantly during development (Wang and Duff, 1997). Shal (Kv4) K⫹ channels (Kv4.1, Kv4.2, and Kv4.3) exhibit Ito-type K⫹ currents, and Ito in rat heart is thought to be composed of Kv4.3 and Kv4.2 channels. In human (and probably canine) heart, the Kv4.3 (not Kv4.2) channel plays a major role in Ito . However, a significant contribution of the Kv1.4 channel is proposed in the endocardial Ito of ferret (and probably rat) ventricle as well as rabbit atrium. The Kv1.4 channel is characterized by the slower recovery kinetics. Consistent with these findings, the developmental changes in rat Ito would result from a consequence of the isoform switch from Kv1.4 to Kv4.2/Kv4.3 (Xu et al., 1996; Shimoni et al., 1997). That is, a relative abundance of Kv1.4 mRNA seen in neonatal rat is attenuated during development, whereas Kv4.2/Kv4.3 predominates in adult. The thyroid hormone (T3) induces the molecular switch of Ito and is thought to play an important role in the postnatal development of Ito (Shimoni et al., 1997). In addition, the basic fibroblast growth factor (bFGF), having tyrosine kinase activity, may also promote the expression of Ito during development (Guo et al., 1995).

FIGURE 12 Peak current–voltage relations of transient outward current (Ito) recorded from rat ventricular cells of different ages. (A) Representative current records from 10-, 5-, and 1-day cells. Voltage clamp pulses were given for 400 msec from a HP of ⫺90 mV to (a) ⫺20 mV, (b) ⫹10 mV, (c) ⫹20 mV, and (d) ⫹40 mV. Tetrodotoxin (TTX) (31 애M) and CdCl (300 애M) were used to block the inward Na⫹ current (INa) and Ca2⫹ current (ICa), respectively. (B) Current–voltage relationships from 16 cells at 1 day of age (䊊), 16 cells at 5 days (䊉), and 18 cells at 10 days (䉭). All data are normalized to cell membrane capacitance and are expressed as mean ⫾ SE. Reproduced, with permission, from Kilborn and Fedida (1990).

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41. Ion Channels

G. Delayed Rectifier K⫹ Currents In heart, delayed rectifier-type (noninactivating) K⫹ currents (IK) are composed of at least three K⫹ currents: (a) a slowly activating delayed rectifier (IKs), (b) a rapidly activating delayed rectifier (IKr), and (c) an ultrarapidly activating delayed rectifier (IKur). In human ventricles, major components of IK consist of IKr and IKs . The molecular clone of IKr is HERG (움 subunit) with MinK or MiRP1 (웁 subunit) (MiRP1: a newly identified MinK-related peptide 1) and that of IKs is KvLQT1 with MinK. [A different kind of IK is present in rat ventricles and forms a noninactivating K⫹ current (sometimes called steadystate K⫹ current). Its molecular candidate includes Kv2.1, Kv1.2, and Kv1.5.] However, IKur is negligible in human ventricular cells, but is dominant in atrial cells. The Kv1.5 channel is thought to form a IKur channel. As Ito in neonatal rat ventricular cells is absent or very small, IKur predominates Ito only during the early neonatal period (Guo et al., 1997b). In cultured neonatal rat ventricular cells, IGF-I promotes the expressions of IKur as well as Kv1.5 protein (Guo et al., 1997a). IKur in ventricular cells decreases during postnatal development, which is associated with a reduction of the Kv1.5 expression. However, no substantial change in Kv1.5 protein is also reported from neonate to adult in the same preparation (Xu et al., 1996). In mouse fetal ventricular cells, IKr is the dominant component of IK, whereas IKs is lacking or very small (Davies et al., 1996; Wang et al., 1996). In early neonate (day-1 to ⫺3), IKs becomes dominant (Fig. 13). However, both components disappear in adult mouse ventricular cells. Moreover, IK,tail (tail current: deactivating current on repolarization) in mouse fetal ventricular cells is abolished completely by dofetilide, a selective IKr blocker, whereas that in early neonatal cells is blocked only partially (suggesting that it is IKs dominant). In adult mouse cells, the predominant K⫹ current is the Ito-type current and is insensitive to dofetilide (Wang et al., 1996). Similarly, IKr in rat ventricular cells is functioning during the fetal period, but is negligible in adult. However, the density of IK (the sum of IKr and IKs) in guinea pig ventricles is smaller in fetal cells than in neonatal and adult cells, suggesting a developmental increase in IK (Kato et al., 1996). No substantial changes in the kinetics and voltage dependency of IK are observed during development.

is observed in early chick embryonic cardiomyocytes (Satoh and Sperelakis, 1993). This current decreases progressively during development and essentially disappears in the late embryonic period (Fig. 14). In neonatal (1–2 days old) rat ventricular cells, Ih is present with a high incidence (more than 90%). The density and the occurrence of Ih decrease during postnatal development, without changing voltage-dependence and ionic selectivity (Cerbai et al., 1999). In contrast, it is also reported that the threshold voltage of Ih in adult cells (when present) shifts extremely in the negative direction, i.e., 앑 ⫺110 mV in adult and 앑 ⫺70 mV in neonate (Robinson et al., 1997). Properties of Ih in rabbit sinoatrial nodal cells differ between neonate and adult (Accili et al., 1997). A larger slope conductance is detected by fully activated current–voltage relationships in neonatal cells. Furthermore, the slope factor of activation curves is greater in the neonate. These findings are in favor of the more rapid diastolic depolarization, although there is a debate concerning this topic (see later). The Ih is called the pacemaker current in adult cardiomyocytes. In Purkinje fibers, Ih plays a key role in pacemaker depolarization during the diastolic phase. In sinoatrial node cells, the contribution of Ih to pacemaker potential is still controversial. This is because the time course of activation of Ih is too slow to account for the high frequency of the pacemaker and because the threshold potential for activation of Ih (close to ⫺70 mV) is beyond the maximum diastolic potential (⫺60 to ⫺70 mV) for nodal cells. Although the time course of decrease in Ih parallels the disappearance of the pacemaker potential in chick embryonic cardiomyocytes, the contribution of Ih to pacemaking may still be small (Satoh and Sperelakis, 1993).

I. Cl⫺ Channels Activity of a large-conductance Cl⫺ channel is recorded in bleb membranes of neonatal rat ventricular cells, but is quasi-completely absent in adult cells. Such channel activities are also evoked by the application of hypotonic medium (producing swelling of the cell) in neonatal cells. However, the occurrence of this channel activity decrease with age and disappear in cells from 15-day-old rats (Coulombe and Coraboeuf, 1992). This swelling (and distention-)-activated Cl⫺ channel would play a role in the volume regulation of neonatal cardiomyocytes.

H. Hyperpolarization-Activated Inward Current

J. Excitation–Contraction (E-C) Coupling

The hyperpolarization-activated inward current (Ih or If), which is mainly carried by Na⫹ and K⫹ ions,

Changes in the E-C coupling process also occur during development of the heart (Fig. 15). The source of

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Ca2⫹ for producing contraction is especially, altered during development. In fetal heart cells, the role of the sarcoplasmic reticulum is minimal so that most of the Ca2⫹ required for contraction is derived from the Ca2⫹ influx through the voltage-dependent Ca2⫹ channels (L type and T type) (i.e., originates from the extracellular space). In neonatal heart cells, the SR matures compared to fetus and plays a role as the source of Ca2⫹ for contraction. Therefore, the Ca2⫹-induced Ca2⫹ release from the SR compartment becomes the more important system for contraction. This is supported by findings that SR Ca2⫹-ATPase (SERCA2) mRNA, as well as its

activity, increases during postnatal development. Furthermore, expression of the SR Ca2⫹ release channel (or ryanodine receptor) in rat or rabbit hearts increases during postnatal growth (Wetzel and Klitzner, 1996; Ramesh et al., 1995). Finally, in adult heart cells, most of the Ca2⫹ for contraction comes from internal SR stores. However, Ca2⫹ influx through the sarcolemma is still the determining factor for contractile force because the Ca2⫹ influx controls the amount of Ca2⫹ released. The spanning protein (foot protein) has been proposed to exist for E-C coupling to work effectively. This

FIGURE 13 Properties of delayed rectifier K⫹ current (IK) in fetal and day-3 neonatal mouse ventricular cells. The HP was ⫺40 mV in both preparations. The duration of depolarization pulses in the day-3 neonatal cell was 10-fold longer than in the fetal cell (5000 vs 500 msec). Representative current traces in fetal (A) and day-3 neonatal (C) cells are shown. The time-dependent K⫹ current (IK-out) increases gradually during depolarization pulses. On repolarization to the HP (of ⫺40 mV), the deactivating K⫹ current (IK-tail) appears. Current– voltage relationships for IK-out (䊉) and IK-tail (䊊) in fetal (B) and day-3 neonatal (D) cells. Current amplitude is normalized to cell membrane capacitance (pA/pF), and data points are expressed as mean ⫾ SD. As shown in (A) and (B), IK in fetal cells activates rapidly and exhibits inward rectification at depolarized potentials, resulting in a voltage-dependent decrease in its IK-out at test potentials ⬎0 mV (a negative slope conductance at voltages positive to 0 mV). These properties are similar to those of IKr. In contrast, in day-3 neonatal cells, IK activates slowly and does not reach a steady state even during a 5000-msec depolarization pulse (C). The current–voltage relationship of its IK-out (D) shows a positive slope conductance throughout the voltage range examined. The properties of slow activation and outward rectification suggest that the current is largely IKs . Reproduced, with permission, from Wang et al. (1996).

41. Ion Channels

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FIGURE 14 Develomental changes of the hyperpolarization-activated inward current (Ih) in young embryonic chick ventricular cells. Test pulses were applied between ⫺40 and ⫺120 mV, in 10-mV increments, from a HP of ⫺30 mV (inset). (A) A large inward current was activated slowly by hyperpolarization in a 3-day-old cell. (B) Smaller Ih in a 10-day-old cell. (C) Further reduced Ih in a 17-day-old cell. (D) Current–voltage relations for Ih current density at the three developmental stages (mean ⫾ SE). Reproduced, with permission, from Satoh and Sperelakis (1993).

protein would couple the L-type Ca2⫹ channel in the T tubule wall membrane to the Ca2⫹ release channel in the SR membrane and may be absent in the immature heart (Cohen and Lederer, 1988). If so, then the release of Ca2⫹ is prevented from any SR that may be present. Thus, Ca2⫹ influx would be the main source of Ca2⫹ for contraction in the immature heart. Maturation of the E-C coupling system requires the physiological level of thyroid hormones. For example, thyroid hormones are essential for the postnatal redistribution of L-type Ca2⫹ channels from nonjunctional sarcolemma to junctional structures, where the Ca2⫹ release channels (ryanodine receptors) in the SR are enriched (Wibo et al., 1995). This structural alteration enables E-C coupling to be operated more efficiently.

K. Na–Ca Exchange Voltage-dependent Ca2⫹ channels are the major source of Ca2⫹ influx, thereby triggering Ca2⫹ release from the SR and producing contraction. In addition to this central mechanism of E-C coupling of the heart, other possibilities include (a) depolarization-induced SR Ca2⫹ release (as observed in skeletal muscle), (b) Ca2⫹ influx via Na⫹ channels (slip-mode conductance), and (c) Na–Ca exchange. Although the unitary flux through the Na–Ca exchanger is perhaps 1000 times

lower than ICa(L) , Ca2⫹ influx via its reverse mode (3 Na⫹ out/ 1 Ca2⫹ in, thereby producing outward current) occurs (i) directly through depolarization and (ii) secondary to the elevation of [Na⫹]i by INa. In addition to the underdeveloped SR in immature hearts, the relative deficiency in ICa is observed especially in immature rabbit heart (see earlier discussion). In this respect, Na–Ca exchange plays a greater role in modulating Ca2⫹ influx in immature cardiomyocytes. For example, Na–Ca exchange protein, as well as mRNA expression, are maximal near the time of birth (i.e., the late fetal/early neonatal period), and decline postnatally in chick, rat, and rabbit hearts (Wetzel and Klitzner, 1996). Similarly, the density of the outward Na–Ca exchange current (i.e., the reverse mode) is highest at birth and decreases during postnatal development (Artman et al., 1995; Haddock et al., 1998). Furthermore, such a Ca2⫹ influx appears to be sufficient to produce contraction in neonate cells but not adult cells (Haddock et al., 1998). It is likely that the abundance of Na–Ca exchange in immature cells compensates for the underdeveloped SR and that its significance in contraction becomes less in association with maturation of the SR. As is the case with maturation of the E-C coupling system, this developmental shift from the Na–Ca exchange-dependent to the SR-dependent Ca2⫹ handling appears to be controlled by T3 (Cernohorsky et al., 1998).

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FIGURE 15 Schematic diagrams showing developmental changes of excitation–contraction (E-C) coupling in adult (A) and immature (B) cardiomyocytes. (A) In adult cells, the sarcoplasmic reticulum (SR) is well developed because both Ca2⫹ release channels (ryanodine receptor) and SR Ca2⫹-ATPase (SERCA2) are abundant. Formation of T tubules brings the sarcolemmal membranes into proximity with the SR, and L-type Ca2⫹ channels (dihydropyridine receptor) in the T tubules interact with SR Ca2⫹ release channels efficiently. Moreover, the spanning protein (which is a protein linking the L-type Ca2⫹ channel with the Ca2⫹ release channel) would be present, allowing Ca2⫹ release from the SR effectively. Therefore, Ca2⫹ released from the SR is greater and more important. (B) In immature cells, the SR is underdeveloped, and the spanning protein is absent. Therefore, Ca2⫹ influx across the sarcolemma plays the more important role in contraction. The relative deficiency of Ca2⫹ handling (release and uptake) by the SR could be compensated by a reciprocal increase in Na–Ca exchangers. Such enhanced Na–Ca exchange activity would produce Ca2⫹ influx via its reverse mode (especially in depolarization and elevated [Na⫹]i). Thick filled or dotted arrows indicate the relative amount of (a) Ca2⫹ influx through L-type Ca2⫹ channels (an upper left arrow, which parts into two ends), (b) Ca2⫹ release from the SR (an arrow located above the SR), and (c) Ca2⫹ influx via reverse mode of the Na–Ca exchanger (an arrow located on the left of the Na–Ca exchanger). Dotted arrow means that the amount of Ca2⫹ flux (or release) is very small or absent.

III. SUMMARY Action potentials and resting potentials in cardiomyocytes are altered greatly during development. In

general, the rate of rise increases, the overshoot increases, the duration decreases, and the RP is hyperpolarized. These electrophysiological alterations are mainly produced by developmental changes in ion channels, i.e., changes in the types of, number of, and kinetic properties of the ion channels. The hyperpolarization of RP during development can be accounted for by the increase in IK(IR) and the resultant decrease in the PNa /PK ratio. This increase in IK(IR) results from changes in single channel conductance, as well as in the number and open probability of the channel. For example, the conductance of early fetal myocytes is much smaller than that of neonate myocytes, thereby contributing to the higher membrane resistance in fetal cells. Because of such a less leaky membrane, the low Na–K pump activity seen in the early embryonic period is sufficient to maintain [K⫹]i as high as that in mature cells. In concert with the increase in PK , Na–K pump activity increases during development. In contrast, the hyperpolarization-activated inward current (Ih), which may affect automaticity, is dominant in early embryonic chick and neonatal rat cardiomyocytes and disappears (or becomes negligible) during development. Muscarinic K⫹ channel, a member of an inward rectifier K⫹ channel family, is abundunt in supraventricular tissues. This channel is characterized by the stimulation of acetylcholine and is often referred to as the AChactivated K⫹ channel. The density of IK,ACh in atrial cells increases from fetus to late neonate and decreases thereafter. With respect to conductance and kinetic properties, no apparent differences are observed between fetus and adult hearts. The ATP-sensitive K⫹ channel, a member of the inward rectifier K⫹ channel family, is present in the embryonic mouse heart. In rabbit ventricular cells, the conductance of the KATP channel appears to be higher in adult compared to neonate. In rat ventricular cells, the current density of IK,ATP increases gradually from the fetal period and reaches its maximal level around the day of birth. In contrast to rabbit hearts, the conductance and kinetic properties are quite similar among fetal, neonatal, and adult rat ventricular cells. However, the developmental differences are evident with respect to the density, open probability, and ATP sensitivity of the channel. A different isoform of cardiac KATP channels may exist in immature cells. The fast Na⫹ current also increases markedly during development, i.e., there are few or no functional fast Na⫹ channels present at the earliest stages and the density of these channels increases progressively during development. The fast Na⫹ current is responsible for the increase in the AP rate of rise (independent of the hyperpolarization of RP). The fast Na⫹ current in embryonic/fetal hearts has a significant sustained (i.e., slow inactivating or steady-

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state) component. The sustained component of the embryonic/fetal Na⫹ current decreases during development, which contributes, at least in part, to the abbreviation of AP duration. Development of Ca2⫹ channels seems more complex. The density of total Ca2⫹ current in chick cardiomyocytes decreases during the developmental period from fetal to neonate. In rat, however, it increases from the fetal to the neonatal period, followed by a substantial decrease in adult. In rabbit and guinea pig cardiomyocytes, the current density in the neonatal period is smaller than that in adult. The total Ca2⫹ current is composed of currents through several different types of channels: L type, T type, and F type. The proportion of the T-type Ca2⫹ channel current in immature cells is generally more than that in mature cells and may actually disappear in adult, i.e., the L-type Ca2⫹ channel current becomes more dominant in mature cells. Appearance of the T-type Ca2⫹ channel is associated with growth rate of the heart. A nifedipine-resistant F-type Ca2⫹ channel current is also present in early fetal cardiomyocytes of rats. Long-lasting openings of L-type Ca2⫹ channels are observed relatively frequently in embryonic chick and fetal rat cardiomyocytes, which are quite unusual in adult cells. The density of the transient outward current (Ito) increases during development. In addition, the recovery from inactivation is slower in immature cells from human atrium and rat ventricle vs adult cells cells. Ito in rat heart is thought to be composed of Kv4.3 and Kv4.2 channels. In human heart, the Kv4.3 (not Kv4.2) channel plays a major role in Ito. However, the Kv1.4 channel, another Ito-type K⫹ channel subunit, is characterized by slower recovery kinetics. The isoform switch from Kv1.4 to Kv4.2/Kv4.3 appears to be responsible for the difference in the recovery kinetics of Ito between neonate and adult. In mouse fetal ventricular cells, IKr is the dominant component of IK, whereas IKs is lacking or very small. In the early neonate, IKs becomes dominant. However, both components disappear in adult mouse ventricular cells. However, the density of IK (the sum of IKr and IKs) in guinea pig ventricles increases during development, and there is no further change during the postnatal period. No substantial changes in the kinetics and voltage dependency of IK are observed during development. IKur in rat ventricular cells is predominant only during the early neonatal period and decreases during postnatal development. Changes of these voltage-gated outward currents (i.e., Ito and IK) affect the duration of AP during development. A swelling-activated, large-conductance Cl⫺ channel is present in neonatal rat ventricular cells. The occurrence of this channel activity decrease with age. This

channel would play a role in the volume regulation of neonatal cardiomyocytes. Because the SR function and/or the essential component for Ca2⫹ release from the SR is immature, Ca2⫹ influx from the extracellular space is the main source of Ca2⫹ for contraction in immature cardiomyocytes. Therefore, Ca2⫹ influx through Ca2⫹ channels is especially important for the excitation–contraction coupling process of fetal cardiomyocytes. Reverse mode of Na–Ca exchange produces Ca2⫹ influx, which appears to contribute to the contraction in immature cells. Expressions and functions of Na–Ca exchange are reduced gradually during postnatal development in association with maturation of the SR. Expressions of ion channels/E-C coupling systems are controlled by neuronal and humoral factors. For example, innervation seems to mimick the developmental changes of both Na⫹ channels and Ca2⫹ channels, probably via 웁-adrenergic/cAMP-dependent and 움-adrenergic dependent signaling pathways, respectively. Thyroid hormones contribute to the redistribution of L-type Ca2⫹ channels from nonjunctional sarcolemma to junctional structures and are required for maturation of the E-C coupling system. A developmental increase and molecular switch of Ito are also controlled by thyroid hormones. Some growth factors, such as bFGF and IGF-I, may promote K⫹ channel expressions. As mentioned previously, ion channels exhibit dramatic changes during development in their structure, function, and distribution. These dynamic alterations determine the electrophysiological properties of heart, which affect the physiological functions, including automaticity, excitability, and contractility. In hypertension and heart failure, the hypertrophy of cardiomyocytes occurs in association with the alterations of ion channels and E-C coupling systems, which have some similarities with those observed in immature hearts (as described in this chapter) (see also Chapter 60). Moreover, a great deal of regulating factors, including neuronal, hormonal, and hemodynamic influences (e.g., catecholamines, renin–angiotensin systems, thyroid hormones, other growth factors, and mechanical stress), are known to be activated under such pathological conditions and to produce hypertrophy of cardiomyocytes. Therefore, developmental changes of ion channels might be regulated by these factors. Further studies are required to clarify the mechanisms by which the developmental changes are controlled.

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Artman, M., Ichikawa, H., Avkiran, M., and Coetzee, W. A. (1995). Na⫹ /Ca2⫹ exchange current density in cardiac myocytes from rabbits and guinea pigs during postnatal development. Am. J. Physiol. 268, H1714–H1722. Cerbai, E., Pino, R., Sartiani, L., and Mugelli, A. (1999). Influence of postnatal-development on If occurrence and properties in neonatal rat ventricular myocytes. Cardiovasc. Res. 42, 416–423. Cernohorsky, J., Kolar, F., Pelouch, V., Korecky, B., and Vetter, R. (1998). Thyroid control of sarcolemmal Na⫹ /Ca2⫹ exchanger and SR Ca2⫹-ATPase in developing rat heart. Am. J. Physiol. 275, H264–H273. Chen, F., Wetzel, G. T., Friedman, W. F., and Klitzner, T. S. (1992). ATP-sensitive potassium channels in neonatal and adult rabbit ventricular myocytes. Pediatr. Res. 32, 230–235. Cohen, N. M., and Lederer, W. J. (1988). Changes in the calcium current of rat heart ventricular myocytes during development. J. Physiol. (Lond.) 406, 115–146. Conforti, L., Tohse, N., and Sperelakis, N. (1993). Tetrodotoxin-sensitive sodium current in rat fetal ventricular myocytes: Contribution to the plateau phase of action potential. J. Mol. Cell. Cardiol. 25, 159–173. Coulombe, A., and Coraboeuf, E. (1992). Large-conductance chloride channels of new-born rat cardiac myocytes are activated by hypotonic media. Pflu¨. Arch. 422, 143–150. Crumb, W. J., Jr., Pigott, J. D., and Clarkson, C. W. (1995). Comparison of Ito in young and adult human atrial myocytes: Evidence for developmental changes. Am. J. Physiol. 268, H1335–H1342. Davies, M. P., An, R. H., Doevendans, P., Kubalak, S., Chien, K. R., and Kass, R. S. (1996). Developmental changes in ionic channel activity in the embryonic murine heart. Circ. Res. 78, 15–25. Felipe, A., Knittle, T. J., Doyle, K. L., and Tamkun, M. M. (1994). Primary structure and differential expression during development and pregnancy of a novel voltage-gated sodium channel in the mouse. J. Biol. Chem. 269, 30125–30131. Guo, W., Kada, K., Kamiya, K., and Toyama, J. (1997a). IGF-I regulates K⫹-channel expression of cultured neonatal rat ventricular myocytes. Am. J. Physiol. 272, H2599–H2606. Guo, W., Kamiya, K., and Toyama, J. (1995). bFGF promotes functional expressions of transient outward currents in cultured neonatal rat ventricular cells. Pflu¨. Arch. 430, 1015–1017. Guo, W., Kamiya, K., Liu, W., and Toyama, J. (1997b). Developmental changes of the ultrarapid delayed rectifier K⫹ current in rat ventricular myocytes. Pflu¨. Arch. 433, 442–445. Haddock, P. S., Artman, M., and Coetzee, W. A. (1998). Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes. Pflu¨. Arch. 435, 789–795. Huynh, T. V., Chen, F., Wetzel, G. T., Friedman, W. F., and Klitzner, T. S. (1992). Developmental changes in membrane Ca2⫹ and K⫹ currents in fetal, neonatal, and adult rabbit ventricular myocytes. Circ. Res. 70, 508–515. Iijima, T., and Pappano, A. J. (1979). Ontogenetic increase of the maximal rate of rise of the chick embryonic heart action potential: Relationship to voltage, time and tetrodotoxin. Circ. Res. 44, 358–367. Josephson, I. R., and Sperelakis, N. (1989). Tetrodotoxin differentially blocks peak and steady-state sodium channel currents in early embryonic chick ventricular myocytes. Pflu¨. Arch. 414, 354–359. Kato, Y., Masumiya, H., Agata, N., Tanaka, H., and Shigenobu, K. (1996). Developmental changes in action potential and membrane currents in fetal, neonatal and adult guinea-pig ventricular myocytes. J. Mol. Cell. Cardiol. 28, 1515–1522. Kilborn, M. J., and Fedida D. (1990). A study of the developmental changes in outward currents of rat ventricular myocytes. J. Physiol. (Lond.) 430, 37–60.

Kojima, M., Sada, H., and Sperelakis, N. (1990). Developmental changes in beta-adrenergic and cholinergic interactions on calcium-dependent slow action potentials in rat ventricular muscles. Br. J. Pharmacol. 99, 327–333. Lucchesi, P. A., and Sweadner, K. J. (1991). Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle: Conservation of biphasic ouabain affinity. J. Biol. Chem. 266, 9327– 9331. Masuda, H., and Sperelakis, N. (1993). Inwardly-rectifying potassium current in rat fetal and neonatal ventricular cardiomyocytes. Am. J. Physiol. 265, H1107–H1111. Masuda, H., Sumii K., and Sperelakis N. (1995). Long openings of calcium channels in fetal rat ventricular cardiomyocytes. Pflu¨. Arch. 429, 595–597. Osaka, T., and Joyner, R. W. (1991). Developmental changes in calcium currents of rabbit ventricular cells. Circ. Res. 68, 788– 796. Pignier, C., Fares, N., and Potreau, D. (1998). Effects of adrenergic stimulation on postnatal development and calcium current in newborn rat cardiomyocytes in primary culture. J. Cardiovasc. Pharmacol. 31, 262–270. Ramesh, V., Kresch, M. J., Katz, A. M., and Kim, D. H. (1995). Characterization of Ca2⫹-release channels in fetal and adult rat hearts. Am. J. Physiol. 269, H778–H782. Renaud, J. F., Kazazoglou, T., Lombet, A., Chicheportiche, R., Jaimovich, E., Romey, G., and Lazdunski, M. (1983). The Na⫹ channel in mammalian cardiac cells. J. Biol. Chem. 258, 8799– 8805. Robinson, R. B., Yu, H., Chang, F., and Cohen, I. S. (1997). Developmental change in the voltage-dependence of the pacemaker current, If, in rat ventricle cells. Pflu¨. Arch. 433, 533–535. Sada, H., Ban, T., Fujita, T., Ebina, Y., and Sperelakis, N. (1995). Developmental change in fast Na⫹ channel properties in embryonic chick ventricular heart cells. Can. J. Physiol. Pharmacol. 73, 1475– 1484. Sa´nchez-Chapula, J., Elizalde, A., Navarro-Polanco, R., and Barajas, H. (1994). Differences in outward currents between neonatal and adult rabbit ventricular cells. Am. J. Physiol. 266, H1184–H1194. Satoh, H., and Sperelakis, N. (1993). Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: Developmental changes and modulation by isoproterenol and carbachol. Eur. J. Pharmacol. 240, 283–290. Shimoni, Y., Fiset, C., Clark, R. B., Dixon, J. E., McKinnon, D., and Giles, W. R. (1997). Thyroid hormone regulates postnatal expression of transient K⫹ channel isoforms in rat ventricle. J. Physiol. (Lond.) 500, 65–73. Sperelakis, N. (1972). (Na⫹, K⫹)-ATPase activity of embryonic chick heart and skeletal muscles as a function of age. Biochim. Biophys. Acta 266, 230–237. Sperelakis, N., and Haddad, G. E. (1995). Developmental changes in membrane electrical properties of the heart. In ‘‘Physiology and Pathology of the Heart’’ (N. Sperelakis, MA. ed.), Chap. 35, pp. 669–770. Kluwer Academic, Dordrecht/Norwell MA. Sperelakis, N., and Shigenobu, K. (1972). Changes in membrane properties of chick embryonic hearts during development. J. Gen. Physiol. 60, 430–453. Takano, M., and Noma, A. (1997). Development of muscarinic potassium current in fetal and neonatal rat heart. Am. J. Physiol. 272, H1188–H1195. Tohse, N., Masuda, H., and Sperelakis, N. (1992a). Novel isoform of Ca2⫹ channel in rat fetal cardiomyocytes. J. Physiol. (Lond.) 451, 295–306. Tohse, N., Maszaros, J., and Sperelakis, N. (1992b). Developmental changes in long-opening behavior of L-type Ca2⫹ channels in embryonic chick heart cells. Circ. Res. 71, 376–384.

41. Ion Channels Tohse, N., and Sperelakis, N. (1990). Long-lasting openings of single slow (L-type) Ca2⫹ channels in chick embryonic heart cells. Am. J. Physiol. 259, H639–H642. Wetzel, G. T., and Klitzner, T. S. (1996). Developmental cardiac electrophysiology: Recent advances in cellular physiology. Cardiovasc. Res. 31, E52–E60. Wang, L., and Duff, H. J. (1997). Developmental changes in transient outward current in mouse ventricle. Circ. Res. 81, 120–127. Wang, L., Feng, Z.-P., Kondo, C. S., Sheldon, R. S., and Duff, H. J. (1996). Developmental changes in the delayed rectifier K⫹ channels in mouse heart. Circ. Res. 79, 79–85. Wibo, M., Kolar, F., Zheng, L., and Godfraind, T. (1995). Influence of thyroid status on postnatal maturation of calcium channels, 웁-adrenoceptors and cation transport ATPases in rat ventricular tissue. J. Mol. Cell. Cardiol. 27, 1731–1743.

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42 Aging of the Cardiovascular System EDWARD G. LAKATTA, YING-YING ZHOU, and RUI-PING XIAO Laboratory of Cardiovascular Science Gerontology Research Center National Institute on Aging National Institutes of Health Baltimore, Maryland 21224

MARVIN BOLUYT Department of Movement Science Laboratory of Molecular Kinesiology University of Michigan Ann Arbor, Michigan 48109

I. INTRODUCTION

tion of the aging variable into our current understanding of the function of the heart and vasculature in normal and pathologic states. Primary emphasis is given to studies of cardiovascular structure function in humans and animal models over the adult range, i.e., from adulthood to senescence.

The proportion of older persons that constitute populations worldwide is increasing. It is estimated that by the year 2035, nearly one in four individuals will be 65 years of age or older. Cardiovascular diseases, e.g., coronary artery disease, atherosclerosis, and hypertension, and chronic heart failure that ensues reach epidemic proportions among older persons. One reason for this is that specific pathophysiological mechanisms that cause these diseases in older individuals are superimposed on heart and vascular substrates that are modified by an evolving aging process per se. Thus changes in cardiovascular structure and function in older persons with cardiovascular diseases are not solely due to the disease per se, but reflect age–disease interactions! In this regard, quantitative information on age-associated alterations in cardiovascular structure and function in the absence of disease is essential in order to define the specific characteristics of the cardiovascular aging process that render it the major risk factor for cardiovascular disease, and eventually to target relevant age-associated changes for therapeutic intervention. This chapter summarizes the results of studies that have investigated the effect of aging on some aspects of cardiovascular structure/function discussed elsewhere in this volume. This approach appears optimal for integra-

Heart Physiology and Pathophysiology, Fourth Edition

II. CARDIOVASCULAR STRUCTURE AND FUNCTION AT REST A. Cardiac Structure with Advancing Age in Humans Differences in cardiovascular structure and function between older and younger humans have been described extensively in the literature. However, confusion often arises in the interpretation of these differences because of a failure to acknowledge, or to control for, interactions among age, disease, and lifestyle. Genetic components of aging, disease, and lifestyle, which presently remain largely unknown, likely complicate the picture further. Since the early 1980s, a sustained effort has been applied to characterize effects of aging in health on multiple aspects of cardiovascular structure and function in specific populations, e.g., the Baltimore Longitudinal Study on Aging (BLSA). In these studies, community-

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dwelling, volunteer participants are screened rigorously to detect both clinical and occult cardiovascular disease and are characterized with respect to lifestyle, e.g., exercise habits. Cross-sectional studies of sedentary BLSA volunteer subjects without cardiovascular disease indicate that the left ventricular (LV) wall thickness, measured via Mmode (one-dimensional) echocardiography, increases progressively with age in both sexes (Gerstenblith et al., 1977). The LV cavity size at end diastole and end systole, measured in the upright, seated position by gated cardiac blood-pool scans of technetium-labeled red cells, increases moderately with age in healthy, normotensive, sedentary BLSA men but does not vary with age in BLSA women (Fleg et al., 1995). The age-associated increase in left ventricular wall thickness with aging in mostly due to an increase in the average myocyte size. In some older, hospitalized patients without apparent cardiovascular disease, and in whom LV mass decreased with age, cardiac myocyte enlargement was found at autopsy, concurrently with an estimated decrease in myocyte number. An increase in the amount and a change in the physical properties of collagen (due to crosslinking) also occur within the myocardium with aging. However, the cardiac muscle-to-collagen ratio in the older heart either remains constant or increases. There is an increase in elastic and collagenous tissue in all parts of the conduction system with advancing age. Fat accumulates around the sinoatrial (SA) node, sometimes producing a partial or complete separation of the node from the atrial musculature. Beginning by age 60 there is a pronounced decrease in the number of pacemaker cells in the SA node, and by age 75 less than 10% of the cell number found in the young adult remains. A variable degree of calcification of the left side of the cardiac skeleton, which includes the aortic and mitral annuli, the central fibrous body, and the summit of the interventricular septum, occurs with aging. Because of their proximity to these structures, the atrioventricular (AV) node, AV bundle, bifurcation, and proximal left and right bundle branches may be affected by this process.

B. Cardiac Structure with Aging in Animals While numerous studies have documented that structural and functional properties of cardiac muscle isolated from human hearts are very much like those of muscle isolated from hearts of most other mammals, it remains to be documented that the effect of aging is similar in all species. Thus, some caution is advisable when extrapolating data from animal models to human. In some cases, however, similar age-associated changes have been observed across a wide range of species, including humans,

and in these instances some degree of extrapolation to the human aging model may be justified. The vast majority of studies of cardiac aging have employed the rat model. Twenty-four-month-old cageconfined Wistar rats are commonly referred to as ‘‘senescent,’’ because 50% colony mortality occurs at approximately that age, but this differs markedly among strains. In most strains of rats the senescent heart exhibits a moderate LV mass hypertrophy (25%) compared to hearts from young and middle-aged animals (Yin et al., 1980, 1982). This can occur in the absence of arterial hypertension (Rothbaum et al., 1973). The aged rodent, unlike humans, does not exhibit an increase LV wall thickness, rather the increase in LV mass is attributable to an increase in LV cavity size. The average LV collagen content doubles between adulthood and senescence (Weisfeldt et al., 1971). Fibronectin is increased markedly in senescent compared to adult hearts (Table I), and an increased expression of fibronectin in the senescent heart is likely part of an overall increase in the proportion of extracellular matrix as evidenced by the increase in collagen content (Anversa et al., 1990). The expression of collagen genes undergoes a dramatic decrease from development to adulthood and then increases only slightly in the senescent heart. While the relatively high level of collagen mRNA in the hearts of developing rats does not result in the accumulation of collagen, the barely detectable increase in collagen mRNA observed in senescence is associated with a twofold increase in hydroxyproline and collagen content compared to levels in adult hearts (Weisfeldt et al., 1971). Despite the increase in collagen content with fibrosis, there is a disparity among studies in the literature with respect to whether age-associated changes occur in passive myocardial stiffness (Lakatta et al., 1982). The majority of the increase in left ventricular mass with aging in apparently healthy rodents, however, as in humans, is due to myocardial cell enlargement. In individual myocytes isolated from rats of 2, 6–9, and 24–26 months of age, the average myocyte length increases by 20% between 2 and 24–26 months of age, but the average slack sarcomere length does not change (Fraticelli et al., 1989). The average volume of individual cells approximately doubles over this age range. Evidence indicates that the number of myocytes within the heart decreases with aging due to both necrosis and apoptosis, with the former predominating (Fleg et al., 1995; Anversa et al., 1990; Kajstura et al., 1996).

C. Cardiac Function in Humans at Rest 1. Heart Rate and Rhythm Supine, basal heart rate does not differ among younger and older individuals. Studies of a large number

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42. Aging of the Cardiovascular System

TABLE I Myocardial Changes with Adult Aging in Rodentsa Structural change ⇑ Myocyte size

Prolonged contraction

⇓ Myocyte number

Diminished contraction velocity

Diminished 웁-adrenergic contractile response

⇑ Myocardial stiffness

⇓ Growth response ⇓ Heat shock response

Molecular mechanisms

Prolonged cytosolic Ca2⫹ transient ⇓ SR Ca2⫹ pumping rate ⇓ Pump site density

Prolonged action potential

⇑ Matrix connective tissue

Ionic, biophysical/biochemical mechanism(s)

Functional change

⇓ ICa inactivation ⇑ Na–Ca exchange protein ⇓ ITo density ⇓ 움-MHC protein ⇑ 웁-MHC protein ⇓ Myosin ATPase activity ⇓ RXR 웂 protein ⇓ Thyroid receptor 웁웂1 protein ⇓ Acyclase No change in Gi activation No change in BARK activity ⇓ TNI phospholamban ⇓ Phospholamban phosphorylation ⇓ ICa augmentation ⇓ Cai transient augmentation ⇑ Enkephalin peptides ⇑ Hydroxyl proline content ⇑ Activity of myocardial RAS ⇑ Atrial naturetic peptide

⇓ SERCA2 mRNA No change in calsequestrin mRNA No change in RYR2 mRNA ⇑ Na/Ca exchanger mRNA ⇓ 움-MHC mRNA ⇑ 웁-MHC mRNA No change in actin mRNA ⇓ RXR 웂 protein ⇓ Thyroid receptor 웁1 protein ⇓ 웁1AR mRNA No change in BARK1 mRNA

⇑ Proenkephalin mRNA ⇑ ⇑ ⇑ ⇑ ⇓ ⇓

Collagen mRNA Fibronectin mRNA AT1R mRNA Atrial natriuretic peptide mRNA Induction of immediate early genes Activation of HSF

a SR, sarcoplasmic reticulum; SERCA, sarco/endoplasmic reticulum calcium ATPase; Ca2⫹, calcium ions; MHC, myosin heavy chain; mRNA, messenger RNA; RXR, retinoid X receptor; BAR, 웁-adrenergic receptor; BARK, 웁-adrenergic receptor kinase; G1 , inhibitory G-protein; TNI, troponin-I; ICa , calcium influx; Cai , intracellular calcium concentration; HSF, heat shock factor; RYR2 , cardiac ryanodine receptor; AT1R, angiotensin AT-1 receptor; RAS, renin–angiotensin system; Acyclase, adenylate cyclase.

of rigorously screened, healthy BLSA volunteers, however, indicate that in the sitting position, the heart rate decreases slightly with age in both men and women (Fleg et al., 1995). The respiratory variation of the resting heart rate becomes diminished with advancing age, as does the spontaneous variation in heart rate measured over a 24-hr period via Holter monitoring or via spectral analysis (Byrne et al., 1996). The decreased variation in heart rate is thought to be influenced by age-associated changes in both parasympathetic and sympathetic modulation. Whether the intrinsic sinus rate, i.e., that measured in the presence of both sympathetic and parasympathetic blockade, is diminished significantly with age is unknown. A modest prolongation of the P-R interval occurs with aging in healthy BLSA individuals (Fleg et al., 1990) and is localized to the proximal P-R segment, probably reflecting delay within the atrioventricular junction. An increase in both supraventricular and ventricular premature beats occurs in healthy older BLSA men and women compared to their younger counterparts (Fleg et al., 1982).

2. Diastolic Function The peak rate at which the LV fills with blood during early diastole is reduced markedly (50%) with aging between the ages of 20 and 80 years. Despite the popular notion that LV compliance decreases with aging, this parameter, in fact, has not been measured in healthy humans, as simultaneous measurements of pressure and volume at end diastole have not been made. Whether atrial or LV pressures during the early filling period or at end diastole differ in healthy younger and older individuals is also presently unknown. The time course of isovolumic myocardial relaxation, measured noninvasively as the time between aortic valve closure and mitral valve opening, becomes prolonged (40% increase) with adult aging in both men and women. While the reduced early filling rate with aging may be due, in part, to ageassociated alterations in LV wall structure or passive properties, it is likely that reduced early filling also reflects, in part, the age-associated prolonged relaxation phase of cardiac muscle contraction (see later). Asynchrony of relengthening among ventricular segments

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VIII. Developmental Changes and Aging

also increases with aging and contributes to the reduction in the filling rate. The age-associated reduction in the early filling rate does not result in a reduced end diastolic volume in healthy older individuals, as greater filling occurs later in diastole, particularly during the atrial contraction (Swinne et al., 1992). The enhanced atrial contribution to ventricular filling with advancing age is associated with left atrial enlargement (Gerstenblith et al., 1977) and a more forceful atrial contraction, which is the basis of an audible fourth heart sound in most healthy older individuals. 3. Myocardial Contractile Function and Cardiac Pump Performance and Output Because an interaction of multiple factors regulates cardiac performance, the intrinsic myocardial contractile function cannot be easily and unequivocally determined noninvasively in situ. However, a crude index of myocardial contractility, the ratio of end systolic arterial pressure to end systolic volume (ESVI), is not reduced at rest with age in either healthy BLSA men or women. The LV ejection fraction (LVEF) is also not altered with aging in healthy BLSA men or women at rest. The seated, resting stroke volume indexed to body size (SVI) in BLSA males in increased due to a slight increase in the LV end diastolic volume index (EDVI). Thus, in healthy older BLSA men, although, as noted earlier, the upright, seated resting heart rate decreases slightly, the cardiac output indexed to body size (CI) is not reduced because SVI is increased due to end diastolic dilatation (Fleg et al., 1995). In contrast to men, CI in the sitting position at rest is decreased slightly in older versus younger healthy BLSA women, as neither resting EDVI nor SVI increases with age to compensate for the modest reduction in heart rate (Fleg et al., 1995). These gender differences appear, in part, to be due to differences in body composition and thus in demand for blood flow in men and women. Specifically, the apparent gender difference in CI, in part, may be an artifact of normalization of cardiac output to body surface area, as the proportion of body fat increases with age in women to a greater extent than in men and is not accounted for in this normalization.

D. Cardiac Muscle Function with Aging in Rodents Most studies of intact rodents have observed that the heart rate decreases with age but that heart function is largely maintained, except the response to pressor stress, until very late in the life span, when disease processes originating in other organs may conspire with undefined aging processes to compromise function.

Most insights of the effect of age on the regulation of intrinsic cardiac muscle performance come from studies of isolated hearts or cardiac muscle cells isolated from the hearts of rodents. Coordinated changes in several key steps of excitation–contraction coupling and its regulation by cell surface receptor stimulation occur with aging (Table I). In general, the kinetics of cellular reactions that underlie the heart beat are reduced in the senescent versus the younger adult. In rodent cardiac muscle, the action potential, the transient increase in cytosolic Ca2⫹, Cai , evoked by the action potential, and the contraction are all prolonged with aging (Fig. 1; Orchard et al., 1985). The ionic basis of the action potential prolongation with aging has been studied in single cardiac myocytes. While the resting membrane potential is unaltered with adult aging, the repolarization of the action potential is consistently prolonged in left ventricular myocytes isolated from senescent rat hearts compared with those from younger hearts (Walker et al., 1993). The prolongation of the action potential can be attributed to either a decrease in the outward currents, i.e., inwardly rectifying (IK1), transient outward (Ito), or sustained (IK) K⫹ current, and/or an increase in the inward currents, i.e., L-type Ca2⫹ current (ICa,L) or inward Na⫹ –Ca2⫹ exchange current (INa-Ca). Although the magnitude of IK1 or IK does not change with aging (Walker et al., 1993), there is a significant decrease in peak Ito density at a late stage of the life span, which

FIGURE 1 Typical transmembrane action potentials (A) and simultaneously measured isometric contractions (B) in right ventricular papillary/muscles isolated from adult (7-month) and senescent (24month) rat hearts. Redrawn from Wei et al. (1984). (C) Aequorin luminescence in representative cardiac muscles from adult (6-month) and senescent (24-month) rats. From Orchard and Lakatta (1985). (D) Velocity of Ca2⫹ accumulation in isolated sarcoplasmic reticular vesicles decreases with adult age. From Froehlich et al. (1978).

42. Aging of the Cardiovascular System

may significantly prolong the repolarization of the action potential (Walker et al., 1993). It has been shown that the increase in the thyroid hormone level after birth may lead to the developmental switch of K⫹ channel isoforms and subsequently to an increase in Ito density and a decrease in action potential duration. Because advanced age is associated with changes in the thyroid hormone level or its related receptors (Long et al., 1999) and the aged heart recapitulates the fetal phenotype in many aspects (see later), whether K⫹ channels undergo a similar switch back to the fetal isoforms merits further study. During aging, the magnitude of the L-type Ca2⫹ channel current (ICa,L) becomes significantly increased, in parallel with the enlargement of cardiac myocytes, resulting in a unaltered ICa,L density (Walker et al., 1993; Xiao et al., 1994). However, the inactivation of ICa,L is significantly prolonged, which produces a larger Ca2⫹ current integral and thus a greater Ca2⫹ influx via ICa,L in senescent vs adult myocytes (Walker et al., 1993). Prolongation of the action potential duration with aging may also relate to a prolonged Cai transient (see later), as Ca2⫹ extrusion via the Na⫹ –Ca2⫹ exchanger during the time of action potential repolarization produces an inward current and prolongs the action potential repolarization time of rat cardiac cells. The Cai transient that follows sarcolemmal depolarization is governed by several Ca2⫹ cycling processes involved in cardiac excitation–contraction coupling, e.g., Ca2⫹ influx and efflux through the sarcolemmal L-type Ca2⫹ channel and Na⫹ –Ca2⫹ exchanger, Ca2⫹ release and uptake by the sarcoplasmic reticulum (SR) Ca2⫹ release channel (ryanodine receptor, RyR), and the Ca2⫹ pump. Like most other alterations during aging, those in Ca2⫹ cycling in aged heart are not just a general degeneration but relate to specific changes in gene expression level, protein level, and function. Ca2⫹ influx via sarcolemmal L-type Ca2⫹ channels is increased due to the slower inactivation of ICa,L and prolongation of action potential, but the Cai transient amplitude, which is mainly determined by Ca2⫹-induced Ca2⫹ release from SR, is not altered by age (at least at low stimulation frequencies). Because both mRNA and protein levels of the cardiac ryanodine receptor (RyR2) remain constant over a broad age range (4–24 months) in rat heart (Xu et al., 1998), the inability of a larger Ca2⫹ influx through the L-type Ca2⫹ channel to elicit a larger Cai transient in the senescent heart may be attributed to the altered function of the normally expressed RyR2, to the disturbance of the architecture in the local response elements (suboptimal spacing between L-type Ca2⫹ channel and RyR2) in enlarged senescent myocytes, as is the case in some hypertrophied and failing myocytes, or to a reduction in the SR Ca2⫹ load. A most striking finding in aged heart is the downregulation of mRNA abun-

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dance and the density of the cardiac SR Ca2⫹ pump (SERCA2a) (Tate et al., 1996; Taffet et al., 1993), which is associated with a diminished SR Ca2⫹ sequestration rate (Fig. 1) and prolongation in Cai transient (Tate et al., 1996; Taffet et al., 1993). In contrast, there is no agerelated difference in the amount of the SR Ca2⫹ pump regulatory protein, phospholamban (PLB), or the SR Ca2⫹ storage protein, calsequestrin (Xu et al., 1998). It is of note that in skeletal muscle the function of DHPR and RyR1, as well as SR Ca2⫹ uptake, is diminished during aging. It is also noteworthy that, in the hearts of exercise-trained old rats, the decay of the Cai transient is accelerated (Gwathmey et al., 1990, Taffet et al., 1996), the rate of SR Ca2⫹ uptake is enhanced along with increases in both mRNA and protein level of SERCA2, and relaxation is accelerated as compared to old sedentary rat controls (Tate et al., 1996). Further, because RyR and PLB are known to be modulated by phosphorylation, alterations of Ca2⫹ release and sequestration in the aged cardiomyocyte, especially during stress, may be due to the age-associated dysfunction of neurohormonal regulation (see later) and/or downregulation of Ca2⫹, calmodulin-dependent protein kinase (CaMKII) (Xu et al., 1998). The cardiac Na⫹ –Ca2⫹ exchanger (NCX1) serves as the main transsarcolemmal Ca2⫹ extrusion mechanism. It has been suggested that the Na⫹ –Ca2⫹ exchanger is more active in ejecting Ca2⫹ from cells of older versus younger hearts during diastole, and an increased exchanger expression may compensate partly for a reduced SR pump function. The supporting evidence is that the abundance of cardiac Na⫹ –Ca2⫹ exchanger transcripts increases 앑50% in senescent (24 month) compared to the adult (6 month) rat heart. Because both SERCA2 and NCX genes are shown to be regulated by the thyroid hormone, the alterations mentioned earlier may also be related to the age-associated change in thyroid hormone and its related receptors (see later). While the decay rate of the Cai transient decreases with aging, there are no data to indicate that the cytosolic [Ca2⫹] at end diastole changes with age. The altered pattern of Ca2⫹ regulation in the older heart permits a prolonged and efficient force bearing capacity enabling the continued ejection of blood during late systole, a beneficial adaptation with respect to enhanced vascular stiffness and early reflected pulse waves (see later; (Vaitkevicius et al., 1993; Lakatta, 1993b; Fleg, unpublished observations). However, altered Ca2⫹ handling in the older heart renders it more prone to spontaneous Ca2⫹ oscillations and Ca2⫹-dependent arrhythmias (Hano et al., 1995). Myofilaments, the end effectors of cardiac E-C coupling, are also strikingly modified during aging. Although the Ca2⫹ sensitivity of myofilament ATPase ac-

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VIII. Developmental Changes and Aging

tivity does not change with adult age, the Ca2⫹-activated ATPase activity of purified myosin preparations declines progressively throughout the entire age range. This ATPase activity is modulated, in part, by the myosin heavy chain (MHC) isoform composition of the myocardium. Both mRNA and protein levels of 움-MHC (V1 isoform) decrease, whereas those of 웁-MHC (V3 isoform) increase during adult aging (Fig. 2). Because 움-MHC has a higher ATPase activity than 웁-MHC, the switching of MHC isoforms with advancing adult age may underlie, in part, the decline in the velocity of shortening in lightly loaded isotonic contractions with aging and may also be related to the prolongation of

time to peak tension in isometric contractions in cardiac muscle from older versus young adult rat. It is noteworthy that 웁-MHC is energy efficient so that the switching of MHC isoforms, however, may be considered as a compensatory mechanism and helpful in maintaining cardiac function for the aged heart. There is no detectable change in other myofilament proteins such as troponin I, troponin T, tropomysin, C protein, myosin light chain, and actin in older rat heart. As discussed previously, gene expression of several major effectors in cardiac E-C coupling such as MHC, SERCA, NCX, and probably some K⫹ channels are regulated, in part, by thyroxine. For example, both

FIGURE 2 (A) Average values for 움 and 웁 myosin heavy chain mRNA/18S rRNA of

individual hearts measured by dot blot analysis (n ⫽ 11, 6, 10, and 10 for ages 6 weeks and 6, 18, and 24 months, respectively). From O’Neil et al. (1991). (B) 움 and 웁 myosin heavy chain proteins (V1 and V3 isoforms) of hearts of the same rat strain. From Effron et al. (1987). (C) Ca2⫹-activated myosin ATPase activity of Wistar rat hearts decreases with age. From Effron et al. (1987). (D) The velocity of shortening during lightly loaded isotonic contractions in isolated cardiac muscle from younger and older rats decreases with aging. From Capasso et al. (1983). (E) Left ventricular actin isoforms (cardiac versus skeletal) do not change with aging in the Wistar rat. From Carrier et al. (1992).

42. Aging of the Cardiovascular System

움- and 웁-MHC genes have thyroid hormone responsive elements (TREs) in the 5⬘-flanking region. MHC gene expression is regulated, in part, by thyroxine via the binding of thyroid hormone receptors (THRs) to thyroid response elements (TREs) in the 5⬘-flanking regions of the genes. Binding of THRs to the TRE of 움-MHC upregulates its expression, whereas binding to 웁-MHC TRE inhibits transcription. THRs are members of a superfamily of receptors that include retinoic acid receptors (RARs), vitamin D receptors, and retinoid X receptors (RXRs). In many cases, RXR–THR heterodimers bind with much higher affinity than THR homodimers to TREs. It might be postulated, therefore, that a reduction in the number of THRs or RXRs might contribute to the downregulation of 움-MHC with age. In fact, a significant reduction (approximately 50%) between 6 and 24 months has been observed in the levels of THR웁1 and RXR웂 proteins in hearts of senescent rats (Long et al., 1999). Levels of mRNA encoding these receptor subtypes are similarly depressed in hearts of older rats, suggesting that the age-associated decreases in THR웁1 and RXR웂 are regulated transcriptionally.

E. Vascular Structure and Function in Humans at Rest Vascular changes occur with aging in sedentary BLSA volunteers considered to be otherwise healthy (Fig. 3). The large elastic arteries become dilated and exhibit an increase in intimal–medial thickness (Figs. 3A and 3B). The formation of a thicker intima is believed by some epidemiologists to herald preclinical features of the atherosclerotic process. Diffuse age-associated changes occur within the media of conduit arteries. Chemical analyses indicate a relative decrease of elastin and an increase of collagen. The glycoprotein component of elastic fibrils decreases and eventually disappears. It has been hypothesized that both an increased elastase activity with aging, as observed in aortae isolated from humans, and an age-associated increase in Ca2⫹ and cholesterol deposition on elastin, rendering the latter more susceptible to elastase activity, contribute to elastin fragmentation or reduction in its content with aging. Although these intramural processes cannot be explained readily on the basis of atherosclerosis, they may contribute to the age-associated increases in the risk for atherosclerosis due to their influence on the mechanical properties of the vascular wall. Whereas the macroscopic changes in vascular cells and matrix of large vessels in humans have been well described, the specific molecular mechanisms that lead to vascular stiffening and a thickened intima remain to be elucidated. Dilation of large arteries by the relaxation of smooth muscle tone

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following 웁-adrenergic stimulation, and via endothelial mediated signals, becomes reduced with aging. The just-described age-associated changes in matrix structure and smooth muscle tone lead to arterial stiffening, which leads to an enhanced pulse wave velocity (Fig. 3C) and to early reflected pulse waves that produce a late augmentation in central systolic arterial pressure (Fig. 3D). Diastolic pressure amplification due to the normal occurrence of reflected waves in diastole becomes reduced in older individuals, which may have substantial clinical implications when the occurrence of coronary atherosclerosis and cardiac hypertrophy, common comorbidities of diseases in older individual hypertensives, jeopardizes coronary flow. As a result of arterial stiffening and early reflected pulse waves, the average systolic blood pressure within a healthy, normotensive population increases (within the normal range by clinical convention) with aging, whether measured in a cross-sectional study design or longitudinally. Many individuals show little or no longitudinal increase in systolic pressure and thus age-associated increases in blood pressure are neither universal nor inevitable. The average increase in diastolic pressure with aging is modest and is not as marked as the average increase in systolic pressure. The age-associated increase in diastolic pressure appears to plateau around midlife and may decrease thereafter due to a continued increase in large vessel stiffness and reflected pulse waves. (Reflected pulse waves due to vascular stiffening act to reduce diastolic pressure, all else equal.) However, the late decline in diastolic pressure to the end diastolic level, in large part, reflects the properties peripheral vascular resistance (PVR). An increase in PVR accompanies aging in some, but not all, studies in the literature and may, in part, be secondary to a reduction in skeletal muscle mass with aging and its concomitant reduction in capillary density. In carefully screened healthy BLSA men PVR, measured at rest, increases minimally with aging in men and modestly in women (Fig. 3E; Fleg et al., 1995). 1. Vascular Component of LV Afterload For a given EDV the SV is determined by the extent to which the heart empties, as reflected in the ESV. The ESV is determined, in part, by the cardiac afterload, or opposition to blood flow from the heart, and the level of myocardial contractility or effectiveness of excitation–contraction coupling within cardiac cells. The cardiac afterload originates from both the vasculature and the heart itself. The vascular afterload is made up of four components: arterial elastance, inertance, reflectance, and resistance. The altered characteristics of the vasculature with aging (Fig. 3; Gerstenblith et al.,

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VIII. Developmental Changes and Aging

FIGURE 3 Changes in elastic artery structure and function during aging in healthy normotensive humans. (A) Carotid artery wall thickness increases with advancing age in healthy men and women of the Baltimore Longitudinal Study of Aging (BLSA) who are free from coronary disease. From Nagai et al. (1998). (B) Aortic root diameter, measured via M-mode echocardiography, increases with age in healthy BLSA men and women. From Gerstenblith et al. (1977). (C) Aortic pulse wave velocity, an index of aortic stiffness, increases with age in healthy BLSA participants. From Vaitkevicius et al. (1993). (D) A late peak in systolic pressure occurs with aging in healthy BLSA individuals and leads to an increase in the augmentation index (AGI) of the carotid pulse pressure. This late augmentation of systolic pressure is attributable to early reflected pulse waves caused by the increasing aortic stiffness with increasing age. From Vaitkevicius et al. (1993). (E) Systemic vascular resistance at rest, derived from cardiac output, measured via gated blood pressure and mean arterial pressure in healthy BLSA men and women. From Fleg et al. (1995).

1977; Fleg, unpublished observations), i.e., reduced arterial compliance, early reflected pulse waves, and, to a lesser degree, increases in PVR, increase the vascular load faced by the heart during each beat. Additionally, as the static column of blood within the aorta at the time at which the aortic valve opens must be accelerated prior to blood ejection from the heart, there is also an

inertial component to the vascular load. Like the other three components, this, too, is altered during aging, as the dilated aorta and large conduit arteries contain a greater volume of blood to be accelerated at the onset of left ventricular ejection of each heart beat. A cardiac component of ventricular afterload is determined by the ventricular size throughout the cardiac

42. Aging of the Cardiovascular System

cycle. As noted in the upright seated position, the EDVI is increased modestly (15%) in healthy sedentary BLSA men but not in women. The ESVI at rest is also increased modestly in men but not in women. The aforementioned age-associated vascular changes (Fig. 3; Vaitkevicius et al., 1993; Fleg, unpublished observations) fall below the present ‘‘clinical threshold’’ for classification as risks for disease and are thus presently perceived as ‘‘normal aging’’ by the medical profession. There is mounting evidence that age-associated increases in arterial stiffness and pressure can be modified by lifestyle or diet. The sodium sensitivity of arterial pressure increases with aging, and many epidemiologists believe that a difference in dietary NaCl accounts for at least some of the differences in blood pressure changes with aging observed among different populations. Additionally, when dietary NaCl salt is reduced, the expected age-associated increase in pulse wave velocity, an index of aortic stiffening, is not observed. Physical conditioning also appears to lessen the vascular stiffening associated with aging, e.g., the augmentation of the late systolic peak in arterial pressure, an index of arterial stiffness, is related inversely to aerobic capacity in sedentary BLSA volunteers; this arterial stiffness index increases only about half as much in senior, endurance trained athletes as it does in sedentary, agematched controls. Therefore, similar to a chronic reduction of NaCl intake, regular exercise may exert a modulatory effect on the stiffening of large arteries with aging.

F. Vascular Changes with Aging in Rodents 1. Vascular Changes in Vivo Unlike cardiac myocytes, vascular smooth muscle and endothelial cells are not terminally differentiated. Vascular smooth muscle cells are subject to phenotypic modulation, during which they revert to a proliferative, secretory and migratory mode and undergo modifications of their contractile apparatus and control of Ca2⫹ cycling by cell surface receptors. This ‘‘modulated’’ vascular smooth muscle phenotype repairs vascular damage and participates in vascular pathologies such as hypertension and atherosclerosis. Age-associated macroscopic changes within large blood vessels in rodents are similar in many ways to those that occur in humans (Table II). Aortic remodeling during aging in rodents consists of dilation, medial thickening, and formation of an intima. Both in vivo and in vitro studies indicate that the aorta stiffens with advancing age. Chronic morphological and biochemical modifications in the aortic intima of aging rats, i.e., fragmentation of the internal elastic membrane and intimal thickening and localized increases in growth factors and

745

TABLE II Changes in the Aorta with Aging in Rodents Diameter ⇑ Wall thickness Intima ⇑ Thickness; VSM cells and matrix ⇑ TGF웁; ⇑ MMPII levels and activity ⇑ ICAM expression Media ⇑ Thickness VSM cells ⇑ Size; ⇓ number Change in intracellular intermediary filaments Matrix ⇑ Collagen amount; ⇑ cross-linking 2⬚ nonenzymatic glycation ⇑ Fibronectin Elastin; fragmented and calcified Exaggerated wound repair response

collagenase activity, appear as a muted version of those chronic alterations associated with chronic hypertension or with transient changes that occur in response to acute mechanical injury, e.g., following balloon angioplasty. The thickened intima in older rats is composed of matrix molecules, including collagen and proteoglycan, and VSM cells and contains markedly higher levels of MMP-2, TGF-웁, and ICAM-1 than younger vessels (Li et al., 1997). Intimal growth during aging in the absence of experimental injury, in some ways, resembles neointimal formation in response to arterial balloon catheterinduced injury. In fact, neointimal growth in response to endothelial injury is enhanced markedly in old vs young rats and this response is due to factors intrinsic to the vessel wall. It has been shown that PDGF-웁 receptor mRNA in rat aorta increases with aging. Additionally, both latent and activated forms of MMP-2 are greater in the aortae of old than in young rats. The cytokine TGF-웁 accumulates in the same regions of intima of old rats as MMP-2. TGF-웁 suppresses protease activity and activates tissue inhibitors of metalloproteinase, is a potent factor for the synthesis of extracellular matrix porteins, and its expression can lead to excessive fibrosis. Accumulation of TGF-웁 in the aortic wall of aged rats occurs with adult aging and may account for a concomitant increase in fibronectin, which itself has a diverse effect on SMC phenotype properties. Some evidence indicates that the collagenolytic and antiproliferative actions of TGF-웁 decrease with aging. Mutations in the R2 TGF-웁 receptor in atheroslcerosis, as in neoplasia, appear to play a role in a reduction in TGF-웁 proliferative effects with a concomitant shift to increased fibroproliferative effects. It is noteworthy that both fibronectin and TGF-웁 expression are regulated by angiotensin II. That chronic administration of an angiotensin-converting enzyme inhibitor substantially

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VIII. Developmental Changes and Aging

reduces and delays the arterial stiffening, matrix, and intimal changes with aging or hypertension suggests that age-associated changes in the vascular angiotensin regulation may have a role in the age-associated changes observed in TGF-웁 and fibronectin. A regional increase in ICAM-1 observed in the intima of aged aortae might also be related to the augmented levels of TGF-웁, as the later is known to induce the synthesis of cell adhesion receptors, which may lead to increased adhesion and interaction of cells with the surrounding extracellular matrix. Prior studies have shown that human endothelial cell senescence in vitro is accompanied by increased mRNA and protein of ICAM and an enhanced capacity to bind monocytes. Ample evidence indicates age-associated discontinuities of the internal elastic lamina in the aorta in the absence of externally imposed experimental injury. Degradation of elastin by elastases and gelatinases acting as elastase may contribute to such elastin membrane breaks. Both MMP-2 and MMP-9 (another type IV gelatinase) exhibit elastase activity, as does metalloelastase cloned from macrophages. In the aged aortae, MMP-2 accumulates in the area surrounding SMCs located in the vicinity of breaks in the internal elastic lamina and along elastic laminae throughout the media (Li et al., 1997), suggesting that MMP-2, in addition to possibly being implicated in the migration of SMC as noted earlier may also have a role in fragmentation of the elastic laminae with aging.

III. CARDIOVASCULAR RESERVE CAPACITY A. Seated Upright Dynamic Exercise Response in Humans The peak work rate and oxygen consumption of healthy, sedentary BLSA men and women during upright, seated cycle ergometry declines approximately 50% with advancing age between 20 and 90 years and is attributable to approximate declines of 30% in cardiac output and 20% in oxygen utilization. The ageassociated decrease in peak cardiac output is due entirely to a reduction in heart rate, as the stroke volume does not decline with age in either gender (Fig. 4). However, the manner in which stroke volume is achieved during exercise varies dramatically with aging. The EDVI increases during vigorous exercise in older but not in younger men and women (Fig. 5A). However, because the ESV in older persons fails to become reduced to the same extent as in their younger counterparts (Fig. 5B), the EF decreases (Fig. 5C) and the SV is not greater in older vs younger persons. In other words, whereas the Frank Starling mechanism is utilized

FIGURE 4 The age-associated decline in cardiac index during peak cycle exercise in healthy BLSA men and women is due to a reduction in peak heart rate, as the stroke volume index (normalized to body surface area) does not decline with aging. From Fleg et al. (1995).

in older persons during exercise, its effectiveness is reduced due to a failure for the LV in older persons to empty to the extent to which it does in younger ones. Thus, the older heart, while contracting from a larger preload at all levels of exercise than the younger heart, does not deliver a stroke volume that exceeds that of the younger heart (Fig. 6). The deficiency in LV ESV reduction during exercise in healthy older individuals can result from an ageassociated decrease in the myocardial contractility or to an increase in afterload. While the pulsatile determinants of ventricular afterload (Fig. 3; Vaitkevicius et al., 1993; Lakatta, 1993b; Fleg, unpublished observations) are increased with aging at rest, they have not been characterized during exercise with respect to age. However, the PVR decline during exercise is less in healthy older vs healthy younger men and women (Fleg et al., 1995). The index of myocardial contractility (ESVI/ SBP), while unchanged with aging at rest, during vigorous exercise decreases with age (Fig. 5D). Finally, the LV response to a pressor stress in the presence of 웁-adrenergic blockade becomes reduced with aging, suggesting an age-associated decline in intrinsic myocardial contractility (Yin et al., 1978).

B. Acute 웁-Adrenergic Modulation of Cardiovascular Performance in Humans The exercise hemodynamic profile of elderly individuals in Fig. 5 and 6 is strikingly similar to that of younger subjects who exercise in the presence of a 웁-adrenergic

42. Aging of the Cardiovascular System

FIGURE 5 End diastolic (A) and end systolic (B) volume indices during peak upright cycle exercise in healthy men and women. From Fleg et al. (1995). The left ventricular contractility index systolic blood pressure (SBP)/end systolic volume index (ESVI) (C) and ejection fraction (D) at seated rest and during peak upright cycle exercise in healthy BLSA males of a broad age range.

FIGURE 6 Stroke volume index as a function of end diastolic volume index at rest (R) and during graded cycle workloads (numbers) to the maximum workload (max) in the upright seated position in healthy BLSA men in the presence and absence of 웁-adrenergic blockade affected by propranolol. From Fleg et al. (1994, 1995).

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VIII. Developmental Changes and Aging

blockade. Age-associated differences in the SVI vs LV EDVI relationship (Fig. 6), in the LV early diastolic filling rate, (Schulman et al., 1992), and in heart rate (Fleg et al., 1995) during exercise in BLSA subjects are abolished when this exercise is performed during a 웁-adrenergic blockade (Fleg et al., 1994). In contrast, the maximum heart rate that can be elicited by external electric pacing, which is far in excess of that elicited by isoproterenol infusion, is not age associated. Thus, one of the most prominent changes in the cardiovascular response to exercise stress that occurs with aging in healthy BLSA subjects vigorously screened to exclude occult disease and highly motivated to perform exercise is a reduction in the efficacy of the 웁-adrenergic modulation of cardiovascular function. The consensus view is that resting sympathetic nervous activity increases progressively with aging, although there is no age-related difference in nerve firing rates in skeletal muscle sympathetic efferents in response to the application of stressors in older persons. Plasma levels of norepinephrine and epinephrine increase with age due to enhanced spillover into the circulation and to reduced clearance (Fleg et al., 1985; Esler et al., 1995). The increased spillover does not occur from all body organs, but is increased within the heart and is thought to be due, at least, in part, to a reduced

reuptake following release. The net result is likely an enhanced postsynaptic receptor occupancy by the neurotransmitter, leading to receptor desensitization. Deficits in 웁-adrenergic signaling with aging are attributable, in large part, to a reduced postsynaptic response (Lakatta, 1993). Abundant evidence indicates that myocardial, vascular, and heart rate responses to infusions of 웁-adrenergic agonists decline with age (Lakatta, 1993). 웁-adrenergic receptor stimulation elicits less of an increase in ejection fraction in healthy older men compared to young men (Fig. 7B) and to a reduced in contractile augmentation in cardiac muscle (White et al., 1994) isolated from human hearts (Figs. 7C and 7D). The effect of bolus infusions of 웁-adrenergic agonists to increase the heart rate diminishes with advancing age in humans (Fig. 7A) and in experimental animals.

C. Acute 웁-Adrenergic Modulation of Cardiac Cell Function in Rodents In contrast to humans, a series of in vitro studies in rat cardiac synaptosomes have demonstrated an ageassociated reduction of NE release in response to an increase in intracellular [Ca2⫹] by electrical stimulation, high extracellular [Ca2⫹]o or high [K⫹]o , but not in re-

FIGURE 7 (A) The response of heart rate in healthy BLSA men to incremental concentrations of the 웁-adrenergic agonist, isoproterenol. (B) The response of ejection fraction to isoproterenol in healthy males. From Stratton et al. (1992). (C) The augmentation of tension development by isoproterenol in left ventricular trabeculae from donor hearts that were not transplanted is reduced with aging. From White et al. (1978). (D) The effect of isoproterenol in myocytes isolated from young and old human hearts removed during transplantation classified by the New York Heart Association (NYHA) status of the donor. From Harding et al. (1992).

42. Aging of the Cardiovascular System

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FIGURE 8 (A) The effect of norepinephrine on the maximum rate of isometric tension development in isolated trabeculae from hearts of varying age. From Lakatta et al. (1975). (B) Velocity of cell shortening and (C) the maximum rate of increases of the Indo-1 fluorescence transient, an index of sarcoplasmic reticulum Ca2⫹ release into the cytosol, during the electrically stimulated twitch in single cardiac myocytes isolated from the hearts of rats of varying ages and loaded with the fluorescent probe, Indo-1. From Xiao et al. (1994). (D) The effect of norepinephrine in increasing the L-type sarcolemmal channel current (Ica) across a range of activating steps to different membrane potentials in single vascular cells declines with aging; the norepinephine concentration was 1 ⫻ 10⫺7 M. From Xiao et al. (1994). (E) Effect of norepinephrine (NE) in increasing the phosphorylation of troponin I (TNI) in suspensions of heart cells isolated from hearts of rats of varying age. From Sakai et al. (1989).

sponse to calcium-independent stimulation by tyramine. The mechanism underlying the species-dependent difference in cardiac NE spillover and reuptake is presently unclear. Studies in isolated LV muscle and in individual rat ventricular cardiocytes (Figs. 8A and 8B) indicate that, similar to humans, a reduced contractile response to 웁AR stimulation also occurs with aging. A deficient response to 웁1AR stimulation in cardiac myocytes from senescent rats is due to a failure of the intracellular Ca2⫹

transient to increase to the same extent to which it increases in cells from younger adult hearts (Fig. 8C). The blunted increase in the Ca2⫹ transient in cells from the aged heart is attributable to a decrease in the ability of 웁1-AR stimulation to increase L-type sarcolemmal Ca2⫹ channel availability versus that in younger adult heart cells (Fig. 8D). These deficits in cardiac cell function in response to 웁1-adrenergic receptor stimulation are caused by deficits in the receptor-linked signaling pathway.

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D. Age-Associated Alterations in Cardiac 웁-AR Signaling Functional and radio-ligand binding studies have shown that both 웁1-AR and 웁2-AR coexist in cardiac myocytes of many mammalian species, including human, although 웁1-AR dominates under normal conditions (Xiao et al., 1993; Altschuld et al., 1995). Since the early 1980s, a large body of evidence has demonstrated that the cardiac response to 웁-AR stimulation decreases in aged hearts and that there is a positive correlation between increased plasma catecholamine levels and the degree of diminution of the 웁-AR response. Mechanistic studies that have focused on the classic ‘‘receptor-Gsadenylyl cyclase-cAMP-PKA’’ signaling cascade have observed multiple defects in this signaling pathway. However, evidence indicates that whereas 웁1-AR stimulation couples only to the Gs-mediated signaling cascade, 웁2-AR dually couples to Gs and pertussis toxin (PTX)-sensitive inhibitory G-proteins (Gi) and that the coupling of 웁2-AR to Gi protein functionally opposes the Gs-mediated contractile response (Zhou et al., 1997). In addition, G-protein-coupled receptor kinases (GRKs) play an important role in 웁-AR desensitization (Anversa et al., 1990). Thus, new information on the potential involvement of negative regulators of 웁-AR signaling, e.g., Gi and GRKs in age-associated alterations in 웁-AR responsiveness, has begun to emerge. 1. ␤-AR Density There is general consensus that total 웁-AR density in cardiomyocytes or myocardium of both human and animals becomes reduced with aging. However, whether aging causes a selective downregulation of myocardial 웁-AR subtypes (웁1 vs 웁2) is controversial. In human explanted ventricles, a significant age-associated reduction in 웁1-AR without a loss in 웁2-AR has been reported; in contrast, in human right atrium, the density of total 웁-AR is not changed with aging. Similarly, in rat hearts, some studies have shown that the density of 웁1-AR is selectively decreased with aging in the absence of changes in the 웁2-AR, whereas other studies have shown that both 웁1-AR and 웁2-AR are nonselectively decreased by 앑30% in aged rat ventricular myocytes and myocardium. 2. Changes in Cardiac Adenylyl Cyclase Activity with Aging The defects of 웁-AR signaling occur not only at the receptor level, but also at postreceptor signaling steps, as evidenced by the fact that cardiac adenylyl cyclase responses to the stimulation of both 웁-AR subtypes, as well as to NaF (a G-protein activator) or to forskolin (a

diterpene derivative of the India plant Coleus forskolin, which activates adenylyl cyclase in a G-protein-sensitive manner), are decreased markedly with aging. Moreover, the number of cardiac catalytic units and/or the capacity to activate the catalytic units clearly decreases with aging (Scarpace, 1990; Bohm et al., 1993), which appears to be responsible for the age-associated decrease in the maximal 웁-AR-stimulated adenylyl cyclase activation. 3. Changes of Gs and Gi Proteins with Aging It is well established that sympathetic and parasympathetic stimulation, the most important cardiac regulatory systems, are mediated by adenylyl cyclase stimulatory G-proteins (Gs) and inhibitory G-proteins (Gi), respectively. Thus, an increase in Gi or a decrease in Gs or an imbalance between Gs and Gi might contribute to the overall age-associated suppression of cardiac 웁AR modulation. In human hearts, a significant decrease in cholera toxin (CTX)-mediated Gs ribosylation (White et al., 1994) and Gs mRNA has been observed in some studies, whereas others have not found a change in Gs abundance, as assayed by Western blot or CTX-catalyzed ribosylation during aging. Similarly, whether Gi is changed with aging is also controversial. In human ventricles, neither Gi abundance nor PTX-mediated Gi ribosylation is increased with aging (White et al., 1994), but more recent studies in human right atrium show that Gi amount is correlated negatively with the age of patients in the absence of a change in Gs (Brodde et al., 1995). In rat ventricles, an age-associated Gs gene expression has been observed (Bohm et al., 1993). Some studies in rat hearts have shown that both Gi protein level and PTX-mediated Gi ribosylation increase with aging, but others have shown that Gi is apparently not altered with aging. More recent studies have found no evidence for an age-associated difference in the abundance of membranous G움i, suggesting that the age effect on 웁-AR signaling is not caused by an alteration in PTX-sensitive G-protein (Gi /Go) abundance or functional activity (Xiao et al., 1998). 4. ␤ARK1 and GRK5 Abundance or Functional Activity in Aged Hearts Several characteristics are shared by both the aged and the failing heart in human and animal models. In both states, deficits in 웁-AR-mediated contractile response are associated with significant increases in circulating catecholamines (Lakatta, 1993b). It is well accepted that a prolonged agonist stimulation desensitizes 웁-ARs. In chronic heart failure in humans and animals, the severity of the heart failure is closely correlated to an increase in 웁ARK1, a dominant cardiac GRK. Thus,

42. Aging of the Cardiovascular System

it is reasonable to assume that 웁-AR desensitization might be involved in the age-associated cardiac 웁-AR dysfunction. In this regard, it has been proposed that 웁-ARs in aged animals may be already partially desensitized under basal conditions (Scarpace, 1988). However, more recent studies in both rat ventricular myocytes and myocardium have shown that neither the enzymatic activity nor the protein or mRNA levels of 웁ARK1 or GRK5 are altered with aging (Xiao et al., 1998). It is noteworthy in this regard that in failing hearts in both humans and animals the amount and activity of Gi proteins are consistently found to be markedly elevated, providing additional evidence to indicate that distinct mechanisms may be involved in the desensitization of 웁-adrenergic signaling in heart failure versus that during normal cardiac aging. Thus, while the current thought is that aging and heart failure have many features in common, they are different in very important ways. In contrast to the heart during aging in health, the diminished 웁-AR responsiveness in failing hearts is associated with (1) a selective downregulation of 웁1-AR, (2) a progressive increase in Gi protein amount and activity, and (3) an increase in 웁ARK1, (4) but no alterations in adenylyl cyclase catalytic activity. These differences indicate that some fundamental differences exist in the underlying mechanisms of different 웁AR signaling of heart failure and normal cardiac senescence. 5. Changes in Cardiac Membrane Fluidity Alterations in the membrane fluidity have been shown to affect membrane signal transduction profoundly. Increased membrane fluidity enhances the coupling efficiency of 웁-ARs to G-proteins. In contrast, decreased membrane fluidity reduces the interaction between 웁-ARs and G-proteins, and thus decreases 웁-AR responsiveness. In aged rat cardiac sarcolemmal membranes, polyunsaturated fatty acids are decreased remarkably, whereas saturated fatty acids are increased, resulting in a reduction in membrane fluidity. In addition to heart, similar age-associated changes in membrane fluidity have been identified in blood vessels and several other tissues. Thus, changes in membrane biophysical properties may be a general mechanism underlying the age-associated defects of cardiovascular 웁-AR signaling. Further studies are required to directly determine the potential role of membrane fluidity in the ageassociated dysfunction of 웁-AR signaling. In summary, studies indicate a marked age-associated reduction in both 웁1-AR and 웁2-AR signaling efficiency in humans and animal hearts. The suppression of the cardiac response to 웁-AR stimulation is associated with a significant downregulation of 웁-AR density and a decrease in the agonist-stimulated adenylyl cyclase activity. In addition, most studies have shown an age-related

751

imbalance of Gi and Gs, which may contribute to the diminished 웁-AR responsiveness in the senescent heart. In contrast, neither 웁ARK1 nor GRK5 is elevated significantly in aged hearts. 6. Other G-Protein-Coupled Receptors in Aged Hearts a. 움1-Adrenergic Receptor Signaling in Aged Hearts In addition to the 웁-AR system, 움1-AR signaling is another important component involved in the regulation of cardiac reserve capacity. Several studies in rat ventricular myocytes or myocardium have shown that, similar to 웁-AR stimulation, the 움1-AR-mediated contractile response is decreased with aging. The age-associated depression of the 움1-AR contractile response may result from an impairment in the translocation of PKC (both PKC움 and PKC␧ isoforms), the key second messenger of 움1-AR signaling, perhaps due to reductions in RACK anchoring proteins in aged hearts. However, unlike 웁-AR stimulation, cellular and molecular mechanisms underlying the age-associated defects of cardiac 움1-AR function remain unknown. Potential changes in 움1-AR density, its G-protein coupling, and downstream signaling merit further investigation. b. M2-Muscarinic Receptor Stimulation with Aging Increasing evidence has indicated that aging is associated with a decrease in cardiac parasympathetic activity. In human right atria, M2-receptor density is correlated negatively with the age of patients, and the effects of carbachol in inhibiting the forskolin-induced positive inotropic response or increase in adenylyl cyclase activity are markedly less potent in aged human atria (Brodde et al., 1998). The muscarinic receptor agonistmediated antiadrenergic effect is reduced significantly in senescent rats relative to adults rats and is accompanied by a reduction in cardiac M2-receptor density (Brodde et al., 1998). In addition, the negative chronotropic effects of muscarinic agonists are diminished in aged rats. Taken together, both M2-receptor density and receptor function decline with aging, although the exact mechanisms underlying the age-associated reduction in cardiac M2-receptor signaling remain to be examined. c. A1-Adenosine Receptor Stimulation in Aged Hearts The A1-adenosine receptor, another Gi-coupled receptor, is critically involved in cardiac remodeling and preconditioning protection of the heart from reperfusion damage. Similar to M2-muscarinic receptors, A1-receptor stimulation potently antagonizes 웁-ARmediated positive inotropic, lusitropic, and chronotropic effects in ventricles of many mammalian species. However, unlike the M2-receptor, A1-receptor-medi-

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ated antiadrenergic effects are enhanced in aged rat hearts, associated with an increase in adenosine levels. The increased A1-receptor antiadrenergic effect may, in part, contribute to the age-associated reduction in cardiac 웁-AR stimulation. 7. Age-Related Dysfunctions of Autonomic Regulation of the Peripheral Vascular System Adrenergic modulation is one of the most important regulatory mechanisms of blood pressure. It is well established that activation of 움-AR, particularly 움1-AR, induces blood vessel contraction, whereas 웁-AR stimulation causes blood vessel relaxation. Several subtypes of adrenergic receptor are present in blood vessels, including 움1-, 움2-, 웁1-, and 웁2-AR. Receptor populations vary between arteries and veins, and within arteries with the distance from the heart and with the transition from conduction to resistance vessels. An 움-AR constriction effect is greater in large vessels than its effects in medium vessels, which, in turn, is greater than that in small size coronary arteries. In contrast, the potency of the 웁-AR-mediated relaxant effect varies in an inverse order relative to that of 움-AR stimulation. A common age-associated vascular dysfunction is a decreased 웁-AR response. Aging may affect the 웁2-AR more than the 웁1-AR-mediated relaxation response in rat vasculature. The age effects on vascular 웁-AR responsiveness are mediated by mechanisms similar to those reported in the heart, including a reduction in 웁-AR density, a dysfunction of adenylyl cyclase and a decrease in the ratio of Gs /Gi proteins due to an increase in Gi and a decrease in Gs protein, and a uncoupling of 웁-ARs to Gs protein. In addition, a reduced membrane fluidity of vascular smooth muscle cells may also play an important role in the age-associated diminution in blood vessel response to 웁-AR stimulation. In summary, data from many different preparations from a variety of species, including human, demonstrate that the autonomic modulation of vasculature is affected by aging, and in particular, that the 웁-AR-mediated vascular dilation response is impaired markedly. Future studies are required to further characterize age-associated changes in specific adrenergic receptor subtypes and interactions among them.

IV. CHRONIC HYPERTENSION MIMICS ACCELERATED AGING A. Humans In humans with clinical hypertension, the same vascular and cardiac changes observed with aging in normo-

tensive humans occur at a younger age, and in some instances are exaggerated. The similarities between aging and hypertension in this regard are so striking that aging has been referred to as ‘‘muted hypertension’’ or hypertension as ‘‘accelerated aging.’’ Thus, changes in the large arteries, LV wall thickness, myocardial relaxation and filling parameters, and the diminished response to 웁-adrenergic stimulation in both normotensive persons during aging and hypertensive patients at any age appear to form a continuum, and clinical distinction between normotensive and hypertensive persons may be somewhat artificial, although clinically useful with regard to risk for cardiovascular morbidity and mortality. However, in hypertensive patients, some changes occur with aging that are not observed in normotensives. In persons with mixed hypertension, the PVR increases substantially with aging, in contrast to the modest changes in PVR in normotensives (Fig. 3E; Fleg et al., 1995). Thus, in hypertensives, an increase in PVR elevates diastolic and mean arterial pressures and plays a greater role in vascular loading of the heart than in normotensives. Additionally, in older hypertensive men, resting stroke volume and cardiac output are not maintained at the levels measured in younger hypertensive men.

B. Animal Models The multiple interrelated changes in cardiac structure, excitation, myofilament activation, contraction mechanisms, and gene expression that occur gradually in aging in rodents (Table I) can be interpreted to be, at least in part, as adaptive in nature, as these also occur in an accelerated fashion in the hypertrophied myocardium of younger animals adapted to experimentally induced chronic ventricular pressure overload (Tables III and IV), and many are reversible following removal of the hypertensive stimulus. Additionally, similar reductions in the cellular RNA concentration and the rate of protein synthesis are observed with aging and chronic myocardial overload in the rat model. A similar pattern of gene expression occurs in hypertensive young animals, during aging in normotensive animals, and to some extent in neonatal heart cells exposed to growth factors (for a review, see Lakatta, 1993b). In addition to a reduction in SERCA and a shift in MHC expression, the expression of many other genes changes with aging (Tables III and IV). A striking increase occurs in the expression of the atrial naturetic factor (ANF) gene in the LV (Fig. 9A; Younes et al., 1995). The concentration of ANF in LV of older rats is increased and is closely related to the magnitude of the LV/body weight ratio (Younes et al., 1995), suggesting that the elevated expression of ANF in the senescent LV

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42. Aging of the Cardiovascular System

may be a secondary consequence of the age-associated myocyte hypertrophy. Work in cultured neonatal cardiac myocytes suggests that ANF may inhibit hypertrophic growth. The structural and functional implications of this finding for adult and senescent hearts remain to be evaluated. The expression of the preproenkephalin (PNK) gene is upregulated five-fold with advancing age (Fig. 9B; Boluyt et al., 1993), resulting in an increased accumulation of the opioid peptides, methionine enkephalin and leucine-enkephalin (LE) (Boluyt et al., 1993; Caffrey et al., 1994). In other cell types, opioid peptides have an antiproliferative effect. Thus, it is of interest that PNK expression is downregulated in the acute phase of adaptation to aortic constriction (Boluyt, Crow, and Lakatta, unpublished observations) or isoproterenol infusion. Also, the inhibitory effects of opioid peptides on cardiac contraction parameters may contribute to age-associated functional changes. In particular, the specific inhibition by LE of 웁-AR-stimulated augmentation

TABLE III Chronic Alterations in Cardiac Structure/Function in Aging and Experimental Left Ventricular Pressure Loading Structural or functional measure Myocardial cell size Collagen content Twitch duration Myosin isozyme composition SR Ca2⫹ pumping rate Cai transient duration Myofilament Ca2⫹ sensitivity Action potential repolarization time 웁-adrenergic intropic response Cardiac glycoside response Threshold for Ca2⫹ overload

Experimental LV pressure loading (rodent)

Normotensive aging (rodent)

⇑ ⇑ ⇑ ⇓움⇑웁 ⇓ ⇑ ⇔ ⇑

⇑ ⇑ ⇑ ⇓움⇑웁 ⇓ ⇑ ⇔ ⇑

⇓ ⇓ ⇓

⇓ ⇓ ⇓

TABLE IV Altered Myocardial Gene Expression: Steady mRNA Levels in Advanced Age, Hypertension, or after Growth Factorsa Growth factor b Rodent

SERCA Calsequestrin Phospholamban 움-Myosin heavy chain 웁-Myosin heavy chain 웁-Tropomyosin 움-Skeletal actin Atrial naturetic factor Proenkephalin Gs움 웁-adrenergic receptor Fibronectin Collagen type I Collagen type III Angiotensinogen Angiotensin-converting enzyme Angiotensin1 receptor Retinoid X receptors 움 웁 웃 Thyroid receptors 움1 움2 웁1 a

FGF

Aging

Hypertension

TGF␤

Acidic

Basic

⇓ ⇔

⇓ ⇔ ⇑ (rabbit) ⇓ ⇑ ⇑c ⇑c ⇑ ⇓c

⇓

⇓

⇓

⇓ ⇑

⇓ ⇑

⇓ ⇑

⇑ ⇑

⇓ ⇑

⇑ ⇑

⇓ ⇑ ⇓ ⇓ ⇑ ⇑ ⇓ ⇓ ⇑ ⇑ ⇑ ⇑ ⇑ ⇑

⇑ ⇑ ⇑ ⇑ ⇑ ⇑

⇔ ⇔ ⇓ ⇔ ⇔ ⇓

See Boluyt and Lakatta (1998) and Lakatta (1993) for review. In neonatal cultured cardiomyctes. c Only transient changes occur in situ following cardiac pressure loading. b

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VIII. Developmental Changes and Aging

FIGURE 9 (A) Atrial natriuretic factor (ANF) mRNA levels in left ventricles (LV) of male Wistar rats. From Younes et al. (1995). (B) Preproenkephalin (PNK) mRNA levels in left ventricles of male Wistar rats. From Boluyt et al. (1993).

of contraction dynamics (Pepe et al., 1995) strongly suggests that the opioidergic system may coregulate the age-associated reduction in the 웁-adrenergic responsiveness of the heart noted earlier. It is tempting to speculate that this nearly identical

pattern of change in gene expression during pressureoverload hypertrophy and aging is indicative of a common set of factors that regulate transcription and the resultant cellular adaptations. This particular constellation of shifts in gene expression (Table IV) appears to be adaptive, in that it allows for an energy-efficient and prolonged contraction. In the hypertensive rodent heart it can be inferred that these changes in gene expression permit functional adaptations in response to an increased vascular ‘‘afterload.’’ Possible stimuli for these changes with normotensive aging are listed in Table V. The array of adaptations that occur in the myocardium with advancing age may be induced by mechanical and biochemical events arising within the heart, as well as those imposed from outside the heart. All of the mechanical factors listed in Table V directly or indirectly increase the stretch of individual myocytes. Therefore, autocrine actions of angiotensin II are likely to be important determinants of age-associated myocyte hypertrophy and adaptation (Sadoshima et al., 1993). Paracrine and endocrine signals are also likely to be involved, e.g., the small ageassociated reduction in circulating thyroid hormone or reductions in THR웁 and RXR웂 (Long et al., 1999). As noted earlier, physical conditioning has been shown to reverse the prolonged contraction duration of rat myocardium (Spurgeon et al., 1983) and reductions in SERCA gene expression (Tate et al., 1996), suggesting that reduced physical activity with aging contributes to some myocardial changes. Reduced glucose tolerance also occurs in aged rats, as in humans, and could possibly contribute to some of the changes noted previously. Finally, an increase in the total time that cardiac cells are exposed to elevated levels of intracellular calcium, due to the slowed reuptake of calcium by the sarcoplasmic reticulum, may contribute to multiple changes in the heart with advancing age, possibly by activating calcineurin, a phosphatase inhibitor, that promotes cardiac hypertrophy.

TABLE V Possible Stimuli for Age-Associated Cardiac Myocyte Adaptations Mechanical factors

Biochemical factors

Intramyocardial 1. Loss of cells, leading to stretch and an increased workload on remaining myocytes 2. Increased levels of fibrosis, leading to stiffness, thereby increasing work

Intramyocardial 1. Autocrine growth factors produced by heart cells, such as angiotensin II 2. Paracrine growth factors from cardiac fibroblasts, endothelial and smooth muscle cells, including local renin–angiotensin system components 3. Calcium loading

Extramyocardial 1. Increased arterial impedance due to stiffening of conduit arteries

Extramyocardial 1. Alterations in thyroid status 2. Physical deconditioning 3. Reduced glucose tolerance

42. Aging of the Cardiovascular System

V. ADAPTIVE RESPONSE OF THE OLDER RAT HEART TO CHRONIC STRESS The chronic adaptive cardiovascular reserve capacity appears to become diminished with advancing age. Thus, responses to mechanical stresses that evoke substantial myocardial hypertrophy (e.g., pressure or volume overload) appear, in many instances, to be reduced in the senescent heart. Mechanisms responsible for this reduced adaptive reserve are not known. The reserves of some cardiac adaptations, e.g., increases in myocyte or heart size, become utilized during ‘‘normal’’ aging (Tables I, III, and IV) such that a further utilization of these mechanisms in response to experimental stresses may be limited. A similar loss of adaptive capacity to pressure overload stress is observed in younger rats that have utilized a part of their reserve capacity prior to a growth factor challenge. For example, hearts of younger rats that have established hypertrophy due to aortic constriction exhibit blunted growth responses (e.g., immediate-early gene induction and incorporation of labeled precursors into protein). Evidence shows that transcriptional events associated with hypertrophy are altered with advancing age. For example, the nuclear-binding activity of the transcription factor NF-␬B was increased twofold in extracts from hearts of older versus young adult mice, whereas that of another transcription factor, Sp1, was diminished. Because these transcription factors each influence expression of a number of genes, they may contribute to the pattern of gene expression observed in the hearts of senescent rodents as well as dictate the limits of adaptive responses. The induction of both immediate-early genes and later responding genes that are expressed during the hypertrophic response is blunted in hearts of aged rats after aortic constriction. Similarly, the induction of heat shock 70 protein genes in response to either ischemia or heat shock is reduced in hearts of senescent rats (reviewed in Boluyt and Lakatta, 1998).

VI. HEART FAILURE IN THE AGING, SPONTANEOUSLY HYPERTENSIVE RAT (SHR) The chronic stress of hypertrophy with advancing age in the spontaneously hypertensive rat is eventually followed by heart failure. The transition from compensated hypertrophy to failure in the SHR seems to demonstrate quite well the consequences of an interaction between ‘‘normal aging’’ and disease. Although elevated blood pressure and marked cardiac hypertrophy are established during the first quartile of the rats lifespan, function remains well compensated through the second and third quartiles. Young adult and middleaged SHR exhibit compensated cardiac hypertrophy,

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whereas depressed contractile function and increased fibrosis are observed in senescent SHR. Despite prominent changes in function and the pathological evidence of failure, there is no additional increase in LV mass of failing hearts. Papillary muscles from SHR-F display increased fibrosis coupled with impaired contraction parameters. Each of these factors is related inversely to myocyte fractional area, suggesting that myocytes are lost and replaced with extracellular matrix. This altered structural profile of papillary muscles is coupled to a depression in contraction amplitude. When contraction amplitude in papillary muscles of SHR-F is normalized to myocyte fractional area, however, the decrement in function is abolished, suggesting that myocyte loss coupled to replacement fibrosis contributes substantially to the reduction in contractile function. One mechanism that has been proposed to account for myocyte loss during the transition to failure is apoptotic cell death. It has been suggested that apoptosis, as evidenced by fragmented DNA in the nucleus of myocytes, detected by end-labeling immunocytochemistry, appears during the transition from stable hypertrophy to heart failure in the SHR (Li et al., 1997). Heart failure in the senescent SHR is also associated with an exacerbation of the shifet toward 웁-MHC mRNA and protein. Disappearance of the 움-MHC protein during the transition to failure may account, at least in part, for the inability of cardiac muscle from failing heart to respond to the stimulation of 웁-adrenergic receptors. A marked increased in collagen and fibronectin mRNA levels occurs during the transition to failure in ventricles of SHR rats (Boluyt et al., 1994), and in situ hybridization suggests that collagen mRNA in failing hearts is localized to interstitial areas. In the failing hearts of SHRs that exhibit markedly increased levels of fibronectin and collagen gene expression, a small, but significant, increase in TGF-웁1 mRNA levels is observed in both ventricles. The upregulation of TGF-웁1 gene expression in the failing SHR heart is consistent with a stimulatory role for this growth factor, leading to accumulation of the extracellular matrix. While the accumulated effects of long-term hypertension and the genetic nature of the model cannot be dismissed, it seems appropriate to hypothesize that the effects of ‘‘normal aging’’ reduce the reserve capacity of the heart for adaptation in the SHR and conspire with hypertensive disease processes to decrease the chances of survival. Taken together with human, animal, and molecular data presented earlier, ‘‘normal aging’’ merits consideration as both a risk factor and a therapeutic target in age-associated cardiovascular disease. In this regard, treatment of SHR rats with an angiotensinconverting enzyme inhibitor (ACEI) from the age of 12 months completely prevents the development of heart

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failure, and markedly alters the pattern of gene expression (Brooks et al., 1997).

VII. SUMMARY A. An Integrated View of Age-Associated Changes in Cardiovascular Structure and Function in Humans at Rest The major age-associated changes, or lack thereof, in several aspects of cardiovascular function at rest in sedentary humans are listed in Table VI and are illustrated in Fig. 10. A unified interpretation of the cardiac changes observed in Fig. 10 suggests that these are adaptive, i.e., they occur in response to arterial changes that occur with aging. Arterial stiffening (Gerstenblith et al., 1977; Fleg et al., 1995; Vaitkevicius et al., 1993; Fleg, unpublished observations) leads to an enhanced pulse wave velocity, which produces a late augmentation in systolic arterial pressure. This augmentation in the pulse pressure causes aortic dilatation; an increase in LV wall thickness (Fig. 3; Gerstenblith et al., 1977) results from an increased vascular impedance (Gerstenblith et al., 1977; Fleg et al., 1995; Vaitkevicius et al., 1993; Fleg unpublished observations). The increase in LV wall thickness moderates the increase in LV wall tension occurring secondary to increased vascular loading (and in men to the modest increase in LV EDVI). A prolonged myocardial contraction, which maintains a normal ejection time in the presence of the late augmentation of aortic impedance due to early reflected pulse waves, also contributes to the maintenance of ejection fraction at rest; otherwise the increase in the vascular

TABLE VI Changes in Cardiac Output Regulation between 20 and 80 Years of Age in Healthy Humans at Seated Rest Cardiac indexa Heart rate Stroke volume Preload EDVa Early filling Late filling Afterload Compliance Reflected waves Inertance PVR Contractility Ejection fraction LV mass a

No change ⇓ (10%) ⇑ (10%) ⇑ (12%) ⇓ ⇑ ⇓ ⇑ ⇑ ⇔ No change No change ⇑

Females differ from males; see text.

loading of the myocardium in late systole would lead to premature closure of the aortic valve. Thus, systolic cardiac pump function at rest in clinically normotensive humans is not altered substantially by age. The disadvantage of prolonged contractile activation is that myocardial relaxation is relatively more incomplete in older than younger individuals at the time of mitral valve opening. This is one factor that causes the early LV filling rate to be reduced in older individuals. Structural changes and functional heterogeneity occurring within the LV with aging may also contribute to this reduction in peak LV filling rate. However, a concomitant adaptation—left atrial enlargement and an enhanced atrial contribution to ventricular filling—compensates for the reduced early filling and, in part, maintains a mildly increased LV EDVI. Whereas the cardiac structural changes with aging may be interpreted as resulting from increased vascular loading of the heart in both genders, and in men, also to an increase in LV EDVI, a decrease in effective 웁-adrenergic stimulation of both the heart and vasculature occurs with aging (see later) and may be implicated in the associated myocardial changes, in part, via a reduction in the heart rate at rest in the sitting position and during stress, e.g., routine activities of daily life or exercise. In addition, potential age-associated changes in tissue levels or activities of growth factors, e.g., catecholamines, angiotensin II, endothelin, TGFB, and FGF, that influence myocardial or vascular cells or their extracellular matrices may have a role in the schema depicted in Fig. 9.

B. Cardiovascular Reserve Function in Humans The major effects of age on cardiovascular performance during the stress of exhaustive upright exercise are listed in Table VII. Aerobic capacity declines with advancing age in individuals without cardiac disease, which can be attributed, in part, to a decrement in cardiac reserve and in part to peripheral factors, e.g., to an increase in body fat and a decrease in muscle mass with age. While heart rate during exercise is lower in healthy older versus younger individuals, cardiac dilatation at end diastole and end systole occurs in older subjects. Thus, in health, older individuals do not exhibit a compromised EDVI due to a ‘‘stiff heart’’ even during exercise. Whereas the age-associated increase in EDVI preserves SVI during exercise in such individuals, the increase in ejection fraction is blunted due to a failure of the older healthy heart to empty as completely as the younger heart. This failure to reduce ESVI results from a combination of factors that relate to augmented vascular afterload, reduced intrinsic myocardial contractility, and reduced augmentation of contractility by

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Arterial stiffening

Arterial systolic and pulse pressure

Pulse wave velocity Early reflected waves Late peak in systolic pressure

Aortic root size Aortic wall thickness

Aortic impedance and LV loading LV wall tension

LV hypertrophy

Prolonged myocardial contraction

L atrial size Atrial filling

Normalization of LV wall tension

Myocardial contraction velocity

Prolonged force bearing capacity

Slightly increased end diastolic volume

Preserved end systolic volume and ejection fraction

Energetic efficiency

Maintenance of ejection time

Early diastolic filling rate

FIGURE 10 Arterial and cardiac changes that occur with aging in healthy humans. One interpretation of the constellation (flow of arrows) is that vascular changes lead to cardiac structural and functional alterations that maintain cardiac function. LV, left ventricular. From Lakatta (1993b).

웁-adrenergic stimulation. This same hemodynamic pattern occurring during exhaustive exercise in healthy older humans, i.e., a reduced exercise heart rate and greater cardiac dilatation at end diastole and end systole, occurs in individuals of any age who exercise in the presence of 웁-adrenergic blockade. In fact, when perspectives from studies that range from measurements of the stress response in intact humans to measurements

TABLE VII Changes in Aerobic Capacity and Cardiac Regulation between 20 and 80 Years of Age in Healthy Men and Women during Exhaustive Upright Exercise Oxygen consumption (A-V)O2 Cardiac index Heart rate Stroke volume Preload EDV Afterload Vascular (PVR) Cardiac (ESV) Cardiac (EDV) Contractility Ejection fraction Plasma catecholamines Cardiac and vascular responses to 웁-adrenergic stimulation

⇓ (50%) ⇓ (25%) ⇓ (25%) ⇓ (25%) No change ⇑ (30%) ⇑ ⇑ ⇑ ⇓ ⇓ ⇑ ⇓

(30%) (275%) (30%) (60%) (15%)

of subcellular biochemistry in animal models are integrated, a diminished responsiveness to 웁-adrenergic modulation is among the most notable changes that occur in the cardiovascular system with advancing age. Alterations in cardiovascular function that exceed the identified limits for age-associated changes for healthy elderly individuals are most likely manifestations of interactions of aging, per se, with age-associated changes of severe physical deconditioning or the presence of cardiovascular disease, which are, unfortunately, so prevalent within our population.

C. Rodents: Structure and Function Cellular and molecular mechanisms that account for age-associated changes in myocardial performance have been studied largely in rodents. In the normotensive rat, cardiac fibrosis increases with aging, the number of myocytes decreases, and myocyte size increases. Variable degrees of left ventricular hypertrophy occur, depending on the rodent strain, and this is due to ventricular dilatation with an apparent preservation of normal ventricular wall thickness. Functional, biophysical/ biochemical, pharmacological, and molecular changes have been observed in the aging rat heart. There are coordinated changes in several key steps of excitation– contraction coupling that result in a prolonged Cai transient and a prolonged contraction. The altered cellular profile, which results in a contraction that exhibits a

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reduced velocity and a prolonged time course, is energy efficient and prolonged contraction permits continued ejection for a longer period. However, aged myocardium (and that exposed chronically to pressure overload) is more susceptible to Ca2⫹ overload and spontaneous sarcoplasmic reticular Ca2⫹ release than young adult myocardium, and aged myocardium demonstrates a reduced Ca2⫹ threshold for diastolic after depolarizations and for ventricular fibrillation. The former is caused by and the latter is preceded by an increase in spontaneous oscillatory Ca2⫹ release from the sarcoplasmic reticulum. Studies in isolated left ventricular muscle and in individual rat ventricular cardiocytes, similar to recent studies in humans, indicate that a reduced contractile response to 웁1-AR stimulation occurs with aging. This is due to failure of the intracellular Ca2⫹ transient to increase in cells of senescent hearts to the same extent to which it increase in cells from younger adult hearts. The blunted increase in the Ca2⫹ transient in cells from the aged heart is attributed to a decrease in the ability of 웁1-AR stimulation to increase L-type sarcolemmal Ca2⫹ channel availability in cells from senescent versus younger adult hearts. The richly documented ageassociated reduction in the postsynaptic response of myocardial cells to 웁1-AR stimulation appears to be due to multiple changes in molecular and biochemical receptor coupling and postreceptor mechanisms rather than to a major modification of a single rate-limiting step, as might occur, for example, in a genetic defect. The multiple changes in cardiac excitation, myofilament activation, contraction mechanisms, and gene expression that occur with aging are interrelated. Many of these can be interpreted as adaptive in nature because they also occur in the hypertrophied myocardium of younger animals adapted to experimentally induced chronic hypertension. Some evidence suggests that the adaptive response to chronic passive loading declines with aging, possibly in part because some of the adaptive capacity of the heart is used as a response to the aging process per se. Age-associated macroscopic changes within large blood vessels in rodents are similar in many ways to those that occur in humans and consist of dilation, medial thickening, and formation of an intima. Chronic morphological and biochemical modifications within the aortic intima of aging rats, i.e., fragmentation of the internal elastic membrane, intimal thickening, and increases in collagen, proteoglycan, fibronectin, MMP-2, TGF웁, and ICAM-1, appear as a muted version of these alterations associated with chronic hypertension or with transient changes that occur in response to acute mechanical injury, e.g., after balloon angioplasty. That chronic administration of an angiotensin-converting en-

zyme inhibitor reduces and delays the matrix and intimal changes with aging or hypertension substantially suggests that age-associated changes in the vascular angiotensin regulation may have a role in the age-associated changes observed in TGF웁 and fibronectin. A regional increase in ICAM-1 observed in the intima of aged aortae may also be related to the augmented levels of TGF웁, as the latter is known to induce the synthesis of cell adhesion receptors, which may lead to increased adhesion and interaction of cells with the surrounding extracellular matrix. Research into specific molecular mechanisms that lead to intimal thickening and disruption of vascular elastin with increasing age is a crucial component of the aging research agenda.

Acknowledgments The authors thank Christina Link and Sharon Wright for the secretarial support in the preparation of this chapter.

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43 Changes in Autonomic Responsiveness during Development RICHARD B. ROBINSON, MICHAEL R. ROSEN, and SUSAN F. STEINBERG Departments of Pharmacology, Medicine, and Pediatrics Columbia University New York, New York 10032

I. INTRODUCTION

among the limbs and neurohumors of the autonomic nervous system. Further, autonomic responsiveness is not static, but rather changes both in the context of normal development and in the response to disease processes. These changes can occur in any element of the signaling cascade, from the most proximal (the receptor) to the most distal (the ion channel or other targets). Importantly, many (but certainly not all) disease-induced changes in signaling molecule expression serve to recapitulate a neonatal phenotype. Thus, elucidation of the developmental changes in autonomic signaling and the mechanisms that regulate these changes are important in their own right, as well as for the potential insights they may provide to pathological processes. This chapter therefore describes the age-dependent changes in 움-adrenergic and 웁-adrenergic receptor signal cascades and cardiac autonomic responsiveness, as well as briefly discussing potential developmental contributions of parasympathetic and other nonadrenergic cascades.

For many years, the role of the autonomic nervous system in the control of cardiac rhythm was thought to be rather straightforward (Pappano, 1977). The parasympathetic nervous system and its mediator, acetylcholine, were thought to exert an inhibitory function (depressing automaticity and atrioventricular conduction), and the sympathetic nervous system and epinephrine and norepinephrine were thought to be excitatory, enhancing automaticity and speeding atrioventricular conduction. In the context of cardiac rhythm modulation per se, the important sympathetic actions were thought to be 웁 (and in particular 웁1)-adrenergic receptor modulated; only a minor role, if any, was consigned to the 움-adrenergic receptor system. The major complicating factor in the picture was that of accentuated antagonism, whereby the parasympathetic limb of the autonomic nervous system was shown to have enhanced effects in the presence of preexisting sympathetic tone (Pappano, 1977; Levy, 1971). A number of observations have served to modify this view of autonomic control. First, pharmacological and molecular biological data establish the presence of multiple 움-adrenergic, 웁-adrenergic, and muscarinic receptor subtypes linked to an elaborate network of second messenger signaling mechanisms and distinct functional responses. Second, it is now realized that additional humoral substances may be released neurally, including adenosine, serotonin, and peptides such as neuropeptide Y, that have demonstrable and important effects on cardiac rhythm. Finally, it is recognized that an increasingly complex set of interactions exist

Heart Physiology and Pathophysiology, Fourth Edition

II. CELLULAR PATHWAYS MEDIATING AUTONOMIC RESPONSIVENESS Under normal physiological conditions, catecholamines induce positive inotropic, chronotropic, and lusitropic (relaxant) responses in the heart through a 웁1-receptor-activated pathway that involves the stimulatory GTP regulatory protein (Gs), activation of adenylyl cyclase, accumulation of cAMP, stimulation of cAMP-dependent protein kinase A (PKA), and phosphorylation of key target proteins [including the L-type

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calcium channel, phospholamban (PLB), and troponin I (TNI)]. However, cardiac myocytes also express 움1- and 웁2-adrenergic receptors that link through a complex network of G-protein-dependent signaling cascades to important changes in cardiac contractile and electrophysiological function. G-proteins are heterotrimers consisting of a guanine nucleotide-binding 움 subunit and a tightly but noncovalently associated dimer of 웁 and 웂 subunits (for a review, see Hamm, 1998). G-proteins are classified according to the identity of their 움 subunits, which are broadly grouped into four classes: G움s , G움i , G움q , and G움12/13 . G움q mediates 움-receptor signaling through phospholipase C, whereas G움s and G움i figure most prominently in 웁-receptor subtype signaling as they couple to the stimulation or inhibition of adenylyl cyclase enzyme activity, respectively. Historically, G-protein function was attributed entirely to the actions of the freed 움 subunit. For Gi , this was accomplished largely with pertussis toxin (PTX), which catalyzes the ADPribosylation of G움i, a covalent modification that blocks G움i –receptor interactions and thereby functions to inactivate signal transduction pathways mediated by 움i subunits. However, more recent studies established that G-protein activation is a bifurcating process, with the liberated 웁웂 dimers also controlling various effector functions (either alone or in concert with the liberated 움 subunits). Additional properties that have been relegated to the 웁웂 dimer include directing the fidelity of G-protein-coupled receptor–effector interactions and facilitating the agonist-dependent 웁-adrenergic receptor phosphorylation/desensitization process by recruiting the G-protein-coupled receptor kinase (GRK or 웁ARK) to the plasma membrane (Ray et al., 1996; Clapham and Neer, 1993). G-proteins are critically regulated by both subunit dissociation (움-웁웂) and guanine nucleotide exchange cycles. The latter is a highly regulated process; the lifetime of the active, GTP-bound form of the 움 subunit depends on the rate of the 움-subunit GTPase, which is an intrinsic property of the 움 subunit itself, but can also be modified by the GTPase-activating protein (GAP) activity of certain downstream effector proteins such as phospholipase C웁 (Paulssen et al., 1996) and adenylyl cyclase (Scholich et al., 1999) or (for all subgroups of 움 subunits except G움s) regulators of G-protein signaling (RGS) proteins (Berman and Gilman, 1998; Guan and Han, 1999). Thus, for receptors that display the potential to act through multiple G-proteins (such as 움1- and 웁2-adrenergic receptors), multiple factors may dictate specificity, including the diverse pathways activated by distinct 움 and 웁웂 subunits and the actions of RGS proteins to modulate the guanine nucleotide exchange cycle kinetics of individual G움 subunits. Mammalian cells ex-

press over 20 distinct RGS proteins. Cardiomyocytes isolated from rat heart are reported to express mRNAs for 10 RGS proteins, with evidence for regulation at the mRNA level during development and in certain cardiac hypertrophy and/or failure models (Kardestuncer et al., 1998; Zhang et al., 1998). Insofar as large changes in mRNA are likely to be associated with coordinate changes in the cognate protein, this would be predicted to provide a potential mechanism for quantitatively distinct responses to agonist even in cells endowed with identical receptor/G-protein composition (and could, at least in theory, be germane to adrenergic receptor signaling in cardiomyocytes). There currently are five known species of 웁 subunit and more than twice that number of 웂 subunits (Ray et al., 1995, 1996; Morishita et al., 1995). 웁 subunits are the most conserved of the G-protein subunits, whereas 웂 subunits are structurally quite divergent (Simon et al., 1991; Cali et al., Robishaw, 1992). Theoretically, an enormous number of different 웁웂 subunit combinations could be assembled, although there appear to be preferred associations between 웁 and 웂 subunits such that G-proteins purified from different tissues differ with respect to their 웂 subunits (Gautam et al., 1990). Evidence shows that cardiomyocytes express at least two distinct species of 웁 (웁1 and 웁2) and four different species of 웂 (웂3 , 웂5 , 웂7 , and 웂12) subunits at the protein level; one of these 웂 subunits (웂3) is expressed in neonatal, but not adult, cardiomyocytes (Hansen et al., 1995; Morishita et al., 1995). One of the cardiac 웂 subunits (웂7) was shown to play a specific role in the activation of adenylyl cyclase by isoproterenol, but not by prostaglandin E1 (Wang et al., 1997). These results emphasize the potential (and as yet unevaluated) importance of the 웂 subunits as specific facilitators (or restrictors) of receptor–effector coupling and suggest (the as yet unexplored notion) that changes in 웂 subunit expression may critically influence 웁-receptor responses in cardiomyocytes. The adenylyl cyclase enzyme is the target for activation by 웁-adrenergic receptors. The mammalian adenylyl cyclase gene family currently contains nine members; types V and VI are the predominant isoforms detected in cardiac tissue (reviewed in Ishikawa and Homcy, 1997). These isoforms have been designated the cardiac subclass of adenylyl cyclase on the basis of their structural homology (65% homology to each other and ⬍40% homology to other isoforms of adenylyl cyclase) and similar patterns of regulation by cofactors. Normal cardiac ontogenic development is associated with a reciprocal change in steady-state levels of mRNAs encoding type V and type VI adenylyl cyclase isoforms; the abundance of transcripts for type V adenylyl cyclase increases and type VI adenylyl cyclase

43. Changes in Autonomic Responsiveness

decreases with age (Tobise et al., 1994; Espinasse et al., 1995). In the context of evidence that the abundance of the adenylyl cyclase enzyme (a relatively sparse membrane protein) may set the limit on transmembrane 웁-adrenergic receptor signaling (Gao et al., 1998), it is intriguing to speculate that the abundance of these adenylyl cyclase isoforms calibrates the sensitivity of the 웁-adrenergic receptor pathway. Moreover, the adenylyl cyclase isoform composition of the cell membrane can critically influence cAMP accumulation; all membranebound forms of adenylyl cyclase are activated by G움s , but they exhibit markedly different patterns of regulation by other cofactors such as 웁웂 subunits, calcium, and protein kinase C (Taussig and Gilman, 1995; Ishikawa et al., 1997). Thus, changes in the ratio of individual adenylyl cyclase isoforms may alter the capability of the cell to receive and integrate signals via cAMP.

A. 움-Adrenergic Cascade 움1-adrenergic receptors constitute a multigene family with three distinct 움1-adrenergic receptor subtypes; these can be distinguished pharmacologically on the basis of their sensitivity to subtype-selective antagonists (Graham et al., 1996; Hieble et al., 1995). The 움1c clone corresponds to the 움1A pharmacologic subtype (henceforth called 움1A/c); it displays a high affinity for the agonist N - [5 - (4,5 - dihydro - 1H - imidazol - 2 - yl) - 2 hydroxy - 5,6,7,8 - tetrahydronaphthalen - 1 - yl]methane sulfonamide hydrobromide (A61603) and the competitive antagonists WB-4101 (WB) and 5-methylurapidil (5-MU), but only low sensitivity to irreversible inactivation by the alkylating agent chloroethylclonidine (CEC). The cloned 움1b-adrenergic receptor is the molecular equivalent of the 움1B pharmacologic subtype; it exhibits a low affinity for WB and 5-MU and is irreversibly inactivated by CEC. The 움1d clone displays a high affinity for WB and an affinity for 5-MU and sensitivity to inactivation by CEC that is intermediate between the 움1A/c and the 움1b clones. The 5HT1A-receptor partial agonist, BMY 7378 (BMY), is a selective competitive antagonist at the 움1D-adrenergic receptor (Goetz et al., 1995). Studies indicate that all three molecular forms of the 움1-adrenergic receptor are expressed at the mRNA level in neonatal rat ventricular myocytes (Stewart et al., 1994; Rokosh et al., 1996). Radioligand-binding experiments have extended these findings to reveal 움1receptor heterogeneity at the protein level in these cells (Rokosh et al., 1996; del Balzo et al., 1990). Age-dependent differences in total 움1-adrenergic receptor protein levels have been identified in several species, including the rat, dog, and sheep. Although the magnitude of the decline in 움1-adrenergic receptor density is highly variable (50% in rat vs 10-fold in dog), the proportion

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of total 움1 receptors that can be identified pharmacologically as the 움1B subtype (with CEC) is similar in the neonatal and adult heart (del Balzo et al., 1990). The 움1A/c receptor links to the Gq-dependent activation of phospholipase C, which results in the formation of the two second messengers IP3 and diacylglycerol (DAG). DAG in turn activates protein kinase C (PKC). Cardiomyocytes coexpress multiple PKC isoforms, including the calcium-sensitive PKC움, the novel PKC웃 and PKC␧, and the atypical PKC␭. Most investigators report the selective activation of nPKC isoforms (PKC웃 and PKC␧) by 움1 receptors (Puce´at et al., 1994; Clerk et al., 1994); PKC움 or PKC␭ are not activated following catecholamine stimulation. Developmental repression of PKC isoform expression also has been noted. Protein and mRNA levels for PKC움, PKC웃, and PKC␧ decline during the first 2 weeks of postnatal life (Rybin and Steinberg, 1994; Rybin et al., 1997). While sympathetic innervation of cardiomyocytes does not induce detectable changes in PKC isoform expression, thyroid hormone represses PKC움 and PKC␧ expression in neonatal cardiomyocytes; these results suggest that the postnatal surge in the thyroid hormone may contribute to the developmental regulation of this enzyme (Rybin and Steinberg, 1996). PKC␭ is expressed at high levels in the fetal heart; its developmental down-regulation occurs at a much earlier developmental stage via unknown mechanisms (Rybin et al., 1997). Although the agedependent changes in PKC isoform expression could in theory translate into changes in 움1-receptor responses, instances where this occurs have not been reported. Although 움1B receptors couple to the stimulation of phospholipase C in noncardiomyocytes, in most cardiomyocyte systems, phospholipase C activation is observed only with the 움1A/c receptor. What then is the effector mechanism for the 움1B receptor? A series of studies largely performed in dog and rat cardiomyocytes link the 움1B receptor to a PTX-sensitive G-protein. Although both 움1B receptors and PTX-sensitive Gproteins are present in the noninnervated neonatal heart, their ability to interact productively requires the maturation of sympathetic innervation (Steinberg et al., 1996). Functional consequences of 움1B-receptor activation are observed only in the mature heart. In the ventricle (rat) or Purkinje fibers (canine) of the adult heart, 움-adrenergic agonists slow spontaneous rate via an 움1B-adrenergic receptor coupled to a PTXsensitive G-protein. This inhibitory response is absent in the newborn heart. The ontogeny of this response can be accelerated or slowed developmentally by treating newborn animals with nerve growth factor or its antibody to speed or delay, respectively, sympathetic innervation of the heart (Fig. 1) (Malfatto et al., 1990). Other experiments employed a cell culture system to

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FIGURE 1 Fraction of neonatal rat septal preparations that exhibited a negative (filled bar) or positive (open bar) chronotropic response to phenylephrine (10⫺8-10⫺7) as a function of postpartum age. (Bottom) Results are shown for rats treated from birth with daily subcutaneous injections of nerve growth factor (NGF) or NGF antibody (Ab) until day 10. After Ab treatment, fewer animals exhibited a negative chronotropic response, and the fraction that did so was comparable to 5to 6-day-old control animals. In contrast, the response of the NGF group was similar to that of 3-week-old control animals. Reprinted with permission from Malfatto et al. (1990).

directly test the role of innervation and explore its mechanism of action (for review, see Robinson, 1996). The model used was the neonatal rat cardiac myocyte in tissue culture alone or in coculture with sympathetic ganglion cells. This model was viewed as appropriate because preliminary studies of adult and neonatal rat ventricles showed, respectively, an 움1-adrenergic-induced decrease and increase in automaticity. That the myocyte cultures were nerve free and the cocultures were innervated was demonstrated in studies of ultrastructure—showing close nerve–muscle apposition in the latter—and in studies using tyramine, which induced the release of catecholamines in the latter but not in the former. In pure muscle cultures, phenylephrine increased automaticity and this was blocked by prazosin. In twothirds of the cocultures, phenylephrine decreased automaticity; in the other one-third the rate increased. Both responses were blocked by prazosin. Propranolol, atropine, or adenosine deaminase did not block the inhibition of automaticity, indicating that the response was unrelated to an action at presynaptic 움1 receptors located on sympathetic, parasympathetic, or purinergic neurons. These results suggested that a factor released

by sympathetic nerves during sustained innervation in some way modified the response to 움-adrenergic stimulation, leading this to change qualitatively from excitation to inhibition of automaticity (Robinson, 1996). That the factor was not norepinephrine was suggested by the absence of a correlation between the endogenous norepinephrine levels in cocultures and the appearance of inhibition of automaticity. Subsequent experiments tested whether neuropeptide Y (NPY), a peptide present in and released from cardiac sympathetic neurons, could account for the action of innervation (Sun et al., 1991). Sustained growth of cultures in the presence of 100 nM NPY resulted in expression of a negative chronotropic 움-adrenergic response, whereas acute exposure did not (Fig. 2). That this was the mechanism by which sympathetic innervation also altered 움-adrenergic responsiveness was demonstrated in another series of experiments. In this case, innervated cultures were exposed either acutely or chronically to the NPY antagonist PYX-2. Chronically treated cultures failed to develop the inhibitory response to phenylephrine typical of the innervated preparation, but acutely treated cells responded normally (Fig. 2). Thus, the action of sympathetic neurons to alter 움-adrenergic chronotropic responsiveness from excitatory to inhibitory appears to be mediated by neurally released NPY. Additional studies indicate that NPY is acting via a Y2-specific NPY receptor on the myocyte (Sun et al., 1998). While the distal element(s) of this inhibitory cascade is not fully elucidated, it appears the end target is the Na/K pump. This is an electrogenic pump that removes three Na⫹ ions from the cell for every two K⫹ ions that are transported into the cell, creating a net negative charge and thereby hyperpolarizing the cell. 움-Adrenergic agonists have been shown to stimulate the pump (Shah et al., 1988) and the associated hyperpolarization would be expected to inhibit automaticity. Additional experiments using ion-sensitive microelectrodes have quantified a decrease in intracellular Na⫹ activity induced by the actions of 움-adrenergic agonists in canine Purkinje fibers (Zaza et al., 1990). This decrease occurs only in those fibers in which automaticity decreases and is linearly proportional to the decrease. Finally, there are multiple pump isoforms and their expression is regulated developmentally, but it is not known at this time if the different isoforms differ in their sensitivity to activation by 움-adrenergic agonists. 움-adrenergic receptors do not alter transmembrane potential or automaticity in rabbit sinus node. Similarly, 움-adrenergic stimulation does not inhibit automaticity in canine sinus node. Moreover, when canine Purkinje fibers are depolarized to approximately ⫺60 mV with intracellular current injection, there is no longer any

43. Changes in Autonomic Responsiveness

FIGURE 2 Effects of NPY and an NPY antagonist on the 움1-adrenergic chronotropic response in neonatal rat ventricle cultures. (Top) Effect of growing cultures in the sustained presence of NPY (䊊) on the phenylephrine dose–response relation. There is a positive chronotropic response in control muscle cultures (䊉), whereas NPYtreated cultures show a negative chronotropic response. (Bottom) Effect of culture condition on the percentage of cultures exhibiting a positive or negative chronotropic response to phenylephrine (over the concentration range 10⫺9-10⫺6 M). Muscle cultures (left) treated acutely with NPY fail to show any negative response (some cultures were nonresponding), 24-hr NPY treatment leads to a small percentage showing a negative response, and 96-hr treatment results in a greater percentage possessing the negative response. Innervated cultures (right) normally exhibit a negative chronotropic response. Acute exposure to the NPY antagonist PYX-2 does not alter this phenylephrine response, but a 96-hr exposure results in the loss of the negative chronotropic response from most of the innervated cultures. Reprinted with permission from Sun et al. (1991).

inhibition of automaticity and, in some instances, automaticity increases. This same effect of 움-adrenergic receptor stimulation to increase automaticity in depolarized preparations was demonstrated in bariumdepolarized fibers (Amerini et al., 1984) and in K⫹depolarized innervated ventricular myocytes (Han et al., 1990). These results can be interpreted as evidence for a voltage-dependent component of 움-adrenergic receptor inhibition of automaticity or an effect of 움-adrenergic stimulation to differentially modulate pacemaker currents at high or low levels of membrane potential.

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What of the increase in automaticity occurring both at high and at low levels of membrane potential? Both are blocked by WB 4101 (an antagonist that is selective for 움1A/c and 움1d adrenergic receptors), which also inhibits the 움-receptor-dependent hydrolysis of phosphoinositides in heart muscle (del Balzo et al., 1990). This had led to the hypothesis that the WB 4101-sensitive 움1adrenergic receptor pathway, perhaps through an IP3 or DAG-dependent action to modulate [Ca2⫹]i, increases automaticity. Although available evidence suggests that [Ca2⫹]i does not increase appreciably in the presence of 움-adrenergic agonists, the observation that the effect of 움-adrenergic agonists to increase automaticity in fibers having high membrane potentials is blocked by ryanodine suggests that calcium-dependent processes may be contributory. However, in depolarized fibers exposed to simulated ‘‘ischemic’’ solutions (p02 ⬍ 25 mm Hg; pH 6.8; [K⫹]o ⫽ 10 mM), 움-adrenergic agonists induce an increase in automaticity that is blocked by WB 4101, and not CEC, but is unaffected by ryanodine (Anyukhovsky et al., 1992). Manipulations that increase (ACh) or decrease (Ba2⫹) K⫹ conductance, respectively, decrease or increase the 움-adrenergic effect on automaticity in these depolarized fibers. This has led to the thought that an action to decrease K conductance may be important to the 움1-adrenergic effect on automaticity at low membrane potentials. This observation is supported by experiments in disaggregated Purkinje myocytes showing that 움1-adrenergic stimulation inhibits background gK, an action that would tend to enhance the pacemaker rate. The physiological and pathological importance of the 움1-adrenergic receptor signaling cascade to cardiac electrophsyiologic function appears to be minor as compared to 웁-adrenergic receptor actions. In the setting of normal cardiac electrophysiology, the effect of 움adrenergic receptor stimulation on sinus node function is at most trivial (Hewett and Rosen, 1985), except in settings where peripheral actions of 움-adrenergic receptor stimulation to elevate the blood pressure result in a vagal reflex to slow the sinus rate (see Section IV). At the level of secondary pacemakers of the heart (specialized conducting fibers outside the sinus node), the action of 움-adrenergic receptor stimulation to enhance Na/K pump function would tend to suppress the expression of automatic activity. In a sense, this would be predicted to be protective, insofar as the catecholaminedependent activation of 웁-adrenergic receptors would serve to enhance pacemaker activity at all levels of the mature and sympathetically innervated heart, but be counterbalanced by the 움-adrenergic receptor action to suppress all pacemakers, save those in the sinus node, and increase the likelihood that the sinus node will remain the dominant pacemaker.

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A series of experiments by Corr and associates in the 1970s (e.g., Sheridan et al., 1980) demonstrated that in anesthetized cats subjected to coronary occlusion and reperfusion, the ventricular arrhythmias that occurred in the setting of cardiac pathology were suppressed by 움1-adrenergic blockade. It should be noted that these results were seen in animal studies; to date there is no convincing evidence that 움-adrenergic receptor mechanisms are important to ischemia- and reperfusioninduced arrhythmias in human subjects. Of perhaps more importance to cardiac pathology is the role of 움1-adrenergic receptor stimulation to prolong repolarization (Rosen et al., 1984). In conditions such as the congenital long QT syndrome, where there is excess prolongation of repolarization as a result of increased inward Na current or decreased repolarizing K current (e.g., Roden et al., 1996), the 움-adrenergic receptordependent actions of released norepinephrine to further prolong repolarization might conceivably play a role in triggering the early afterdepolarizations that have been proposed as the ultimate determinants of the torsades de pointes arrhythmia. Again, the effect of 웁-adrenergic receptor stimulation is likely more important, given the great success seen in treating these arrhythmias with 웁 blockade. 움 blockade has not been reported in any series of patients, but the success of left sympathectomy in some patients in whom 웁 blockade is unsuccessful suggests that an 움-adrenergic receptor trigger might be involved here (Roden et al., 1996).

B. 웁-Adrenergic Receptor Cascade 웁1-adrenergic receptors modulate cardiac contractility exclusively through a cAMP-dependent mechanism. However, the mechanism(s) underlying 웁2-receptor actions in cardiac myocytes is less straightforward. While 웁2-adrenergic receptors can also link to the activation of adenylyl cyclase [in fact, they inherently couple to the activation of adenylyl cyclase more effectively than 웁1-adrenergic receptors (Levy et al., 1993; Green et al., 1992; Bristow et al., 1989)], the relationship between 웁2receptor-dependent increases in cAMP accumulation and inotropic responses in the heart is tenuous. This has led to speculations that 웁2 receptors might couple to distinct compartments of cAMP and/or cAMPindependent inotropic mechanisms (Bristow et al., 1986, 1989). Studies in neonatal and adult rat ventricular myocytes further bolster the notion that the signaling pathways mediating 웁1- and 웁2-adrenergic receptordependent modulation of contractile function must differ. These studies reveal important age-dependent differences in 웁2-receptor coupling to more distal elements in the signaling cascade that lead to profound age-

dependent differences in the mechanism for 웁2-receptor-dependent modulation of contractile function. In neonatal rat ventricular myocytes, stimulation of 웁1-adrenergic receptors leads to increased intracellular cAMP accumulation, enhanced phosphorylation of PLB and TNI, and an increase in the amplitude and an acceleration of the kinetics of the calcium and motion transients. All of these responses are also elicited by 웁2-adrenergic receptor stimulation with zinterol (Kuznetsov et al., 1995) (Fig. 3). While it would be reasonable to assume that 웁1- and 웁2-adrenergic receptors activate identical cAMP-dependent pathways in neonatal myocytes, studies indicate that this is not the case, as muscarinic receptors interfere with the action of 웁-adrenergic receptor subtypes via distinct inhibitory pathways. Carbachol blocks 웁1-adrenergic receptor-dependent inotropic and lusitropic responses in neonatal rat ventricular myocytes by preventing the rise in cAMP. In contrast, carbachol acts through M2-muscarinic receptors to block the 웁2-adrenergic receptor-dependent phosphorylation of PLB and TNI and the associated positive lusitropic response without interfering with cAMP accumulation or the positive inotropic response (Fig. 3). Although there is precedent to speculate that a Gi-dependent pathway, leading to stimulation of a protein phosphatase and local inactivation of the cAMP signal at cytosolic and sarcoplasmic reticular PKA target proteins, provides a mechanism for the actions of carbachol, direct evidence is still lacking. Nevertheless, these studies indicate that at the level of cAMP accumulation, the pathway activated by the 웁2-adrenergic receptor is relatively refractory to inhibitory modulation by the parasympathetic limb of the autonomic nervous system. The molecular basis for differences in signaling properties of 웁1- and 웁2-adrenergic receptors can only be speculated upon at present. The previous observation that purinergic receptors specifically target to the activation of type V (and not types IV or VI) adenylyl cyclase and are refractory to inhibitory modulation by adenosine (Puce´at et al., 1998) could constitute a potential mechanism for differences in signaling by individual 웁-adrenergic receptor subtypes. However, compartmentalization of receptor subtypes and/or adenylyl cyclase isoforms to membrane subdomains (including caveolae) could also provide a mechanism for differential susceptibility to inhibitory modulation. Similar to the scenario in neonatal rat cardiomyocytes, 웁1-adrenergic receptor stimulation with isoproterenol induces a robust increase in cAMP accumulation that is associated with an increase in the amplitude and an acceleration of the kinetics of both the calcium transient and the twitch in adult rat ventricular myocytes. In contrast, only high concentrations of the 웁2-adrenergic agonist zinterol (10⫺5 M) modulate contractile function

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FIGURE 3 Distinct inhibitory effects of carbachol on signaling by 웁1 and 웁2 receptors. (Left) Myocytes were challenged for 5 min with isoproterenol (ISO; 10⫺9 M) or zinterol (ZIN; 10⫺7 M), and cAMP accumulation in the absence (open bars) or in the presence (filled bars) of 10⫺6 M carbachol (CCH, starting 5 min prior to 웁receptor stimulation) was determined. (Right) Representative tracings illustrating the responses of myocytes driven electrically at 1 Hz and challenged with ISO (10⫺9 M) or ZIN (10⫺7 M), without or with carbachol (10⫺6 M), starting 5 min prior to 웁-receptor stimulation. Myocyte shortening was recorded as micrometers of motion of a glass microsphere on the cell surface. Because the motion of only one portion of the neonatal myocytes is monitored (rather than total cell length), the position of the microsphere before electrical stimulation is set to zero (diastole) and motion relative to the diastolic position is reported. Signal averaged motion transients before (solid line) and after (dashed line) stimulation with 웁 agonists are superimposed for comparison. Reprinted with permission from Aprigliano et al. (1997).

in adult rat ventricular myocytes. On the basis of this developmental difference in sensitivity to the cellular actions of 웁2-adrenergic receptors, it has been speculated that 웁2-adrenergic receptors (which would be activated by circulating epinephrine) may play a more important role in mediating the response to catecholamines in the noninnervated neonatal (or transplanted adult) than the innervated adult heart (Kuznetsov et al., 1995). The age-dependent difference in the sensitivity of 웁2-adrenergic receptors to agonist-induced activation is not due to a higher proportion of 웁2-adrenergic receptors in the neonatal heart; 웁2-receptors comprise a minor proportion of the total 웁-adrenergic receptor population in both neonatal and adult heart preparations (Kuznetsov et al., 1995). Activation of 웁2-adrenergic receptors generally increases the amplitude of the calcium transient and twitch in adult cardiomyoytes (Xiao and Lakatta, 1993; Kuznetsov et al., 1995). However, 웁2-adrenergic receptors do not accelerate the relaxation kinetics of the calcium transient and twitch (i.e., the response is quite distinct from the cAMP-dependent positive lusitropic response

typically observed following activation of 웁1-receptors; Fig. 4). The literature on whether zinterol elevates intracellular cAMP in adult rat cardiomyocytes is conflicting. In the presence of a phosphodiesterase inhibitor to prevent cAMP breakdown, two laboratories report preferential stimulation of cAMP formation by 웁1-, and not 웁2-, adrenergic receptors (Kuznetsov et al., 1995; Laflamme and Becker, 1998) (see Fig. 4). In contrast, another laboratory reports that when cAMP breakdown is not inhibited by a phosphodiesterase inhibitor, 웁1and 웁2-adrenergic receptors both induce a rise in intracellular cAMP content; but the magnitude of these responses is quite modest (Xiao et al., 1994). It has been suggested that 웁2-adrenergic receptors induce a rise in cAMP that is most pronounced in the soluble compartment of the cell (Xiao et al., 1994), where it would be dissociated from changes in calcium and contractile function. There also is controversy as to the role of cAMP in the functional response to 웁2-adrenergic receptor stimulation. The evidence that 웁2-adrenergic receptordependent increases in the amplitude of the calcium

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FIGURE 4 Distinct 웁2-adrenergic receptor responses in neonatal and adult rat ventricular cardiomyocytes. (Left) Effects of zinterol on cAMP accumulation in neonatal and adult rat ventricular myocytes. The significant, concentration-dependent increase in cAMP accumulation induced by zinterol in the newborn contrasts with the minimal increase in cAMP in the adult. (Right) Representative tracings comparing the effect of 웁2adrenergic receptor activation on the amplitude and kinetics of cell contraction in neonatal and adult ventricular cardiomyocytes. 웁2-receptor activation was accomplished with zinterol, following a 5-min pretreatment with the 웁1-receptor blocker CGP20712A (10⫺7 M, to prevent any 웁1-receptor activation). The top tracing illustrates typical results obtained in a neonatal myocyte, where a low concentration of zinterol increases the amplitude and accelerates the kinetics of the twitch. As in Fig. 3, in neonatal myocytes, the motion of only one portion of the cell (rather than total cell length) is measured. The bottom tracing illustrates a typical result obtained in adult cells, where 웁2-receptor activation requires a 100-fold higher concentration of zinterol and is associated with an increase in the amplitude of the twitch, but a delay in its relaxation kinetics. In adult myocyte records, total cell length is reported. In each case, cell shortening is recorded as microns of motion and is represented as a downward deflection. Reprinted with permission from Kuznetsov et al. (1995).

transient and contraction are not accompanied by an acceleration in the kinetics of relaxation or any significant phosphorylation of PLB (Xiao et al., 1993; Kuznetsov et al., 1995) could suggest that 웁2-adrenergic receptor agonists, modulate cardiac excitation–contraction coupling via a cAMP-independent mechanism. Indeed, experiments with H7 (at concentrations predicted to inhibit PKA, protein kinase C, and cGMP-dependent protein kinase) are consistent with this formulation; this inhibitor blocks 웁1-adrenergic receptor-dependent positive inotropic and lusitropic responses, but does not interfere with the mechanical response to 웁2-adrenergic receptor agonists (Jiang and Steinberg, 1997). Studies demonstrate that 웁2-adrenergic receptors activate a mechanism that would lead to an increase in myofilament responsiveness to calcium. The effect of 웁2-adrenergic receptor agonists to increase twitch amplitude is associated with a brisk intracellular alkalinization over

a similar time course. This response is blocked by the removal of bicarbonate from the extracellular buffer; it is not blocked by hexamethyleneamiloride and therefore is not likely to be mediated by the Na/H exchanger (Jiang et al., 1997). On the basis of these results, it can be argued that 웁2-adrenergic receptors elevate pHi, leading to increased myofibrillar calcium sensitivity as a mechanism for the 웁2-adrenergic receptor-dependent positive inotropic response. An alternative model has also been proposed to account for the 웁2-adrenergic receptor-dependent positive inotropic response in adult rat ventricular cardiomyocytes. Xiao and colleagues have reported that the 웁2-adrenergic receptor-dependent increase of L-type calcium current is blocked by the inhibitory cAMP analogues Rp-cAMPS and Rp-CPT-cAMPS (Zhou et al., 1997). These results have been taken as evidence that the functional response to 웁2-adrenergic receptors is

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mediated by a cAMP pathway that is compartmentalized to the sarcolemma (Kuschel et al., 1999). These investigators have presented further evidence that PTX pretreatment potentiates the response to 웁2 (but not 웁1) agonists (i.e., that 웁2-adrenergic receptors in adult ventricular myocytes couple to both Gs and a PTXsensitive Gi protein that selectively dampens the cellular response to 웁2-adrenergic receptor agonists). The actions of PTX are reported to include a marked upward and leftward shift in the dose–response curve for the effects of zinterol on contraction amplitude (Xiao et al., 1995) and a de novo positive lusitropic response, which is associated with PLB phosphorylation (Kuschel et al., 1999). This has been taken as evidence that 웁2-adrenergic receptor-dependent elevations in intracellular cAMP are localized and activate only L-type calcium channels that are adjacent to receptors at the plasma membrane; the cAMP signal is transmitted to other regions in the cell only following treatment with PTX. This model is supported by evidence that 웁-adrenergic receptor activation leads to a compartmentalized elevation of cAMP (only in the vicinity of the 웁-adrenergic receptor) in frog ventricular myocytes (Jurevicius and Fischmeister, 1996). Evidence suggests that activation of a phosphatase could constitute a molecular mechanism(s) to restrict the actions of cAMP generated in response to 웁2 (but not 웁1) activation (Kuschel et al., 1999). However, this would not explain the effects of PTX to increase 웁2-receptor affinity for agonists, suggesting that further studies are warranted. Finally, there has been interest in the question of 웁3-adrenergic receptor actions in cardiomyocytes. 웁3adrenergic receptors are potential targets for antiobesity and antidiabetic drugs, as they function to activate thermogenesis and lipolysis in adipose tissue and regulate the physiologic properties of the gastrointestinal tract. However, studies indicate that 웁3-adrenergic receptors are also present in human cardiomyocytes where, in contrast to 웁1- and 웁2-adrenergic receptors, they inhibit contractile function (Gauthier et al., 1996). The mechanism for the negative inotropic response appears to involve a PTX-sensitive G-protein and the activation of a nitric oxide synthase pathway (Gauthier et al., 1998). 웁3-adrenergic receptor expression is species dependent. 웁3-adrenergic receptors generally are not detected in rat cardiomyocytes. Nevertheless, under certain experimental conditions (submaximal agonist concentrations, rapid pacing rates), the nonselective 웁-adrenergic receptor agonist isoproterenol activates an endogenous NO pathway that blunts the 웁-adrenergic receptor-dependent inotropic response (Kelly et al., 1996). Therefore, an NO pathway and, at least in some species, a cardiac 웁3-adrenergic receptor subtype must be considered in the analysis of catecholamine action in the heart.

III. DEVELOPMENTAL REGULATION OF ION CHANNELS AND ITS RELATIONSHIP TO ADRENERGIC STIMULATION Among the critical end points of autonomic signaling are the ion channels that underlie the cardiac action potential and control the heartbeat. A number of ion channels that are sensitive to modulation by adrenergic and muscarinic receptor agonists and other neurotransmitters or neuropeptides are also strongly regulated developmentally, resulting in significant age-dependent differences in the availability of targets for the autonomic cascades.

A. L-Type Ca Current Osaka and Joyner (1991) reported a postnatal increase in L-type Ca current (Ica,L) density in the rabbit ventricle with no change in the voltage dependence of activation or inactivation. There was a modest increase in the rate of inactivation with age that was suggested to be secondary to the increased current density and associated Ca-dependent inactivation. A similar postnatal increase in ICa,L density is observed in the rat ventricle, where it has been attributed to the influence of sympathetic innervation during development. Sympathetic innervation appears to exert its effect via neurally released NPY (Protas and Robinson, 1999), although evidence suggests a role for neurally released norepinephrine acting at 웁-adrenergic receptors as well (Maki et al., 1996). 웁-adrenergic receptor stimulation of ICa,L is also developmentally regulated. Sperelakis and colleagues demonstrated that in the rat ventricle isoproterenol stimulation of ICa,L increases during the late fetal period, is fully effective in the neonate, and decreases during postnatal development. They also suggested that at least part of the stimulatory effect of isoproterenol was cAMP independent, particularly in the neonatal rat. Further, they found the isoproterenol response to be more sensitive to accentuated antagonism by carbachol in the neonate than adult and associated this with decreased activity of a PTX-sensitive G-protein (Katsube et al., 1996, 1998). Qualitatively similar results are observed in the rabbit, albeit with a somewhat different time course. In this species the 웁-adrenergic receptor agonist isoproterenol is less effective than forskolin or intracellular cAMP at stimulating the current in the neonate, but equally effective with these other agents in the adult. It has been suggested that this difference arises from an age-dependent reduction in tonic inhibition of adenylyl cyclase by Gi (Osaka and Joyner, 1992), consistent with the observation that accentuated antagonism is more marked in the neonatal than adult rabbit ventricle

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(Osaka et al., 1993). It should be mentioned that these reports of greater accentuated antagonism of isoproterenol-stimulated ICa,L in the neonate are difficult to reconcile with other reports of a greater 웁2-adrenergic receptor contribution to contractility in the neonate and the relative insensitivity of this cascade to accentuated antagonism. This dichotomy will need to be resolved in the future. Finally, although profound 웁-adrenergic effects on Ca currents have been noted, no major role has been seen for 움-adrenergic receptor stimulation. Indeed, the greatest magnitude of effect in any study has been an approximate 10% increase in the aequorin signal recorded from cardiac cells by Endoh and Blinks (1988). However, there have been reports of greater 움-adrenergic responsiveness of the L-type current in neonatal than adult ventricle (Liu et al., 1994).

B. Transient Outward Current In the rat ventricle the 4-aminopyridine-sensitive transient outward potassium current, Ito (sometimes referred to as Ito1), is largely responsible for repolarization and its inhibition results in action potential prolongation. In other species, such as the dog, Ito contributes to the brief but transient repolarization (notch) at the beginning of the plateau. In this case, inhibition of the current can actually shorten action potential duration by elevating the plateau and causing greater activation of the delayed rectifier. 움-adrenergic receptor agonists inhibit Ito in rat ventricle and prolong repolarization. 움adrenergic agonists also prolong action potential duration in canine ventricle, but here the effect appears to be through inhibition of IKs . Not only would inhibition of Ito not be predicted to prolong action potential duration in the canine ventricle, but direct voltage clamp studies demonstrate no effect of 움-adrenergic receptor agonists on canine ventricular Ito . This does not reflect a simple species difference between rat and canine heart because Ito in canine Purkinje myocytes is inhibited by 움-adrenergic receptor agonists (Robinson et al., 2000). Thus, the ineffectiveness in canine ventricle probably reflects either a distinct 움-adrenergic receptor signaling cascade or a difference in the end target (i.e., the ion channel). Supporting the latter possibility is evidence that rat ventricular Ito is predominantly the Kv4.2 isoform, whereas the canine ventricle largely expresses the Kv4.3 isoform (Dixon et al., 1996). In addition, there is strong developmental regulation of Ito. The current is absent or small at birth, in both the rat and the canine ventricle. In the canine ventricle, it matures approximately 2 months postnatal. The current is also downregulated readily by disease and, for example, is reduced markedly in myocytes isolated from

the surviving epicardial border zone of hearts 5 days postmyocardial infarction (Lue and Boyden, 1992). In summary then, the impact of 움-adrenergic receptor modulation of Ito is complex. For modulation to occur, the current must be present, which largely eliminates both immature and diseased hearts. The current must be responsive to modulation, which eliminates some species and/or cardiac regions. Even then, whether the effect of Ito inhibition is shortening or lengthening of the repolarization time course will depend on the interactions of Ito with other currents active during the cardiac plateau.

C. Delayed Rectifier Current The delayed rectifier current, IK, contributes to the repolarization of the action potential. It consists of two components, IKr and IKs, with the relative contributions varying with tissue and species. The IKs component is generally considered to be responsive to 웁-adrenergic receptor stimulation, and this component has been reported to change developmentally (cf. Nerbonne, 1998). There are also developmental differences in the electrophysiological response to 웁-adrenergic receptor agonists, which, together with other data, suggest diversity in the signaling cascades coupled to individual ionic channels. In canine epicardium, 웁-adrenergic receptor stimulation increases the plateau at all ages studied, but action potential duration is shortened only in adults (Charpentier et al., 1996). The increase in plateau amplitude is explained by the potentiation of L-type calcium current, which is increased by 웁-adrenergic receptor stimulation in both young and adult canine ventricle. In contrast, IK is increased by 웁-adrenergic receptor stimulation only in adult canine ventricular myocytes; neonatal cells are unresponsive (Fig. 5). This age-related difference in the 웁-adrenergic receptor modulation of IK probably explains, at least in part, the absence of isoproterenol-induced shortening of the action potential in young dogs. Furthermore, the fact that isoproterenol elevates the plateau and increases ICa,L in ventricular myocytes from young dogs demonstrates that there is not a general lesion in the 웁-adrenergic cascade at an early age. Most of the single cell data to date on 웁-adrenergic receptor modulation of IK come from the adult guinea pig ventricle, and data in this tissue also suggest diversity between 웁-adrenergic receptor modulation of IK and ICa,L . For example, although both IK and ICa,L are increased by isoproterenol, forskolin, and cAMP at temperatures of 30⬚C or greater, only ICa,L responds at room temperature (Walsh et al., 1989). In addition, it has been reported that the two currents respond differently to 웁1- and 웁2-receptor subtype activation (Iijima et al.,

43. Changes in Autonomic Responsiveness

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FIGURE 5 Effect of isoproterenol on the delayed rectifier current in an adult and a 23-dayold canine epicardial cell. (A) Records from an adult cell. On the left are currents recorded during voltage steps to 10, 30, and 50 mV from a holding potential of ⫺40 mV before and after isoproterenol (Iso, 10⫺7 M). On the right are isochronal (at 3400 msec) current density–voltage relationships in control (䊊) and isoproterenol (䊉). (B) Records from a young cell. Left and right panels are as described in (A). Note the absence of an isoproterenol response at this age. Reprinted with permission from Charpentier et al. (1996).

1990), with the nonselective agonist isoproterenol being equieffective but a 웁1-receptor selective agonist preferentially enhancing ICa,L . In addition, there are species-specific differences in the nature of the 웁-adrenergic receptor modulation of IK that suggest additional complexity. In guinea pig, one observes both voltage-independent and voltagedependent enhancement of IK , depending on whether the channel is phosphorylated by protein kinase C or protein kinase A, respectively (Walsh and Kass, 1991). In contrast to the voltage dependence of PKA activation of IK in the guinea pig, there is no such voltage dependence in adult canine ventricular myocytes. Thus there appear to be species-specific differences in the extent of voltage dependence of the response, which may re-

late, in part, to the location of the phosphorylation site(s) of the channel in each tissue. The effect of 움-adrenergic receptor agonists on the delayed rectifier has been studied in the guinea pig and rat. In the former species, IK is increased and the action potential is accelerated. In the latter species, IK is decreased and the action potential is prolonged. The effect of 움-adrenergic receptor agonists on action potential repolarization has also been studied in canine Purkinje fibers (Lee and Rosen, 1994). Here, 4-aminopyridine was used to block the transient outward current, Ito . Nonetheless, an 움 agonist still prolonged repolarization. In contrast, the IK blocker, WY48986, completely attenuated the 움-adrenergic receptor-dependent prolongation of action potential duration. This observation is

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more consistent with an 움1-adrenergic receptor-dependent decrease in IK than any other single mechanism.

D. Pacemaker Current In the sinoatrial node, the primary current contributing to automaticity and one of the currents most sensitive to both adrenergic and muscarinic agonists is the pacemaker current, If. Limited information on the developmental regulation of this current and its autonomic sensitivity in the sinotrial node is available. Current density is modestly greater in neonate than adult, but the maximal response to isoproterenol is unaffected; the dose-dependent response to either isoproterenol or acetylcholine has not been studied as a function of age. A greater developmental change in If is seen in the ventricle. Originally this current was thought to be absent in ventricular myocytes. However, it is now recognized that the current is present in both newborn and adult ventricle, but with markedly different voltage dependence. In the neonatal rat, the current activates when the cell membrane potential is negative to ⫺70 mV, whereas in the adult the threshold for activation is ⫺113 mV (Robinson et al., 1997). Similar negative activation values have been observed in adult guinea pig and dog ventricle myocytes. Interestingly, more positive activation, at physiological voltages, is seen in cardiac disease (both the spontaneously hypertensive rat and the failing human heart; e.g., Cerbai et al., 1997). Because 웁-adrenergic receptor agonists enhance the current by shifting its activation to more positive potentials and because muscarinic agonists inhibit the current by shifting its activation negative, the appearance of the current at physiologic voltages in the normal neonatal ventricle and the diseased adult ventricle suggests a greater potential contribution to autonomic responsiveness in these preparations than in the normal adult ventricle. In addition, it is now known that there are at least four isoforms of the pacemaker channel, termed HCN1– HCN4, and that the relative abundance of message for the different isoforms varies with the region of the heart and development (Shi et al., 1999). Expression data to date suggest isoform-specific differences in the maximal voltage shift induced by cAMP. Thus, the age-dependent change in isoform expression may turn out to be linked to a developmental difference in autonomic sensitivity.

E. Other Currents One also should not ignore developmental changes in other currents, even if they are not major targets of autonomic modulation. For example, IK1 increases

postnatally in the ventricle. In the neonate, the reduced IK1 density and corresponding increase in specific membrane resistance will magnify the effect of any change in If activation. In contrast, in the adult ventricle, where IK1 density is greater, even a significant positive shift of If may have only a modest effect on the slope of diastolic depolarization. As another example, the neonatal rabbit sinoatrial node action potential has a strong contribution from a Na current that is absent in the adult (Baruscotti et al., 1996), where the action potential is largely Ca dependent. The contribution of ICa,L is reduced in the neonatal sinus node due largely to the presence of the Na current. Because Na currents are, in general, less affected than ICa,L by autonomic agonists, this potentially impacts the developmental autonomic modulation of the sinoatrial node.

IV. PARASYMPATHETIC AND OTHER NONADRENERGIC SIGNALING CASCADES With respect to cardiac function, the parasympathetic limb of the autonomic nervous system is largely described in the context of inhibition of automaticity and suppression of AV nodal conduction via M2-receptors. However, non-M2 excitatory actions have been reported in the neonatal ventricle, and the ontogeny of sympathetic innervation may play a role in the age-dependent loss of this response (see Robinson, 1996). It is clear that in the mature heart, the M2-agonist action of acetylcholine is the major determinant of expression of parasympathetic function. At the level of the sinus node and of secondary atrial pacemakers, the effect of M2 stimulation is to decrease the slope of phase 4 depolarization. This occurs via at least two mechanisms (DiFrancesco et al., 1989): the first, seen at nanomolar concentrations of acetylcholine (ACh), is to decrease the pacemaker current, If . The second, at micromolar concentrations, is to increase IK,ACh , thereby hyperpolarizing the membrane, generating the net outward current, and accelerating repolarization while decreasing the automatic rate. It is of interest that the acceleration of repolarization may have an important role in a subset of cardiac arrhythmias; i.e., a small percentage of instances of atrial fibrillation occur as the result of parasympathetic stimulation, apparently as a result of the shortened atrial effective refractory period that accompanies the acceleration of repolarization (Coumel et al., 1978). At the level of the AV node, parasympathetic stimulation suppresses conduction markedly, thereby slowing the propagation of impulses from atria to ventricles. This is the result of the effect of ACh to decrease the density of L-type Ca current, which is the major determinant of the AV nodal action potential upstroke.

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It is important to stress that some actions of acetylcholine can result from direct modulation of channels by ligand-activated M2 receptors (see Quarmby and Hartzell, 1995). However, an equally if not more important action of parasympathetic stimulation involves an interaction with 웁-adrenergic receptors and is referred to as accentuated antagonism (Levy, 1977; Lindemann and Watanabe, 1984). In this scenario, the 웁-adrenergic receptor-dependent modulation of pacemaker, Ca, and IKs currents (leading to changes in pacemaker rate, increases in contractility and AV nodal conduction, and acceleration of repolarization) is attenuated by M2-muscarinic receptors. This can occur via a Gi-dependent mechanism to attenuate cAMP accumulation in response to 웁-adrenergic receptor activation or result from an action at some target downstream from cAMP in the signaling cascade (and may differ for 웁1- and 웁2adrenergic receptors, as noted earlier). This mechanism is of especial importance in the ventricles where little parasympathetic effect is expressed except in the situation of preexisting 웁-adrenergic receptor stimulation. In addition, as discussed earlier, there are clear developmental changes in the extent of counterbalance between inhibitory muscarinic and excitatory 웁-adrenergic receptor responses (accentuated antagonism). In addition to norepinephrine and acetylcholine, the autonomic nervous system releases a number of other substances locally or into the circulation. There is virtually no information on age-dependent changes in the acute actions of these neuropeptides or other nonadrenergic/nonmuscarinic neurotransmitters on the cardiovascular system. Nonetheless, there are several reasons to believe that these substances may be important in understanding developmental changes in cardiac autonomic responsiveness. First, as outlined earlier, at least one of these substances (NPY) acts as a long-term or trophic modulator of autonomic responsiveness, with its release at the time of cardiac sympathetic innervation contributing to changes in both the 움-adrenergic receptor signaling cascade and the developmental regulation of currents that are the end target of autonomic signals. Moreover, NPY has been shown to suppress the function of the pacemaker current If (Chang et al., 1994). In other words, release of this peptide as a result of sympathetic stimulation provides an internal brake on the effect of the adrenergic receptor neurohumor, norepinephrine, to increase the pacemaker current, the slope of phase 4, and, conceivably, the pacemaker rate. The counterpart of this situation in the parasympathetic nervous system is seen via the vagal release of vasoactive intestinal peptide (VIP). The effect of this peptide is to increase If and pacemaker rate (Chang et al., 1994): again the presence of an internal brake is seen, as one that would limit the effect of acetylcholine to excessively

suppress the sinus node and other pacemakers. Although experiments with animal models have suggested that the effects of both neuropeptides are expressed physiologically, these results have not been consistent, rendering the ultimate importance of neuropeptide release to pacemaker function still somewhat uncertain (see Shvilkin et al., 1994; Sosunov et al., 1996).

V. SUMMARY There are pronounced age-dependent changes in autonomic responsiveness of the heart during postnatal development. In general, these differences result in greater excitatory responses in the young heart. For example, the 움-adrenergic receptor response is entirely excitatory (via 움1A/c or 움1d receptors) in the neonate, but is both excitatory and inhibitory (via 움1B receptors) in the adult. The 웁-adrenergic receptor response is excitatory at both ages, but is contributed to both by 웁1 and 웁2 receptors in the neonate but largely by 웁1 in the adult. Muscarinic responsiveness is both excitatory (via M1 or M3 receptors) and inhibitory (via M2 receptors) in the neonate, but is entirely inhibitory in the adult. Given that parasympathetic innervation of the heart precedes sympathetic innervation, this may be a mechanism to avoid excess inhibitory influence during a transient period of parasympathetic/sympathetic imbalance.

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44 Inotropic Mechanisms in Cardiac Muscle DONALD M. BERS Department of Physiology Loyola University Chicago Maywood, Illinois 60153

I. INTRODUCTION

II. MAJOR CELLULAR TARGETS

The major ways to alter the inotropic state in the heart are to modulate the cellular Ca transient or alter how the myofilaments respond to the Ca transient. Thus, it is useful to start the discussion by reviewing the basic Ca fluxes that occur during the cardiac contraction– relaxation cycle (see Fig. 1; Bers, 1991). During the cardiac action potential (AP), L-type Ca channels are activated and Ca entering the cell via the Ca current (ICa) can trigger Ca release from the sarcoplasmic reticulum (SR) and, to a certain extent, can contribute to activation of the myofilaments directly (more on this later). Ca can also enter the cell via Na/Ca exchange during the action potential, but under normal physiological conditions the amount of Ca entering via this pathway is very small compared to the amount entering via ICa. Ca entry plus the amount released by the SR via Ca-induced Ca release (CICR) raises cytosolic [Ca] ([Ca]i) and, as [Ca]i rises, it binds to numerous Ca buffers in the cytosol. One of the most prominent and functionally important cytosolic Ca buffers is the thin filament protein troponin C (TnC). When Ca binds to TnC it activates the myofilaments to produce a contraction. For relaxation to occur (so the heart can refill), [Ca]i must decline so that Ca dissociates from TnC, turning off the contractile machinery. Four Ca transport processes remove Ca from the cytosol: (1) the SR Ca-ATPase, (2) sarcolemmal Na/Ca exchange, (3) the sarcolemmal Ca-ATPase, and (4) the mitochondrial Ca uniporter. The SR Ca-ATPase and the Na/Ca exchanger are the most important quantitatively and the relative contributions will be discussed further later.

The main ways to enhance cardiac contractility are to either increase the force produced by the myofilaments for a given Ca transient or to increase the amplitude or duration of the Ca transients. These will be discussed in this order.

Heart Physiology and Pathophysiology, Fourth Edition

A. Myofilament Ca Sensitivity Myofilaments are responsible for transducing the [Ca]i signal to a mechanical one as force development, shortening, or both (as in a normal heart beat). The molecular mechanism of how the myofilaments work is discussed Chapter 30. Here, myofilament force will be considered a simple function of [Ca]i . Figure 2 (thick Normal curve) shows a typical sigmoid [Ca]i dependence of force for cardiac myofilaments. Thus, at higher [Ca]i , greater force can be achieved and that is why alteration of the Ca transient is a powerful way to modulate contractility. However, for a given Ca transient (lets say from 0.1 to 1 애M), the myofilament force can be altered. If the average myofilament crossbridge force is increased (without changing the shape of the [Ca]i dependence), the maximal force will be higher and force will also be higher at any given [Ca]i . A second way to increase the myofilament response to a given Ca transient is to increase the sensitivity to [Ca]i . This shifts the whole curve to the left but parallel to the Normal curve, again resulting in higher force for any [Ca]i . Many inotropic agents produce this type of shift in myofilament Ca sensitivity. A potential pitfall with this particular strategy is that the heart may not completely relax

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IX. Cardioactive Drugs

FIGURE 1 Ca regulation in ventricular myocytes. The schematic diagram indicates various Ca transport pathways, which bring Ca into and remove Ca out of the cytosol during normal Ca handling in ventricular myocytes. See text for details (RyR, ryanodine receptor; Myofil, myofilaments; Mito, mitochondria).

at diastolic [Ca]i of 150–200 nM (see Fig. 2). This would not only create increased diastolic stiffness and limit refilling, but it would also result in substantial energy utilization during diastole (as crossbridge cycling uses cellular ATP) and thus increase the demand for coronary blood flow. A third way to modulate responsiveness of myofilaments to [Ca]i is to alter the cooperativity. The normal curve in Fig. 2 has a Hill coefficient of 2, which is indicative of the steepness of the curve or how much Ca facilitates the further activation of the myofilaments by Ca (i.e., cooperativity) The equation for the curves in

FIGURE 2 Activation of cardiac myofilaments by Ca. The thick curve is the normal situation (with Kd ⫽ 1.6 애M Ca and n ⫽ 2), whereas thin solid curves illustrate increases in either maximal force (Tmax ⫽ 127%) or myofilament Ca sensitivity (from Kd ⫽ 1.6 to 0.56 애M). The broken curve shows an increase in Hill coefficient (n ⫽ 4) and an increase Kd (from 1.6 to 0.8 애M).

Fig. 2 is tension ⫽ Tmax /(1 ⫹(Kd /[Ca]i)n), where Tmax is the maximum tension, Kd is the [Ca]i for half-maximal activation, and n is the Hill coefficient. It is clear that the degree of cooperativity in response to Ca can change under certain conditions and an increased cooperativity could also increase force for a given [Ca]i . Indeed, evidence suggests that the physiological Hill coefficient in intact heart muscle is much higher than 2 (Yue et al., 1986). A Hill coefficient of 2 is shown in the normal curve in Fig. 2 and is based on results in isolated myofilaments or skinned fibers. The example in Fig. 2 (broken line) shows an increase in the Hill coefficient to 4 (increased cooperativity) combined with a slight decrease in Kd (increased Ca affinity). This specific combination of effects has the potential advantage of not elevating diastolic force, but enhancing the myofilament activation greatly for a given systolic [Ca]i . Increasing sarcomere length also increases force development due to a combination of greater thick–thin filament overlap as well as increased myofilament Ca sensitivity (allowing more crossbridges to interact). However, this is normally not considered an altered inotropic state, but rather a heterometric regulation of cardiac force (or Starling’s law of the heart). The inotropic state or contractility refers to changes in contractile force that are independent of changes in sarcomere length. The kinetics of crossbridge cycling can also affect the rate of cardiac muscle shortening and thus the rate of ejection of blood in the heart. Thus there are several ways in which altered myofilament characteristics can change the inotropic state in the heart.

44. Inotropic Mechanisms

B. Altered Ca Fluxes Because the amplitude and time course of the Ca transient are the driving functions for the behavior of the myofilaments in terms of contraction and relaxation, it is important to consider how alterations in the Ca transient may occur. As indicated in Fig. 1, several key Ca transport systems should be considered. 1. Ca Influx The two primary pathways for Ca influx into ventricular myocytes are via voltage-dependent Ca channels (as ICa) and via Na/Ca exchange. Both L-type and T-type Ca channels have been reported in cardiac myocytes (Bean, 1989; Bers, 1991). L-type ICa activates at membrane potentials (Em) above ⫺40 mV (more positive than for T-type ICa) and inactivates more slowly than T-type ICa. However, most ventricular myocytes only express L-type ICa, and this is undoubtedly the most functionally important with respect to both total transsarcolemmal Ca influx and also in controlling E-C coupling. Thus, further discussion will assume that ICa refers to ICa via L-type Ca channels. The crucial role in E-C coupling is also underscored by the apparent colocalization of L-type Ca channels (or dihydropyridine receptors) with the SR Ca release channel (or ryanodine receptor) and the central role of CICR in the regulation of SR Ca release. ICa is activated very rapidly by depolarization during the cardiac AP and this early Ca entry activates SR Ca release (via local CICR). ICa also inactivates in a voltage and Ca dependent manner during the AP and Ca transient. Most of the inactivation of ICa during the AP is due to Ca-dependent inactivation, and this results from both the Ca entering via the Ca channel itself as well as the Ca released from the nearby SR Ca release channels (ryanodine receptors; Puglisi et al., 1999). Ca entry and Ca release contribute about equally to this shut off of the Ca channel during the AP. This creates a negative feedback, whereby if SR Ca release is very large (e.g., due to high cellular and SR Ca load), then ICa turns off faster, thereby preventing further loading of the cell with Ca. This may be relevant in the context of preventing cellular Ca overload (see later). Conversely, if SR Ca release (or influx) is low, then ICa will not turn off as rapidly, which will tend to increase Ca influx (and cellular Ca load). Thus things that either increase ICa amplitude or reduce inactivation of ICa will increase Ca entry and tend to increase cellular Ca content (some of which can be released subsequently from the SR). Increasing ICa can thereby be positively inotropic by increasing (1) Ca entry, (2) the signal that triggers SR Ca release, and

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(3) the cellular and SR Ca content at subsequent beats. Indeed, the amount of SR Ca release (for a given SR Ca load) is a graded function of the amplitude of the ICa trigger (Beuckelmann and Wier, 1989). These factors make the modulation of ICa a potentially powerful means of modulating inotropic state. Ca can also enter via Na/Ca exchange, although the amounts under physiological conditions may be small. Na/Ca exchange transports three Na ions for one Ca ion so that there is net movement of one positive charge per cycle in the direction of Na transport. Thus Ca influx via Na/Ca exchange is an outward current (INa/Ca) and Na/Ca exchange is sensitive to Em (as well as the Na and Ca concentration gradients across the membrane). Because there is a large electrochemical driving force for both Ca and Na entry into cells, there is an Em where net transport is zero (i.e., where the electrochemical potential in the [Ca] gradient (2Em ⫺2ECa) is exactly the same as that in the [Na] gradient for three Na ions (3Em ⫺3ENa). This is like a reversal potential for an ion channel, and this ENa/Ca is typically about ⫺35 mV in a normal resting myocyte (see Fig. 3A). Thus at very positive Em during the peak of the AP, Ca influx via Na/Ca exchange is favored, whereas at rest (Em ⫽ ⫺80 mV) Ca efflux is favored. However, as local [Ca]i rises as a consequence of both Ca entry via ICa and SR Ca release, ENa/Ca becomes much more positive and Na/Ca exchange can shift to net extrusion of Ca. This probably limits Ca influx via Na/Ca exchange to a very small amount very early in the AP. As will be seen later, when [Na]i is elevated, Na/Ca exchange may bring in more Ca during the AP and this can be appreciated by comparing Fig. 3B to Fig. 3A. Either enhancing Ca influx or inhibiting Ca efflux via Na/Ca exchange can alter cellular and SR Ca load dramatically and thereby have major effects on contractility. Ca entry via Na/Ca exchange may also be able to trigger SR Ca release, but the physiological role of this mechanism remains controversial at normal [Na]i . 2. SR Ca Load and Release The amount of Ca released from the SR is one of the most critical determinants of the amplitude of the Ca transient. If there is more Ca available for release, the same trigger for SR Ca release would be expected to produce proportionally more Ca release. While this is true, there is also a potent regulatory effect of SR Ca content on SR Ca release (Bassani et al., 1995), i.e., increasing SR Ca load also increases the fraction of SR Ca released for a given ICa trigger. When the SR Ca content is relatively low, fractional SR Ca release can be extremely small, as if SR Ca release is nonfunctional (tending to increase SR Ca load at subsequent beats as

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Ca content would rise). When the SR Ca load is extremely high, SR Ca release appears to occur spontaneously (even without an ICa trigger), such that it may be the SR Ca load itself that triggers these ‘‘spontaneous’’ SR Ca release events that reflect cellular Ca overload. It should also be noted that even at rest there is a finite probability that localized SR Ca release events occur, and these are referred to as Ca sparks (Cheng et al., 1993). The probability of these Ca sparks occurring is increased at a higher [Ca]i or SR Ca load (Satoh et al., 1997), and during the normal twitch these Ca spark events are synchronized in time throughout the cell by the ICa trigger during the AP. This is what constitutes the normal Ca transient. When the SR Ca load and/or [Ca]i at rest is high enough, these focal Ca sparks can activate SR Ca release at adjacent regions, resulting in Ca waves that propagate through the entire cell. These are the hallmark of Ca overload and can be arrhythmogenic (see later). Thus, the amount of Ca stored in the SR plays a crucial role in governing the amplitude of the Ca transient. The SR Ca load is a result of the rate of SR Ca release (primarily due to Ca sparks or more global Ca release events) and SR Ca uptake (mediated by the SR Ca-ATPase). The forward rate of Ca transport by the SR Ca-ATPase depends on the [Ca]i in a manner analogous to the dependence of tension on [Ca]i in Fig. 2. However, the SR Ca-ATPase is an enzyme that will also exhibit a reverse reaction when the concentration of product is high (in this case as intra-SR [Ca] rises during [Ca]i decline). Thus, the SR Ca pump has both a forward Ca uptake mode as well as a backward Ca backflux mode (Shannon et al., 2000). In this context the concentration of ATP (vs ADP) in the vicinity of

the SR Ca-ATPase is also important in determining the net Ca transport rate by the SR Ca-ATPase. For example, if there is an increase of the ratio of [ADP]/ [ATP] during energetic compromise, this would decrease the rate and extent of SR Ca uptake. Phospholamban (PLB) is an important endogenous inhibitor of the SR Ca-ATPase and it shifts the [Ca] dependence of forward Ca pumping toward higher [Ca]i (Koss and Kranias, 1996). That is to say, it takes a much higher [Ca]i to reach the same Ca transport rate in the presence of PLB. Physiologically, this inhibitory effect of PLB can be relieved when PLB is phosphorylated by either cAMP-dependent protein kinase (PKA) or Ca–calmodulin-dependent protein kinase (CaMKII). It is thought that phosphorylation of PLB decreases the interaction between PLB and the SR Ca-ATPase (derepressing the Ca pump). Indeed, when the PLB gene is ablated in the PLB knockout mouse, cardiac relaxation and [Ca]i decline are accelerated greatly and the SR Ca content is increased (Luo et al., 1994; Li et al., 1998). Thus, PLB is an important modulator of SR Ca content because PLB phosphorylation can increase forward SR Ca-ATPase greatly, such that with unaltered leak the SR Ca content can be enhanced greatly (with the consequent enhancement of the Ca transient). 3. Ca Removal during Relaxation and Diastole The four mechanisms for Ca extrusion from the cytosol during relaxation indicated in Fig. 1 (SR Ca-ATPase, Na/Ca exchange, sarcolemmal Ca-ATPase, and mitochondrial Ca uniporter) contribute differentially to the time course of [Ca]i decline. The relative contributions also differ in different cardiac muscle tissues (Bassani

FIGURE 3 Changes in Na/Ca exchange reversal potential (ENa/Ca) during the rabbit ventricular AP. This schematic diagram shows how ENa/Ca is expected to change during the AP for two different levels of [Na]i (8.9 and 12.7 mM). For an activity coefficient for Na of 앑0.78, these correspond to Na activities of 7 and 10 mM. When Em is positive to ENa/Ca, Ca influx via the Na/Ca exchanger is favored thermodynamically (shaded areas). When Em is negative to ENa/Ca , Ca extrusion is favored (i.e., at all other times). Resting [Ca]i ⫽ 150 nM, and [Ca]o ⫽ 2 mM, and [Na]o ⫽ 140 mM for both traces and peak [Ca]i are as indicated. The [Ca]i trace reaches a peak at 40 msec after the action potential begins (after Bers, 1987, 1991).

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FIGURE 4 Integrated Ca fluxes during twitch relaxation in (A) rabbit and (B) rat ventricular myocytes. Free [Ca]i during relaxation of a normal twitch was used as a driving function so that Ca flux via each system could be calculated with respect to time, using the [Ca]i dependence of transport (in 애mol/liter cytosol/sec) as measured when each system was studied in isolation. SR, SR Ca-ATPase; NaCaX, Na/Ca exchange. The slow systems are a combination of sarcolemmal (SL) Ca-ATPase and mitochondrial (Mito) Ca uniporter. Percentages indicate the fraction of the total Ca removal from the cytosol attributable to each system when they interact dynamically in the cell (based on Bassani et al., 1994).

et al., 1994). Figure 4A shows that in rabbit ventricular myocytes, the SR Ca-ATPase is responsible for removing 70% of the activator Ca from the cytosol, whereas the Na/Ca exchange removes 28%, leaving about 1% each for the sarcolemmal Ca-ATPase and mitochondrial Ca uniporter (referred to collectively as the slow systems). In rat ventricles, the SR Ca-ATPase is more potent (probably due to more pump molecules per unit cell volume) and the Na/Ca exchanger is weaker, resulting in a balance of 92:7:1% for SR Ca-ATPase, Na/ Ca exchange, and slow systems (see Fig. 4B). In mouse ventricles the situation is quantitatively similar to rat, whereas guinea pig, ferret, and human ventricles are more similar to rabbits in this regard. It is important to keep in mind that if 28% of the activator Ca in rabbits is extruded from the cell by Na/ Ca exchange at a steady-state twitch, there must be a similar amount of Ca entry (e.g., via ICa) at each beat. Otherwise there would be progressive loss or gain of cellular Ca (i.e., this would not be a steady state). Indeed this seems to be the case and in rabbits there is more Ca entry during an AP and less SR Ca release than in rats (Yuan et al., 1996; Bers, 2000). 4. Ca Transient Kinetics and Cytosolic Ca Buffering In addition to varying the amount of Ca entering the cell or released by the SR, changes in the time course of the Ca transient can alter force production. For example, if [Ca]i remains high for a longer than normal period of time, this will prolong the time during which the myofilaments can generate force. Indeed, a longer ‘‘active state’’ would allow the myofilaments to come closer to steady state with respect to force development at a given [Ca]i . This can increase contractility. The [Ca]i is also heavily buffered. It takes the addition of about

50–100 애mol Ca/liter cytosol to raise [Ca]i from 100 to 700 nM during a twitch (Berlin et al., 1994). This is a buffering power of about 100:1. While some of the major cytosolic Ca buffers are well known and functionally important for contractility (e.g., 70 애M TnC, 40 애M SR Ca-ATPase sites), there are many other buffers that contribute to this (Bers, 2000). Thus if one were to reduce the concentration of a particular cytosolic Ca buffer, then the same Ca influx and release would raise [Ca]i more and could increase force production as well. Indeed, in neonatal rat ventricular myocytes, Ca buffering is much lower than in adult (Bassani et al., 1998), such that less Ca is required to raise [Ca]i to the same degree (although part of the lower Ca buffering is due to a lower concentration of myofilament proteins). It can be appreciated that there are many potential targets for inotropic intervention in heart. Many of these are dynamically interacting, and changes in one system can lead to changes in other systems. At this point it is practical to take a look at a few different mechanisms that can alter cardiac contractility in order to synthesize some of this information in a practical context.

III. 웁-ADRENERGIC AGONISTS Activation of the sympathetic nervous system causes the release of norepinephrine in the heart and this can potently activate 웁-adrenergic receptors (Fig. 5). Activation of the 웁-adrenergic receptor activates a G-protein (Gs), which then activates adenylate cyclase (AC), which produces cyclic AMP (cAMP) from ATP. Activated Gs has also been suggested to directly stimulate ICa to some extent. The cAMP produced by adenylate cyclase binds to the regulatory subunit of PKA and activates the catalytic subunit of the kinase. Several

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FIGURE 5 웁-adrenergic activation of Ca transporters and myofilaments in cardiac muscle. Norepinephrine (Norepi) is indicated to activate the 웁-adrenergic receptor, causing the GTP-binding protein (Gs) to stimulate adenylate cyclase (AC) and possibly also ICa directly (dotted lines). The cAMP produced activates protein kinase A (PKA) by allowing the regulatory subunit (Reg) to dissociate from the catalytic subunit. Broken lines indicate the targets for PKA phosphorylation (see text).

PKA targets in cardiac myocytes are relevant to the inotropic state. PKA-dependent phosphorylation of the sarcolemmal Ca channel increases ICa dramatically. This increases Ca influx (contributing to myofilament activation and SR Ca loading at subsequent pulses) and also increases the trigger for SR Ca release. PKA also phosphorylates PLB, which increases the rate of SR Ca pumping. This speeds relaxation and also tends to increase the SR Ca content. The former effect would tend to curtail the amplitude and duration of the Ca transient, whereas the latter would increase the amount of SR Ca available for release as well as the fraction released by a given ICa trigger. The SR Ca release channel is also a target for PKA phosphorylation, where it can increase the open probability of the RyR for a given Ca activating signal (Valdivia et al., 1995). This can also increase the fractional release of SR Ca content. PKA-dependent phosphorylation of the myofilaments occurs mainly at troponin I (TnI) on the thin filament and this reduces myofilament Ca sensitivity, such that a given Ca transient would not produce as much force. However, phosphorylation of TnI increases the rate of Ca dissociation from the myofilaments and this may contribute to the enhanced rate of relaxation seen with 웁-adrenergic agonists. The PKA-stimulated increase in ICa, SR Ca content, and SR Ca release are sufficient to increase the ampli-

tude of the Ca transient tremendously. This effect more than makes up for the decreased myofilament Ca sensitivity as a consequence of TnI phosphorylation, such that a substantial increase in force is observed. A faster relaxation rate due to PLB and TnI phosphorylation is also important physiologically, as 웁-adrenergic agonists also increase the heart rate. Thus faster relaxation allows more complete ventricular filling between beats at higher frequencies.

IV. 움-ADRENERGIC ACTIVATION There are also 움1-adrenergic receptors on cardiac myocytes, and activation of these can also increase the Ca transient amplitude and inotropic state, although the receptor transduction is different from that for 웁adrenergic agonists. Activation of 움-adrenergic receptors activates a different G-protein, which stimulates phospholipase C to produce diacylglycerol and inositol1,4,5-trisphosphate (IP3). While production of IP3 can lead to release of SR Ca in many cell types (and there are IP3 receptor channels in myocytes), it is controversial whether this contributes to the enhanced Ca transients in ventricular myocytes exposed to 움-adrenergic activation. The diacylglycerol produced, however, activates protein kinase C (PKC), which can lead to enhanced

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Ca transients as well as an increase in myofilament Ca sensitivity. PKC activation can stimulate Na/H exchange in myocytes greatly (see Fig. 1), which can result in intracellular alkalosis that increases myofilament Ca sensitivity. It is less clear how PKC activation increases the Ca transient, but there are many ways that alkalosis could shift Ca homeostatis (e.g., increased [Na]i, stimulation of SR Ca-ATPase). In summary, 움-adrenergic activation increases both the Ca transient (not as much as 웁-adrenergic activation) and myofilament Ca sensitivity (opposite to that of 웁-adrenergic activation), such that both effects contribute to increased contractility without changing the time course of relaxation appreciably (again in contrast to 웁-adrenergic activation). Physiological catecholamine release activates both 움- and 웁adrenergic receptors, but the main inotropic response is due to 웁-adrenergic receptors. The extent of 움-adrenergic activation varies in different cardiac preparations and can be as high as one-third of the overall inotropic response to norepinephrine.

V. HYPOTHERMIC INOTROPY Lowering the temperature of cardiac muscle or myocytes from 37 to 23–30⬚C has the surprising effect of increasing contractility dramatically (on the order of 500%; Bers, 1991). What makes this surprising is that cooling decreases the amplitude of ICa and also reduces the myofilament Ca sensitivity and maximal force, such that reduced force might have been expected. However, cooling slows inactivation of ICa such that the integrated Ca entry during the AP is essentially unaffected by cooling (Puglisi et al., 1999). Even more crucial is that the rate of Ca removal from the cytosol is slowed greatly upon cooling, thereby prolonging the duration of the Ca transient and active state greatly. Indeed, cooling slows the SR Ca-ATPase and Na/Ca exchange, so the competition during relaxation remains rather similar (Puglisi et al., 1996). The lower temperature also slows the Na/K ATPase and raises [Na]i . This further slows Ca extrusion from the cell via Na/Ca exchange and may contribute to an increase in SR Ca content and even increased diastolic [Ca]i (Shattock and Bers, 1987). This, along with the slowed Ca removal, may contribute to the larger Ca transient at lower temperature. These effects are sufficient to overcome the depressant effect of reduced ICa and myofilament Ca sensitivity at cooler temperatures. Cooling may also prolong the phase of SR Ca release, as it increases single ryanodine receptor channel open times (Sitsepesan et al., 1991). This may also explain why cooling cardiac muscle rapidly to near 0⬚C produces contractures which are indicative of SR Ca content

(Bers et al., 1989). That is, cooling to 0⬚C produces very long SR Ca release channel openings and suppresses Ca transport strongly by the SR Ca-ATPase and Na/ Ca exchange, so that the entire SR Ca content can be dumped to the cytosol, and the resulting [Ca]i or contraction used as an index of SR Ca load.

VI. CARDIAC GLYCOSIDES Digitalis glycosides such as digoxin and ouabain are selective inhibitors of the Na/K ATPase in the sarcolemma and have been used as cardiotonic agents for over 200 years. Of course for most of this time the inotropic effects were not understood from a mechanistic standpoint. At a simple level the mechanism can be described as follows. When glycoside binds to some modest fraction of Na/K ATPase molecules, these pumps are inhibited, thereby reducing the rate of Na extrusion from the cell. For a given amount of Na influx, this results in an increase in [Na]i . The higher [Na]i will tend to reduce Ca extrusion and favor Ca entry via the Na/Ca exchanger. The increased cellular and SR Ca levels result in the increase in Ca transients observed and the inotropic effect. Because the Na/Ca exchange stoichiometry is 3Na:1Ca, very small changes in [Na]i can have very large effects on Ca fluxes and inotropic state. Thus, several aspects can contribute to the enhanced Ca transients observed, which will be considered here in a bit more detail.

A. Increased Diastolic [Ca]i Because the Na/Ca exchanger in cardiac myocytes is so dominant over the sarcolemmal Ca-ATPase in transporting Ca out of the cell, it is the main mechanism responsible for maintaining the high electrochemical gradient for [Ca] across the sarcolemma. Again, the steep dependence of Na/Ca exchange function on [Na]i (due to the stoichiometry with respect to Ca) means that a small rise in [Na]i could have a dramatic effect on resting [Ca]i . For example, if the Na/Ca exchange were to come to complete equilibrium (which it may not be able to achieve) at resting [Na]i ⫽ 10 mM and Em ⫽ ⫺80 mV, it could theoretically lower [Ca]i to 36 nM. If [Na]i were to rise by only 3 mM, the minimum [Ca]i obtainable would more than double that (80 nM). While this kind of effect can contribute to an elevated diastolic force and its energetic consequences (as discussed earlier), elevated diastolic [Ca]i may also contribute to the inotropic effect of digitalis glycosides. This is because increased diastolic [Ca]i in the range below the threshold for generating contractile force can, in a sense, preactivate the myofilament response. Thus, if diastolic

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[Ca]i increases to just below the threshold for force development (e.g., from 100 to 200 nM in Fig. 2), the same increment in added Ca (from the SR and Ca influx) will raise [Ca]i and force to a higher level.

B. Increased SR Ca Content An increase in diastolic [Ca]i, average [Ca]i during the Ca transient, and slowed Ca extrusion of Ca from the cell by Na/Ca exchange all contribute to the increased SR Ca load observed in the presence of cardiac glycosides. The increased SR Ca content makes more Ca available for release, but also increases the fractional SR Ca release for a given Ca influx trigger. Furthermore, with elevated [Na]i there is greater Ca influx via Na/Ca exchange and, under these conditions, this Ca influx may become a more significant contributor (along with ICa) to the triggering of SR Ca release (Litwin et al., 1998). Thus, multiple factors contribute synergistically to the greater SR Ca release as [Na]i rises during glycoside inotropy. As the SR Ca load gets higher and higher, it reaches a point (Ca overload) where the intra-SR Ca level may cause ‘‘spontaneous’’ SR Ca release events and contractions. This limits the extent of glycoside inotropy and can result in serious problems (see Section VII).

C. Increased Ca Influx via Na/Ca Exchange As mentioned previously, greater Ca influx via Na/ Ca exchange at high [Na]i may allow this pathway to be more important in triggering SR Ca release. Ca entry via Na/Ca exchange is normally limited to the very early phase of the AP (Fig. 3A). However, when [Na]i is elevated (Fig. 3B), Ca influx may continue for most of the AP. Even with the SR rendered nonfunctional (by exposure to ryanodine and caffeine), cardiac glycosides can be strongly inotropic and the Ca influx via Na/ Ca exchange with each contraction can be increased substantially (Bers, 1987). It should be kept in mind, however, that preventing SR Ca release will actually increase the amount of Ca influx via Na/Ca exchange. This is because the rise in [Ca]i due to SR Ca release normally shifts the ENa/Ca to more positive potentials (see Fig. 3) and favors Ca efflux more and Ca influx less. Moreover, if peak [Ca]i is higher than shown in Fig. 3B, one can readily envisage the ENa/Ca curve exceeding the Em curve and favoring net Ca extrusion for much of the AP duration (as in Fig. 3A). Indeed, both scenarios can be seen during glycoside inotropy (Bers, 1987, 1991). Thus, while unidirectional Ca influx via Na/ Ca exchange is more favored by elevation of [Na]i, the situation is complicated because Ca transport via the Na/Ca exchanger is also sensitive to the higher Ca tran-

sient, which would push Ca transport in the other direction.

D. Slowed Ca Extrusion Of course the same thermodynamic shift that favors Ca entry also slows unidirectional Ca extrusion. Because this is the main way for Ca to exit the cell and must essentially be in balance with the total Ca influx (due to both ICa and INa/Ca), it is important to appreciate that the intrinsic depression of Ca extrusion by high [Na]i is offset by the higher [Ca]i driving greater Ca efflux, especially upon repolarization of the AP. Indeed, something not mentioned so far is that cardiac glycosides typically shorten AP duration, which favors Ca extrusion (because Em falls below ENa/Ca). Some results with an agent that appears to inhibit Ca influx selectively rather than efflux via Na/Ca exchange (KB-R7943) suggest that the [Na]i-dependent inhibition of Ca extrusion by Na/Ca exchange is sufficient to generate glycoside inotropy (Satoh et al., 2000). However, the Ca overload state and consequent arrhythmogenesis may rely on reaching high enough [Na]i that net Ca influx via Na/ Ca exchange is favored more heavily (even at rest). So, while it is difficult to functionally divorce the Ca influx and efflux modes of Na/Ca exchange, the balance of fluxes is crucial to the mechanism of glycoside inotropy and arrhythmogenesis.

VII. Ca OVERLOAD AND SPONTANEOUS SR Ca RELEASE When cardiac glycoside concentrations are increased further beyond the positive inotropic range, there are clear negative inotropic effects. Other consequences of glycosides also become apparent: (1) increased resting force, (2) aftercontractions, and (3) delayed afterdepolarizations (which can trigger arrhythmias). It now seems likely that all of these effects are secondary to cellular Ca overload and spontaneous Ca release from the SR during diastole. When the SR Ca load increases to a certain level, spontaneous SR Ca release events occur, which propagate through the cell as Ca waves. As mentioned earlier, these Ca waves may be triggered by intra-SR [Ca], such that the term ‘‘spontaneous’’ is a bit of a misnomer. A unique aspect of these Ca release events is that they occur at the resting membrane potential, where the Na/ Ca exchnage especially favors Ca extrusion (see Fig. 3). Thus, Ca extrusion via Na/Ca exchange at resting Em helps unload the cell of the Ca overload that caused the SR Ca release in the first place. This constitutes a negative feedback loop and thus helps limit SR Ca load-

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ing. However, the diastolic SR Ca release also produces aftercontractions and delayed afterdepolarizations (DADs, due to Ca-activated currents). These aftercontractions and DADs produced by Ca released during the normal diastolic period have very important negative consequences. These include disturbance of the normal syncytial nature of the heart with respect to both electrical and mechanical activity. These mechanical and electrophysiological aspects are discussed here (in that order). The elevated [Ca]i in some cells causes an increase in the average diastolic [Ca]i and can elevate diastolic force. This would create diastolic stiffness and could limit ventricular filling. In cells where there was a ‘‘spontaneous’’ SR Ca release, E-C coupling may be partially refractory at the moment that the next normally timed systole occurs. This may be due to the refractoriness of Ca-induced Ca release at the ryanodine receptor or even a lowered availability of Ca channels due to Cadependent inactivation and the accompanying DAD. These factors contribute to a reduced Ca transient and contractility from these cells. The mechanical impact is, however, magnified further in the heart because cells that fail to contract effectively constitute an increased series compliance for cells that do contract actively, i.e., when ‘‘normal cells’’ contract, much of their effort goes into stretching the refractory cells in series with them. Thus, energy that could go into systolic pressure development and ejection of blood is wasted in stretching refractory (and relatively compliant cells). This can exacerbate the negative inotropic effect of Ca overload greatly.

vated inward current (Iti) can be abolished by replacing extracellular Na with Li (Delbridge et al., 1995), it seems likely that INa/Ca is by far the most important Ca-activated current in mediating Iti and DADs in mammalian heart. These DADs may also limit Na channel availability when the normally timed beat comes along, further limiting AP duration and contractility. This is because Na channel availability is very sensitive to the Em in the range of resting Em. The biggest electrophysiological danger of these DADs is if the depolarization is large enough to trigger an AP at the wrong time in the wrong place. It is clear that SR Ca release can trigger APs (Capogrossi et al., 1987). This can begin a triggered arrhythmia by propagating AP through an inappropriate and ill-timed conduction pathway through the heart, which can lead to ventricular tachycardia and fibrillation. Evidence suggests that ventricular tachycardia episodes in the intact heart can be initiated by this mechanism (Pogwizd, 1995). Although the Ca overload secondary to Na pump inhibition may have been studied most widely, these same sequelae occur with other causes of cellular Ca overload. These may include 웁-adrenergic agonists, reduced [Na]o, increased Na permeability (e.g., with monensin), elevated [Ca]o, Ca channel agonists, large depolarizations, high—frequency stimulation, decreased membrane Ca or Na permeability barrier, or decrease in energy supply required to maintain normal ionic gradients.

A. Afterdepolarizations and Triggered Arrhythmias

VIII. SUMMARY

The spontaneous SR Ca release also activates ionic currents, known as transient inward current (Iti). There are three known Ca-activated currents in ventricular myocytes: (1) INa/Ca , (2) Ca-activated C1 current [ICl(Ca)], and (3) Ca-activated nonselective monovalent cation current [INS(Ca)]. All of these currents can produce inward current and depolarization at diastolic Em , but their relative importance in mediating DADs is important to address. Although INS(Ca) was one of the earliest Ca-activated currents reported in heart, it turns out that this current is probably a very minor player in Iti and DAD generation. There is stronger evidence that ICl(Ca) exists, but its amplitude seems to vary in different cardiac cells. Moreover, because the reversal potential for this current (ECl) is about ⫺65 mV, the amout of depolarization that this current can produce at negative Em near the resting Em is limited, i.e., ICl(Ca) could not depolarize the Em beyond ECl . Indeed, because the Ca-acti-

The main mechanisms of cardiac inotropy involve either increasing the force production of the myofilaments for a given [Ca]i or increasing the Ca transient amplitude or duration. Myofilament responsiveness can be modified by increasing myofilament Ca sensitivity, maximal force, or cooperativity, as well as crossbridge kinetics. The Ca transient can be enhanced by increasing ICa, Ca entry via Na/Ca exchange, SR Ca-ATPase, SR Ca content, fractional SR Ca release, or slowing Ca removal from the cytosol and lowering cytosolic Ca buffering. The various Ca transport systems interact in a dynamic yet delicate balance to determine the inotropic state. Examples of altered contractility and their underlying mechanisms have been discussed with respect to 움and 웁-adrenergic activation, hypothermia, and cardiac glycosides. These examples illustrate the complex interplay of Ca and contractile force regulation in intact cardiac myocytes. The fundamental basis for cellular Ca overload, spontaneous SR Ca release, and how this leads

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to both mechanical and electrophysiological dysfunction (including triggered arrhythmias) have also been discussed in this context.

Bibliography Bassani, J. W. M., Bassani, R. A., and Bers, D. M. (1994). Relaxation in rabbit and rat cardiac cells: Species-dependent differences in cellular mechanisms. J. Physiol. 476, 279–293. Bassani, J. W. M., Yuan, W., and Bers, D. M. (1995). Fractional SR Ca release is altered by trigger Ca and SR Ca content in cardiac myocytes. Am. J. Physiol. 268, C1313–C1319. Bassani, R. A., Shannon, T. R., and Bers, D. M. (1998). Passive Ca2⫹ binding in ventricular myocardium of neonatal and adult rats. Cell Calcium 23, 433–442. Bean, B. P. (1989). Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51, 367–384. Berlin J. R., Bassani, J. W. M., and Bers, D. M. (1994). Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. Biophys. J. 67, 1775–1787. Bers, D. M., (1987). Mechanisms contributing to the cardiac inotropic effect of Na-pump inhibition and reduction of extracellular Na. J. Gen. Physiol. 90, 479–504. Bers, D. M. (1991). ‘‘Excitation-Contraction Coupling and Cardiac Contractile Force.’’ Kluwer Academic Press, Dordrecht, Netherlands. Bers, D. M. (2000). Regulation of cellular calcium in cardiac myocytes. In ‘‘Handbook of Physiology’’ (E. Page, H. A. Fozzard, and R. J. Solaro, eds.) Oxford Univ. Press. Bers, D. M., Bridge, J. H. B., and Spitzer, K. W. (1989). Intracellular Ca transients during rapid cooling contractures in guinea-pig ventricular myocytes. J. Physiol. 417, 537–553. Beuckelmann, D. J., and Wier, W. G. (1989). Sodium-calcium exchange in guinea-pig cardiac cells: Exchange current and changes in intracellular Ca2⫹. J. Physiol. 414, 499–520. Capogrossi, M. C., Houser, S., Bahinski, A., and Lakatta, E. G. (1987). Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential. Circ. Res. 61, 498–503. Cheng H., Lederer, W. J., and Cannell, M. B. (1993). Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle. Science 262, 740–744. Delbridge, L. M., Bassani, J. W. M., and Bers, D. M. (1996). Steadystate twitch Ca fluxes and cytosolic Ca buffering in rabbit ventricular myocytes. Am. J. Physiol. 39, C192–C199. Endoh, M., and Blinks, J. R. (1988). Actions of sympathomimetic amines on the Ca2⫹ transients and contractions of rabbit myocardium: Reciprocal changes in myofibrillar responsiveness to Ca2⫹ mediated through 움- and 웁-adrenoceptors. Circ. Res. 62, 247–265.

Koss, K. L., and Kranias, E. G. (1996). Phospholamban: A prominent regulator of myocardial contractility. Circ. Res. 79, 1059–1063. Li, L., Chu, G., Kranias, E. G., and Bers, D. M. (1998). Cardiac myocyte calcium transport in phospholamban knockout mouse: Relaxation and endogenous CaMKII effects. Am. J. Physiol. 274, H1335–H1347. Litwin, S. E., Li, J., and Bridge, J. H. (1998). Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophys J. 75, 359–371. Luo, W., Grupp, I. L., Harrer, J., Ponniah, S., Grupp, G., Duffy, J. J., Doetschman, T., and Kranias, E. G. (1994). Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of 웁-agonist stimulation. Circ. Res. 75, 401–409. Pogwizd, S. M. (1995). Nonreentrant mechanisms underlying spontaneous ventricular arrhythmias in a model of nonischemic heart failure in rabbits. Circulation 92, 1034–1048. Puglisi, J. L., Bassani, R. A., Bassani, J. W. M., Amin, J. N., and Bers, D. M. (1996). Temperature and the relative contributions of Ca transport systems in cardiac myocyte relaxation. Am. J. Physiol. 270, H1772–H1778. Puglisi, J. L., Yuan, W., Bassani, J. W. M., and Bers, D. M. (1999). Ca influx through Ca channels in rabbit ventricular myocytes during action potential clamp: Influence of temperature. Circ. Res. 85, e7–e16. Satoh, H., Blatter, L. A., and Bers, D. M. (1997). Effects of [Ca]i, Ca2⫹ load and rest on Ca2⫹ spark frequency in ventricular myocytes. Am. J. Physiol. 272, H657–H668. Satoh, H., Ginsburg, K. S., Qing, K., Terada, H., Hayashi, H., and Bers, D. M. (2000). KB-R7943 block of Ca2⫹ influx via Na⫹ /Ca2⫹ exchange does not alter twitches or glycoside inotropy, but prevents Ca2⫹ overload in rat ventricular myocytes. Circulation. 101, 1441–1446. Shannon, T. R., Ginsburg, K. S., and Bers, D. M. (2000). Reverse mode of the SR Ca-pump and load-dependent Ca decline in voltage clamped cardiac ventricular myocytes. Biophys. J. 78, 322–333. Shattock, M. J., and Bers, D. M. (1987). The inotropic response to hypothermia and the temperature-dependence of ryanodine action in isolated rabbit and rat ventricular muscle: Implications for EC coupling. Circ. Res. 61, 761–771. Sitsapesan, R., Montgomery, R. A. P., MacLeod, K. T., and Williams, A. J. (1991). Sheep cardiac sarcoplasmic reticulum calcium-release channels: Modification of conductance and gating by temperature. J. Physiol. 434, 469–488. Valdivia H. H., Kaplan, J. H., Ellis-Davies, G. C. R., and Lederer, W. J., (1995). Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2⫹ and phosphorylation. Science 267, 1997–1999. Yuan, W., Ginsburg, K. S., and Bers, D. M. (1996). Comparison of sarcolemmal Ca channel current in rabbit and rat ventricular myocytes. J. Physiol. 493, 733–746. Yue, D. T., Marban, E., and Wier, W. G. (1986). Relationship between force and intracellular [Ca2⫹] in tetanized mammalian heart muscle. J. Gen. Physiol. 87, 223–242.

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45 Mechanisms of Action of Calcium Antagonists HELMUT A. TRITTHART Institut fu¨r Medizinische Physik und Biophysik Karl-Franzens-Universita¨t Graz A-8010 Graz, Austria

I. INTRODUCTION

properties (for review, see Janis et al., 1987) was well recognized, and Fleckenstein’s initial classification of calcium antagonists aimed to distinguish clearly between nonspecific calcium antagonism and calcium antagonism by compounds having outstanding specificity (group A) or satisfactory specificity (group B) (Fleckenstein, 1983). As shown in Fig. 1, calcium antagonists of group A (verapamil, gallopamil, nifedipine, and diltiazem) are capable of inhibiting calcium-dependent excitation–contraction coupling in the mammalian ventricular myocardium by 90% or more before the fast Na⫹ influx, which occurs during the rising phase of the action potential, is also affected, i.e., before dV/dtmax is diminished. Group B antagonists (prenylamine, fendiline, terodiline, perhexiline, and caroverine) are somewhat less specific, and it was found that potent, Na⫹ antagonistic effects (dV/dtmax decrease) occurred at concentrations that reduced the Ca2⫹-dependent isometric tension development in papillary muscles by about 50 to 70% (Hondeghem et al., 1977). At the time of this first classification, it was not known that the dV/dtmax decrease is not a wholy reliable measure of Na⫹ current inhibition (Hondeghem, 1977) because Na⫹ current inhibition is usually not only dose but also strongly rate dependent (Hondeghem et al., 1977). Further calmodulin inhibition, which was found to be an additional property of some calcium blockers such as fendiline flunarizine and bepridil (Lugner et al., 1984), exerts not only marked smooth muscle relaxation but also a Na⫹ antagonistic effect (Ichikawa et al., 1991). Although initial criteria for the classification of calcium antagonists were unrelated to their chemical constitution or to their binding efficacy and did not include

The term ‘‘calcium antagonism’’ was coined by A. Fleckenstein in the late sixties to describe the mode of action of a new group of compounds that apparently mimicked the effect of Ca2⫹ withdrawal on excitation and contraction of cardiac and smooth muscle fibers (Fleckenstein et al., 1968; Gru¨n et al., 1969). The initial, key observation was that verapamil blocked contraction of the myocardium and the high-energy phosphate utilization related to contraction but produced little change in the action potential (Fleckenstein et al., 1968, 1969). This specific inhibition of cardiac contraction was a new finding but in line with the early discovery by Mines in 1913 that, although perfusion with Ca2⫹-free solution stopped the contraction, the surface electrical activity of the heart was still present. Incomplete inhibition of contraction but a decrease in amplitude was a well-known effect of a great variety of compounds exerting a negative inotropic effect, e.g., 웁-adrenoceptor blocking agents, membrane stabilizing and antiarrhythmic compounds, barbiturates. Hence, Fleckenstein’s interpretation of these new findings and the hypothesis of a selective calcium antagonistic mode of action for this new group of compounds were not initially accepted in pharmacology and physiology. It should be noted, however, that regenerative membrane depolarizations recorded from crustacean muscle in sodium-free solutions had, already in 1953, led to the suggestion that a voltage-gated calcium influx exists (Fatt et al., 1953). That a variety of other chemical structures, at least in higher concentrations, also possess calcium antagonistic

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FIGURE 1 Negative inotropic effects of the calcium antagonists gallopamil (A, C) and nifedipine (B). Note that the maximum rate of rise of the action potentials (dV/dt in lower half of A and in B) is not reduced by the calcium antagonists. A and B are experiments with guinea pig papillary muscles (35⬚C, rate of stimulation 0.5 Hz). In (C) the inhibition of tension development, due to antagonism of calcium, by gallopamil in cat ventricular myocardium is antagonized by the addition of cardiac glycosides, by stimulation of 웁-adrenoceptors with epinephrine, or by increase in the extracellular calcium concentration. Data from Fleckenstein et al. (1969); see Janis et al. (1987) for review.

smooth muscle effects, the clinically now widely established agents diltiazem, nifedipine, and verapamil and closely related compounds all proved to belong to group A of Fleckenstein’s first classification. Even more convincing was the pharmacological line of evidence for a highly specific mode of action of calcium antagonists and for the lack of 웁-adrenoceptor blocking activities. Originally, these new compounds were considered to be 웁-adrenoceptor blockers, but Fleckenstein and others (Singh et al., 1972) presented evidence that their calcium

antagonists lacked 웁-adrenoceptor blocking effects. In fact, the relaxation and inhibition of excitation of the smooth muscle fibers of the myometrium by calcium antagonists (Tritthart et al., 1970) were very opposite effects to that expected for 웁-adrenoceptor blockers (see Fig. 2). It was found later that calcium antagonists relax essentially all smooth muscle fibers and also inhibit excitation. The most prominent effects were found to occur in the coronary system and in the peripheral vascular bed (see Fig. 2).

45. Calcium Antagonists

FIGURE 2 Calcium antagonists inhibit spontaneous transmembrane electrical activity (measured by sucrose gap technique) and tension development in rat myometrial strips [A; see Tritthart et al. (1970)]. Excitation and excitation contraction coupling is inhibited by calcium antagonists throughout smooth muscle fibers, whereas 웁-adrenoceptor blockers predominantly promote contractions (B). In (C) Relaxation of potassium-depolarized coronary strips is shown for Ca2⫹-withdrawal (left) or for calcium antagonist addition (right). In the presence of calcium antagonists, an increase of extracellular Ca2⫹ partially restores contractile tension. In freshly-dissected coronary arteries, rhythmic spontaneous contractions of large amplitude are common or can be initiated or supported by a variety of factors (e.g., potassium. see D). Calcium antagonists are very effective inhibitors of these spontaneous or induced vasospastic activities (D). Trace N was recorded 13 min after the addition of nifedipine. Potassium-induced contractile activities under control conditions and after the addition of nifedipine are superimposed.

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At this early stage (1972/1973) of definition of Ca2⫹ antagonistic properties, the following criteria had been delineated by Fleckenstein’s laboratory: 1. Inhibition of excitation and of excitation– contraction coupling of uterine and vascular smooth muscle (Gru¨n et al., 1969; Tritthart et al., 1970). 2. Inhibition of [45Ca2⫹] uptake into the myocardium and of the Ca2⫹ influx, measured by sucrose gap voltage clamp (Janke et al., 1970; Tritthart et al., 1972). 3. Inhibition of contraction of the ventricular myocardium and antagonism of this effect by Ca2⫹ ions, 웁-adrenoceptor stimulation, or cardiac glycosides (Fleckenstein et al., 1968, 1969; Volkmann et al., 1973) (see Fig. 1). 4. Inhibition of Ca2⫹-dependent action potentials in partially depolarized ventricular myocardium by inhibitors of the transmembrane Ca2⫹ inflow and antagonism of this effect by promotors of Ca2⫹ inflow (Tritthart et al., 1973; Volkmann et al., 1973). 5. Inhibition of cardiac pacemaker activity, of atrioventricular conduction velocity, and prolongation of refractoriness (Fleckenstein, 1983) (see Fig. 3). In parallel with these studies, the clinical testing of calcium antagonistic compounds started as early as 1963 in Europe and Japan and, up to 1972, covered the following topics: angina pectoris (Cardoe, 1971; Hayase et al.,

1972; Kaltenbach, 1970; Kobayashi et al., 1972; Neumann et al., 1966; Rowe et al., 1971; Sandler et al., 1968), supraventricular tachyarrhythmias (Bender 1967; Brichard and Zimmermann 1970; Hanna and Schmid 1970; Schamroth 1971; and Sacks and Kennelly 1972), and hypertension (Vaughan-Neil et al., 1972). There was one report of tocolytic effects (Weidinger et al., 1971) and one of asystole (Krikler et al., 1972). These early studies on the cardiovascular and smooth muscle effects of Ca2⫹ antagonists were consistent with Fleckenstein’s hypothesis of a specific inhibition of the permeation of Ca2⫹ ions through sarcolemmal calcium channels. Calcium-selective ion channels as receptor sites of action of calcium antagonists was a concept that became more and more accepted with the further characterization of channel subtypes by a new technique (called patch clamp) that allowed the measurement of ionic currents through single membrane channels (Hamill et al., 1981; Sakmann et al., 1983). The term calcium antagonists became refined successively more specifically as calcium entry or calcium channel blockers, as slow channel blockers, and, finally, as L-type calcium channel blockers. It should be noted, however, that these drugs do not produce an irreversible block but rather a reversible inhibition of calcium channel activities. In addition, advances in the understanding of the genetic regulation of ion channels (Salkoff et al., 1986; Tanabe et al., 1988), of their amino acid sequences, and of their probable configurations strongly promoted the

FIGURE 3 Inhibitory effects of verapamil on the isolated AV node of the rabbit (Tritthart et al. (1971). Beat frequency decreases due to elevation of threshold potentials and retardation of slow diastolic depolarizations. In addition, the rate of rise (dV/dt) and overshoot of AV-action potentials are reduced by a calcium antagonist. The electrical activity of embryonic chick heart cells resembles that of SA and of AV nodal cells in adult hearts, and calcium antagonists completely inhibit the spontaneous discharge in these cells (B). Slow action potentials recorded in 20 mM Ko⫹ of guinea pig papillary muscles are blocked by a calcium antagonist (C). This complete inhibition of slow and Ca2⫹-dependent action potentials by gallopamil (C) is overcome by periods of rest of sufficient duration (right, change of stimulation rate from 1 Hz to 1/min).

45. Calcium Antagonists

concept of Ca2⫹ ion channels as likely key receptor sites for the modulation of cardiovascular functions (Mikami et al., 1989). The application of voltage and patch clamp techniques to isolated cardiac or vascular smooth muscle cells, the use of fluorescent calcium-sensitive dyes, and, last but not least, the utilization of calcium channel blockers became very useful tools in studying the role of calcium ions in the cardiovascular system, including pathophysiological changes. However, it soon became evident that calcium channels are ubiquitous features of other excitable cells. They have an important function not only in vertebrate smooth and cardiac muscle, but also in embryonic skeletal muscle, neuron somata, synaptic terminals, sensory receptors, and a wide range of cells that secrete hormones, neuromodulators, or neurotransmitters. Calcium channels have also been shown to exist in a number of nonexcitable cells such as glial cells (Barres et al., 1988), myeloma cells (Fukushima et al., 1983), osteoblasts (Chesnoy-Marchai et al., 1988), fibroblasts (Chen et al., 1988), and tomato cells (Gelli et al., 1997), with some of the latter being modulated by growth factors and oncogenes. Calcium channels, are not as highly conserved as, for instance, sodium channels, but vary rather widely between different tissues in properties such as their conductance, voltage dependence, inactivation mechanism, inhibition by ions, blockers, or toxins, and modulation by receptors, G-proteins, or second messengers. In analogy to conventional pharmacological characterizations, the key criterion for the classification of voltagedependent calcium channels (L-, N-, T-, P/Q-, R-types; see Chapter 13) is their pharmacological specificity and, where available, information concerning separate genetic control. T channel expression can be inhibited by transforming genes without the simultaneous modification of L-channel expression (Tsien, 1983). This chapter focuses on L-type and, in part, on T-type channels only; for an overview of calcium channels in a variety of cell membranes, see Kostyuk (1989) and Tsien (1983, 1987). A major monograph (Fleckenstein, 1983) and other reviews shed light on the historical aspects of the discovery of drugs acting on calcium channels (Janis et al., 1987; Triggle et al., 1989).

II. CALCIUM ION CHANNELS Julius Bernstein correctly hypothesized in 1902 that excitable cells maintain an intracellular potential as a result of their membranes being selectively permeable to potassium ions. Selective permeability exists because of aqueous pores, called channels, formed by protein macromolecules that span the membrane and through

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which ions can permeate the hydrophobic lipid membrane. The property of K⫹ selectivity is related to the presence of just two amino acid residues in the ion channel protein (Heginbotham et al., 1992). Selectivity is a distinct property of ion channels for which they are particularly named, i.e., a calcium channel selects for Ca2⫹ ions and this preferred ion passes through the channel at least a hundred times more easily than other ions. Channels accomplish ion selectivity by either steric means (narrow portion excluding larger ions) or by the binding strength of the selected ion to sites within the channel. Calcium channels do not simply repel other ions, such as Na⫹, as large currents of Na⫹ or of other alkaline ions flow through calcium channels in the absence of Ca2⫹ ions (for review, see Tsien et al., 1987), but these currents are blocked by micromolar amounts of Ca2⫹ ions. Calcium channels are both very selective and highly permeable. During channel opening, fluxes of 10–100 thousand Ca2⫹ ions per millisecond were observed (Isenberg et al., 1982; Reuter et al., 1982). The calcium channel is somewhat unique in its selectivity when compared to other ionic channels, e.g., in Na⫹ channels the permeability for K⫹ is 0.1, for Ca2⫹ 0.05 of the Na⫹ permeability (defined as 1), whereas the Na⫹ permeability of Ca2⫹ channels is usually calculated to be 0.001. Models that account for the ionic selectivity of calcium channels incorporate specific multiple binding properties of the channel for Ca2⫹ (Hess et al., 1984) and Ca2⫹induced transformation from strongly to weakly binding states (Lus et al., 1990). It is likely that two Ca2⫹ ions are bound to two neighboring high-affinity binding sites (four glutamine acids residues). Ca2⫹ binding to both sites modulates the binding toward a very fast binding and release state, enabling Ca2⫹ ions to penetrate the channel very quickly one by one without endangering the ionic selectivity. Ca2⫹ is rather exceptional among permeant ions in that Ca2⫹ ions themselves serve crucial functions within the cell and inside the membrane in addition to merely carrying electrical charge. Calcium ions are ubiquitous intracellular second messengers in an environment of a normally very low calcium concentration (Carafoli, 1987), and the need is obvious for high selectivity and powerful transmembrane signaling via high Ca2⫹ permeability pathways. Intracellular Ca2⫹ modulates other ion channels, such as Ca2⫹-activated potassium, chloride, or nonselective channels, leading to complex electrical responses. Elevated intracellular Ca2⫹ levels in cardiac or smooth muscle cells play a role in various pathophysiological processes (see Chapter 53). In addition, Ca2⫹dependent enzymes, such as protein kinase C or Ca2⫹ – calmodulin kinases, are most probably influenced by calcium channel openings, and, via modulation of other

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channels and receptors, these enzymes will then contribute to the complexity of electrical and nonelectrical activities following the modulation of calcium channel activity. Macromolecules spanning the lipid membrane and forming the Ca2⫹ channel may conceptually be considered as enzymes that reduce the energy for transmembrane ion diffusion and, thus, enhance the rate of diffusion by a factor of 1015 to 1020. In fact, like enzymes, channels display substrate specifity in the form of ionic selectivity. Furthermore competitive inhibition is seen with substrate analogues, namly for Ca2⫹ channels, other divalent ions, or blockers. Like enzymes, calcium channels have the capability of undergoing rapid conformational changes, i.e., changes between open and conducting, or closed and nonconducting, states of the channel. Conductivity of a single Ca channel has a constant value in the open state under constant experimental conditions. Hence, key factors governing the calcium current strength in an individual cell are (I) the likelihood of stoichastic channel opening reactions, (II) the duration of channel opening, and (III) the density of active channels. Very little is known about up- and downregulation of calcium channels in humans and about the homeostasis of calcium channel density, e.g., during chronic drug administration (Gengo et al., 1988), cardiac failure (Schwinger et al., 1999), hypertension (Chatelain et al., 1984), or cardiomyopathy (Finkel et al., 1988). After prolonged treatment with calcium channel blockers, only a few rebound effects following the cessation of therapy have been reported so far (Triggle et al., 1987). This probably indicates that channel density is little affected, if at all, by calcium antagonists (Schwinger et al., 1999). In summary, all agents or factors that modify activation or function of voltage-dependent calcium channels alter, predominantly, the probability and duration of open times of activated channels and, as a result, change the peak calcium currents (cf. Fig. 4). Prolonged treatment may, nevertheless, alter intracellular Ca2⫹ homeostasis and thus cause a variety of indirect effects.

III. VOLTAGE AND ION EFFECTS ON CALCIUM CHANNELS The single channel calcium current has been shown to flicker between ‘‘on’’ and ‘‘off’’ states with a time course that is considerably faster than that of the activation of the macroscopic current (Reuter et al., 1982). These rapid transitions between open and closed states allow the investigator to combine them into one state, the active state. This can be modulated by a comparatively slowly developing process that converts the channel to a temporarily inactive and nonconductive form

different from the closed state of the activated channel. The complex kinetics of calcium channel activation and inactivation can be modeled by assuming that activation starts from two inactivated states (left and right in Fig. 5). Figure 5 shows that all transitions leading to the left inactive state are Ca2⫹ and voltage dependent, whereas the other transitions are simply voltage dependent. This model (Carbone et al., 1989) additionally accounts for the voltage and Ca2⫹ dependency of the recovery from inactivation, for the biphasic inactivation time course, and for the Ca2⫹ dependency of the inactivation rate (Campbell et al., 1988). Voltage-dependent activation and inactivation of calcium channels imply that, at the molecular level, charged groups (‘‘gates’’) move in response to the alteration of the transmembrane electrical field. Although gating currents could, so far, not be resolved at the single channel level, it is likely that they contain components for activation and inactivation, as well as for opening and closing of individual channels. Voltage-sensitive calcium channels may either require a large amount of membrane depolarization to become activated (L, N, P, class) or are activated with minor amounts of depolarization (low threshold). These latter channels are called T (for transient) channels. Transient T-type calcium channels inactivate rapidly, whereas long-lasting L-type channels exhibit a slow, time-dependent inactivation. However, due to the Ca2⫹ dependency of inactivation (see later), L channels may inactivate quite rapidly under physiological conditions, i.e., when Ca2⫹ ions are present instead of the more permeable Ba2⫹ ions often used experimentally as charge carriers (half-times of 20–100 msec). Inactivation of T channels develops monoexponentially, is complete, and appears to be strictly voltage dependent and is thus independent of the type of chargecarrying ion or the amount of Ca2⫹ entry into the cell. Maximal slope conductances in cell-attached patches were found to be 3.4 to 6.8 pS (100 mM Cao2⫹ ), with T channels being equally permeable to Ca2⫹ and Ba2⫹ ions (Nilius et al., 1985). In contrast, the high-voltageactivated L-type of calcium channels carry Ba2⫹ ions nearly twice as effectively as they do Ca2⫹ ions (Bean, 1985) and have much higher conductances: 22 to 28 pS, in 110 mM Bao2⫹ in cardiac fibers, and 20 to 25 pS, in 110 mM Bao2⫹ , in smooth muscle fibers (Yatani et al., 1987). T channels are dominant in embryonic cells (McCobb et al., 1989), and, because pacemaker discharge activities are characteristic for embryonic heart cells (see Chapter 41), it is not surprising that T channels are found predominantly in those atrial and ventricular cells that exhibit an automatic discharge capability, such as pacemaker cells (Tseng et al., 1989). T channels are also found in smooth muscle cells (Yatani et al., 1987; Aaronson et al., 1986). T channels, at resting membrane poten-

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FIGURE 4 Transmembrane calcium current through L-type Ca2⫹ channels. (A) whole cell voltage clamp. The initial depolarization to ⫺40 mV elicits a fast Na⫹ current with rapid inactivation. The ensuing depolarization activates the calcium inward current. This whole cell calcium current is the result of accumulated single channel openings as indicated in (B) and can be resolved by patch clamp experiments (B). Following a depolarization, the probability of calcium channel opening, as well as the open duration in individual channels, is reduced by calcium antagonists. Thus, the depolarization-induced transmembrane Ca2⫹ current is diminished by calcium antagonists.

tials, are probably inactivated, but they may be activated following hyperpolarization and, thus, modulate the discharge rate of pacemaker cells and excitability as well as oscillatory and bursting activities in smooth muscle. Calcium channels greatly prefer Ca2⫹, Ba2⫹, and Sr2⫹ over all other alkaline ions for which a permeant ratio as low as 0.001 or less has been calculated. The different types of calcium channels share a similar permeability for alkaline earth ions with the exception that the Mg2⫹ ion is nearly impermeant. The T channel is equally permeable to Ca2⫹ and Ba2⫹ with a small preference for Sr2⫹ (Aaronson et al., 1986), whereas the L-type calcium channel has a clear preference for Ba2⫹ ions (Bean, 1985). Competitive inhibitors of calcium currents are La3⫹, Co2⫹, Ni2⫹, and Cd2⫹. T channels are potently blocked by Ni2⫹ and are less potently blocked by Cd2⫹. Cadmium is a much more potent blocker of the L-type channel (Lansman et al., 1986). Mn2⫹, Zn2⫹, and Mg2⫹ often act as competitive inhibitors of calcium currents but can carry current in some cells. The blockade of calcium currents by Mg2⫹ was discovered early (Hartzell et al., 1989) and could be attributed to reduced mean open times (Lansman et al., 1986), most probably due to the entry of Mg2⫹ ions. When Ca2⫹ ions are lacking, Mg2⫹ ions can occupy the channel for some time, interdicting the passage of permeant ions and, thus, resembling in part the blocking action of Ca2⫹ ions, which opposes the passage of Na⫹ or Li⫹ ions through calcium

channels. An increase in the concentration of intracellular Mg2⫹ to the millimolar range inhibits L-type calcium channels (Hartzell et al., 1989, Agus et al., 1989). A phosphorylation-independent, direct activation of calcium channels by Mg2⫹-nucleotide complexes has been reported (O’Rourke et al., 1992). The addition of adenosine triphosphate (ATP) to the intracellular solution reduces the rate of irreversible ‘‘run down’’ of calcium current in dialyzed cells and increases calcium current when endogenous ATP production is inhibited (Taniguchi et al., 1983). In guinea pig ventricular myocytes, alkaline pH enhanced L-type calcium current and acidic pH reduced it to about 60 to 70% of that at pH 7.5 (Prod’hom et al., 1987). These findings are consistent with the titration of negatively charged groups in the inner or outer membrane surface, which control not only gating, but also ion permeation of the channel. Lowering pH inside the cell caused both activation and inactivation curves to be shifted toward less negative potentials as well as a decrease in channel opening probability (half-maximum inhibition at pH 6.6; Kaibara et al., 1988).

IV. CHANNEL INHIBITORS The compounds and agents that affect the activity of voltage-dependent calcium channels can conveniently

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be grouped into three categories: (A) inorganic ions, (B) channel inhibitors and openers, and (C) naturally occurring regulators. The main focus of this chapter is on groups A and B of calcium channel modulators. Group C is discussed in Chapters 34, 35, 36, 37, and 43. ‘‘Corpora non agunt nisi fixata,’’ i.e., compounds act only when bound to some structure (Ehrlich, 1910). This receptor concept raises many questions regarding the location and type of binding sites for Ca2⫹ antagonists, access to these sites, structure–activity relations, and binding and allosteric modulation of channel function and of affinity to binding sites. However, first of all comes the question whether calcium channels act as true receptors or only have some features that resemble those of receptors. Chemical heterogeneities of the most widely studied calcium antagonists, nifedipine, verapamil, and diltiazem, were obvious and a common receptor for their inhibitory action appeared highly unlikely. Consequently, Fleckenstein’s intention to introduce the new pharmacological principle of calcium antagonism was seriously hampered by the fact that it was necessary to have a group of different receptor sites that were all responsible for the same mode of action. In addition, indirect evidence attributed a calcium blocking property to a very large number of other structures, which contributed to the confusion. That a multiplicity of binding sites are found on the L channel is, today, not so surprising, as the 움1 subunit of the calcium channel, the site of binding of Ca2⫹ antagonists, has a sequence highly homologous with that of the sodium channel, which exhibits at least five distinct binding sites (Strichartz et al., 1987). Nevertheless, it is surprising that evidence for the existence of endogenous inhibitory ligands for calcium channels is still missing. There are only a few reports of calcium channel modulation by endogenous peptides (Callewaert et al., 1989; Hallstro¨m et al., 1991; Janis et al., 1987), but no specific inhibition of calcium antagonist binding has been reported for any of the known neurotransmitters or peptide hormones. There are only a few compounds, none very specific, that block T channels. These include octanol (Llinas et al., 1986), amiloride (Tang et al., 1988), and phenytoin (Twombly et al., 1988). Among calcium antagonists, flunarizine and nicardipine were reported to inhibit T channels in vascular smooth muscle (Kuga et al., 1990), as were felodipine and cinnarizine in atria (Van Skiver et al., 1985). As already seen with sodium channels, T channels are inhibited by almost all compounds that inhibit L-type channels, provided that the concentration is high enough. There are few specific toxins such as kurtoxine (but see Furukawa et al., 1992) or calcium antagonists available that block T channels only, or even with sufficient selectivity to study T-channel function. Hence, no extensive pharmacology for T channels has

yet been developed and the contribution of T channels to normal and disturbed cardiovascular functions is not at all well defined. The nondihydropyridin T-channel blocker Mibefradil promised to be therapeutically useful by producing vasodilation and bradycardia without negative inotropy. The MACH 1 study (Mortality Assessment in Congestive Heart Failure) led to market withdrawal due to cytochrome inhibition (P450 isoenzymes) and serious side effects of comedication (Levine, 1997).

V. L-TYPE CALCIUM CHANNELS: STRUCTURE, LIGANDS, BINDING, AND FUNCTION Calcium channel ligands are compounds that act by binding directly to voltage-sensitive calcium channels and not to an allosteric site or to some nearby or distant receptors. This binding will decrease (or increase) the movement of Ca2⫹ ions through the channel without alteration of the intrinsic permeability in the open state but by modulation of the open state probability and duration. The overall calcium current through channels in a cell (I) is the product of active channel number (n), the probability of a channel being in the open state (P), and the unitary current through a single calcium channel (i), i.e., I ⫽ n.P.i. These unitary currents (i) are probably events that are mediated by the 움1 subunit of the channel molecule, as calcium currents can be reconstituted by the dihydropyridine receptor 움1 subunit alone (Tanabe et al., 1988; Mikami et al., 1989; Perez-Reyes et al., 1989). Reconstitution experiments showed that the purified dihydropyridine binding site is the functional calcium transmembrane pore (Hymel et al., 1988). Linings of the conduction pores are probably formed by localized stretches of only 20 to 25 amino acid residues repeated in each subunit. Point mutations can lead to alterations of ion selectivity, conductance, and affinity for blocking agents. Hence, binding sites for Ca2⫹ antagonists are most likely located in close proximity to the channel outer and inner mouth and to calcium binding sites within the core of the channel. The number of discrete binding sites for Ca2⫹ antagonists is, most likely, not only linked allosterically, one to another, but also to the structures of the channel governing permeation and gating. Figure 5 shows the main components of the L-type calcium channel. Four tightly coupled subunits, 움1 , 움2 , 웁, 웂, and 웃, form the channel complex (cf. Fig. 5), the primary structure of each subunit has been determined, and 움1 , 움2 , and 웁 cDNAs have been used to characterize

45. Calcium Antagonists

797

FIGURE 5 Schematic model of the L-type Ca2⫹ channel. The part forming the transmembrane pore and water conduit is the 움1 subunit, which is also the site of binding of calcium antagonists. Models of kinetic channels transitions usually include open, closed, activated, and inactivated states in order to describe channel functions. The extremely fast transition between open and closed states of activated channels allows the separation of these fast transitions from the slow processes of Ca2⫹ channel activation and inactivation, both being voltage and/or Ca2⫹ dependent.

transcripts expressed in various tissues (Perez-Reyes et al., 1989, 1986). The 움1 subunit contains channelforming structures (Hymel et al., 1988; Mikami et al., 1989; Mori et al., 1991) as well as various binding sites of Ca2⫹ antagonistic compounds. The cytoplasmic side of the 움1 subunit is a good substrate for protein kinase A phosphorylation, and the 웁 subunit may also be a site of cAMP-dependent phosphorylation (Nastainczky et al., 1987) (see Chapters 13, 34, and 38 for second messenger and G-protein-mediated alterations of channel function). The key receptor in modulating cardiac contractility is the 웁-adrenoceptor. The human heart has 80% 웁1 and 20% 웁2 receptors and both are linked to Gs proteins. In addition, H2 , glucagon, prostanoid (EP2 DP and IP), 5HT4 and VIP receptors are also linked to Gs proteins, but exert less than 50% of the effects of 웁-adrenoceptors on contractility. Effector cascade 웁-adrenoceptor–Gs protein–Ca2⫹ channels uses two ways to modulate Ca2⫹ channels; a direct one and an indirect one via adenylate cyclase, cAMP, and protein kinase A, leading to channel phosphorylation. Both effects of G-proteins increase the open probability of Ca2⫹ channels strongly, and this effect can antagonize the inhibitory effects of calcium antagonists. In ischemia and cardiac failure, a modulation of Ca2⫹ channels via 움1 receptors and Gq/II proteins (which are also activated via endothelin EtA , EtB , and angiotensin II–AT1 receptors) seems to be importand. The cAMP-mediated stimulation of Ca2⫹ channels can be dimished, even producing negative inotropic effects, by agonists acting on receptors that couple the Gi/o proteins, e.g., muscarinic (M2), adenosine (A1), or neuropeptide Y (Y1-6) receptors (indirect negative inotropic effects). The calcium channel has at least seven possibely separate binding sites for calcium antagonists of structurally

distinct chemical classes (Miller, 1992), including the sites for 1,4-dihydropyridines, phenylalkylamines, and benzothiazepines. Depending on the position of the binding site, different avenues of access may be used by ligands: (I) directly from the extracellular aqueous environment, (II) using the water conduit of the open channel, (III) by lateral diffusion after partitioning into the membrane bilayer, or (IV) after crossing the cell membrane from the intracellular aqueous environment. Most calcium antagonists are some hundred times more concentrated in the cell membrane than in the ambient aqueous environment and are usually more potent when added to the external side of the cell membrane. Electrophysiological and binding studies showed that the affinity increased with membrane depolarization (Schilling et al., 1986), indicating the preferential binding of calcium antagonists to the inactivated state of the channel. The ‘‘modulated receptor’’ hypothesis (see Chapter 48) was the first to depict the binding of a channel inhibitor to a receptor in a transmembrane channel as being modulated by the state of the channel. Nifedipine has a high binding affinity to Ca2⫹ channels at low membrane potentials. Smooth muscles cells have lower resting membrane potentials than cardiac fibers. A higher solubility of nifedipine in the membranes of smooth muscle cells and the low membrane potential may both be responsible for the observation that nifedipine is more effective on the vasculature than on the heart. Verapamil and other members of the phenylalkylamine group and diltiazem exert strongly rate-dependent inhibitions of calcium currents. In contrast to the uncharged 1,4-dihydropyridines at physiological pH, these compounds are charged and their access to the binding site is not predominantly via the lipophilic membrane pathway, but rather through open channels. These observations of dependency on the rate of excitation

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may be attributed either to preferential binding to the open state or to easier access through open channels to binding sites of the inactivated channel. Calcium antagonists bound to their receptor site in the channel-forming 움1 subunit do not alter the amplitude of the unitary channel current but allosterically lessen the activation of channels and, hence, decrease the probability of channel openings. Positive and negative allosteric interactions between different binding sites as well as between binding sites and the calcium pore have also been found, e.g., diltiazem and (⫺)devapamil inhibit or increase 1,4-dihydropyridine binding, depending on the temperature (Kanaya et al., 1981). The S-enantiomer of 5-nitro-1,4-dihydropyridine acts as a channel activator, increasing opening probability and open state duration, but can also act as a channel inhibitor depending on the membrane potential (Kass, 1987). Transformation of the mode of action of this calcium channel-activating compound into an inhibitory action was observed by allosteric interaction from another binding site, i.e., following fendiline binding (Schreibmayer et al., 1992).

VI. THERAPEUTIC ASPECTS OF CALCIUM CHANNEL LIGANDS As described in detail in other chapters, the most important therapeutic sites of action of calcium antagonists are the L-type channels in both vascular smooth muscle and cardiac cells. Effects on other calcium channels, as well as on other receptors and channels, at dosages employed in the clinic, rarely reach borderline clinical significance. This statement applies only to the first generation of calcium antagonists; the actions of second-generation compounds reach beyond the cardiovascular system, e.g., nimodipine is used in the treatment of neurological deficits following subarachnoid hemorrhage (Pickard et al., 1989), and future drug developments are aimed at channel, tissue, and/or disease selectivity. Among the borderline effects of classical calcium antagonists is the nifedipine inhibition of aldosterone secretion (Hiramatsu et al., 1982). Also, a mild but significant reduction of platelet aggregation and prolongation of bleeding time, which is probably due to inhibition of the transmembrane Ca2⫹ inflow in thrombocytes, has been reported (Dale et al., 1983). Beneficial effects of calcium channel blockers in arterial thrombosis, in deep vein thrombosis, in pulmonary emboli, in vasculities, in thrombotic thrombocytopenic purpura, and in thrombocythemia have been reported (Ahn et al., 1987). Experiments on smooth muscle cells without intact membranes, so-called ‘‘skinned’’ fibers, indicated that some calcium antagonists have intracellular sites of ac-

tion. Fendiline, bepridil, and flunarizine are potent inhibitors of calmodulin (Lugner et al., 1984) and, thus, of smooth muscle contraction regardless of the source of calcium activating the contraction. To what extent calmodulin antagonism of some calcium channel blockers contributes to the therapeutically important, smooth muscle relaxation needs to be evaluated. This intracellular site of action of Ca2⫹ channel blockers is different from the membrane site of action in every pharmacokinetic and pharmacodynamic aspect studied so far. In cancer therapy, calcium antagonists have beneficial effects when used in combination with certain anticancer compounds. This action is probably related to inhibition of the multidrug resistance channel phenotype (Baeyens, 1988). A similar mechanism may be responsible for the verapamil-induced reversal of chloroquine resistance in malaria (Martin et al., 1987). Features of the classical calcium antagonists relevant to the acute and chronic treatment of cardiovascular diseases, as well as their clinical efficacy, have been reviewed extensively (Fleckenstein et al., 1983; Janis et al., 1987; Sperelakis et al., 1985); only selected aspects related to physiology and pathophysiology are briefly summarized here. The inhibitory effects of calcium antagonists on excitation and excitation–contraction coupling in vascular smooth muscle fibers explain the successful and widespread use of calcium antagonists against hypertension, in hypertensive crisis, in angina, and, in Prinzmetal angina, against coronary vasospasm. In vascular smooth muscle cells, all calcium antagonists exhibit the highest efficacy against vasospastic activities (Fig. 2), are effective against depolarization-induced contractions, and are least effective against receptor-operated channel activities and the resultant transmitter-induced vasoconstrictions. Accordingly, in the coronary system, calcium antagonists are very effective against vasospastic activities and they dilate large extramural coronary vessels but do not alter peripheral coronary resistance or even cause ‘‘coronary steal’’ effects (Braunwald, 1981). More than 90% of patients with angina pectoris have parts of the large extramural vessels narrowed to less than half the normal diameter and, hence, vasodilatation in these areas of increased resistance will be effective in increasing the blood supply, and the concomitant reduction in myocardial oxygen demand will effectively reduce myocardial ischemia. The cardiac effects of calcium antagonists given directly into coronary blood vessels differ markedly from those following general systemic administration (see Table I). When given systemically, calcium antagonists lower vascular resistance and blood pressure (Fleckenstein, 1983) and interfere with autoregulative vasoconstriction and, thus, alter the circulatory reflex control

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45. Calcium Antagonists

FIGURE 6 (A) Chemical structures of compounds in the classical first generation of calcium antagonists (not shown is gallopamil, a methoxyderivative of verapamil). (B) Calcium antagonists suppress the potassiuminduced contractions of pig coronary strips. The tension induced by 43 mM KCl within 40 min is taken as 100%, and the dose-dependent decrease of tension is shown for different calcium antagonists and for papaverine (average values from 15 individual experiments for each concentration).

of the heart. Diltiazem and verapamil have been reported to reduce baroreceptor reflex sensitivity (Millard et al., 1982). Stimulation of 웁-adrenoceptors in the myocardium is a physiological opponent of the inhibitory effects of calcium antagonists. Agents that stimulate adenylate cyclase in cardiac cells, or cAMP and its analogues, all stimulate the cardiac L current by increasing the probability and the duration of channel openings, whereas inhibitors of adenylate cyclase or muscarinic (M2) agonists decrease the cAMP-increased calcium current (Reuter, 1983). The direct cardiac effects of calcium antagonists on SA nodal dominant pacemaker impulse generation (heart rate) and conduction (SA and

AV nodal conduction) are therefore modified significantly in vivo by the autonomic nervous system, and the negative inotropic and chronotropic cardiodepressant effects, unless profound, are self-limiting. Whenever a decrease in cardiac output leads to a disproportionale fall in blood pressure, the reflex release of sympathetic transmitters will counteract the drug effect and make the drug safe to use in vivo. The reduction of contractility to the minimum level necessary for normal pump function in combination with a reduced heart rate adjusted to the workload and vasadilator effects reducing afterload and preload and the improved oxygen supply make the pumping activity of the heart more economic and reduce

TABLE I Differences of in Vivo or in Vitro Applications of Calcium Antagonistsa Ca2ⴙ antagonists applied In vitro or intracoronary Sinus rate

Decrease: N, V, D

Sinus node recovery time

Prolongation: N, V, D: ⫹⫹

Atrioventricular conduction time (AH duration)

VD: strongly rate-dependent prolongation of AH duration, Wenckebach-type blocks N: no rate dependency of prolongation of AH duration, almost no blocks VD: ⫹⫹ prolongation N: minor or no prolongation Verapamil V

Effective refractory period of AV conduction Nifedipine N

In vivo at rest N: ⫹, V, D: ⫺/⫹ during exercise N: ⫺/⫹, V, D: ⫺ N, V, D: ⫺/⫹ silk sinus node syndrome: N, V, D: ⫹⫹⫹ VD: prolongation of AH duration, Wenckebachtype blocks N: no or minor AH prolongation no blocks VD: prolongation Wenckebach-type blocks N: no prolongation, no blocks Diltiazem D

a Generation and conduction of the cardiac impulse are influenced by calcium antagonists. Only in in vitro experiments or following intracoronary administration do these direct inhibitory effects of calcium antagonists become evident. The baroreceptor reflex control of cardiac activities is stimulated by the vascular effects of calcium antagonists and significantly modifies these direct, inhibitory effects.

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the risk of angina. In this way, calcium antagonists improve cardiac function even in patients with failing left ventricles (Ferlinz, 1981). Even more surprising, patients with stable angina receiving oral 웁-adrenoceptor blockers showed an increased cardiac output with nifedipine but no change in cardiac output after verapamil (Winniford et al., 1982). When given by the intracoronary route, the direct effects of all Ca2⫹ antagonists are a decrease in heart rate and a slowing of sinoatrial conduction. Among heart cells, sinus node cells have the lowest membrane potential, and, accordingly, the most prominent inhibitory effect of nifedipine is in the sinus node. Less marked effects are seen in the AV node. In healthy people, nifedipine leaves the heart rate unchanged or even, due to induced baroreceptor reflexes, slightly increased, indicating a complete compensation or even an overcompensation of the direct and inhibitory effects of nifedipine on the sinus node. This reflex increase in the heart rate is not advantageous in patients with angina pectoris. In the ‘‘sick sinus node syndrome,’’ compensation by 웁-adrenoceptor stimulation is no longer effective and, accordingly, all Ca2⫹ antagonists may lead to life-threatening bradycardia or asystole and are, therefore, strictly contraindicated. The inhibitory effects of nifedipine are not dependent on the rate of heart activity or on channel openings as are those of verapamil and diltiazem. Thus, the effects of nifedipine may be completely compensated by the cAMP-mediated activation of calcium channels, whereas the rate-dependent part of the overall inhibitory action of verapamil or diltiazem is less likely to be compensated by baroreceptor reflex responses. Direct and antiarrhythmic effects of Ca2⫹ antagonists, verapamil and diltiazem, prolong the intranodal conduction time (AH duration) and lengthen antegrade and retrograde effective and functional refractory periods in a strongly dose- and rate-dependent manner, finally causing a Wenckebach-type block of conduction. As a rational basis for the clinical use of Ca2⫹ antagonists in the management of supraventricular arrhythmias, Singh et al. (1972) have proposed that among their fourth class of antiarrhythmic drugs there is a type 1 subgroup, which includes verapamil and diltiazem, that prolong AV nodal conduction and refractoriness and are moderate vasodilatators, and a type 2 subgroup, which includes nifedipine, with almost no direct electrophysiological effects in the heart and causing potent peripheral vasodilation.

VII. SUMMARY Transsarcolemmal Ca2⫹ ion movements play a pivotal role in excitation–contraction coupling in heart and in

smooth muscle fibres. They contribute significantly to the transmembrane currents initiating and sustaining action potentials. The pioneering work of Fleckenstein was the first indication that the modulation of transsarcolemmal Ca2⫹ currents is possible in a very selective way. The introduction of calcium antagonistic compounds strongly improved the understanding of the Ca2⫹ dependency of many physiological functions of the heart as well as of some cardiovascular diseases. Hence, many clinical studies were initiated and their results confirmed the new pharmacological principle of calcium antagonism. These new compounds were also very helpful tools for the study of transmembrane Ca2⫹ currents throughout biology and medicine. This chapter briefly outlined the history of the discovery of calcium antagonists. The verification of Ca2⫹-selective membrane channels as receptor sites for calcium antagonists and the advent of new methods to study single channel opening heralded a molecular dimension of the physiology and pharmacology of Ca2⫹ channels that comprised an analysis of structure, function, ligand binding, and channel modulation by receptors or second messengers. The distinct features of L-type and T-type channels, both of which are present in the cardiovascular system, relative to conductance, activation, and inactivation processes, are described. The two channels differ significantly in their ion preference, inhibition by divalent ions or blockers, and in their voltage dependence. The present concepts of structure and function of Ltype channels, of their ionic selectivity, and their modulation by other ions, including Mg2⫹, and by pH allow an interesting synoptic view on the likely sites and the modes of action of calcium antagonists. Ligand binding is necessarily dependent on the pathway of access to and on the affinity for the binding site: the latter may very depending on the state of the channel. There are a number of distinct sites for the different chemical classes of calcium antagonists on the 움1 subunit, and the modulation of channel activity most probably takes place by allosteric interactions with the key structures of channel function (activation, gating). In this way the molecular mode of action of calcium antagonists can be analyzed very precisely and the decrease in the total transmembrane current of a cell can be attributed to a reduction in the probability and or duration of the open state of Ca2⫹ channels without any simultaneous change in the unitary current amplitude of the individual open channel. All direct effects of calcium antagonists on cardiac cells are modified significantly by baroreceptor reflex responses induced by concurrent events in the cardiovascular system, particularly blood pressure changes, that may follow the systemic administration of these compounds. 웁-adrenoceptor stimulation is the main

45. Calcium Antagonists

physiological opponent of calcium antagonistic effects and, unless this is compromised, the pumping function, even of failing hearts, is usually not endangered by the direct negative inotropic effects of calcium antagonists. In a similar fashion, the potential bradycardiac effect of calcium antagonists is hardly detectable in vivo (exception: ‘‘sick sinus node syndrome’’). Calcium antagonists prove to be most effective against vasospastic activities and against depolarization-induced contractions of smooth muscle and least effective against transmitterinduced smooth muscle contractions. Nifedipine is more effective on blood vessels than verapamil and diltiazem. There are some interesting effects of calcium antagonists that are not related to effects on L-type channels (e.g., in cancer treatment). However, the future trend of the steadily increasing spectrum of clinical applications for the next generation of calcium antagonists will be focused on the refined selectivities of such compounds with respect to channel types and subtypes, tissue-specific expression of channels, and in relation to specific changes associated with cardiovascular diseases.

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Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflu¨g. Arch. 391, 85–100. Hanna, C., and Schmid, J. R. (1970). Antiarrhythmic actions of coronary vasodilator agents papaverine, dioxyline and verapamil. Arch. Int. Pharmacol. 185, 228–233. Hartzell, H. C., and White, R. E. (1989). Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes. J. Gen. Physiol. 94(4), 745–767. Hayase, S., Hirakawa, S., Hosokawa, S., Mori, N., Kanyama, S., and Iwasa, M. (1972). Hemodynamic and therapeutic effects of BAY a 1040 on the patients with ischemic heart disease. Arzneim Forsch/ Drug Res. 22, 370–373. Heginbotham, L., Abramson, T., and MacKinnon, R. (1992). A functional connection between the pores of distantly related ion channels as revealed by mutant K⫹-channels. Science 258, 1152–1155. Hess, P., and Tsien, R. W. (1984). Mechanism of ion permeation through calcium channels. Nature 309, 453–456. Hiramatsu, K., Yamagishi, F., Kubota, T., and Yamada, T. (1982). Acute effects of the calcium antagonist, nifedipine, on blood pressure, pulse rate and the renin-angiotensin-aldosterone system in patients with essential hypertension. Am. Heart J. 104, 1346–1350. Hondeghem, L. M. (1978). Validity of Vmax as a measure of the sodium current in cardiac and nervous tissues. Biophys. J. 23, 147–152. Hondeghem, L. M., and Katzung, B. G. (1977). Time and voltage dependent interaction of antiarrhythmic drugs with cardiac sodium channels. Biochim. Biophys. Acta 472, 373–398. Hymel, L., Striessnig, J., Glossmann, H., and Schindler, H. (1988). Purified skeletal muscle 1-4 dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers. Proc. Natl. Acad. Sci. USA 85, 4290–4294. Ichikawa, M., Urayama, M., and Matsumoto, G. (1991). Anticalmodulin drugs block the sodium gating current of squid axons. J. Membr. Biol. 120(3), 211–222. Isenberg, G., and Klo¨ckner, U. (1982). Calcium currents of isolated bovine ventricular myocytes are fast and of large amplitude. Pflu¨g. Arch. 395, 30–41. Janis, R. A., Silver, P., and Trigger, D. J. (1987). Drug action and cellular calcium regulation. Adv. Drug Res. 16, 309–591. Janke, J., Fleckenstein, A., and Jaedike, W. (1970). Hemmung der Isoproterenol-induzierten Ca2⫹-45 Netto-Aufnahme in das Ventrikelmyokard durch Ca2⫹-antagonistische Hemmstoffe der elektromechanischen Koppelung (Isoptin-Verapamil, Iproveratril und Substanz D600). Pflu¨g. Arch. Physiol. 316, 10. Kaibara, M., and Kameyama, M. (1988). Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea pig. J. Physiol. 403, 621–640. Kaltenbach, M. (1970). Medikamento¨se Therapie der Angina pectoris. Pru¨fung verschiedener Medikamente mit Hilfe von Arbeitsversuchen. Arnzeim Forsch/Drug Res. 20, 1304–1310. Kanaya, S., and Katzung, B. G. (1981). Rate- and voltage-dependent block of slow responses and calcium current by diltiazem. Circulation 64(4, II), IV 274. Kass, R. S. (1987). Voltage-dependent modulation of cardiac calcium channel current by optical isomers of Bay K8644: Implications for channel gating. Circ. Res. 61, 11–15. Kobayashi, T., Ito, Y., and Tawara, I. (1972). Clinical experience with a new coronary active substance (BAY a 1040). Arzneim Forsch/ Drug Res. 22, 380–389. Kostyuk, P. G. (1989). Diversity of calcium ion channels in cellular membranes. Neuroscience 28(2), 253–261. Krikler, D., and Spurell, R. A. (1972). Asystole after verapamil. Br. Med. J. 2, 405.

Kuga, T., Sadoshima, J., Tomoike, H., Kanaide, N., and Nakamura, M. (1990). Action of Ca2⫹ antagonists on two types of Ca2⫹ channels in rat aorta smooth muscle cells in primary culture. Circ. Res. 67(2), 469–480. Lansman, J. B., Hess, P., and Tsien, R. W. (1986). Blockage of current through single calcium channels by cadmium, magnesium and calcium. J. Gen. Physiol. 88, 321–347. Levine, T. B. (1997). The design of the Mortality Assessment in Congestive Heart Failure Trial (MACH-1, mibefradil). Clin. Cardiol. 20(4), 320–326. Llinas, R., and Yarom, Y. (1986). Specific blockage of the low threshold calcium channel by high molecular weight alcohols. Soc. Neurosci. Abs. 12, 174. Lugner, C., Follenius, A., Gerard, D., and Stoclet, J. C. (1984). Bepridil and flunarizine as calmodulin inhibitors. Eur. J. Pharmacol. 98, 157–161. Lux, H. D., Carbone, E., and Zucker, H. (1990). Na⫹ currents through low voltage activated Ca2⫹ channels of chick sensory neurones; block by external Ca2⫹ and Mg2⫹. J. Physiol. 430, 159–188. Martin, S. K., Oduola, A. M., and Milhous, W. K. (1987). Reversal of chloroquine resistance in plasmodium falciparum by verapamil. Science 235, 899–901. McCobb, D. P., Best, P. M., and Beam, K. G. (1989). Development alters the expression of calcium currents in chick limb motoneurons. Neuron 2, 1633–1643. Mikami, A. K., Imoto, T., Tanabe, T., Niidome, Y., Mori, H., Takeshima, S., Narumiya, S., and Numa, S. (1989). Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 340, 230–233. Millard, R. W., Gabel, M., Fowler, N. O., and Schwartz, A. (1982). Baroreceptor reflex sensitivity reduced by diltiazem and verapamil. Fed. Proc. 41, 57959. Miller, R. J. (1992). Voltage-sensitive Ca2⫹ channels. J. Biol. Chem. 267(3), 1403–1406. Mori, Y., Friedrich, T., Kim, M. S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., Mikoshiba, K., Imoto, K., Tanabe, T., and Numa, S. (1991). Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350, 398–402. Nastainczky, W., Ro¨hrkasten, W., Sieber, M., Rudolph, C., Scha¨chtele, C., Marme, D., and Hofmann, F. (1987). Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C. Eur. J. Biochem. 169(1), 137–142. Neumann, M., and Luisada, A. A. (1966). Double blind evaluation of orally administered iproveratril in patients with angina pectoris. Am. J. Med. Sci. 251, 552–556. Nilius, B., Hess, P., Lansmann, J. B., and Tsien, R. W. (1985). A novel type of cardiac calcium channel in ventricular cells. Nature 316, 443–446. O’Rourke, B., Backx, P. H., and Marban, E. (1992). Phosphorylationindependent modulation of L-type calcium channels by magnesium-nucleotide complexes. Science 257, 245–248. Perez-Reyes, E., Kim, S. H., Lacerda, A. E., Horne, W., Wei, X., Rampe, D., Campbell, K. P., Brown, A. M., and Birnbaumer, L. (1989). Induction of calcium currents by the expression of the alpha1-subunit of the dihydropyridine receptor from skeletal muscle. Nature 340, 233–236. Pickard, J. D., Murray, G. D., Illingworth, R., Shaw, M. D. M., Teasdale, G. M., Foy, P. M., Humphrey, P. R. D., Lang, D. A., Nelson, R., Richards, P., Sinar, J., Bailey, S., and Skene, A. (1989). Effect of oral nimodipine on cerebral infarction and outcome after subraarchnoid haemorrhage: British aneurysm nimodipine trial. Br. Med. J. 298, 636–642.

45. Calcium Antagonists Prod’hom, B., Pietrobon, D., and Hess, P. (1987). Direct measurement of proton transferates to a group controlling the dihydropyridinesensitive Ca-channel. Nature 329, 243–246. Reuter, H., Stevens, C. F., Tsien, R. W., and Yellen, G. (1982). Properties of single calcium channels in cardiac cell culture. Nature 297, 501–504. Reuter, H. (1983). Calcium channel modulation of neurotransmitters, enzymes and drugs. Nature 301, 569–574. Rowe, G. G., Stenlund, R. R., Thomsen, J. H., Corliss, R. J., and Sialer, S. (1971). The systemic and coronary hemodynamic effects of iproveratril. Arch. Int. Pharmacodyn. Ther. 193, 381–390. Sacks, H., and Kennelly, B. M. (1972). Verapamil in cardiac arrhythmias. Br. Med. J. 2, 716. Sakmann, B., and Neher, E. (1983). ‘‘Single Channel Recording.’’ Plenum Press, New York. Salkoff, L. B., and Tanouye, M. A. (1986). Genetics of ion channels. Physiol. Rev. 66(2), 301–329. Sandler, G., Clayton, G. A., and Thornicroft, S. G. (1968). Clinical evaluation of verapamil in angina pectoris. Br. Med. J. 224–227. Schamroth, L. (1971). Immediate effects of intravenous verapamil on atrial fibrillation. Cardiovasc. Res. 5, 419–424. Schilling, W. P., and Drewe, J. A. (1986). Voltage-sensitive nitrendipine binding in isolated cardiac sarcolemma preparation. J. Biol. Chem. 261, 2750–2758. Schreibmayer, W., Tripathi, O., and Tritthart, H. A. (1992). Kinetic modulation of guinea-pig cardiac L-type calcium channels by fendiline and reversal of the effects of Bay K8644. Br. J. Pharmacol. 106, 151–156. Schwinger, R. H. G., Hoischen, S., Reuter, H., and Hullin, R. (1999). Regional expression and functional characterization of the L-type Ca2⫹-channel in myocardium from patients with end-stage heart failure and in non-failing human hearts. J. Mol. Cardiol. 31, 283–296. Singh, B. N., Vaughan, Williams, E. M. (1972). A fourth class of antiarrhythmic action: Effects of Verapamil on Quabain toxicity on atrial and ventricular intracellular potentials and other features of cardiac function. Cardiovasc. Res. 6, 109–119. Sperelakis, N., and Caulfield, J. B. (1985). ‘‘Calcium Antagonists.’’ Nijhoff, Martinus. Strichartz, G., Rando, T., and Wang, G. R. (1987). An integrated view of the molecular toxicology of sodium channel gating in excitable cells. Annu. Rev. Neurosci. 10, 237–267. Tanabe, T., Beam, K. G., Powell, J. A., and Numa, S. (1988). Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 336, 134–139. Tang, C. M., Presser, F., and Morad, M. (1988). Amiloride selectively blocks the low threshold (T) calcium channel. Science 240, 213–215. Taniguchi, J., Noma, A., and Irisawa, H. (1983). Modification of the cardiac action potential by intracellular injection of adenosin triphosphate and related substances in guinea pig single ventricular cells. Circ. Res. 53, 131–139.

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46 Cyclic Nucleotides and Protein Phosphorylation in Vascular Smooth Muscle Relaxation GIOVANNI M. PITARI, DONALD H. MAURICE,*† BRIAN M. BENNETT,* and SCOTT A. WALDMAN Departments of Medicine and Biochemistry and Molecular Pharmacology Division of Clinical Pharmacology Thomas Jefferson University Philadelphia, Pennsylvania 19107 Departments of †Pathology and *Pharmacology and Toxicology Faculty of Medicine Queen’s University Kingston, Ontario Canada K7L 3N6

I. INTRODUCTION

influences on the intracellular concentration of the others. However, which of the several possible interacting mechanisms predominates in mediating vascular smooth muscle relaxation in a variety of physiological or pathophysiological conditions remains to be better defined. This chapter concentrates on defining the mechanisms by which cyclic AMP and cyclic GMP regulate vascular tone. These mechanisms are defined by available experimental evidence, focusing on their relationships to the regulation of [Ca2⫹]i and on the Ca2⫹ sensitivity of the contractile apparatus. Although vascular smooth muscle function is the focus of this chapter, evidence obtained with other types of smooth muscle and, in some cases, with tissues other than smooth muscle is evaluated. It is hoped that this review permits the reader to appreciate the complexity of vascular smooth muscle function at the molecular level and the gaps in understanding that remain to be filled.

Vascular smooth muscle contractility is regulated by a complex balance between a variety of antagonistic and synergistic signal transduction pathways and intracellular second messenger molecules. Contraction is initiated by increases in the concentration of intracellular calcium ([Ca2⫹]i), which is regulated by the interplay of a variety of systems, including receptor- and voltageoperated Ca2⫹ channels, other ion channels, and receptor-mediated increases in the metabolism of signaltransducing phospholipids. Calcium interacts with a number of receptor proteins, particularly calmodulin, which alters the activity of other enzymes, ultimately resulting in increases in the activity of actin-activated myosin ATPase and smooth muscle contraction. Relaxation of vascular smooth muscle involves reducing [Ca2⫹]i by increasing the efflux of this cation out of the cell, decreasing its influx, or increasing its intracellular sequestration. Additionally, the sensitivity of the contractile apparatus to Ca2⫹ may be decreased so that there is a decreased contractile response at any given intracellular concentration of Ca2⫹. Smooth muscle relaxation is mediated by the intracellular second messengers cyclic AMP and cyclic GMP. The production and metabolism of these cyclic nucleotides and the regulation of intracellular concentrations of Ca2⫹ are highly interdependent. Experimental evidence suggests that each of these messenger molecules exerts regulatory

Heart Physiology and Pathophysiology, Fourth Edition

II. GUANYLYL CYCLASE, CYCLIC GMP, AND CYCLIC GMP-DEPENDENT PROTEIN KINASE A. Cyclic GMP Low levels of cyclic GMP in biological systems slowed progress in understanding the biochemistry and physiology of this cyclic nucleotide. The observations

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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that cyclic AMP accumulation was associated with vascular relaxation stimulated by catecholamines and that cholinergic-mediated vasoconstriction was associated with cyclic GMP accumulation misled many to suggest that cyclic AMP was responsible for relaxation, whereas cyclic GMP mediated smooth muscle contraction. This hypothesis was challenged when increases in cyclic GMP stimulated by cholinergic agents were detected only after the onset of smooth muscle contraction. The proposal that cyclic GMP was involved in the regulation of smooth muscle relaxation followed the observation of increased cyclic GMP levels associated with the relaxation of ductus deferens, tracheal, and vascular smooth muscle to glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). In the ensuing years, the involvement of cyclic GMP in vascular smooth muscle relaxation has been firmly established. It is generally accepted that the vasodilator effects of a number of compounds and therapeutic agents, in addition to endogenous substances such as endothelium-derived relaxing factor (EDRF) and atrial natriuretic peptides (ANPs), are mediated by increased intracellular accumulation of this cyclic nucleotide. Guanylyl cyclase, which catalyzes the formation of cyclic GMP from GTP, exists in both a cytosolic and a particulate or membrane-bound form in most tissues. These isoenzymes differ in their kinetic, physicochemical, and antigenic properties. Furthermore, these isoenzyme forms are regulated by different agents: the soluble enzyme is activated by the nitrovasodilators, EDRF, protoporphyrin IX, arachidonate, and carbon monoxide (CO), whereas particulate guanylyl cyclases are generally activated by hemin and selectively activated by peptide ligands, including Escherichia coli heat-stable enterotoxin, guanylin, and natriuretic peptides (1, 2).

B. Soluble Guanylyl Cyclase Soluble guanylyl cyclase has been purified to homogeneity from a variety of tissues. This protein can be purified in association with heme as a prosthetic group, which is required for enzyme activation by nitric oxide (NO). The immunopurified enzyme from rat lung exists as a heterodimer of 82 kDa (움1) and 70 kDa (웁1) subunits (SDS gels), whereas that of bovine lung consists of 움1 and 웁1 subunits of 73 and 70 kDa. Both 움1 and 웁1 subunits have been cloned and sequenced and have deduced molecular masses of 77.5 and 70 kDa, respectively. Sequence analysis of 움1 subunits from rat and bovine lung indicates that they are identical. A second 웁 subunit (웁2) has been isolated from rat kidney and has substantial sequence homology with the 웁1 subunit from lung. The 웁2 subunit possesses an additional 86 amino acids at the carboxyl-terminal, which contains

the consensus recognition sequence for isoprenylation/ carboxy-methylation, suggesting that it may associate with the plasma membrane. Other subunits have been isolated in mammals. An 움2 subunit, exhibiting 40% sequence homology with the 움1 subunit, has been identified in human fetal brain and rat kidney and mapped on human chromosome 11. Two new subunits of 81 and 70 kDa, 움3 and 웁3, respectively, have been identified in adult human brain. However, due to their high homology with 움1 and 웁1 subunits, they have been considered as species variants of the same subtypes. The carboxylterminal regions of the 움 and 웁 subunits possess a high degree of sequence homology with each other and share partial sequence homology with the putative catalytic domains of particulate guanylyl cyclase and of adenylyl cyclase. Expression of either subunit alone in COS-7 or L cells did not result in guanylyl cyclase activity, whereas coexpression of the two subunits yielded a catalytically active enzyme that could be stimulated by SNP. Thus, while each subunit appears to contain a catalytic domain, the presence of both subunits is required for basal and stimulated enzyme activity. This observation is reinforced by the coexpression of mRNA coding for the 움 and 웁 subunits in various tissues and by the colocalization to the same human chromosome of the 움3 and 웁3 subunits. Structural studies of adenylyl cyclase suggest that soluble guanylyl cyclase could function as a heterodimer containing a single catalytic site and a sibling regulatory site. Although 웁1 subunits are expressed ubiquitously, they have a tissue-specific association with different 움 subunits. To date, an enzymatically active complex with 웁2 has not been found. These data support the suggestion that the tissue-specific expression of different subunits is one mechanism regulating soluble guanylyl cyclase signaling (2, 3). Early studies of the kinetic properties of guanylyl cyclase utilized GTPase inhibitors to maintain constant substrate concentrations. One of these inhibitors, sodium azide, was a potent activator of soluble guanylyl cyclase. This fortuitous observation established a relationship between nitrovasodilators and cyclic GMP. In addition to azide, other agents activate soluble guanylyl cyclase, such as SNP, organic nitrates, sodium nitrite, hydroxylamine, phenylhydrazine, nitrosoamines, nitrosoureas, amyl nitrite, nicorandil, and molsidomine. Nitrovasodilators activate soluble guanylyl cyclase in broken cell preparations from numerous tissues. It has been suggested that these agents require either enzymatic or nonenzymatic conversion to NO or S-nitrosothiols to activate guanylyl cyclase (4). Thus, NO has been proposed as the proximal activator of guanylyl cyclase by these agents, and its formation may represent the final common pathway by which diverse nitrovasodilators influence enzyme activity and vascular motility.

46. Cyclic Nucleotides and Protein Phosphorylation

NO activates soluble guanylyl cyclase from all tissues tested. Maximum activation by NO is similar to that with other nitrovasodilators and is not additive with those agents. In 1980, Furchgott and Zawadzki (5) described a class of vasodilators that required intact endothelium to express their vasorelaxant effects. In these studies with rabbit aorta, relaxation stimulated by acetylcholine was dependent on the interaction of the hormone with endothelial cells. Indeed, many other agents have been shown to require endothelial cells to promote relaxation, including histamine, bradykinin, the calcium ionophore A23187, ATP, and thrombin. Data suggested that these agents stimulated the release of a factor from endothelial cells (EDRF) that subsequently acted on smooth muscle cells to induce relaxation. Soon thereafter, Rapoport and Murad demonstrated that endothelium-dependent vasodilators, acetylcholine, A23187, and histamine elevated cyclic GMP in rat thoracic aorta (1). Subsequent to these and many other studies, NO was identified as the molecule that accounted for the biological properties of EDRF, and the enzyme that catalyzes the formation of NO from L-arginine (NO synthase) was purified from endothelial cells and cloned. Whereas it appears that NO is the sole nitrogen oxidecontaining species generated by NO synthase, differences in some of the properties of EDRF and NO suggest that NO is released from endothelial cells in a stabilized form such as S-nitrosothiol or a nitrosyl–iron complex with thiol ligands. Moreover, an endotheliumderived hyperpolarizing factor (EDHF) has been proposed to participate in the acetylcholine-mediated relaxation of some vascular beds (6). Regardless of the chemical nature of the released form of EDRF, it is generally accepted that NO is the proximal activator of vascular smooth muscle guanylyl cyclase by endothelium-dependent vasodilators (1, 2). It is now apparent that endothelial cells respond to stimuli such as shear stress and pulsatile flow and that EDRF release is an important determinant of vascular tone under normal physiological conditions. In addition, vascular responsiveness in pathophysiological conditions such as hypertension and atherosclerosis may involve a reduction in EDRF-mediated vasodilator tone, whereas the induction of NO synthase in vascular smooth muscle and endothelial cells is likely a major determinant of the pathological vasodilation seen in endotoxic shock. There is a general consensus that other endogenous ligands, besides NO, could directly regulate soluble guanylyl cyclase. Among these, CO has been proposed as a biological gaseous messenger analogous to NO, mediating the inhibition of platelet aggregation, vascu-

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lar relaxation, and neurotransmission via soluble guanylyl cyclase activation. Thus, in porcine distal pulmonary artery, endothelium-dependent relaxation, in the presence of NO-synthase inhibition, was reversed by blocking heme oxygenase-2, a constitutive subtype of CO-synthase. However, the precise role of CO in vivo remains unclear, as the potency of this gas for activating soluble guanylyl cyclase is low; however, only low concentrations are produced by cells and tissues (3). Whereas the enzymatic basis for endogenous NO formation by endothelial cells is now established, the mechanism of metabolic activation of therapeutic nitrovasodilators such as organic nitrates remains to be fully characterized. The biotransformation of organic nitrates (e.g., GTN) to either inorganic nitrite alone or to nitrite and NO can occur by a variety of enzymatic and nonenzymatic pathways, including glutathione S-transferases, cytochromes P450, hemoproteins such as hemoglobin and myoglobin, and various thiol compounds. Biotransformation of GTN by vascular glutathione S-transferases and cytochrome P450 has been demonstrated, and both enzyme systems have been implicated in mediating, at least in part, the vasodilator effects of GTN (7). With respect to the cytochrome P450–cytochrome P450 reductase system, the cytochrome P450 substrate/ inhibitor, 7-ethoxyresorufin, and the flavoprotein inhibitor diphenyleneiodonium (DPI) inhibited the GTNinduced relaxation of isolated rat aorta, concomitant with inhibition of both cyclic GMP accumulation and vascular biotransformation of GTN (7). This suggested a significant role of the cytochrome P450–cytochrome P450 reductase system in mediating the conversion of GTN to an activator (presumably NO) of guanylyl cyclase. In addition, incubation of rat hepatic microsomes with GTN resulted in the NADPH-dependent formation of an activator of rat aortic guanylyl cyclase, and this activation could be inhibited by inhibiting cytochrome P450 reductase with DPI (8). Furthermore, in whole animal studies, DPI inhibited the blood pressure-lowering effect of GTN (8). Tolerance to the vasodilator effects of organic nitrates following prolonged exposure to these agents is associated with both decreased vascular biotransformation and decreased cyclic GMP accumulation. Although desensitization of guanylyl cyclase to activation by SNP, GTN, and NO is observed in broken cell preparations from GTN-tolerant blood vessels (9; Fig. 1), vasodilation by SNP and endothelium-dependent vasodilators is affected only modestly compared to the loss of vasodilator activity of GTN (10). However, biotransformation of GTN is decreased in tolerant tissues, and in cells allowed to recover from GTN tolerance, GTN biotransformation and cyclic GMP responses returned toward control values (11). Thus, the metabolic activation system for

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FIGURE 1 Effect of GTN tolerance on activation of crude soluble guanylyl cyclase by SNP, GTN, D-isoidide dinitrate (D-IIDN), and L-isoidide dinitrate (L-IIDN). Rat aortas were divided in half and treated with either diluent (control) or 0.5 mM GTN for 1 hr, washed for 1 hr, and then homogenized. The 105,000 g supernatant fraction was used for the determination of guanylyl cyclase activity. Reproduced from Bennett et al. (9), with permission. Copyright  1988, American Heart Association.

organic nitrates in vascular smooth muscle appears to be the primary target for the tolerance-inducing effects of these drugs. Agents that generate NO by other mechanisms appear capable of activating guanylyl cyclase to a degree sufficient to permit vasodilation, even though a substantial portion of vascular guanylyl cyclase has been inactivated during GTN tolerance development. In addition, continuous exposure of bovine and rat vascular smooth muscle cells to nitrovasodilators suppressed the expression of cyclic GMP-dependent protein kinase (PKG) type I움, presumably contributing to the diminished responsiveness of smooth muscle cells to relaxation (12).

C. Particulate Guanylyl Cyclase (PGCs) Particulate guanylyl cyclases are a family of single transmembrane domain proteins (13). They contain an extracellular domain that confers ligand specificity, whereas the cytoplasmic portion of the enzyme contains protein tyrosine kinase-like and cyclase catalytic domains. Various studies suggest that all members of PGCs function as homodimers, sharing two catalytic sites. Three mammalian isoforms (designated GC-A, GC-B,

and GC-C) have been identified and sequenced with deduced molecular masses of 114–121 kDa and Mr values of 120–180 kDa on SDS gels. GC-A and GC-B isoforms exhibit considerable sequence homology, especially within the carboxyl-terminal cytosolic region. GC-A binds and is activated by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), with a higher affinity for ANP. GC-B is activated by low concentrations of a C-type natriuretic peptide and by higher concentrations of ANP and BNP. GC-C has considerably less sequence homology with GC-A and GC-B, especially across the extracellular domain. This isoform binds and is activated by the E. coli heat-stable enterotoxin and by the endogenous peptides guanylin and uroguanylin. Four other members of GCs, orphan receptors lacking ligands, have been identified in mammals. Three, expressed in sensory tissues, include GC-D in the olfactory mucosa and GC-E and GC-F in retina. GC-G was identified in various peripheral tissues such as lung, kidney, intestine, and skeletal muscle. Natriuretic peptides and GC have well-defined roles in mediating vascular smooth muscle relaxation. The vasorelaxant properties of ANP and related peptides in vitro are endothelium independent, occur in the absence of extracellular Ca2⫹, similarly to SNP, and are preceded by cyclic GMP accumulation and PKG activation. In contrast to nitrovasodilators, ANP specifically activated particulate guanylyl cyclase in tissues and cultured cells from blood vessels, kidney, and adrenal gland (14). Other studies have examined the effects of ANP on protein phosphorylation and Ca2⫹ dynamics in vascular smooth muscle (see later) and are for the most part consistent with a role for cyclic GMP in mediating the relaxant effects of ANP. Further suggestions for a pivotal role of the ANP/GC-A pathway in regulating cardiovascular homeostasis derive from genetic studies. Thus, genes for both ANP and GC-A have been disrupted: homozygous ANP- or GC-A-null mice had elevated blood pressure, insensitive to salt, and developed massive cardiac hypertrophy (13). Moreover, in transgenic mouse models, overexpression of ANP induced a 9- to 10-fold elevation of circulating ANP levels and chronic hypotension, whereas overexpression of GC-A produced a decrease in blood pressure that was inversely related to GC-A gene copy number (13). These reports suggest the presence, in mammals, of a unique and conserved function for the modulation of vascular tone, represented by the ANP/GC-A/cyclic GMP pathway.

D. Cyclic GMP-Dependent Protein Kinase Agents that elevate cyclic GMP, including nitrovasodilators, endothelium-dependent vasodilators, and

46. Cyclic Nucleotides and Protein Phosphorylation

ANP, activate PKG (1). This activation and subsequent phosphorylation of key proteins has been suggested as the mechanism underlying the regulation of vascular smooth muscle tone by these agents. In contrast to the broad distribution of cyclic AMP-dependent protein kinase (PKA) in mammalian tissues, the distribution of PKG is more limited. In an early study, appreciable amounts of the enzyme were detected in smooth muscle, cerebellum, heart, lung, and platelets. In mammals, two different forms of PKG have been identified: the soluble type I and the particulate type II. Smooth muscle cells highly express only PKG I, whereas PKG II is abundant in brain and intestine (15). PKG I comprises two identical subunits of 76.3 kDa, each of which possesses a catalytic domain in the carboxyl-terminal region. The amino-terminal region contains the subunit dimerization site, autophosphorylation sites that are thought to inhibit the catalytic domain, a hinge region, and two binding sites for cyclic GMP. The two sites differ in their kinetics and cyclic nucleotide analogue selectivities, and binding of cyclic GMP at both sites is required for full activation of the enzyme. The enzyme can be activated by cyclic AMP, but requires at least 10-fold higher concentrations of this cyclic nucleotide. Two isoforms of PKG I, designated type I움 and type I웁, have been purified from bovine aorta, porcine coronary artery, and bovine tracheal smooth muscle, and also are present in human aorta. The two isoforms are present in approximately equal amounts in these tissues. The type I웁 isoform differs from type I움 only in the amino-terminal region of the protein. The two isoforms also differ with respect to autophosphorylation sites, dissociation kinetics of cyclic GMP, and the concentration of cyclic GMP required for half-maximal activation. This presumably reflects differences in the amino-terminal sequence. The potency of various cyclic GMP analogues for activating the type I움 isoform correlated with the potency for relaxing porcine coronary artery, suggesting that this enzyme may be the relevant isoform mediating the relaxant effects of cyclic GMP (16). Southern and Northern analyses of human tissues revealed that the two isoforms of PKG I could arise from alternative splicing of a single gene assigned to chromosome 10 and that while PKG I웁 was mainly detected only in the uterus, PKG I움 was highly expressed in aorta, heart, kidney, and adrenals (15). A variety of studies have been performed to elucidate the endogenous protein substrates of PKG in vascular smooth muscle and to correlate phosphorylation of these proteins with the functional changes known to occur following increases in cyclic GMP accumulation. Early studies focused on the endogenous phosphorylation in broken cell preparations of rabbit aorta and

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cultured rabbit vascular smooth muscle cells exposed to cyclic GMP. Endogenous protein phosphorylation could only be demonstrated in the particulate fraction (1). Three proteins of 250, 130, and 85 kDa (designated G0 , G1 , and G2) exhibited increased phosphorylation, with G1 being the major PKG substrate. G2 is thought to be a degradation product of G1 . The similar molecular sizes of G1 and the plasma membrane Ca2⫹-pumping ATPase suggested that the Ca2⫹-pumping ATPase might be a substrate for PKG. Using a Ca2⫹-pumping ATPase preparation purified from bovine aortic smooth muscle by calmodulin affinity chromatography and reconstituted into phospholipid vesicles, exogenous PKG was found to phosphorylate a 135-kDa protein of identical mobility to that of the Ca2⫹-pumping ATPase (17). Furthermore, PKG was found to stimulate Ca2⫹ uptake into these proteoliposomes. The authors suggested that cyclic GMP regulation of the plasma membrane Ca2⫹ pump involved phosphorylation of the pump itself. Results of a number of other studies argue against the plasma membrane Ca2⫹-pumping ATPase being a substrate for PKG. Vrolix et al. (18) utilized a Ca2⫹-pumping ATPase preparation from pig stomach smooth muscle purified by calmodulin affinity chromatography. However, the membranes from which the ATPase was solubilized were first extracted with 0.6 M KCl rather than the 1.2 M KCl used by Furakawa and Nakamura (17). This resulted in extraction of a 130-kDa PKG substrate (presumably G1) from the crude membrane fraction concomitant with less 32P labeling of the 130-kDa protein in the Ca2⫹-pumping ATPase preparation (18). The authors concluded that the 130-kDa protein in the ATPase preparation was not the ATPase itself, but a contaminant protein that comigrated with the ATPase. They suggested that this 130-kDa protein was myosin light chain kinase (MLCK), based on cross-reactivity with antibodies to chicken gizzard MLCK. In other study, however, MLCK and the G1 protein from porcine aortic smooth muscle differed in molecular mass by 13–20 kDa and had quite different solubility properties (19). Purification of the Ca2⫹-pumping ATPase from porcine aortic smooth muscle also utilized calmodulin affinity chromatography (20). In these studies, a high salt wash of the affinity gel prior to elution of the ATPase resulted in the removal of all PKG substrates from the Ca2⫹-pumping ATPase preparation, providing further evidence that the Ca2⫹-pumping ATPase is not a PKG substrate. The studies of Baltensperger et al. (19) utilized an enriched plasma membrane fraction from porcine aorta that contained endogenous PKG activity and demonstrated ATP-dependent Ca2⫹ uptake. Electrophoretic analysis demonstrated that the G1 phosphoprotein and

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the acyl phosphate intermediate of the Ca2⫹-pumping ATPase had different Mr values. Furthermore, overlay experiments using 125I-calmodulin demonstrated interaction with the Ca2⫹-pumping ATPase, but not with the G1 protein (18). The Ca2⫹ uptake properties of the plasma membrane vesicles were found to be independent of the phosphorylation level of G1, suggesting that the pumping activity of the ATPase was not regulated by G1 phosphorylation. In a subsequent study (19), it was found that the G1 phosphoprotein could be crosslinked oxidatively to plasma membrane-bound actin. It was proposed that the G1 protein might act as a membrane attachment protein for actin and could therefore play a regulatory role in cytoskeletal reorganization, leading to vascular smooth muscle relaxation. Taken together, the balance of evidence suggests that the G1 protein is not the Ca2⫹-pumping ATPase and that the Ca2⫹-pumping ATPase is not a PKG substrate. A role for the 240-kDa PKG substrate (G0) in the regulation of the plasma membrane Ca2⫹-pumping ATPase has been described (21). As discussed in another section of this review, Koga et al. (22) suggested that the 240-kDa protein G0 might be an 1,4,5-inositol triphosphate (IP3) receptor isoform. In subsequent studies, Komalavias and Lincoln (23) supported this suggestion, arguing for a crucial role of PKG-mediated IP3 receptor phosphorylation in cyclic nucleotidedependent vascular relaxation in vivo. However, a highly purified preparation of Ca2⫹-pumping ATPase from porcine aortic smooth muscle, in which the type I IP3 receptor was removed by specific immunoprecipitation, was found to be specifically stimulated by PKG I움 without direct phosphorylation of the pump (21). In addition to the plasma membrane PKG substrates G0, G1, and G2, a number of investigators have demonstrated the PKG-dependent phosphorylation of vascular smooth muscle phospholamban (24–26). In sarcoplasmic reticulum membrane fractions of bovine pulmonary artery, exogenous PKG phosphorylated phospholamban, which was associated with an increase in Ca2⫹ uptake by the membrane fraction (27). Also, phospholamban was phosphorylated in microsomes from cultured rat aortic smooth muscle cells following stimulation with cyclic GMP or ANP or after the addition of purified PKG (25). Huggins et al. (24) demonstrated the phosphorylation of phospholamban in microsomes from sheep pulmonary artery after exposure to exogenous PKG. However, in intact rabbit aorta, 32P labeling of phospholamban could not be demonstrated in tissues after a 10-min exposure to a relatively high concentration of SNP (150 nM). The authors concluded that there is a functional separation between increases in cyclic GMP and

phospholamban phosphorylation, possibly due to inaccessibility of PKG to phospholamban in intact cells or to the action of phospholamban phosphatase or phosphodiesterase in close proximity to phospholamban (24). In contrast to the just-described study, phosphorylation of phospholamban in intact cultured rat aortic smooth muscle cells was observed after exposure to 100 nM SNP or ANP, with maximal phosphorylation occurring after 1 min (26). The increase in phospholamban phosphorylation was associated with an increase in Ca2⫹-activated ATPase activity in membranes isolated from stimulated cells. Furthermore, studies using confocal laser-scanning microscopy indicated that PKG and phospholamban were localized to the same cellular regions (26). These results suggest that PKG may indeed be located in close proximity to phospholamban in situ and that phosphorylation of phospholamban by PKG with subsequent activation of the sarcoplasmic reticulum Ca2⫹-pumping ATPase may be one mechanism by which cyclic GMP mediates a reduction in [Ca2⫹]i. Relatively few other studies in addition to those just noted have been performed using intact cells. Rapoport et al. (28) examined cyclic GMP-dependent protein phosphorylation in rat aortic strips. A variety of agents were tested, including SNP, 8-bromo-cyclic GMP, and the endothelium-dependent vasodilator, acetylcholine. All of these agents produced a qualitatively similar pattern of protein phosphorylation. The pattern of phosphorylation observed with acetylcholine was abolished if the endothelium was removed from vessels prior to exposure to the relaxant. In these studies, phosphorylation of proteins was observed in both soluble and particulate compartments. Altered phosphorylation of nine proteins was observed. While most proteins had increased phosphorylation, two were decreased in their phosphorylation. The molecular sizes of those proteins demonstrating increased phosphorylation ranged from 49 to 21 kDa. Proteins demonstrating decreased phosphorylation had molecular sizes of 22 kDa and were later identified as the phosphorylatable light chains of myosin. Other PKG substrates have been identified in bovine aortic smooth muscle cells in studies examining the protein phosphorylation activity of purified PKG on heat or acid tissue extracts (29). Seven of these protein substrates of 40, 33, 28, 25, 24, 23, and 22 kDa exhibited a relative specificity for PKG. In summary, there has been progress in identifying some of the protein substrates of PKG in vascular smooth muscle. Inhibition of Ca2⫹ transients through activation of Ca2⫹-pumping ATPases via PKG-dependent phosphorylation of G0 as well as phospholamban

46. Cyclic Nucleotides and Protein Phosphorylation

phosphorylation in the sarcoplasmic reticulum provide explanations for the effects of cyclic GMP on Ca2⫹ mobilization, efflux, and sequestration (see later), whereas a role of G1 as a membrane attachment protein for actin might at least partially explain relaxant effects of cyclic GMP, which are independent of changes in [Ca2⫹]i. What is still lacking is knowledge of the relative role of the identified PKG substrates in mediating vascular relaxation and the identity and function of other PKG substrates.

III. ADENYLYL CYCLASE, CYCLIC AMP, AND CYCLIC AMP-DEPENDENT PROTEIN KINASE A. Cyclic AMP There is substantial evidence that elevations of intracellular cyclic AMP concentrations are associated with relaxation of vascular smooth muscle (30). Thus, hormones and autacoids that interact with specific receptors such as 웁-adrenoceptor agonists, adenosine, prostaglandins, vasoactive intestinal peptide, and glucagon induce smooth muscle relaxation by increasing intracellular cyclic AMP. Similarly, agents that directly activate adenylyl cyclase and increase the synthesis of intracellular cyclic AMP, such as forskolin, or cell-permeant structural analogues of cyclic AMP relax vascular smooth muscle (30).

B. Adenylyl Cyclase Since the discovery of cyclic AMP, the activity of adenylyl cyclase, the enzyme that catalyzes the synthesis of this second messenger from ATP, has been studied intensely. Gilman and colleagues were first to clone a cDNA for this protein from a bovine brain cDNA library. Since this initial success, no fewer than 10 isoforms of mammalian adenylyl cyclase have been cloned from various tissues with these individual isoforms being referred to as AC1–AC10, respectively (31–35). Sequence analysis has identified a common overall structure, as well as several highly conserved domains. Thus, each adenylyl cyclase is predicted to contain two hydrophobic domains (M1 and M2), each of which traverses the plasma membrane six times and two large cytoplasmic domains that form the catalytic center of the molecule. Although the sequence identity within the cytoplasmic domains of the various isoforms is high (50–80%), the overall sequence homology among the 10 adenylyl cyclases is much lower (⬍30%). Experiments using molecular biological and biochemical approaches have shown that most isoforms of adenylyl cyclase are

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expressed in many tissues (i.e., brain, heart, liver), albeit at different levels and perhaps more importantly by different cells within these tissues. Indeed, several very elegant studies have demonstrated that although there is evidence that mRNA for all adenylyl cyclases are expressed in cellularly heterogeneous tissues, such as brain, the cellular expression pattern of the individual adenylyl cyclase gene products is distinct and, in many cases, nonoverlapping (36, 37). While hormones, neurotransmitters, and drugs can stimulate or inhibit adenylyl cyclase activity in a receptor and GTP-binding protein (G-protein)-dependent manner (31–35), evidence has shown that the activities of the individual isoforms can be regulated by several distinct mechanisms. Several excellent reviews have been published that detail the molecular basis of the regulation of these individual isoforms of adenylyl cyclase (31–35). Briefly, expression of each of the 10 cloned adenylyl cyclase isoforms leads to G움s-stimulated activities, a finding consistent with the initial model of G-protein-regulated adenylyl cyclase activity. In similar studies, inhibition of adenylyl cyclase activity by G움i was found to be adenylyl cyclase isozyme specific and was also more pronounced with some activators (i.e., forskolin or Ca2⫹ /calmodulin) than for others (i.e., G움s). 웁웂-subunit dimers of heterotrimeric G-proteins were shown to stimulate directly AC2 and AC4 and to inhibit AC1. Submicromolar Ca2⫹ inhibits two adenylyl cyclase isozymes (AC5 and AC6), whereas at higher concentrations, Ca2⫹ interacts with calmodulin to stimulate three other isoforms (AC1, AC3, and AC8). Adenylyl cyclase activity is also regulated by protein phosphorylation by various protein kinases (31–35). Phosphorylation of AC5 and AC6 by PKA inhibits these enzymes, whereas protein kinase C (PKC) phosphorylation of AC1, AC2, AC3, and AC7 activates these isoforms. Studies using broken cell preparations of isolated blood vessels, or of cultured vascular smooth muscle cells, have demonstrated that these tissues express G움s-, forskolin-, or Ca2⫹ /calmodulin-stimulated adenylyl cyclase activities and, in some instances, a Ca2⫹-inhibited adenylyl cyclase activity (38–40). Although these data are consistent with the expression of several distinct adenylyl cyclases in vascular smooth muscle cells, virtually nothing is known about the adenylyl cyclase isoforms expressed in blood vessels or if differential expression of adenylyl cyclases influences the responses of blood vessels to vasoactive substances. Differences in both basal and forskolin-stimulated adenylyl cyclase activities of blood vessels have been reported. As such, it is perhaps likely that differences in adenylyl cyclase expression will, at least in part, account for variations in the responses of distinct blood vessels to vaso-

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active agents that act by regulating adenylyl cyclase activity.

C. Cyclic AMP-Dependent Protein Kinase Two forms of PKA, which differ in the characteristics of their regulatory subunits, have been described. Type I and type II kinase are composed of two regulatory (RI and RII) and two catalytic subunits (30). These kinases are tightly regulated, and the catalytic subunits are maintained in the inactive conformation by the regulatory subunits in the absence of cyclic AMP. Each regulatory subunit binds two molecules of cyclic AMP that induce conformational changes in that subunit, stabilizing a structure that has a lower affinity for the catalytic subunits. Binding of four cyclic AMP molecules results in the dissociation of the tetrameric kinase into a regulatory subunit dimer and two active catalytic monomeric subunits (30). Monomeric catalytic subunits are active and participate in the regulation of intracellular processes by phosphorylating target proteins (30). Isoenzymes of PKA, corresponding to type I and type II kinase, have been identified in several blood vesselderived smooth muscles. Whereas the PKA-RI complex is essentially cytosolic, the PKA-RII is almost entirely particulate in most tissues. The mechanism by which PKA-RII localizes to particulate structures in cells has been elucidated (41). Thus, this complex is immobilized by interaction with members of an increasingly large and complex family of proteins referred to as PKAanchoring proteins (AKAPs) (41). Although AKAPs have been studied in many cell types, AKAPs expressed in vascular smooth muscle have yet to be elucidated. A role for PKA in mediating vascular smooth muscle relaxation has been suggested by correlating increased enzyme activity with physiological responses. Thus, relaxation of bovine coronary arteries by isoproterenol was associated with increases in type II kinase activity (42). Activation of type II kinase and relaxation was inhibited by the 웁-adrenoceptor antagonist propranolol. Similarly, isoproterenol and forskolin increased intracellular concentrations of cyclic AMP, activated PKA, and induced relaxation in bovine coronary arteries precontracted with KCl (43). Furthermore, adenosine produced a time- and concentration-dependent increase in PKA activity and relaxation in bovine circumflex arteries (44). Identification of the proteins phosphorylated by PKA has led to attempts to correlate changes in the phosphorylation of proteins with alterations in [Ca2⫹]i concentrations. Membrane proteins ranging in size from 11 to 256 kDa have been identified in various blood vessels as undergoing alterations in phosphorylation as a result of cyclic AMP accumulation or PKA activation. More

recently, identification of some of these proteins has been achieved. Phospholamban is a regulatory protein that inhibits the activity of the 100-kDa Ca2⫹-pumping ATPase responsible for the sequestration of intracellular Ca2⫹ in sarcoplasmic reticulum. This protein has been identified in vascular smooth muscle from a variety of sources and has been shown to be a substrate for PKA (45). These data suggest that one mechanism by which cyclic AMP may induce vascular smooth muscle relaxation involves activating PKA, resulting in the phosphorylation of phospholamban, which subsequently dissociates from the Ca2⫹-pumping ATPase. This dissociation removes the inhibition mediated by phospholamban, activating the transporter, increasing the sequestration of Ca2⫹ into the sarcoplasmic reticulum, and lowering [Ca2⫹]i. It is noteworthy that data supporting this model involving PKA were obtained in studies of cell-free systems. Interestingly, studies on intact cells in vitro suggest that agents that increase intracellular cyclic AMP concentrations in vascular smooth muscle result in the activation of PKG, which phosphorylates phospholamban on the same serine residues as PKA, resulting in relaxation. Thus, regardless of the kinase involved, cyclic AMP and GMP both appear to regulate vascular smooth muscle relaxation by a convergent mechanism involving phospholamban and increased Ca2⫹ sequestration into sarcoplasmic reticulum. Cyclic AMP has also been reported to increase the activity of a sarcolemmal Ca2⫹-pumping ATPase, which results in the extrusion of Ca2⫹, lowering the intracellular concentration of this cation, and relaxation. Activation of the sarcolemmal Ca2⫹-pumping ATPase by cyclic AMP or PKA has been reported in microsomes purified from rat mesenteric arteries and porcine aorta (46). Interestingly, PKA phosphorylates the 130-kDa plasma membrane Ca2⫹-pumping ATPase in erythrocytes, decreasing the Km and increasing the Vmax of this enzyme. These data suggest that another mechanism by which PKA mediates vascular smooth muscle relaxation is by phosphorylating the sarcolemmal Ca2⫹-pumping ATPase and increased extrusion of Ca2⫹. Another target for phosphorylation by cyclic AMP and PKA is myosin light chain kinase (MLCK). The mechanism by which MLCK phosphorylation reduces the Ca2⫹ sensitivity of the contractile apparatus is discussed in another section of this review.

IV. CYCLIC NUCLEOTIDE PHOSPHODIESTERASES The reaction catalyzed by cyclic nucleotide phosphodiesterase (PDE) involves the hydrolysis of 3⬘,5⬘ cyclic nucleotides to yield 5⬘ nucleotides and terminates cyclic

46. Cyclic Nucleotides and Protein Phosphorylation

nucleotide-mediated signaling. These enzymes form a 10 family multiprotein group of enzymes in which no fewer than 19 distinct genes encode more than 30 different isoforms (47–51). At the molecular level, each family member contains a highly conserved stretch of approximately 260 amino acids, situated within the carboxyl-terminal half of these enzymes, that encodes the catalytic domain. Although the catalytic domains of these enzymes share significant homologies, amino- and carboxyl-terminal sequences of individual PDE families are very different and contain regulatory domains (47– 51). Most of the information available concerning PDE structure, function, and expression has come from molecular cloning studies (47, 48). Three PDE1 genes encode Ca2⫹ /calmodulin-activated enzymes that can preferentially hydrolyze cyclic GMP (PDE1A and PDE1B) or both cyclic GMP and cyclic AMP (PDE1C). One PDE2 gene encodes an enzyme that can hydrolyze both cyclic AMP or cyclic GMP and which is stimulated by binding of cyclic GMP to amino-terminal regulatory sites (47). In contrast, two PDE3 genes encode enzymes that can also hydrolyze both cyclic AMP and cyclic GMP, but which are inhibited by cyclic GMP (47, 49). In PDE3, cyclic GMP inhibits by competing for cyclic AMP binding to the catalytic domain, and not via distinct regulatory sequences. Four PDE4 genes encode cyclic AMP-specific enzymes that are inhibited by the antidepressant drug Rolipram and some of its structural analogues (47, 50). One PDE5 gene encodes a cyclic GMP-specific enzyme, which, like PDE2, is stimulated by cyclic GMP binding to amino-terminal regulatory domains (47, 51). Three PDE6 genes lead to the expression of cyclic GMP-specific PDEs, which are expressed exclusively in cells of the retina. The catalytic activity of these enzymes is regulated by cyclic GMP, in a manner similar to that described previously for PDE2 and PDE5, as well as by interaction with the 움 subunit of the heterotrimeric G-protein transducin (47). Although the possible expression of five distinct PDE families in cells made analysis of cyclic nucleotide-mediated effects complex, data indicate that at least four other PDE families exist, although much less is known about the expression pattern and regulation of these enzymes. Thus, cloning studies identified one PDE7 gene and two PDE8 genes encoding the cyclic AMP-specific enzyme (47, 52) and one PDE9 gene and one PDE10 gene encoding enzymes that preferentially catabolize the breakdown of cyclic GMP (53, 54). Interestingly, the breakdown of cyclic GMP by PDE10 is inhibited competitively by cyclic AMP, a mechanism similar to that described for the inhibition of cyclic AMP breakdown by cyclic GMP for PDE3 enzymes (54–56). PDE activity can be regulated by protein phosphorylation (47–51). Indeed, certain variants of PDE1, PDE2, PDE3, PDE4,

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and PDE5 have been reported to be substrates of protein kinase C, PKA, PKG, or of yet uncharacterized kinases, and the effects of phosphorylation on catalysis, or on binding of cofactors (i.e., calmodulin), are enzyme specific. In addition, the expression of certain PDEs appears to be regulated in some cell types. The most extensively studied regulator of PDE expression is cyclic AMP. Indeed, in most studies, prolonged increases in cyclic AMP increase overall cyclic nucleotide PDE activity, with effects on individual enzymes being cell specific (47–51).

A. Cyclic Nucleotide Phosphodiesterases in Blood Vessels PDE isoenzyme activity profiles based on the effects of selective inhibitors have been generated using homogenates of blood vessels from several species, including human, bovine, rabbit, pig, dog, guinea pig, and rat (57). Although one of the most thorough studies to date identified five distinct PDE activities (PDE1–PDE5) in porcine aortic VSMC, this is not necessarily a common finding. Thus, aorta isolated from different species do not have identical cyclic nucleotide PDE activity profiles, and different blood vessels isolated from the same species can express very different patterns of activity. For example, although PDE2 is not abundant in rat aorta, this enzyme is highly expressed in rat pulmonary artery. 1. PDE1 Calmodulin-dependent cyclic nucleotide PDE activity has been detected in extracts from blood vesselderived smooth muscle from several species, although the relative contribution has varied between sources (57). This finding is undoubtedly due to species differences in addition to differences in the expression of the three PDE1 gene products in different blood vessels. Vinpocetine (TCV-3B), a relatively selective inhibitor of PDE1A and PDE1B, but a poor inhibitor of PDE1C, causes relaxation of blood vessels and an increase in cyclic GMP. In addition, relaxation by agents that activate soluble or particulate guanylyl cyclases are potentiated by vinpocetine (57). 2. PDE2 The amount of PDE2 activity detected in blood vessel smooth muscle extracts is low, although the enzyme is expressed in blood vessel endothelial cells (57). Consistent with the very small amounts of this activity detected in blood vessel-derived smooth muscle, selective PDE2 inhibitors do not relax blood vessels, nor do they po-

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tentiate the effects of activators of guanylyl cyclases. The abundance of PDE2 in endothelial cells may implicate a role for this enzyme in the regulation of blood vessel function by those cells. 3. PDE3 PDE3s are distinguished from other PDEs by their high affinity for both cyclic AMP and cyclic GMP (Km 0.1–1 애M) and their Vmax for cyclic AMP, which is 10-fold higher than that for cyclic GMP (47, 49). The Km for cyclic AMP indicates that these enzymes could be important in controlling basal as well as stimulated levels of cyclic AMP. PDE3 activity has been purified from several blood vessel extracts and accounts for 앑50% of the total cyclic AMP hydrolyzing potential (47, 49, 57). Two different genes encoding PDE3 have been identified (PDE3A and PDE3B) and, contrary to initial reports (47, 49), both gene products are expressed in several tissues, including blood vessel smooth muscle (58). Because of the kinetics of cyclic GMP hydrolysis by PDE3, endogenous cyclic GMP can inhibit cyclic AMP hydrolysis by this enzyme in certain tissues, including blood platelets, smooth muscle, and heart (55, 56). PDE3 activity also is inhibited by agents that stimulate myocardial contractility, inhibit platelet aggregation, and relax vascular and airway smooth muscle (e.g., cilostamide, enoximone, lixazinone), and studies support a role for PDE3 inhibition in the effects of these compounds (47, 49, 57). Attesting to the importance of PDE3 in cyclic AMP hydrolysis, inhibitors of this enzyme markedly potentiate relaxant effects of activators of adenylyl cyclase. 4. PDE4 These enzymes hydrolyze cyclic AMP with a Km between 1 and 4 애M and as such would primarily be responsible for hydrolyzing cyclic AMP following hormone stimulation of cyclic AMP synthesis (47, 50, 57). PDE4 activity has been detected in blood vessel extracts from several species, accounting for 앑20–30% of total cyclic AMP hydrolyzing activity. In contrast to PDE3 inhibitors, PDE4 inhibitors are generally weak relaxants of blood vessels. Indeed, in several studies, the IC50 values for relaxation of endothelium-denuded rat aorta by PDE4 inhibitors, such as Rolipram and Ro 20-1724, were severalfold higher than IC50 values for inhibition of the isolated enzyme. Studies have shown that PDE4 inhibitors were much more effective relaxants of blood vessels with intact endothelium (59). The molecular basis of this effect is thought to be related to an increased role for PDE4 under conditions in which PDE3 was inhibited by cyclic GMP. Consistent with this, PDE3

inhibitors also potentiate the effects of PDE4 inhibitors. PDE4 inhibitors were shown to be more effective than PDE3 inhibitors at potentiating the forskolin-mediated inhibition of smooth muscle proliferation and migration (60). The therapeutic potential of these agents at controlling the proliferative and migratory potential of smooth muscle cells has not been assessed. 5. PDE5 A Ca2⫹ /calmodulin-insensitive cyclic GMP-specific PDE activity has been demonstrated in several extracts of blood vessels from several species, with the relative percentage attributed to this activity ranging from 15 to 50% in different studies (47, 51, 57). The ability to selectively and potently inhibit PDE5 with the novel anti-impotence agent, Sildenafil, has led to a significant increase in the level of interest in this enzyme (51). Sildenafil and other inhibitors of PDE5, such as zaprinast and dipyridamole, increase intracellular cyclic GMP in intact blood vessels at concentrations that specifically inhibit PDE5 activity (57). In keeping with an important role for PDE5 in the regulation of smooth muscle contractility, relaxation by PDE5 inhibitors is endothelium dependent and was inhibited by L-NGmonomethylarginine. Also, PDE5 inhibitors could potentiate the relaxant effects of activators of both soluble and particulate guanylyl cyclases (57). 6. PDE7–PDE10 There is, at present, no information available about the relative abundance of these enzymes in blood vessels nor on their roles, if any, on cyclic nucleotide-mediated relaxations of blood vessels. Obviously, further work will be required to assess if these enzymes are important in smooth muscle relaxation and if inhibitors of these enzymes can modulate blood vessel function.

V. MECHANISMS OF VASCULAR RELAXATION A. Vascular Smooth Muscle Contraction Discussions concerning the molecular mechanisms underlying vascular smooth muscle relaxation are predicated on an understanding of those mechanisms mediating contraction. Because the primary focus of this chapter is smooth muscle relaxation, this brief review of contraction serves only to highlight those mechanisms in order to put into context mechanisms mediating relaxation. The reader is referred to other chapters for a more complete discussion of the mechanisms underlying vascular smooth muscle contraction.

46. Cyclic Nucleotides and Protein Phosphorylation

Smooth muscle contractility and relaxation are intimately dependent on the [Ca2⫹]i. Thus, contraction is dependent on an increase in [Ca2⫹]i, increased sensitivity of the contractile apparatus to [Ca2⫹]i, or both. In contrast, relaxation is dependent on a decrease in [Ca2⫹]i, a decreased sensitivity of the contractile apparatus to [Ca2⫹]i, or both. In addition, relaxation can be mediated by the uncoupling of myosin light chain phosphorylation from force generation. [Ca2⫹]i can be increased by two major mechanisms: electromechanical and pharmacomechanical coupling. In electromechanical coupling, [Ca2⫹]i is regulated by the depolarization of smooth muscle cells. Depolarization activates L-type Ca2⫹ channels, which mediate the influx of Ca2⫹, increase [Ca2⫹]i, and induce contraction. Pharmacomechanical coupling refers to contraction initiated by ligand–receptor interaction at the extracellular surface of the sarcolemma and the stimulation of transmembrane signaling mechanisms. Increased [Ca2⫹]i resulting from electromechanical or pharmacomechanical coupling binds to and activates calmodulin in vascular smooth muscle cells. The Ca2⫹ / calmodulin complex binds to MLCK, decreasing the autoinhibition characteristic of this enzyme. Activated MLCK phosphorylates the 20-kDa regulatory light chain of myosin (MLC20) on serine 19, increasing the actin-activated myosin ATPase activity. This promotes crossbridge cycling between thick and thin filaments, force generation, and smooth muscle contraction. Although force development in vascular smooth muscle is accompanied by an increase in the level of myosin phosphorylation, the maintenance of isometric tension can be preserved, despite a reduction in the level of MLC20 phosphorylation and [Ca2⫹]i . These data are consistent with the suggestion that the Ca2⫹dependent phosphorylation of MLC20 initiates an initial rapid cycling of crossbridges and development of force. In contrast, as the muscle approaches the sustained phase of isometric contraction, [Ca2⫹]i and MLC20 phosphorylation decrease, and rapidly cycling crossbridges give way to stable latch bridges. These latch bridges are dependent on adequate [Ca2⫹]i, although with a greater sensitivity to that ion than is found during the transient phase of force generation (61). Because both force development and force maintenance are subject to regulation by [Ca2⫹]i, both processes are expected to be altered by cyclic nucleotide-induced changes in [Ca2⫹]i. Several mechanisms have been suggested that might participate in pharmacomechanical coupling. Thus, contractile agonists, such as norepinephrine, bind to their G-protein-coupled receptors (GPCR) and activate sarcolemmal phospholipase C웁 (PLC웁), increasing the metabolism of membrane phosphatidyl inositides. This increase in metabolism results in the production of

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1,4,5-inositol triphosphate and diacylglycerol (DAG). Increases in IP3 result in the release of Ca2⫹ from the sarcoplasmic reticulum, increases in [Ca2⫹]i, and contraction. Contractile agonists may also increase the influx of Ca2⫹ by activating L-type Ca2⫹ channels or nonspecific ion channels in the sarcolemma in the absence of changes in membrane potential. As indicated earlier, agonists can also mediate contraction by increasing the sensitivity of the contractile apparatus to Ca2⫹. Evidence that agonist-coupled G-proteins mediate this Ca2⫹ sensitization has emerged through the use of plasma membrane-permeabilizing agents such as staphylococcal 움-toxin, which allow clamping of [Ca2⫹]i, while maintaining intact receptor and signal transduction systems (62, 63). In these and other studies, agonists such as norepinephrine, or the nonhydrolyzable GTP analogue GTP웂S, increased the Ca2⫹ sensitivity of the myofilaments (i.e., shifted the pCa2⫹ –tension curve to the left) and increased the level of MLC20 phosphorylation at constant [Ca2⫹]i, an effect mediated by the inhibition of myosin light chain phosphatase (MLCP). More recent studies suggest that the Ca2⫹- sensitizing effect of agonists acting through GPCRs is mediated by the monomeric GTP-binding protein rhoA and its associated kinase, p160ROCK, which is thought to phosphorylate the regulatory subunit of MLCP, resulting in the inhibition of enzyme activity. Thus the Ca2⫹-sensitizing effects of GTP웂S and GPCR agonists are inhibited by bacterial exoenzymes that selectively inactivate rhoA and by the selective p160ROCK antagonist Y-27632 (64). Furthermore, Y-27362 inhibits phenylephrine-induced contraction in intact blood vessels and lowers blood pressure in various animal models of hypertension (64). This is an important finding, as it demonstrates that Ca2⫹ sensitization can contribute to vascular tone in physiological settings in which [Ca2⫹]i has not been experimentally manipulated. What remains to be established is the mechanism by which heterotrimeric G-proteins exert their downstream effects on rhoA activity. A number of studies have examined the role of PKC in Ca2⫹ sensitization. Thus, phorbol esters, which are potent activators of PKC, induce sustained contractions of blood vessels associated with low or only small increases in [Ca2⫹]i and potentiate Ca2⫹-induced contractions in permeabilized vascular preparations. This led to the idea that DAG produced subsequent to agonistinduced activation of PLC웁 and hydrolysis of phosphatidylinositides might activate PKC. In addition, DAG is the product of other lipid metabolic enzymes, including phospholipase D and phospholipases C, which utilize phospholipids other than those containing inositol. Activation of these enzymes could increase DAG in the

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absence of IP3, activate PKC, and induce contraction in the absence of increases in [Ca2⫹]i. The specific target proteins phosphorylated by PKC that increase the sensitivity of the contractile apparatus to Ca2⫹ in situ remain unclear, but candidates include the thin filament proteins caldesmon and calponin and the MLCP inhibitor protein CPI-17. Although it is clear that the activation of PKC can alter the Ca2⫹ sensitivity for contraction per se, the role of PKC in Ca2⫹ sensitization by GPCR agonists and GTP웂S has been controversial (65). Thus in some studies, but not others, PKC inhibitors such as H7 and staurosporine inhibited Ca2⫹ sensitization by agonists and GTP웂S. The selectivity of these inhibitors for PKC has been questioned, and in studies utilizing the highly selective peptide inhibitors, Ca2⫹ sensitization by phorbol esters was inhibited, but that by agonists and GTP웂S was not. In other studies, downregulation of PKC by prolonged exposure to phorbol esters did not block agonist-induced Ca2⫹ sensitization; conversely, Ca2⫹ sensitization by agonists could be downregulated by prolonged exposure to GTP웂S without affecting Ca2⫹ sensitization by phorbol esters. In addition, concentrations of the p160ROCK inhibitor Y-27632 that inhibited agonistand GTP웂S-induced Ca2⫹ sensitization had no effect on Ca2⫹ sensitization induced by phorbol esters. Thus, to the extent that Ca2⫹ sensitization is an important component of the contractile activity of GPCR agonists, PKCmediated Ca2⫹ sensitization does not appear to play a significant role.

B. Effects of Cyclic Nucleotides on the Sensitivity of the Contractile Apparatus to [Ca2⫹]i Studies using permeabilized blood vessel preparations provided some of the first evidence that cyclic nucleotides could alter the Ca2⫹ sensitivity for contraction (66, 67). In Triton X-100-skinned porcine carotid arteries, cyclic AMP and cyclic GMP inhibited Ca2⫹induced contractions (67), and in 움-toxin-permeabilized rat mesenteric artery in which [Ca2⫹]i was controlled and [Ca2⫹]i stores were depleted, both cyclic AMP and cyclic GMP shifted the pCa2⫹ –tension curve to the right (66; Fig. 2). In a more recent study, Ca2⫹ desensitization in permeabilized rat mesenteric artery by 8-bromocyclic AMP and 8-bromo-cyclic GMP was completely reversed by the selective PKG inhibitor Rp-8-bromocGMPS but was unaltered by the PKA inhibitor RpcAMPS, suggesting that PKG activity accounts for Ca2⫹ desensitization induced by both cyclic GMP and cyclic AMP (68). Potential mechanisms whereby cyclic nucleotides could alter the Ca2⫹ sensitivity for contraction in-

FIGURE 2 Effect of cyclic nucleotides on the Ca2⫹ –force relationship in 움-toxin-permeabilized rat superior mesenteric artery. Ca2⫹ –force curves were formed in the absence (䊉) or presence of 30 애M cyclic AMP (䉱) or 30 애M cyclic GMP (䊐). Experiments were performed in the presence of 2 애M ionomycin in order to deplete sarcoplasmic reticulum Ca2⫹ stores. Modified from Nishimura and Van Breemen (66), with permission.

clude inhibition of MLCK, an increase in MLCP activity, or both. Historically, the former mechanism has received the most attention. Both PKA and PKG phosphorylate MLCK in cellfree preparations (69). Phosphorylation of MLCK by PKA decreased the affinity of this enzyme for the Ca2⫹ / calmodulin complex, the ability of this enzyme to phosphorylate MLC20, and, consequently, to induce actinactivated myosin ATPase activity. Phosphorylation of MLCK by PKA and resultant alterations in enzyme activity were dependent on the concentration of Ca2⫹. Thus, in the presence of a high Ca2⫹ concentration, PKA phosphorylated MLCK without altering its activity. In contrast, at a lower Ca2⫹ concentration, PKA phosphorylation of MLCK inhibited the enzyme. Although PKG phosphorylated MLCK, this modification was without effect on the MLCK activity (69). These data suggested a model for the regulation of vascular smooth muscle contractility in which increases in [Ca2⫹]i result in binding of the Ca2⫹ /calmodulin complex to MLCK, altering the sensitivity of MLCK to inhibition by PKA-mediated phosphorylation. Thus, in the absence of elevated [Ca2⫹]i and binding of Ca2⫹ / calmodulin, PKA phosphorylates MLCK at a site that reduces the affinity of this protein for Ca2⫹ /calmodulin, decreases the sensitivity of MLCK to increases in [Ca2⫹]i, and inhibits contraction. In the presence of elevated [Ca2⫹]i and binding of Ca2⫹ /calmodulin, PKA phosphorylates MLCK at a different site, which does not alter its sensitivity to increases in [Ca2⫹]i or its ability to mediate contraction. More recently, data have become available that significantly alter earlier hypotheses. It has been demon-

46. Cyclic Nucleotides and Protein Phosphorylation

strated that MLCK possesses six phosphorylation sites (peptides A–F), defined by proteolytic mapping (70). Of significance, only phosphorylation on peptide A (site A) decreased the sensitivity of MLCK to increases in [Ca2⫹]i. Phosphorylation of peptides B–F did not alter the affinity of MLCK for Ca2⫹ /calmodulin or its ability to phosphorylate MLC20 (70). Furthermore, site A was phosphorylated by Ca2⫹ /calmodulin-dependent protein kinase II (CaM kinase II) and PKA, but not PKG (70– 72). These data suggest that large increases of [Ca2⫹]i activate CaM kinase II, which phosphorylates MLCK at site A, decreasing the sensitivity of MLCK to increases in [Ca2⫹]i. This could serve as an autoinhibitory mechanism to reduce overactivation of the contractile machinery. Similarly, increases in cyclic AMP activate PKA, which phosphorylates MLCK at site A, decreasing the sensitivity of MLCK to increases in [Ca2⫹]i . Increases in cyclic GMP and activation of PKG result in the phosphorylation of MLCK at sites other than site A, and therefore do not alter the sensitivity of MLCK to increases in [Ca2⫹]i or its ability to phosphorylate MLC20. In experiments using purified MLCK bound to Ca2⫹ / calmodulin, phosphorylation of site A by CaM kinase II was inhibited (72). Because the affinity of Ca2⫹ /calmodulin for MLCK is higher than that for CaM kinase II, one would predict that at elevated [Ca2⫹]i , Ca2⫹ / calmodulin would be bound to MLCK and phosphorylation of site A by CaM kinase II would be inhibited. However, evidence suggests that the free Ca2⫹ /calmodulin concentration may be limiting such that not all MLCK would be bound by Ca2⫹ /calmodulin at elevated [Ca2⫹]i, and therefore a fraction of MLCK would be available for site A phosphorylation by CaM kinase II (73). In support of this, several studies have examined MLCK phosphorylation in intact, nonpermeabilized blood vessels (74, 75). Using a porcine carotid artery preparation, cells were loaded with 32PO4 and the phosphorylation of site A was assessed after contraction with histamine. Force, [Ca2⫹]i, MLCK activity, and MLC20 phosphorylation were assessed in parallel experiments. These authors found that MLCK activity was related inversely to site A phosphorylation and that MLCK phosphorylation and activity were dependent only on [Ca2⫹]i, suggesting a functional role for CaM kinase II in situ. Furthermore, during relaxation of histaminecontracted tissues by forskolin, MLCK phosphorylation decreased in proportion to the forskolin-induced decrease in [Ca2⫹]i, suggesting that site A phosphorylation by PKA does not contribute to changes in MLCK activity in intact cells (74). In a subsequent study using the same experimental model, maximal contraction with 10 애M histamine re-

817

sulted in increased force, [Ca2⫹]i, MLC20 phosphorylation, and MLCK site A phosphorylation. Addition of the nitrovasodilators GTN and SNP to histaminecontracted tissues did not affect [Ca2⫹]i but did decrease MLC20 phosphorylation and force, indicating that a decrease in the Ca2⫹ sensitivity for MLC20 phosphorylation had occurred. However, the phosphorylation of MLCK site A in the presence of histamine plus GTN or SNP was not increased over that caused by histamine alone, indicating that the nitrovasodilator -induced decrease in Ca2⫹ sensitivity was not due to changes in MLCK site A phosphorylation (75). In contrast to the results observed using maximally contracted tissue, submaximal contraction with histamine and subsequent exposure to GTN or SNP resulted in decreased [Ca2⫹]i and unaltered Ca2⫹ sensitivity, suggesting that mechanisms that alter [Ca2⫹]i handling play a more prominent role in nitrovasodilator-induced relaxation at moderate levels of contraction. This was supported by other experiments that examined the effects of nitrovasodilators added prior to the contractile agonist. In unstimulated tissues, GTN or SNP caused increased MLCK site A phosphorylation, presumably due to the cyclic GMP-mediated activation of PKA. The contractile response to histamine was attenuated in tissues that had been preincubated with SNP; however the histamine-induced increase in [Ca2⫹]i was also attenuated. Thus, although the phosphorylation of MLCK could have contributed to the altered contractile response, it was concluded that inhibition of the histamine-induced increase in [Ca2⫹]i by nitrovasodilators was a more important determinant (75). With the recently emerging evidence for a role of regulated MLCP activity in the phenomenon of Ca2⫹ sensitization and the availability of MLCP inhibitors, several studies have examined the effects of cyclic nucleotides on MLCP activity. In permeabilized rabbit femoral artery contracted submaximally with Ca2⫹, 8-bromo-cyclic GMP decreased the steady-state level of MLC20 phosphorylation and contractile force, which did not occur if MLCP activity was blocked using microcystin or calyculin A (76). This suggests that cyclic GMP mediates Ca2⫹ sensitization by the activation of MLCP, presumably by the PKG-mediated phosphorylation of MLCP or of a protein that regulates MLCP activity. Data also suggested that MLCK activity was not affected, as in the presence of MLCP inhibitors, 8bromo-cyclic GMP had no affect on the rate of force development or of MLC20 phosphorylation during the Ca2⫹-induced contraction (76). Similar results were obtained in permeabilized rabbit ileum smooth muscle contracted with Ca2⫹ (77), and in addition, 8-bromocyclic GMP inhibited the contraction induced by GTP웂S and inhibited the GTP웂S-induced increase in MLC20

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phosphorylation, effects that are thought to be mediated by the inhibition of MLCP. In addition to the Ca2⫹ desensitization mechanisms discussed earlier, relaxation by cyclic nucleotides may involve an uncoupling of force from MLC20 phosphorylation, i.e., a reduction in force for a given degree of MLC20 phosphorylation. Thus, in histamine-contracted intact porcine carotid artery, relaxation induced by SNP occurred without a proportional decrease in MLC20 phosphorylation. Furthermore, tissues contracted with 1 애M histamine had the same degree of MLC20 phosphorylation as those contracted with 3 애M histamine and then relaxed with SNP (78). Although the basis for the nitrovasodilator-induced uncoupling of force MLC20 phosphorylation is not known, it may involve inhibition of the attachment and cycling of phosphorylated crossbridges or inhibition of the formation of dephosphorylated latch bridges (79).

C. Effects of Cyclic Nucleotides on the Concentration of [Ca2⫹]i Evidence suggests that cyclic nucleotides induce vascular smooth muscle relaxation, in part, by decreasing the [Ca2⫹]i. Early work demonstrated an antagonistic relationship between agents that increased contraction and [Ca2⫹]i and those that induced relaxation and decreased [Ca2⫹]i. Thus, a dose-dependent relationship exists among norepinephrine concentration, contraction, and [Ca2⫹]i in rat aorta (80). Agents that increased intracellular concentrations of cyclic GMP, such as SNP,

were more effective at inducing relaxation at low norepinephrine concentrations. Presumably, this reflects the increased efficacy of cyclic GMP to decrease [Ca2⫹]i when the concentrations of this cation are low. In contrast, when high concentrations of agonist are employed, [Ca2⫹]i is high and agents that increase the concentration of cyclic GMP are ineffective at reducing [Ca2⫹]i below that required for relaxation. Similar results were obtained using agents that induce contraction by electromechanical coupling, such as depolarization by KCl (80). Relaxation by cyclic GMP under these conditions was reduced greatly and was thought to reflect the ability of L-type Ca2⫹ channels to elevate [Ca2⫹]i above the level that can be reduced by cyclic GMP. There are a variety of mechanisms by which cyclic nucleotides could decrease [Ca2⫹]i and induce relaxation (Table I, Fig. 3). An increased efflux of Ca2⫹ out of the cell may be mediated directly by a sarcolemmal Ca2⫹pumping ATPase or indirectly by activation of a Na⫹-K⫹ ATPase, which decreases intracellular Na⫹, activating a sarcolemmal Na⫹ –Ca2⫹ exchanger. Membrane hyperpolarization by the activation of K⫹ channels would also serve to increase Ca2⫹ efflux by Na⫹ –Ca2⫹ exchange. Also, a Ca2⫹-pumping ATPase in the sarcoplasmic reticulum may deplete Ca2⫹ from the cytoplasm by sequestration. In addition, relaxing agents may decrease the influx of Ca2⫹ by regulating L-type Ca2⫹ channels. Finally, relaxing agents may alter the production or the effects of second messengers, which are critical for elevations of [Ca2⫹]i induced by contractile agents and required for vascular smooth muscle contraction. Evi-

TABLE I Mechanisms by which Cyclic Nucleotides May Mediate Vascular Smooth Muscle Relaxation 1. Decreased [Ca2⫹ ]i A. Decreased influx of Ca2⫹ i. Inhibition of L-type Ca2⫹ channels ii. Activation of K channels or Na⫹ /K⫹ ATPase 씮 hyperpolarization 씮 decreased Ca2⫹ influx via potential-dependent Ca2⫹ channels iii. Uncoupling of agonist-induced phosphatidylinositide turnover 씮 decreased Ca2⫹ influx via receptor-operator Ca2⫹ channels iv. Activation of ryanodine receptor channels 씮 increased Ca2⫹ transients 씮 activation of KCa channels 씮 hyperpolarization 씮 decreased Ca2⫹ influx via potential-dependent Ca2⫹ channels B. Increased efflux of Ca2⫹ i. Increased activity of plasma membrane Ca2⫹-pumping ATPase ii. Activation of KCa channels or Na⫹ /K⫹ ATPase 씮 hyperpolarization 씮 increased activity of Na⫹ /Ca2⫹ 씮 exchanger iii. Activation of Na⫹ /K⫹ ATPase 씮 decreased intracellular Na⫹ 씮 increased activity of Na⫹ /Ca2⫹ exchanger C. Increased sequestration of Ca2⫹ i. Increased activity of sarcoplasmic reticulum Ca2⫹-pumping ATPase D. Decreased mobilization of Ca2⫹ i. Uncoupling of agonist-induced phosphatidylinositide turnover 씮 decreased IP3 formation ii. Inhibition of sarcoplasmic reticulum IP3 receptors 씮 decreased IP3 efficacy 2. Decreased sensitivity of the contractile apparatus to Ca2⫹ A. Decreased affinity of myosin light chain kinase for Ca2⫹ –calmodulin 씮 decreased phosphorylation of myosin light chain B. Decreased Ca2⫹ sensitivity of myofibrils C. Altered actin–plasma membrane interaction 씮 cytoskeletal reorganization D. Increased MLCP activity 씮 decreased phosphorylation of MLC20 3. Uncoupling of force from MLC20 phosphorylation

46. Cyclic Nucleotides and Protein Phosphorylation

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FIGURE 3 Proposed mechanisms by which cyclic nucleotides may lower [Ca2⫹]i leading to vascular smooth muscle relaxation. It is assumed that under resting conditions or conditions of low stimulation, there is vectoral transport of Ca2⫹ from the superficial sarcoplasmic reticulum (SR) toward the plasma membrane (PM) where Ca2⫹ is extruded by the Ca2⫹-pumping ATPase (2) and the Na⫹ / Ca2⫹ exchanger (5). Ca2⫹ release from the SR is mediated by IP3sensitive Ca2⫹ channels, Ca2⫹-induced Ca2⫹ release channels (CICR), and passive Ca2⫹ leak. Phosphorylation of phospholamban (Plb) by PKA or PKG would result in stimulation of the SR Ca2⫹-pumping ATPase (1), increasing Ca2⫹ sequestration and providing additional Ca2⫹ for vectoral Ca2⫹ transport. Phosphorylation of the PKG substrate G0 may result in stimulation of the PM Ca2⫹-pumping ATPase (2), leading to increased Ca2⫹ extrusion. Phosphorylation of K⫹ channels (3) by PKA or PKG may result in an increased probability of channel opening. The resulting membrane hyperpolarization would decrease Ca2⫹ entry by potential-dependent Ca2⫹ channels (PDC) and increase Ca2⫹ extrusion by the Na⫹ / Ca2⫹ exchanger (5). Activation of the Na⫹ /K⫹ ATPase (4) by PKA or PKG would result in membrane hyperpolarization and decreased intracellular Na⫹, both of which would increase Ca2⫹ extrusion by the Na⫹ /Ca2⫹ exchanger. Inhibition of agonist-induced phosphatidylinositol turnover by cyclic GMP may involve uncoupling of receptor (R)-G-protein (G)-phospholipase C (PLC) interaction (6), resulting in decreased IP3 formation and decreased Ca2⫹ mobilization from the SR via IP3sensitive Ca2⫹ channels. This uncoupling may also inhibit Ca2⫹ entry via receptor-operated Ca2⫹ channels (ROC).

dence has been obtained for the involvement of cyclic nucleotide in most of these mechanisms lowering [Ca2⫹]i. It is noteworthy that there is significant convergence of the mechanisms by which cyclic AMP and cyclic GMP mediate vascular smooth muscle relaxation. The major mechanism by which cyclic nucleotides alter cellular physiology is through protein phosphorylation on serine and threonine residues mediated by cyclic nucleotidedependent protein kinases. Indeed, as discussed previously, there are kinases that are specifically activated by cyclic AMP or cyclic GMP. Furthermore, these cyclic nucleotide-selective kinases appear to phosphorylate specific, although overlapping, protein substrates within target cells. Finally, both cyclic AMP and cyclic GMPspecific protein kinases are present in vascular smooth muscle and could potentially mediate relaxation. Although these enzymes are selective for cyclic nucleotides, either cyclic AMP or cyclic GMP can activate

each. Indeed, PKG can be activated by cyclic AMP, although at least a 10-fold higher concentration of cyclic AMP compared to cyclic GMP is required for this activation. Similarly, PKA can be activated by cyclic GMP, although a 10-fold higher concentration of cyclic GMP compared to cyclic AMP is required. It is significant that in vascular smooth muscle, the concentration of cyclic AMP is about 10-fold greater than cyclic GMP. Thus, elevations in cyclic AMP could activate both PKA and PKG, whereas cyclic GMP may activate only PKG. Data to support this hypothesis come from studies of vascular smooth muscle cells in culture (81) and of intact rat aorta (82). In particular, primary cultures of rat aortic smooth muscle cells exhibited increases in [Ca2⫹]i when treated with agents that induce contraction by electromechanical (KCl) or pharmacomechanical (arginine vasopressin) coupling. Pretreatment of those cells with agents that increase cyclic GMP concentrations, such as ANP or 8-bromo-cyclic GMP, inhibited

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increases in [Ca2⫹]i induced by KCl or vasopressin. These data suggest that agents that increase intracellular cyclic GMP induce relaxation by decreasing the [Ca2⫹]i , presumably by activating PKG. Interestingly, rat aortic smooth muscle cells passaged several times in culture lost their ability to respond to natriuretic peptides and 8-bromo-cyclic GMP with a decrease in [Ca2⫹]i. This resulted from a loss of PKG in the passaged cells, demonstrated by Western blot analysis using affinitypurified polyclonal antibodies to this protein. These data demonstrate that the ability of cyclic GMP to decrease [Ca2⫹]i was mediated by PKG. Of significance, however, was the observation that in passaged cells the response to agents that increase cyclic AMP was an increase in [Ca2⫹]i rather than a decrease. Thus, in primary cultures but not passaged cells, forskolin pretreatment of cells increased intracellular cyclic AMP and prevented the increase in [Ca2⫹]i induced by vasopressin or KCl. However, both primary cultures and passaged cells contain the same amount of PKA, suggesting that the loss of response to cyclic AMP in passaged cells resulted from the loss of PKG. When PKG was restored to deficient passaged cells, these cells regained their ability to respond to agents that increase intracellular cyclic AMP with a decrease in [Ca2⫹]i (81; Fig. 4). These data demonstrate that in vascular smooth muscle cells, agents that elevate cyclic AMP activate both PKG and PKA. The functional predominance of PKG versus PKA is determined by the spatial distribution and degree of compart-

mental accumulation of cyclic AMP (82). Activation of PKG by agents that increase cyclic AMP concentrations may decrease [Ca2⫹]i. In contrast, the activation of PKA may decrease the Ca2⫹ sensitivity of the contractile apparatus by increasing [Ca2⫹]i, activating Ca2⫹ calmodulin kinase II, and phosphorylating MLCK on peptide A, resulting in relaxation. This hypothetical cross talk between cyclic AMP and cyclic GMP in the cardiovascular system in vivo has been questioned by Pfeifer et al. (83), who demonstrated a specific cyclic GMP dependence of defective smooth muscle relaxation in PKG I-deficient mice. In this model, only cyclic GMP-mediated, but not cyclic AMPmediated vascular smooth muscle relaxation was impaired, supporting the suggestion that these two cyclic nucleotides signal via independent pathways in blood vessels in vivo. 1. Regulation of Calcium Influx As described earlier, electromechanical coupling involves the depolarization of vascular smooth muscle cells and activation of voltage-dependent L-type Ca2⫹ channels, with a resultant increased influx of Ca2⫹ and elevations of [Ca2⫹]i. One mechanism by which cyclic nucleotides could induce relaxation is to alter the influx of Ca2⫹ through these channels. Thus, exposure of rabbit coronary arteries to isoproterenol during Ca2⫹ loading in the presence of KCl resulted in the inhibition of

FIGURE 4 Effect of forskolin on [Ca2⫹]i in cultured rat aortic smooth muscle cells. Cells were loaded with Fura 2/AM and incubated with forskolin (FOR) or diluent (0.95% ethanol) for 5 min before the addition of 50 nM arginine vasopressin (AVP). (A) Cells in primary culture and (B and C) cells in passage 20–23. (A and B) Cells were treated with diluent (䊊), 1 애M forskolin (䉱) or 10 애M forskolin (䊐). (C) Treatments were diluent (䊉), 10 애M forskolin (䊐), or 10 애M forskolin in cells in which G kinase (cGK) had been repleted (䉭). Time courses of experiments were 2 min in A and C and 4 min in B. Reprinted from Lincoln and Cornwell (81), with permission, S. Karger AG, Basel.

46. Cyclic Nucleotides and Protein Phosphorylation

contraction subsequently induced by histamine (84). These data suggest that isoproterenol, an agent that increases intracellular cyclic AMP, decreased [Ca2⫹]i by inhibiting the influx of this cation through potentialoperated Ca2⫹ channels. Similarly, isoproterenol, dibutyryl cyclic AMP, and forskolin inhibited 45Ca2⫹ influx and determined the relaxation of rabbit aortic smooth muscle cells depolarized by incubation in high K⫹ (145 mM), low Na⫹ (0 mM) buffer (85). Indeed, there is controversy concerning the effects of the cyclic AMP/PKA pathway on vascular smooth muscle L-type Ca2⫹ channels. Using patch clamp techniques in vascular smooth muscle cells from rabbit or rat portal vein, Liu et al. (86) reported that application of both 8-bromo-cyclic AMP or purified PKA inhibited L-type Ca2⫹ channel activity. In contrast, Ruisz-Velasco et al. (87) found that isoproterenol, 8-bromo-cyclic AMP, forskolin, and the catalytic subunit of PKA enhanced L-type Ca2⫹ channel activity and this effect was reversed by selective PKA blockers. Studies with smooth muscle from different vertebrate vascular tissues support the hypothesis that an increase in intracellular cyclic AMP enhances L-type Ca2⫹ channel currents (87) and that the effect of cyclic AMP is concentration dependent (86,88). One explanation for these conflicting results is that low concentrations of cyclic AMP activate the L-type Ca2⫹ channel by PKA-mediated phosphorylation, whereas higher concentrations inhibit the channel via crossover activation of PKG, and that both pathways operate in physiological conditions to regulate L-type Ca2⫹-channel current, with the net effect depending on species, tissue, cyclic nucleotide levels, and phosphorylation state of PKA and PKG (88). There is general consensus that cyclic GMP activates PKG and inhibits Ca2⫹ influx into vascular smooth muscle through L-type Ca2⫹ channels (88). NO-releasing agents SNP, GTN, and S-nitroso-N-acetylpenicillamine (SNAP) have been reported to decrease L-type Ca2⫹ channel activity in smooth muscle cells from rat mesenteric artery (87) and human coronary artery (89). Previous studies in blood vessels contracted with agonists or K⫹ demonstrated that SNP, EDRF, or atrial peptides inhibited Ca2⫹ influx with a greater efficacy on agonistinduced Ca2⫹ transients, suggesting that cyclic GMP is better able to modulate Ca2⫹ influx when changes in the membrane potential are minimal (1). Whereas the just described studies suggest that the inhibition of Ca2⫹ influx by PKG could directly inhibit Ca2⫹ channels, other studies suggest that reduced Ca2⫹ influx by cyclic nucleotides may be secondary to hyperpolarization mediated by opening of K⫹ channels. At least four types of K⫹ channels have been described in vascular smooth muscle, including ATP-sensitive (KATP), delayed rectifier (KDR), Ca2⫹-activated (KCa),

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and inward rectifier K⫹ channels (90). Studies support an involvement of the first three types of K⫹ channels in cyclic nucleotide-mediated vascular relaxation. Indeed, cyclic GMP and/or cyclic AMP affect the activity of one or more of these channels, depending on the source and physiology of the vascular tissue and on the experimental conditions employed. Thus, hyperpolarization and relaxation induced by isoproterenol, forskolin, dibutyryl cyclic AMP, adenosine, and prostanoids were inhibited by the selective blockade of KATP channels with glibenclamide (91), KDR channels with 4-aminopyridine (90), or KCa channels with iberiotoxin, charybtotoxin, or tetraethylammonium ion (90). Moreover, phosphorylation of K⫹ channels by cyclic AMP/PKA activation has been suggested based on (a) an increase of the open probability of KDR (92) and KCa (93) after application of the purified catalytic subunit of PKA; (b) prevention of agonist activation of KDR (94) and KCa (93) by the selective blockade of PKA; and (c) decreased KDR- and KCa-mediated vasodilation by PKA blockers (95). In studies of rabbit portal vein myocytes using inside-out and cell-attached patch clamp techniques, the signal transduction mechanism involving isoproterenol/cyclic AMP/PKA increased the activity of a specific subtype of KDR channel, the slowly inactivating and 4-aminopyridine-sensitive 15-pS KDR channel (92), suggesting the possibility of a subtle regulation of vascular tone by cyclic AMP. Furthermore, due to the cyclic AMP-mediated increase in the sensitivity of KCa to low [Ca2⫹]i, these data emphasize the multifunctional role of cyclic AMP in regulating smooth muscle relaxation by alterations in Ca2⫹ influx. Thus, cyclic AMP may initially produce increases in [Ca2⫹]i by directly activating Ltype Ca2⫹ channels and decrease the sensitivity of the contractile apparatus to [Ca2⫹]i, promoting relaxation. In addition, this cyclic nucleotide activates K⫹ channels both by small increases in [Ca2⫹]i and by direct phosphorylation, resulting in hyperpolarization, decreased Ca2⫹ influx through L-type Ca2⫹ channels, decreased [Ca2⫹]i, and relaxation. With respect to cyclic GMP, although modulation of KATP and KDR activities has been reported with agents that increase intracellular cyclic GMP concentrations in vascular tissues (96), many studies stress the crucial role played by KCa channels. Hyperpolarization of the membrane of several vascular tissues by nitrovasodilators has been reported. It has been shown that the NO/cyclic GMP-mediated relaxation of rabbit middle cerebral artery involved either large or small conductance KCa channels (90). Williams et al. (97) reported that 5⬘-GMP, not cyclic GMP, enhanced the activity of KCa channels. However, cyclic GMP directly modulates KCa channels through PKG-dependent phosphorylation in different vascular tissues (98). Similarly, the 움 subunit of the KCa

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IX. Cardioactive Drugs

channel was phosphorylated by PKG at serine 1072 in a heterologous expression system (99). Indeed, rather complex interactions between different factors are involved in the cyclic nucleotide regulation of K⫹ channels and subsequent vasorelaxation in vivo. Thus, in physiological conditions, the net effect of changes in vascular tissue cyclic nucleotide concentrations depends on, among others, the (a) cellular genotype and phenotype; (b) protein spatial distribution and intracellular compartmentalization of the smooth muscle cell; (c) pH and ionic composition of intra- and extracellular fluids; (d) cellular status of protein phosphorylation; and (e) nature and contents of neurotransmitters, hormones, and autocoids in the tissue environment. This explains why experimental models are only approximations of complex in vivo processes, which, due to different methods and materials employed, can lead to confusion in understanding physiological mechanisms. For example, the hypercapnia-induced vasodilating response of rat cerebral arterioles converted from the normally K⫹ channel-independent to a K⫹ channeldependent process in the presence of NO synthase inhibition and cyclic GMP replacement; the authors proposed a reevaluation of the permissive role for cyclic GMP in vasorelaxation mediated by K⫹ channels (100). A rational approach to cyclic nucleotide functions should include characterizing the integration of the various processes in which they play a role. Jaggar et al. (101) described a new mechanism for cyclic nucleotidemediated dilation based on previous findings that the frequency of both ‘‘Ca2⫹ sparks’’ (unitary Ca2⫹ release events) from ryanodine-sensitive Ca2⫹ release (RyR) channels in the sarcoplasmic reticulum and spontaneous transient outward currents (STOCs) from plasma membrane KCa channels was regulated by cyclic GMP/PKG activity in cerebral and coronary vascular tissues. According to this model, in vascular smooth muscle cells the L-type Ca2⫹ channel, the RyR channel, and the KCa channel appear to function as a coupled unit with a negative feedback relationship regulating membrane potential and arterial tone. An increase in sarcoplasmic reticulum [Ca2⫹] and/or in [Ca2⫹]i through L-type Ca2⫹ channels activates RyR channels and KCa channels, increasing ‘‘Ca2⫹ sparks’’ and ‘‘STOCs’’ frequency. The increase in ‘‘Ca2⫹ sparks’’ frequency further activates plasmalemmal KCa channels, leading to membrane hyperpolarization, a reduction in the open probability of L-type Ca2⫹ channels, a reduction in the [Ca2⫹]i, and vasodilation. Cyclic nucleotides act on this circuit via their respective kinases in two ways: directly, increasing the ‘‘STOCs’’ amplitude by phosphorylation of the plasmalemmal KCa channel, and indirectly, determining ‘‘Ca2⫹ sparks’’ elevation in frequency, through activation of sarcoplasmic reticulum Ca2⫹ sequestration and,

possibly, the RyR channel, with subsequent increase in the ‘‘STOCs’’ frequency (101). In summary, evidence shows that both cyclic AMP and cyclic GMP can modulate K⫹ channel activity in vascular smooth muscle. Hyperpolarization resulting from K⫹ channel opening would limit Ca2⫹ influx through plasma membrane Ca2⫹ channels. An action of cyclic nucleotides on K⫹ channels would explain some of the differences observed in agonist and K⫹-contracted tissues, as Ca2⫹ entry through L-type Ca2⫹ channels in depolarized cells would not be expected to be sensitive to small changes in the membrane potential resulting from K⫹ channel activation. 2. Regulation of Calcium Efflux Calcium efflux appears to be mediated by two different mechanisms in vascular smooth muscle cells. One mechanism involves the active transport of Ca2⫹ out of cells by a plasma membrane Ca2⫹-pumping ATPase. The other mechanism by which Ca2⫹ is extruded from smooth muscle cells is a plasmalemmal Na⫹ /Ca2⫹ exchanger that may be coupled to other Na⫹ –K⫹ ATPase or K⫹ channel activity. Thus, activation of Na⫹ –K⫹ ATPase would decrease [Na⫹]i, which in turn would increase the driving force for Ca2⫹ extrusion by the Na⫹ / Ca2⫹ exchanger. The Na⫹ /Ca2⫹ exchanger in vascular smooth muscle appears to be electrogenic and sensitive to changes in membrane potential. Membrane hyperpolarization by either K⫹ channel activation or increased Na⫹ –K⫹ ATPase activity also increases Ca2⫹ extrusion by this exchanger. The quantitative contribution of these different mechanisms to the regulation of Ca2⫹ efflux from vascular smooth muscle cells has not been completely defined. Studies in a variety of systems, including giant squid axons and vascular smooth muscle cells, suggest that the Ca2⫹-pumping ATPase has a higher affinity but lower capacity for Ca2⫹ and probably contributes significantly to maintenance of [Ca2⫹]i at low resting levels of this cation. In contrast, the Na⫹ /Ca2⫹ exchanger appears to have a lower affinity but a higher capacity for Ca2⫹ and probably contributes significantly to the extrusion of this cation at high [Ca2⫹]i generated during contraction (102). However, according to the superficial buffer barrier hypothesis, under resting conditions, vectoral transport of Ca2⫹ from the sarcoplasmic reticulum into the narrow cytoplasmic space between the sarcoplasmic reticulum and the plasma membrane could elevate the local Ca2⫹ concentration to a level that would be amenable to Na⫹ /Ca2⫹ exchange. Earlier data suggested a role for cyclic AMP in regulating the activity of the Ca2⫹-pumping ATPase and relaxation in vascular smooth muscle. Thus, incubation

46. Cyclic Nucleotides and Protein Phosphorylation

of inside-out plasma membrane vesicles purified from rat mesenteric arteries or porcine aorta with PKA purified from the same tissue stimulated the uptake of Ca2⫹ into these vesicles (103). However, more recent studies have demonstrated that neither forskolin nor cyclic AMP analogues evoke significant increases in the efflux of Ca2⫹ by the Ca2⫹-pumping ATPase from cultured rat aortic smooth muscle cells (102). Furthermore, there was no significant phosphorylation of the Ca2⫹-pumping ATPase purified from bovine aortic smooth muscle when this protein was incubated in the presence of ATP and the catalytic subunit of PKA (102). These data suggest that cyclic AMP and PKA do not play significant roles in regulating [Ca2⫹]i by altering the activity of the Ca2⫹-pumping ATPase. In contrast, considerable evidence suggests that cyclic GMP regulates Ca2⫹ efflux by activation of the plasma membrane Ca2⫹-pumping ATPase (17,18,20,102,103). Although a number of biochemical studies have shown that the Ca2⫹-pumping ATPase itself is not a substrate for PKG (see earlier discussion), phosphorylation of G0 by PKG appears to mediate the activation of the Ca2⫹-pumping ATPase (20). In cultured rat aortic smooth muscle cells, SNP, ANP, and 8-bromo-cyclic GMP all increased the component of 45Ca2⫹ efflux that was independent of extracellular Na⫹ (i.e., efflux mediated by the Ca2⫹-pumping ATPase rather than the Na⫹ /Ca2⫹ exchanger) (102). Furthermore, this effect on Ca2⫹ efflux was especially evident at lower [Ca2⫹]i (0.1 애M), suggesting that the cyclic GMP regulation of the Ca2⫹-pumping ATPase could have a significant effect on vascular smooth muscle tone at the [Ca2⫹]i associated with force maintenance (i.e., the lower levels of [Ca2⫹]i seen in the ‘‘latch’’ state). Membrane hyperpolarization resulting from the cyclic nucleotide-mediated activation of K⫹ channels (see earlier discussion) would be expected to increase Ca2⫹ efflux by Na⫹ –Ca2⫹ exchange. A stimulatory effect of cyclic nucleotides on the plasma membrane Na⫹ –K⫹ ATPase would also serve to increase Ca2⫹ extrusion by the exchanger, as this would decrease [Na⫹]i and hyperpolarize the plasma membrane, both of which would increase the driving force of Na⫹ entry. PKA increased Na⫹ –K⫹ ATPase activity and Ca2⫹ uptake in inside-out plasma membrane vesicles prepared from rat aorta (104). The uptake of Ca2⫹ was blocked by ouabain, an inhibitor of Na⫹ –K⫹ ATPase, suggesting that the Na⫹ /Ca2⫹ exchange mechanism coupled to the ATPase was mediating Ca2⫹ uptake. More recently, analogues of cyclic AMP were demonstrated to activate the Na⫹ –K⫹ ATPase in smooth muscle membranes and decrease the [Na⫹]i in vascular smooth muscle cells (102). The reports mentioned previosly of stimulation of the Na⫹ –K⫹ ATPase by cyclic AMP are, however, difficult to recon-

823

cile with the reported inhibition of Na⫹ –K⫹ ATPase activity following phosphorylation of the catalytic subunit of the pump by PKA (105). With respect to cyclic GMP-mediated activation of the Na⫹ –K⫹ ATPase, exposure of rat aortic strips to ouabain or K⫹-free media inhibited relaxation induced by SNP, EDRF, or 8-bromo-cyclic GMP (106). Moreover, in smooth muscle cells isolated from canine pulmonary artery, cyclic GMP activated Na⫹ –K⫹ ATPase through PKG (107). However, in cultured primary rat aortic smooth muscle cells, 8-bromo-cyclic GMP had no effect on [Na⫹]i, in contrast to the 30% reduction in [Na⫹]i caused by dibutyryl- or 8-bromo-cyclic AMP (102). In a subsequent study (108), these investigators demonstrated that ANP or 8-bromo-cyclic GMP stimulated the Na⫹ –Ca2⫹ exchanger independent of an effect on membrane potential or Na⫹ –K⫹ ATPase activity. Na⫹ –Ca2⫹ exchange activity was assessed by the [Na⫹] dependent 45Ca2⫹ efflux from cells under conditions where the Ca2⫹-pumping ATPase was inhibited (pH 8.8 and 20 mM Mg2⫹). This 45Ca2⫹ efflux was augmented by the exposure of cells to ANP or 8-bromo-cyclic GMP. Furthermore, 8-bromo-cyclic GMP had no effect on [Na⫹]i, intracellular pH, or the membrane potential, suggesting that the observed effect on the Na⫹ –Ca2⫹ exchanger was not secondary to alteration of these parameters. Data just described suggest that at low [Ca2⫹]i, the main sarcolemmal extrusion mechanism for maintaining Ca2⫹ is the high-affinity, low-capacity Ca2⫹-pumping ATPase selectively regulated by cyclic GMP. At higher concentrations of [Ca2⫹]i, e.g., those produced by contractile stimuli, the Na⫹ –Ca2⫹ exchanger makes a significantly greater contribution to Ca2⫹ extrusion and can be regulated by both cyclic GMP and cyclic AMP. 3. Regulation of Calcium Sequestration Cyclic nucleotides can decrease [Ca2⫹]i by sequestering this ion in intracellular storage sites. One mechanism by which this occurs is the increased transport of Ca2⫹ into the sarcoplasmic reticulum. Cyclic nucleotidemediated increases in Ca2⫹ sequestration appear to be due to PKA- and/or PKG-dependent phosphorylation of phospholamban. Both cyclic GMP and cyclic AMP increase the rate of phosphorylation of phospholamban in rat cardiomyocytes in culture (109). A role for cyclic AMP in this process was suggested by studies of the amount of Ca2⫹ stored in intracellular sites in rabbit ear and coronary arteries and guinea pig mesenteric arteries (84). Calcium-depleted vessels were loaded with this cation by exposure to KCl and the amount was incorporated into intracellular sites quantified by assessing the amplitude of contraction induced by histamine. Expo-

824

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sure of vessels to isoproterenol during Ca2⫹ loading increased the amplitude of contraction produced by histamine. These data suggested that isoproterenol increased the sequestration of Ca2⫹ into intracellular sites during the loading process. Similar conclusions were reached in studies of the effect of dibutyryl-cyclic AMP on net 45 Ca uptake during Ca2⫹ loading (85). Findings indicate that Ser 16 of phospholamban is a unique phosphorylation site for PKA in vitro and that, in perfused rat hearts, cyclic AMP-elevating agents accelerate relaxation via PKA-dependent phosphorylation of phospholamban (110). PKA phosphorylation of phospholamban increased Ca2⫹ sequestration by increasing both the Ca2⫹ sensitivity and the maximum velocity of net Ca2⫹ uptake into the sarcoplasmic reticulum (111). Moreover, in vascular smooth muscle relaxation, cyclic AMP could also act through PKG rather than PKA, as discussed earlier. Studies utilizing both intact and skinned blood vessel preparations have provided evidence for Ca2⫹ sequestration by agents that increase cyclic GMP. The uptake of 45Ca into saponin-skinned primary rat aortic smooth muscle cells in culture was increased by cyclic GMP (112). In intact blood vessels or vascular smooth muscle cells, SNP, 8-bromo-cyclic GMP, and ANP increased Ca2⫹ sequestration subsequent to the elevation of intracellular cyclic GMP (1, 113). Activation of the cyclic GMP/PKG pathway enhanced Ca2⫹ sequestration in the sarcoplasmic reticulum through a Ca2⫹-ATPase (114). Finally, the abundance of phospholamban in smooth muscle varies with the source of vascular tissue, with significant amounts found in dog, rabbit, and rat aorta and in bovine pulmonary artery and low amounts in porcine aorta and coronary artery. Thus, the relative contribution of phospholamban phosphorylation by PKG to lowering [Ca2⫹]i would also be expected to vary between vascular tissues. Indeed, activation of PKG has no effect on Ca2⫹ uptake into sarcoplasmic reticulumenriched fractions of porcine aorta (103), whereas Ca2⫹ uptake is stimulated by PKG in those fractions from bovine pulmonary artery (27). 4. Regulation of Calcium Mobilization In addition to their effects on lowering [Ca2⫹]i, there is substantial evidence that cyclic nucleotides inhibit Ca2⫹ mobilization. In porcine coronary arteries, agents that increase cyclic AMP inhibited U46619-mediated Ca2⫹ release from the sarcoplasmic reticulum (115). Similarly, agonist-induced increases in [Ca2⫹]i mediated by the release of Ca2⫹ from intracellular stores are attenuated by a number of agents that increase cyclic GMP. Cyclic nucleotides could alter Ca2⫹ release from the sarcoplasmic reticulum by inhibiting either IP3 signaling or ryanodine-sensitive receptors. However, Ca2⫹ release

from intracellular stores induced by caffeine (an activator of ryanodine receptors) was not affected by SNP and EDRF in rat aorta (116) nor by agents that increase cyclic AMP in porcine coronary arteries (115), suggesting that the inhibition of ryanodine receptor Ca2⫹ release may not occur. Moreover, while in rat caudal artery 8-bromo-cyclic GMP regulated Ca2⫹ stores separately from ryanodine-sensitive sites (117), in other vascular districts, cyclic nucleotides increase ‘‘Ca2⫹ sparks’’ frequency via ryanodine receptor channels, as discussed earlier. In contrast, several investigators have demonstrated that cyclic GMP inhibits IP3 signaling by decreasing the amount of IP3 formed during agonist stimulation or blocking the effects of IP3 on the sarcoplasmic reticulum, with subsequent attenuation of Ca2⫹ release. Attenuated Ca2⫹ mobilization due to PKG-mediated phosphorylation of the smooth muscle type I IP3 receptor is supported by the findings that the PKG substrate, G0 protein, closely resembles the IP3 receptor (22) and that phosphorylation of IP3 receptors occurred in primary cultures of rat aortic myoctes and in intact rat aorta in response to the elevation of cyclic nucleotides through PKG (23). However, in skinned vascular smooth muscle cells, Ca2⫹ release from fully loaded Ca2⫹ stores by IP3 was not altered by cyclic GMP (112), and, in Chinese hamster ovary (CHO) cells transfected with PKG type I움, the IP3 receptor was not involved in the regulation of the cytosolic Ca2⫹ level by cyclic GMP (118). With respect to cyclic GMP inhibition of phosphatidylinositide turnover, SNP, EDRF, 8-bromo-cyclic GMP, GTN, and ANP inhibited agonist-induced increases in IP3 formation (119). In subsequent studies, the decreased responsiveness of vascular smooth muscle cells from normotensive rats to IP3 mobilization by angiotensin II, when compared to cells from spontaneously hypertensive rats, was ascribed to an inhibitory influence of cyclic GMP (120). Similarly, cyclic GMP suppressed thrombin-induced IP3 production in CHO cells transfected with PKG I움 (121). The mechanism of attenuated phosphatidylinositide hydrolysis was addressed by Hirata et al. (119), who found that cyclic GMP inhibited both arginine vasopressin-induced GTPase activation and GTP웂S-induced formation of inositol phosphates in homogenates of bovine aortic smooth muscle cells. They proposed that the action of cyclic GMP involved the inhibition of agonistinduced G-protein activation and of coupling between the G-protein and phospholipase C. Of interest is the report of CHO cells transfected with PKG I움 showing that a Gi protein subtype was a substrate for PKG and that 8-bromo-cyclic GMP blocked IP3 production and [Ca2⫹]i increase via a pertussis toxin-sensitive Gi protein (118). However, in other studies, angiotensin II-, phen-

46. Cyclic Nucleotides and Protein Phosphorylation

ylephrine-, or endothelin 1-induced increases in inositol phosphates were not affected by ANP (122) and 8bromo-cyclic GMP (117), and pertussis toxin-sensitive and -insensitive G-proteins were not found to be substrates for PKG in vitro (122).

9.

10.

VI. SUMMARY Data presented herein suggest that cyclic nucleotides can mediate vascular smooth muscle relaxation by a variety of mechanisms involving alterations in intracellular concentrations of Ca2⫹ or the sensitivity of the contractile apparatus to that cation. Which of these mechanisms predominates varies with the experimental conditions, the type of blood vessel studied, and the source of blood vessels. Thus, it is difficult to determine which of these mechanisms participate, and which predominates, in vascular smooth muscle relaxation in vivo. However, it is clear that a complex relationship exists between the regulation of intracellular concentrations of cyclic nucleotides and Ca2⫹ and vascular smooth muscle contractility. This complexity is reflected in the precision with which the contractile tone of vascular smooth muscle regulates total peripheral resistance and regional blood flow to meet physiological requirements. The relative roles of these mechanisms in maintaining vascular tone and their contributions to vascular relaxation under various physiological and pathophysiological conditions will be elucidated as further investigations in this important area are conducted.

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protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta. J. Biol. Chem. 271, 21933–21938. Huggins, J. P., Cook, E. A., Piggott, J. R., Mattinsley, T. J., and England, P. J. (1989). Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle. Biochem. J. 260, 829–835. Sarcevic, B., Brookes, V., Martin, T. J., Kemp, B. E., and Robinson, P. J. (1989). Atrial natriuretic peptide-dependent phosphorylation of smooth muscle cell particulate fraction proteins is mediated by cGMP-dependent protein kinase. J. Biol. Chem. 264, 20648–20654. Cornwell, T. L., Pryzwansky, K. B., Wyatt, T. A., and Lincoln, T. M. (1991). Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol. Pharmacol. 40, 923–931. Raeymaekers, L., Eggermont, J. A., Wuytack, F., and Casteels, R. (1990). Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2⫹ pump of bovine pulmonary artery. Cell Calcium 11, 261–268. Rapoport, R. M., Draznin, M. B., and Murad, F. (1983). Endothelium-dependent relaxation in rat thoracic aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature (London) 306, 174–176. Li, H., Liu, J. P., and Robinson, P. J. (1996). Multiple substrates for cGMP-dependent protein kinase from bovine aortic smooth muscle: Purification of P132. J. Vasc. Res. 33, 99–110. Murray, K. J. (1990). Cyclic AMP and mechanisms of vasodilation. Pharmacal. Ther. 47, 329–345. Sunahara, R. K., Dessauer, C. W., and Gilman, A. G. (1996). Complexity and diversity of mammalian adenylyl cyclases. Annu. Rev. Pharmacol. Toxicol. 36, 461–480. Tesmer, J. J. G., and Sprang, S. R. (1998). The structure, catalytic mechanism and regulation of adenylyl cyclase. Curr. Opin. Struct. Biol. 8, 713–719. Hurley, J. H. (1999). Structure, mechanism and regulation of mammalian adenylyl cyclase. J. Biol. Chem. 274, 7599–7602. Dessauer, C. W., Tesmer, J. J. G., Sprang, S. R., and Gilman, A. G. (1999). The interactions of adenylate cyclases with p-site inhibitors. TIPS 20, 205–210. Simonds, W. F. (1999). G-protein regulation of adenylate cyclase. TIPS 20, 66–73. Mons, N., Yoshimura, M., and Cooper, D. M. (1993). Discrete expression of Ca2⫹ /calmodulin-sensitive and Ca2⫹-insensitive adenylyl cyclases in rat brain. Synapse 14, 51–59. Mons, N., Guillou, J. L., and Jaffard, R. (1999). The role of Ca2⫹ / calmodulin-stimulable adenylyl cyclases as molecular coincidence detectors in memory formation. Cell. Mol. Life Sci. 55, 525–533. Boonen, H. C., Struyker-Boudier, H. A., and De Mey, J. G. (1990). Effects of tertatolol on the responsiveness of isolated femoral, mesenteric, and renal resistance arteries to adrenergic stimuli. J. Cardiovasc. Pharmacol. 15, 124–129. Heesen, B. J., and De Mey, J. G. (1990). Effects of cyclic AMPaffecting agents on contractile reactivity of isolated mesenteric and renal resistance arteries of the rat. Br. J. Pharmacol, 101, 859–864. Tamaki, T., Hasui, K., Shoji, T., Aki, Y., Kiyomoto, H., Iwao, H., and Abe, Y. (1991). Forskolin preferentially dilates the afferent arteriole in the canine kidney. Jpn. J. Pharmacol. 55, 161–164. Colledge, M., and Scott, J. D. (1999). AKAPs: From structure to function. Trends Cell. Biol. 9, 216–221. Silver, P. J., Schmidt-Silver, C., and DiSalvo, J. (1982). Betaadrenergic relaxation and c-AMP kinase activation in coronary areterial smooth muscle. Am. J. Physiol. 242, H177–H184.

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47 K⫹ Channel Openers ARSHAD JAHANGIR,* WIN-KUANG SHEN,* and ANDRE TERZIC*,† †

*Division of Cardiovascular Diseases and Department of Internal Medicine, and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota 55905

I. INTRODUCTION

The primary endogenous ligand of KATP channels is intracellular ATP, which blocks channel activity, whereas intracellular ADP serves as the major channel activator (Noma, 1983). Thus, in normal cells, KATP channels are believed to be silent and open under conditions of metabolic stress with changes in the ATP/ADP ratio (Dzeja and Terzic, 1998). Kir6.2, in conjunction with the regulatory SUR subunit, is responsible for ATP-induced channel inhibition, whereas SUR, which possesses two cytosolic nucleotide-binding domains, governs ADP-induced channel activation (Fig. 2; Nichols et al., 1996; Gribble et al., 1997; Ueda et al., 1997; Tucker et al., 1998; D’hahan et al., 1999ab). Channel transition from the closed ATP-liganded state to the open ADP-liganded state could be catalyzed by an ATPase activity inherent to the channel complex (Dzeja et al., 1999), as well as by phosphotransfer enzymes, such as adenylate kinase (Elvir-Mairena et al., 1996; Dzeja and Terzic, 1998; Carrasco et al., 1999). Moreover, intracellular H⫹ (Vivaudou and Forestier, 1995), lactate (Keung and Li, 1991), and phospholipids (Baukrowitz et al., 1998; Shyng and Nichols, 1998) are all believed to contribute to channel regulation and promote KATP channel opening by decreasing the channel sensitivity to ATP. In conjunction or separately from adenine nucleotide-mediated gating, intracellular diadenosine polyphosphates regulate cardiac KATP channel opening as well (Jovanovic et al., 1997). Diadenosine polyphosphates are normally elevated in heart muscle, but do drop in response to ischemia, which is associated with channel opening (Jovanovic et al., 1998c). In addition, neurohormones, such as adenosine, acetylcholine, calcitonin-gene related peptide, and endothelium-dependent

Potassium channel openers target adenosine triphosphate-sensitive potassium (KATP) channels. These channels couple cellular metabolism with membrane excitability and have been implicated in the regulation of vascular tone and cardioprotection under metabolic stress. Potassium channel openers have a unique therapeutic potential as combined cardioprotective and vasodilatory agents (Fig. 1).

II. KATP CHANNELS KATP channels are distributed in high density in the sarcolemma of cardiomyocytes and vascular smooth muscle cells (Noma, 1983; Nichols and Lederer, 1991; Terzic et al., 1995; Quayle et al., 1997; Aguilar-Bryan and Bryan, 1999; Seino, 1999). The cardiac KATP channel is an octameric complex (Fig. 2). It is composed of the pore-forming inwardly rectifying K⫹ channel, Kir6.2, and the regulatory sulfonylurea-receptor subunit SUR2A, a member of the ATP-binding cassette (ABC) protein family (Inagaki et al., 1995, 1996; Babenko et al., 1998; Okuyama et al., 1998; Lorenz and Terzic, 1999). Vascular KATP channels are believed to be formed by association of the SUR2B isoform with either Kir6.2 itself and/or Kir6.1, a homologous inwardly rectifying channel (Isomoto et al., 1996; Yamada et al., 1997; Repunte et al., 1999). A related channel of unknown structure has also been recognized in the inner membrane of mitochondria (Inoue et al., 1991; Paucek et al., 1992), underscoring the role of KATP channels in signaling networks that transduce metabolic events.

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FIGURE 1 Therapeutic potential of potassium channel openers in the cardiovascular system. KCO, potassium channel openers.

hyperpolarizing factor, regulate KATP channel activity in cardiomyocytes and vasculature (Standen et al., 1989; Daut et al., 1990; Nelson et al., 1990; Terzic et al., 1994).

III. POTASSIUM CHANNEL OPENERS Sulfonylurea drugs are established KATP channel blockers and are used regularly in clinical practice to promote insulin secretion from the pancreas (Ashcroft and Ashcroft, 1992; Brady and Terzic, 1998). Here, emphasis will be placed on potassium channel openers, which activate KATP channels. This chemically diverse

group of agents (Fig. 3) include benzopyrans (e.g., levcromakalim), thioformamides (e.g., aprikalim), nicotinamides (e.g., nicorandil), cyanoguanidines (e.g., pinacidil), pyrimidines (e.g., minoxidil), and benzothiadiazines (e.g., diazoxide). Whereas the common pharmacophore responsible for KATP channel activation remains to be identified, specific binding sites for potassium channel openers have been identified on the SUR subunit of the KATP channel in both vascular smooth muscle (Bray and Quast, 1992) and cardiac muscle (Atwal et al., 1998; Lo¨ffler-Walz and Quast, 1998). Activation of KATP channels by openers is accompanied by an apparent reduction in the sensitivity to ATP inhibition (Thuringer and Escande, 1989; Terzic et al., 1995; Forestier et al., 1996). Potassium channel openers preferentially activate K⫹ channels in smooth muscle cells, leading to membrane hyperpolarization. Thereby, the primary pharmacodynamic effect of potassium channel openers is the relaxation of vascular smooth muscle, resulting in vasodilatation (Quayle et al., 1997; Yokoshiki et al., 1998; Fig. 4). Potassium channel openers also open KATP channels in the myocardium (Fig. 5). The effect is more pronounced during ischemia (Escande et al., 1988; Weiss and Venkatesh, 1993). Opening of cardiac KATP channels shortens the action potential duration, with diminished time available for Ca2⫹ influx. This can reduce the force of

FIGURE 2 The KATP channel complex consists of Kir6.x and SUR subunits. The cardiac KATP channel is composed of Kir6.2 and SUR2A isoforms, whereas the vascular KATP channel may consist of related Kir6.1 and SUR2B subunits. The pore-forming Kir subunit serves as a K⫹ channel, whereas the regulatory SUR subunit serves as a target of drug action. It is believed that four Kir subunits associate with four SUR subunits to generate functional KATP channels. NBF-1 and -2 are nucleotide-binding domains 1 and 2 (with Walker A and B consensus motif) implicated in the channel regulation by intracellular nucleotides. N and C represent the respective amino and carboxy terminus of constitutive channel subunits.

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contraction, but also preserves energy expenditure associated with the maintenance of cellular Ca2⫹ homeostasis (Nichols and Lederer, 1991; Terzic et al., 1995). Potassium channel openers improve the recovery of myocardial function following metabolic insult, decrease loss of adenine nucleotides, and diminish infarct size (Gross and Fryer, 1999). Such a protective effect is independent from changes in collateral blood flow, suggesting a direct cytoprotective action on the cardiomyocyte. In fact, overexpression of cardiac KATP channel genes, Kir6.2 and SUR2A, in conjunction with a potassium channel opener enhances cytoprotection and maintains cellular Ca2⫹ homeostasis under stress (Jovanovic et al., 1998ab, 1999). Although the cardioprotective effect of potassium channel openers has been ascribed to the activation of sarcolemmal KATP channels, newer evidence indicates that the mitochondrial KATP (mitoKATP) channel also contributes (Garlid et al., 1997; Liu et al., 1998; Gross and Fryer, 1999; Holmuhamedov et al., 1999). In fact, potassium channel openers promote K⫹ influx into mitochondria through the opening of mitoKATP channels (Paucek et al., 1992; Garlid et al., 1996). In so doing, openers depolarize the mitochondrial membrane and increase the rate of mitochondrial respiration (Holmuhamedov et al., 1998). Activation of mitoKATP channels by potassium channel openers has been associated with an increase in the number of surviving cardiac cells following ischemia, a delay in the onset of contracture, and an improved postischemic recovery of heart muscle (Garlid et al., 1997; Liu et al., 1998). The mechanism responsible for such a cardioprotective effect of potassium channel openers, at the mitochondrial level, is unknown but may include reduction in mitochondrial Ca2⫹ overload and maintenance of mitochondrial Ca2⫹ homeostasis under stress (Holmuhamedov et al., 1999).

IV. THERAPEUTIC POTENTIAL OF POTASSIUM CHANNEL OPENERS IN CARDIOVASCULAR MEDICINE Due to combined cardioprotective and vasodilatory properties, potassium channel openers are considered in a number of cardiac conditions. These include protecting the myocardium under cardiopulmonary bypass and preserving donor transplant heart (Menasche, 1997; Hebbar et al., 1998), treating ischemic heart disease

FIGURE 3 Classification of potassium channel openers based on chemical structure. The structures of common potassium channel openers—cromakalim, aprikalim, nicorandil, pinacidil, minoxidil, diazoxide, BMS 182264, and BMS 180448—are provided.

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FIGURE 4 Potassium channel openers (KCO) in vascular smooth muscle. KCO promote membrane hyperpolarization, decrease intracellular Ca2⫹, and cause smooth muscle relaxation, resulting in vasodilation.

FIGURE 5 Potassium channel openers (KCO) modulate sarcolemmal and mitochondrial KATP channels in cardiac myocytes. KCO activate sarcolemmal KATP channels, leading to K⫹ efflux, shortening of the action potential duration (APD), and a decrease in Ca2⫹ influx. KATP channels opening in ischemia occur through a change in the ADP/ATP ratio at the channel site. Adenosine (Ado) has been recognized as a channel activator through a direct action via a guanosine triphosphatebinding protein (G). KCO also activate a KATP-related channel in mitochondria. Activation of the mitochondrial KATP channel has been implicated in mitochondrial membrane depolarization and in prevention of mitochondrial Ca2⫹ overload. Cardioprotection could, in principle, be the result of activation of sarcolemmal and/or mitochondrial KATP channels.

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(Gross and Fryer, 1999), hypertension (Anderson, 1992), peripheral vascular disease (Cook and Chapman, 1993), and arrhythmia related to abnormal repolarization (Haverkamp et al., 1995).

troepiploic arteries) used as coronary artery bypass grafts and could be useful in preventing spasm of bypass grafts (Akar et al., 1997).

VIII. SYSTEMIC HYPERTENSION

V. MYOCARDIAL PRESERVATION IN CARDIAC SURGERY Openers of KATP channels may serve as adjuncts or main components in cardioplegic solutions. In several models of surgical ischemia and cardiopulmonary bypass, potassium channel openers, including nicorandil, aprikalim, and pinacidil, provide greater cardioprotection than conventional cardioplegia (Lopez et al., 1996; Menasche, 1997; Hebbar et al., 1998). In particular, when potassium channel openers are given for a brief period, before hypothermic cardioplegic arrest, they preserve ventricular contractile function after rewarming. In this regard, it is proposed that a brief period of potassium channel opener treatment may provide a strategy for myopreservation during prolonged cardioplegic arrest in the setting of cardiac operation (Hebbar et al., 1998).

VI. ISCHEMIC HEART DISEASE

Diazoxide and minoxidil are used for the treatment of severe resistant hypertension and hypertensive emergencies. Due to potential cardioprotective or antiischemic effects and beneficial action on plasma lipids and/or bronchial smooth muscle, openers as antihypertensive agents are an alternative over existing medication.

IX. PULMONARY HYPERTENSION Potassium channel openers have been shown to inhibit hypoxic pulmonary vasoconstriction, which might be of benefit in pulmonary hypertension (Dumas et al., 1996). Potassium channel openers decrease mean pulmonary artery pressure and pulmonary resistance in models of pulmonary hypertension (Oka et al., 1993). Also, a beneficial effect in decreasing pulmonary vascular resistance and reperfusion injury in lung allotransplantation has been reported (Yamashita et al., 1996). Therefore, potassium channel openers appear to have promise in managing conditions associated with increased pulmonary vascular resistance.

Potassium channel openers are suitable in patients with coronary artery disease (Frydman, 1992) and can be used in the management of angina pectoris (Patel et al., 1999; Frampton et al., 1992). Intravenous nicorandil produces coronary dilatation without significantly altering heart rate, blood pressure, and cardiac output (Frydman, 1992; Frampton et al., 1992). In patients with ischemic heart disease, nicorandil attenuates rest and effort angina, prolongs the duration of exercise and the time to onset of angina, ischemic ST-T changes, regional wall motion abnormalities, and perfusion in infarctrelated areas (Ito et al., 1999; Patel et al., 1999). In conjuction with coronary angioplasty, nicorandil preserves microvascular integrity and myocardial viability in patients with acute myocardial infarction (Ito et al., 1999).

Potassium channel openers do not divert blood to nonischemic regions and improve blood flow and oxygen availability to the chronically ischemic muscle. This restores the high-energy phosphate content of a cell and improves muscle performance during ischemia, as observed in models of occlusive arterial disease (Cook and Chapman, 1993). Thus, potassium channel openers may be advantageous in severe peripheral vascular disease.

VII. CORONARY ARTERY SPASM

XI. ARRHYTHMIA

Nicorandil, with potent vasospasmolytic activity, attenuates episodes of variant angina, suppresses ST segment changes, and improves perfusion defects. This suggests effectiveness in relieving vasospasm and improving microcirculatory impairment in patients with vasospastic angina (Kaski, 1995). Levcromakalim and aprikalim also relax conduit arteries (internal mammary and gas-

Potassium channel openers may be antiarrhythmic in certain conditions associated with abnormal repolarization, such as early and delayed afterdepolarization (Haverkamp et al., 1995; Fish et al., 1990). In models of prolonged QT and torsades de pointes, pinacidil and nicorandil are effective in suppressing polymorphic ventricular tachycardia (Fish et al., 1990; Carlsson et al.,

X. PERIPHERAL VASCULAR DISEASE

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1992). In congenital long QT syndrome, nicorandil abolished early afterdepolarization, improved repolarization abnormalities, and prevented recurrence of syncope (Shimizu et al., 1998). In conditions that predispose cardiac tissue to reentrant arrhythmias, such as increased electrical inhomogeneity during ischemia, potassium channel openers may increase the dispersion of refractoriness by the heterogeneous shortening of action potential duration, facilitating reentrant arrhythmias (Wilde and Janse, 1994). However, the induction of life-threatening arrhythmias has not been documented for any of the potassium channel openers tested clinically, and in patients with acute myocardial infarction, a reduction in malignant ventricular arrhythmia with nicorandil has been reported (Ito et al., 1999).

XII. SUMMARY Modulation of KATP channels is a novel pharmacological principle with significant clinical potential (Terzic, 1999). Extensive experimental studies and limited clinical experience point toward the safety and efficacy of potassium channel openers as a class; however, largescale clinical trials are necessary before these novel ion channel modulators are accepted in clinical medicine. Development of openers exhibiting high selectivity for targeted tissues, and possibly disease-dependent efficacy, is a top priority. With continuous refinement in the pharmacokinetic and pharmacodynamic properties of KATP channel modulators, along with progress in pharmacogenetics and pharmacogenomics, individualized patient therapy is the ultimate goal. As of now, the main goal is elucidating the precise role(s) of KATP channels in specific tissues and organelles. This is a necessary step to fully capitalize on the therapeutic potential of modulating such a unique metabolism-sensing ion channel.

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48 Mode of Action of Antiarrhythmic Drugs AUGUSTUS O. GRANT and VIJAY S. CHAUHAN Duke University Medical Center Durham, North Carolina 27710 and University of Western Ontario, Canada

I. INTRODUCTION

tify a single vulnerable parameter to target in order to successfully treat a patient’s arrhythmia. To add to this complexity, a vulnerable parameter should ideally be modifiable at higher heart rates to effectively treat tachyrrhythmias or extrasystoles with minimal proarrhythmic potential. Despite these uncertainties, identification of a vulnerable parameter provides a rationale for drug selection and a better understanding of drug action.

Cardiac arrhythmias arise from abnormalities of impulse initiation or of conduction. Abnormal automaticity and triggered activity are the principal mechanisms of abnormal impulse initiation (Wit and Rosen, 1981). Reentry is primarily the result of abnormal impulse conduction (Mines, 1914). In order to prevent or terminate each of these mechanisms, a drug must influence one or more critical component that is a basis for the arrhythmia. These electrophysiological components are known as vulnerable parameters and have been identified explicitly in the ‘‘Sicilian gambit’’ (Table I) (Task Force of the Working on Arrhythmias of the European Society of Cardiology, 1991). For example, one mechanism for atrial fibrillation involves multiple small reentry circuits, each with a short excitable gap. The vulnerable parameter is the effective refractory period. Appropriate targets are the K⫹ channels that control repolarization. K⫹ channel blockers such as sotalol can reduce the incidence of atrial fibrillation by prolonging the effective refractory period, which prevents reentry by causing the leading edge of the excitable wavefront to collide with the refractory tail. Thus, by modifying the short excitable gap, which is the vulnerable parameter, the arrhythmia can be terminated. Unfortunately, the mechanism of most arrhythmias is difficult to define or has multiple etiologies. One mechanism may initiate an arrhythmia that is sustained by another mechanism. The arrhythmogenic substrate may not be static in disease states such as myocardial ischemia and infarction, and can change the mechanism of morphologically similar arrhythmias. Consequently, it is often difficult to iden-

Heart Physiology and Pathophysiology, Fourth Edition

II. KINETICS OF DRUG BLOCK Modification of a particular vulnerable parameter is achieved by either blocking specific ionic currents arising from channels or pumps or activating selected ionic currents such as those from potassium channels (Table I). Putative drug-binding sites have been identified from the primary structure of many channels. This discussion focuses on drug blockade, as this is the mode of action of most antiarrhythmics. A given drug (D) will block an ion channel or pump based on the following firstorder binding equation (Schwarz, 1986): k⫺1

R ⫹ D S RD k1

where k1 and k⫺1 are the rate constants for drug binding and unbinding, respectively. The ratio of k⫺1 /k1 is the dissociation constant Kd and corresponds to the drug concentration at which 50% of the channels are blocked by the drug. Fractional occupancy (y) of the channel by the drug can be described by

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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IX. Cardioactive Drugs

TABLE I Vaughan Williams Classification of Antiarrhythmic Drug Action Drug class

Antiarrhythmic action

Prototype

1

Local anesthetic class Na⫹ channel blockade

Quinidine

2

Sympatholytic

Propranolol

3

APD prolongation

Amiodarone

4a

‘‘Centrally acting’’

Dilantin

a

Replaced by Ca2⫹ channel-blocking drugs in a more recent classification.

y ⫽ 1/[1 ⫹ Kd /D]

(1)

With a change in drug concentration, the occupancy of the channel relaxes to a new steady state with time constant, ␶ :

␶ ⫽ 1/(k⫺1 ⫹ k[D])

(2)

The rate of change is faster the higher the drug concentration. The change is slowest when the drug is washed out (␶ ⫽ 1/k⫺1). By plotting the inverse of the time constant as a function of [D], a linear relation is obtained from which the two rate constants can be derived.

III. STATE DEPENDENCE OF DRUG BLOCK Voltage-gated channels exist in two principal sets of conformations, conducting and nonconducting. From Fig. 1, a channel in the resting or closed state (C1) is nonconducting (Carmeliet and Mubagwa, 1998). In response to a threshold voltage stimulus, the channel can pass through multiple closed states (Cn) to a preopen state (A) before opening (O). Alternatively, it can activate directly to the conducting open state (O). Once the applied voltage is removed, the channel will deactivate to its original closed state (C1). Many channels also exhibit a second nonconducting state, the inactivated state. After such channels activate, inactivation (I) occurs in a time-dependent manner, despite the maintenance of the applied potential. For channels that are inactivated by depolarizing potentials, both deactivation and recovery from inactivation to the closed state are accelerated with hyperpolarization. The existence of multiple channel conformations is supported by a substantial body of data. The extent of drug block is influenced by the rate of association and dissociation to a particular channel. Binding and unbinding rates in turn will be dependent on the channel conformation or state.

Modulated and guarded receptor models have been proposed as alternative frameworks in which blockade can be examined (Hille, 1977; Hondeghem and Katzung, 1977; Starmer et al., 1984). Both models assume a single receptor site for drug binding in the channel. In the modulated receptor hypothesis, drug affinity for the receptor is dependent on the state of the channel. In contrast, the guarded receptor hypothesis proposes that drug accessibility to its binding site varies with the channel state, whereas the receptor affinity remains constant. Because of the larger number of free parameters, the modulated receptor model is necessarily more complete. However, the guarded receptor model is computational tractable and has proved useful in estimating relative binding parameters of drugs using simple stimulation protocols. The high-affinity states for the interaction of drugs with most ion channels are occupied at depolarized potentials. As open and inactivated states are occupied at depolarized potentials, these are the primary blocked states. The precise blocked state is difficult to define from action potential and macroscopic current measurements. The overlapping time course of currents in naturally occurring preparations makes the measurement of specific ion currents coded by single genes in heterelogous systems more suitable for analysis. Single channel recordings provide the most direct approach for demonstrating open channel block. In addition to the challenge of the technique, the very nature of certain single channel currents makes the measurements difficult. For example, even at room temperature, the mean open time for the sodium channel is about 1 msec (Nettleton and Wang, 1992; Grant and Starmer, 1987). A drug-induced reduction of open time places the required measurement close to the limit of the resolution of the technique. The usual experimental approach has involved prolongation of the single channel open time by enzymatic, chemical, or mutational reduction of the rate of channel inactivation. Open channel block has been studied most thoroughly for the cardiac Na⫹ channel (Kohlhardt and Fichtner, 1988; Kohlhardt et al., 1989; Grant et al., 1993; Carmeliet et al., 1989; Gingrich et al., 1993; Zamponi et al., 1993; Matsuki et al., 1984; Zamponi et al., 1993; Zamponi and French, 1993). The expression of open channel block depends on the kinetics of association and dissociation of drugs with the channel. It is instructive to relate the changes in single channel kinetics to that of the time course of the macroscopic current. These relationships are outlined in Fig. 2 (Carmeliet and Mubagwa, 1998). A ‘‘very fast on/very fast off’’ open channel blocker (e.g., the quaternay ammonium lidocaine analogue QX314) will dissociate from the channel faster than the recording bandwidth of the amplifier.

48. Antiarrhythmic Drug Action

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FIGURE 1 Interaction of drugs with states of a channel showing activation and inactivation. C, rested closed state; Ca, activated, preopen state; O, activated, open state; I, inactivated state. Inactivation is assumed to proceed only from Cr or O states and is an absorbing state. (Upper left) The drug (D) binds to the O state but cannot bind in the rested state (CrD): the drug is ‘‘trapped’’ at/within the channel and activation is necessary for drug dissociation. Trapping is favored by hyperpolarization in a channel that normally is activated on depolarization. (Lower left) The drug (D) binds to the O state but can unbind from the rested state (CrD 씮 Cr ⫹ D) or from the inactivated state (ID 씮 I ⫹ D): activation is not necessary for drug dissociation. Negative potentials will enhance unbinding or recovery from block in a channel normally activated by depolarization. The same scheme can be applied for a channel that does not inactivate. In this case the inactivated state is absent. (Upper right) The drug binds preferentially to the inactivated state: escape can occur from the inactivated state on hyperpolarization (for a channel that activates on depolarization) or from the rested state. (Lower right) Interaction of drugs with the activated, preopen state. The drug is supposed to escape from the channel in the inactivated or closed rested state but other variants are possible. Reproduced from Carmeliet and Mubagwa (1998), Progr. Biophysics Mol. Biol. 70, p. 7, with permission from Elsevier Science.

A reduction in single channel opening duration is not observed. However, apparent single channel conductance will be reduced and macroscopic current will remain unchanged. In contrast, a ‘‘fast on/fast off’’ drug (e.g., disopyramide) will shorten single channel opening duration and produce multiple reopenings, which will prolong the overall burst duration. Peak whole cell current will be reduced because of more frequent nulls (sweeps with no channel openings). Macroscopic current relaxation will also be slower due to longer single channel burst duration. The rate of activation also is slowed. Finally, a ‘‘fast on/slow off’’ drug will reduce single channel open time duration. However, fewer reopenings will be seen because of the slower drug dissociation that will shorten burst duration. The peak whole cell current is reduced because of more frequent nulls. The inactivating channel shows an acceleration of macroscopic current relaxation (Carmeliet and Mu-

bagwa, 1998). Recovery from block begins at the end of the activating potential when the channel can no longer remain open. Hyperpolarizing potentials will increase the rate of return to the closed state, thereby accelerating unblocking. Deactivation can sometimes cause the drug to be trapped in the channel. By closing the activation gate, the drug cannot dissociate readily from the open state, and recovery from block is slowed. If drug trapping occurs, channel reactivation back to the open state is the only means of completing unblocking. For inactivated state blockers, which preferentially bind to the inactivated state, kinetics are typically ‘‘fast on/slow off.’’ Therefore, single channel and whole cell currents may look similar to open-state blockers with the exception that average open channel duration does not change (Fig. 2) (Carmeliet and Mubagwa, 1998). Block can be enhanced in two ways: (1) increasing the number of channels in the inactivated state by applying

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IX. Cardioactive Drugs

FIGURE 2 Predicted changes on single channel and macroscopic currents for an open-state block. Various kinetics of binding (on)/unbinding (off) of the drug are assumed. Microscopic currents are drawn as inward currents. Macroscopic currents are drawn as inactivating inward currents or noninactivating outward currents. (Uppermost row) Very fast on/off kinetics; the burst-like activity induced by the drug cannot be resolved (due to the limited bandwidth of the recording apparatus) and appears as a decrease in channel amplitude. At the macroscopic level, current amplitude is only slightly decreased and slowed. (Second row) Fast on/off kinetics; the drug effect appears as an induction of burst-like activity without a change in channel amplitude. The duration of a burst is prolonged if the blocked channel cannot inactivate, but can be reduced if the blocked channel inactivates. At the macroscopic level, the amplitude of the current is decreased. Inactivation, if present, can be faster or slower, depending on the possibility for the blocked channel to inactivate or not. (Third row) Fast on/slow off kinetics. Channel block results in a decrease of mean open time. Once blocked, the channel remains longer in the blocked state, from where it either recovers (reopenings occur after longer gaps between openings) or has time to reach the inactivated state (no reopening occurs). At the macroscopic level, inactivation of the current is accelerated for a channel that normally inactivates. For a channel that shows only activation, two possibilities exist depending on the relative rates of block onset and of activation. If the rate of block is much faster than that of activation, the current is simply scaled down (as is the case for fast on/fast off). If the rate of block is slower than the rate of activation, the current is slowed in its onset, attains a maximum, and decreases again, with induction of apparent inactivation. Reproduced from Carmeliet and Mubagwa (1998), Progr. Biophysics Mol. Biol. 70, p. 7, with permission from Elsevier Science.

subthreshold depolarizing pulses or (2) maintaining a given number of channels in the inactivated state for a longer period of time by increasing the duration of suprathreshold depolarizing pulses. The hallmark of inactivation state block is a hyperpolarizing shift in the availability curve. It is the result of stabilization of the inactivated state by blockade according to the modulated receptor model. The guarded receptor model suggests that this apparent shift is the direct consequence of blockade independent of channel state (open state or inactivated state), which leaves fewer channels available to activate. Recovery from block is favored by hyperpolarizing potentials that permit greater recovery of inactivated channel to the closed state where less

drug will bind. Table II summarizes the distinguishing features of open and inactivated state blockers. At therapeutic concentrations, drug affinity for the closed or resting state is quite small (Snyders et al., 1992). In fact, the Kd for drug interaction with the resting channel is in the millimolar range, suggesting that drugs typically dissociate from channels in the resting state. Resting state blockers should be inherently cardiotoxic and will not be discussed further. As with drug-free channel states, drug-bound channels can transition among closed, open, and inactivated states in a time- and voltage-dependent manner. Channel blockade will then vary with the drug affinity of each state. For example, an open channel blocker may

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48. Antiarrhythmic Drug Action

TABLE II Harrison Modification of Class 1 Agentsa Class

Action

Agents

1A

Slows dV/dt of phase O Moderate prolongation of repolarization Prolongs PR, QRS, and QT intervals

Quinidine Procainamide Disopyramide

1B

Limited effects on dV/dt of phase O Shortens repolarization Shortens QT in clinical doses Elevated fibrillation threshold

Lidocaine Mexiletine Ethmozine

1C

Markedly slows dV/dt Little effect on repolarization Markedly prolongs PR and QRS on ECG

Flecainide Propafenone

a

Modified from Harrison (1985).

partially dissociate from its binding site after the channel has inactivated from the open conformation.

IV. ACCESS ROUTES AND SITE FOR BLOCKADE The modulated receptor model suggests two access routes for drug–receptor binding and unbinding (Hille, 1977; Hondeghem and Katzung, 1977). Charged drugs pass directly through the hydrophilic channel pore to reach their receptor on the intracellular mouth of the channel. Neutral drugs may also access their binding site via a hydrophobic pathway through the membrane. Membrane transit is considered to be faster than that through the channel pore. Most drugs are weak acids or bases and will exist as charged and uncharged species at physiological pH, allowing use of both access pathways. However, drug access can change based on its pKa and the ambient pH of the cell under normal and pathological conditions, such as ischemia or inflammation. Generally, lipid-soluble drugs and those with lower pKa tend to associate and dissociate faster (Broughton et al., 1984). In addition to drug solubility, drug dissociation rates may also be determined by their molecular dimensions. Those drugs with smaller ‘‘end-on dimension XY’’ tend to have faster dissociation rates (Courtney, 1988).

V. MODULATION OF BLOCK A. Proton Concentration Intra- and extracellular pH may vary because of local processes such as myocardial ischemia or infarction. They may also vary as a result of systemic respiratory or metabolic changes. Intra- and extracellular pH may modulate drug action because of an effect of pH on the drug or on the ion channel. These effects have been

analyzed most thoroughly for the Na⫹ channel (Schwarz et al., 1977; Grant et al., 1982a,b, Zhang and Siegelbaum, 1991). The principal mode of blockade by most drugs is that they cross the membrane in the uncharged form and block the channel from an intracellular site(s) in the charged form. Reducing the extracellular pH decreases the proportion of uncharged lipid soluble moiety. Therefore, the intracellular concentrations of drug will be reduced. In principle, this should reduce the extent of block. However, low extracellular pH decreases the rate of dissociation of drug from the receptor site. Changes in pH also affect block through indirect effects on channel gating. Low pH reduces the membrane potential and shifts the voltage dependence of Na⫹ channel availability to more potentials (Wendt et al., 1993; Zhang and Siegelbaum, 1991). Variations in intra- and extracellular pH have similar effects on the action of Ca2⫹ channel-blocking drugs (Gilliam et al., 1990).

B. Adrenergic Tone 웁-adrenergic agonists augment several ionic currents, including IKs , ICa , If , and ICl (Lewis et al., 1990; Carmeliet and Mubagwa, 1998). Effects of 웁-adrenergic stimulation of Na have been variable, with both increased and decreased current amplitude reported. The conductance of gap junction channels is increased, contributing to an increase in conduction velocity. 웁-adrenergic stimulation reverses the action of class I drugs (Myerburg et al., 1989; Jazayeri et al., 1989). Drugs with intrinsic antiadrenergic action, such as amiodarone and sotolol, are more resistant to reversal. Several antiarrhythmic drug trials have suggested that drugs with significant 웁-adrenergic blocking activity or concomitant 웁-adrenergic blocker administration may increase drug efficacy (Teo et al., 1990).

C. Modes of Ion Channel Blockade: Tonic and Phasic Block Two patterns of block are observed during antiarrhythmic drug exposure: tonic and phasic block (Strichartz, 1973). Tonic block is the reduction in current observed during infrequent stimulation. The membrane current during control is compared with that elicited by the first pulse during drug exposure. Phasic block is the progressive decline in current observed during repetitive stimulation. Phasic block is central to an antiarrhythmic action. Little block is observed with infrequent stimulation. The rapid succession of action potentials occurring during a tachycardia are strongly suppressed. Figure 3 illustrates the principle of the quantitative analysis of phasic block (Grant et al., 1995). The cardiac

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IX. Cardioactive Drugs

FIGURE 3 Schematic representation of the development of drug block during action potential and recovery from block during the interval between successive action potentials. ␶d and ␶r are the time constants for the development of block and recovery from block, respectively. Accumulation of block is observed during the second action potential because the interval between action potentials (␶r) is insufficient to allow full recovery from block. Reproduced from Grant et al. (1995).

cycle may be divided into a period td during action potentials and an interval tr between action potentials. During td, net blocking occurs as the drug binds to the open or inactivated channel states. During tr , there is net drug unbinding with a time constant ␶r . If ␶r is less than four times tr , block accumulates with repetitive depolarization. Eventually, a nonequilibrium steady state is reached in which block accumulated during td is dissipated during tr . Block during the nth pulse, bn , is given by the following recursion relationship (Starmer and Grant, 1985): bn ⫽ bo ⫹ (bo ⫺ bss )exp(⫺n␭) where bo is the initial fraction of blocked channels, bss is the steady-state functional block, and ␭ is the uptake rate. ␭ is related to td and tr according to

␭ ⫽ Y[(td / ␶d ⫹ (tr / ␶r )] If the kinetics of block are determined at multiple rates, 1/ ␶d and 1/ ␶r can be determined (Starmer and Grant, 1985). As the frequency of stimulation is increased (tr smaller), the steady-state level of block increases. This is a termed frequency-dependent block. For some drugs, ␶r is large and block may reach equilibrium at low frequencies of stimulation. As discussed later the differing patterns of frequency-dependent block may account for differences in drug action observed clinically.

VI. CLASSIFICATION OF ANTIARRHYTHMIC DRUGS Quinidine, procainamide, and lidocaine were the only drugs available for the primary treatment of arrhythmias for many years. There was little need or basis for dividing them into specific categories. The 1970s and

1980s witnessed the introduction of a wide range of antiarrhythmic drugs. They exhibited limited structural similarity and, on the basis of the pharmacodymamic effects, there seemed little possibility that they all acted in the same manner to terminate or prevent arrhythmia recurrence. Vaughan Williams (1970) first suggested a scheme to classify antiarrhythmic drug action in 1970. Although data on the electrophysiology basis of arrhythmias and drug action on the currents underlying the generation of the action potential were limited, he indicated that these two factors form a basis for the proposed classification of antiarrhythmic drug actions. Vaughan Williams proposed four classes of antiarrhythmic drug actions, summarized in Table I. Class I drugs had a ‘‘direct membrane action.’’ They reduced the maximum rate of rise, Vmax of the action potential, without a change in the resting potential. They produce a local anesthetic effect on nerves at concentrations tens to hundred times higher than the clinically effective antiarrhythmic level. He inferred that their effects resulted from the interaction of the drug with the inactivation gate of the Na⫹ channel. Class II drugs were sympatholytic. Although antiarrhythmic action was apparent in circumstances in which the role of the sympathetic nervous system appeared central, their mechanism of action was uncertain in other circumstances. The fact that many adrenergic-blocking drugs also had Na⫹ channel class I-blocking properties at high concentrations added to the difficulty in interpreting their action. Many 웁-adrenergic-blocking drugs have an asymmetric center. Adrenergic blocking activity resides in the L isomer, whereas the D and L isomers have similar class I action. It is now apparent that the antiarrhythmic action resides primarily in the L isomer. Drawing from the observation of the rarity of arrhythmias in the hypothyroid state and the associated

48. Antiarrhythmic Drug Action

prolongation of the APD, Vaughan Williams suggested that prolongation of the APD may represent a third class of antiarrhythmic drug action. The APD is the most important determinant of the refractory period and its prolongation should reduce the excitability gap in reentrant circuits and decrease the probability of reentry. Amiodarone produced uniform prolongation of the APD and proved to be a highly effective antiarrhythmic drug. In an attempt to explain the antiarrhythmic effects of diphenylhydantoin (DPH), Vaughan Williams proposed a fourth class of ‘‘centrally acting drugs.’’ However, any antiarrhythmic action of DPH is probably the result of its Na⫹ channel-blocking action. Introduced as a coronary vasodilator, verapamil proved effective against a variety of supraventricular arrhythmias. It did not share any of the previously described classes of action. Singh and Vaughan Williams (1972) suggested that it acted by blockade of transsarcolemmal calcium movements. It replaced DPH as the fourth class of antiarrhythmic action. A number of modifications and alternative schemes of classification have been proposed (Campbell, 1992; Weirich and Antoni, 1990). However, Vaughan Williams’ scheme has proved enduring. A majority of the antiarrhythmic drugs introduced in the late 1970s were of the class I (Na⫹ channel blocking) type. Harrison (1985) suggested that these class I drugs could be subdivided further in classes IA, IB, and IC based on their effects on the action potential upstroke and duration (Table II). A detailed study by Campbell (1983a,b) showed that subgroups IA, IB, and IC blocked the Na⫹ channel with characteristic kinetics: class IB drugs had rapid kinetics of association and dissociation from the Na⫹ channel, class IC drugs had slow kinetics and class IA drugs, intermediate kinetics. The difference in kinetics could account for the variations in the EKG changes observed at therapeutic drug concentrations. Weirich and Antoni (1990) extended the kinetic analysis to consider the drugs’ kinetics, both at plateau and the resting levels of membrane potential. They proposed subgroups of class I drugs very similar to that of Harrison. However, they suggested that the differing patterns at which the saturation of block was observed at high frequencies of stimulation provide a basis for the proarrhythmic effect of drugs with slow kinetics such as flecainide. The Vaughan Williams classification had the appeal of simplicity. It predicted the nature of the adverse cardiac effects that drugs of a certain class would produce. Use of class IA and III drugs carried the risk of torsade de pointes because of significant APD prolongation. Arrhythmias requiring treatment with multiple drugs were best treated with a combination of drugs from different classes or subgroups (Duff et al., 1983). With their blockade of inactivated sodium channels with rapid

843

kinetics, class IB drugs were not likely to be very effective in atrial arrhythmias. The working group on arrhythmias of the European Society of Cardiology met in 1990 to reexamine the classification of antiarrhythmic drugs (Task Force of the Working on Arrhythmias of the European Society of Cardiology, 1991). Their initial effort, termed ‘‘The Sicilian Gambit,’’ critiqued the Vaughan Williams classification and introduced an alternative approach to drug classification. They pointed out that the Vaughan Williams scheme was hybrid: class I and III drugs represented block of ion channels, whereas class II receptor blockade and class III represented a change in an action potential characteristic, the APD. Certain antiarrhythmic drugs, such as digitalis and adenosine, were not covered in the scheme. Potential targets for drug action, such as membrane transporters and gap junctions, were not considered. The Sicilian Gambit considers the mechanisms of the clinical arrhythmias. Based on putative ion channels or receptors playing key roles, vulnerable parameters can be identified. These vulnerable parameters are the targets for drug action. For a given drug, the scheme complies all its actions. The idea of a greater focus on the possible mechanism of the arrhythmia is a positive aspect of the scheme. However, simplicity is sacrificed for a comprehensive account of all the actions of each drug. The scheme has not achieved widespread acceptance. As discussed previously, it does provide a useful framework in which to analyze arrhythmias and their treatment by drugs.

VII. DRUGS WITH CLASS I ACTION: SODIUM CHANNEL BLOCKERS Studies combining site-directed mutagenesis and electrophysiology have provided multiple views of the mechanism of block of the Na⫹ channel that need to be reconciled. Bennett et al. (1995) showed use-dependent block of the cardiac Na⫹ channel by lidocaine is strongly inhibited by the inactivation-disabling mutation IFM/ QQQ. Their conclusions were supported by the studies of Balser et al. (1996), who proposed a refinement of the modulated receptor model in which lidocaine acted as an allosteric enhancer of inactivation. Vedantham and Cannon (1999) used the technique of substituted cysteine accessibility to determine the function of the inactivation gate during block by lidocaine. The critical phenylalanine residue in the IFM triplet was modified to cysteine (IFM/ICM) and the conformation of the inactivation gate was followed by determination of the reactivity of the substituted cysteine with the thiol-modifying agent timethylammonium ethylmethane thiosulfo-

844

IX. Cardioactive Drugs

nate (MTS-ET). They concluded that the inactivation gate was functioning normally during blockade by lidocaine. Building on prior studies of blockers of the Ca2⫹ channel, Ragsdale et al. (1994, 1996) used the technique of alamine scanning mutagenesis to determine the local anesthetic-binding site of the Na⫹ channel. Successive residues in the sixth transmembrane segment of domain IV were mutated to alamine, and sensitivity of the Na⫹ current to block by ethidocaine was determined. A F/A (F1764A, brain II sequence) mutation in the middle of DIV, S6 decreased sensitivity of the Na⫹ channel to 1% of the wild type, An isolucine residue 4 residues 5⬘ to phenylalanine 1764 controlled access to the binding site. They proposed a model in which the local anesthetic molecule binds to the site in the inner vestibule of the pore (Fig. 4). The antiarrhythmic drugs flecainide, lidocaine, and quinidine bind to the same site. However, the change in affinity with the F1764A mutation for quinidine and flecainide binding was only two- to threefold less than that of the wild type. This suggests that other residues may be involved in the binding of these open channel blockers. Mutation of the residues that form the selectivity filler of the Na⫹ channel also influence local anesthetic class drug binding (Sunami et al., 1997). Na⫹ channel-blocking drugs may terminate arrhythmias by a number of mechanisms. They may slow conduction in reentrant arrhythmias with a long excitable

FIGURE 4 Proposed orientation of amino acids in IVS6 with respect to a bound local anesthetic molecule in the ion-conducting pore. Segment SS1–SS2, which also contributes to the pore (27), is shown as well. Amino acids at positions 1760, 1764, and 1771 are shown facing the pore lumen. Reproduced from Ragsdale et al. Science 265, p. 1727. Copyright (1994) American Association for the Advancement of Science.

gap. Areas of unidirectional block may be converted to bidirectional block. Arrhythmias with a short excitable gap may be terminated by a prolongation of refractoriness. Na⫹ channel blockers delay the recovery of the availability of Na⫹ channels during phase 3. Repolarization has to occur to more negative membrane potentials before a sufficient number of channels to support regenerative depolarization become available. Sodium channel blockers are also capable of terminating arrhythmias that result from automaticity. The pacemaker current If may be reduced by Na⫹ channel-blocking drugs. Some of these drugs also block the slow, persistent component of Na⫹ current that contributes to the plateau of the action potential (Wang et al., 1997; An et al., 1996). Blockade of this component of current will abbreviate the action potential duration and decrease the probability of EADs.

VIII. DRUGS WITH CLASS III ACTION: POTASSIUM CHANNEL BLOCKERS At least five K⫹ channels contribute to the repolarizing phase of the action potential. However, potassium channel blockers in clinical use block primarily the rapid (IKr) and slow (IKs) components of the delayed rectifier; IKr is the most common target of drug action. Block of delayed rectifier currents will shift the balance of current during the action potential plateau in the inward direction and prolong the APD. The change in the APD is effected without a reduction of conduction velocity. Therefore, the wavelength for reentry is increased, and its probability is decreased. This antiarrhythmic strategy is particularly useful in arrhythmias with a short excitable gap. The ideal class III antiarrhythmic drug should show the greatest APD prolongation during the rapid rates of a tachycardia. Unfortunately, most K⫹ channel blockers show the opposite effect: APD prolongation is greatest at slow heart rates (Hondeghem and Snyders, 1990). The long APD at slow heart rates increases the likelihood of EAD generation and torsade de pointes. The basis for this reverse use-dependent effect is multifactoral. In the absence of drug, the APD prolongs substantially as the heart rate is decreased (Hoffman and Cranefield, 1960). At slower rates, I Ca2⫹ has more time for recovery from inactivation and so the contribution of inward currents to phases 2 and 3 of the AP is increased (Li and Nattel, 1997). Similarly, at slow rates, time in the depolarized states is reduced and less IKs is activated. These two factors shift the balance of current in the inward direction and prolong the APD. K⫹ channel-blocking drugs do not have a significant effect on the Ca2⫹ channel at

48. Antiarrhythmic Drug Action

therapeutic concentrations. In principle, a K⫹ channelblocking drug can produce a reverse use-dependent effect on the APD if their binding kinetics are favored by diastolic membrane potentials or if they produce a significant differential in block of IKr and Iks . The latter effect is believed to be more important: greater block of IKr , therefore a greater contribution of IKs to repolarization. As IKs exerts a diminished role at slow rates, there is relatively greater APD prolongation at these rates. Delayed rectifier blockers may be divided into a selective group that blocks Ikr and a nonselective group that also blocks Iks . Certain drugs, e.g., quinidine, block all types of K⫹ channels (Colatsky, 1991; Imaizumi and Giles, 1987). Azimilide blocks Ikr and Iks . Methanesufoanilides are selective blockers of Ikr (Sanguinetti and Jurkiewicz, 1990). Sotolol is the prototype of this subgroup of drugs. Sotalol is a racemic mixture, and IKr blocking action resides in both enantiomers (Carmeliet, 1985). In addition, the L isomer is a 웁-adrenergic blocker. Dofetilide, ibutilide, sematilide, and E4031 also belong to this subgroup. Methanesufoanilides block IKr in the open (activated) state (Carmeliet, 1992; Carmeliet and Mubagwa, 1998). Block is not significant at a very positive membrane potential at which channels are predominantly in the inactivated state. Elevation of the concentration of the permeant ion K⫹ reduces block. In AT 1 cells, the Kd for block of IKr by dofetilide increases from 2.7 to 79 nM as [K⫹]0 is raised from 1 to 8 mM (Yang and Roden, 1996). The binding of [H3]dofetilide to ventricular myocytes showed a similar dependence of [Ko⫹] (Duff et al., 1997).

IX. DRUG WITH CLASS IV ACTION: CALCIUM CHANNEL BLOCKERS Blockers of the L-type Ca2⫹ channel are an important group of therapeutic agents, not only in the management of arrhythmias, but also in hypertension, coronary artery, and vasospastic diseases. The major Ca2⫹ channelblocking drugs belong to three structurally distinct classes: dihydropyridines (DHPs), phenylalkylamines (PAA), and benzthiazepines. The three groups of drugs block the L-type Ca2⫹ channel by binding to different interacting sites on the 움 subunit. Only phenylalkalmines (verapamil) and benzthiazepines (diltiazem) are used as antiarrhythmic drugs. This may relate to differences in blocking kinetics among the three classes of drugs. Antiarrhythmic Ca2⫹ channel-blocking drugs are used primarily in supraventricular arrhythmias. In AV node-dependent arrhythmias, they terminate or prevent arrhythmias by inducing block in the slow and/or fast

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pathway. In AV node-independent arrhythmias, Ca2⫹ channel blocking reduces the ventricular response to supraventricular tachycardias. There is a small group of ventricular tachycardias that are also verapamil sensitive. These tachycardias are probably the result of Ca2⫹ channel-dependent triggered activity. The binding sites for all three classes of Ca2⫹-blocking drugs involve D111S5, D111S6, and DIVS6 of the 움 subunit. Single amino acids required for the high-affinity binding of phenylalklamine and dihydropyridines have been identified in these domains (Catterall and Striessnig, 1992; Striessning et al., 1990). Phenylalklamines and dihydropyridines bind to different faces of the DIIIS6 and DIVS6 transmembrane segments of the Ca2⫹ channel 움 subunit. There is allosteric interaction between the binding sites. Benzthiazepines (d-cis-diltiazem) increase the binding affinity of dihydropyridines to their receptor site (Catterall and Striessnig, 1992). However, phenylalkylamines decrease the binding affinity of dihydropyridines. There are also important interactions among the permeant ion, Ca2⫹, and the affinity of the Ca2⫹ blockers for their receptor site(s). Increasing [Ca2⫹]o to the millimolar range increases the affinity of dihydropyridines for their recepor site, and chelating agents such as EGTA and EDTA markedly inhibit binding. In contrast, increasing [Ca2⫹]o above 100 애M decreases the affinity of phenylalkylamines and benthiazepines for their receptor sites. The differing influence of [Ca2⫹]o on the affinity of the Ca2⫹ channel antagonist for their receptor site is potentially important when considering the use of Ca2⫹ solutions to treat toxicity of Ca2⫹blocking drugs. Dihydropyridines fall into two groups with low and high pKa values (Carmeliet & Mubagwa, 1998). Drugs with low pH values (⬍3), such as nifedipine, nisoldipine, nitrendipine, and nimoldipine, are neutral at physiological pH. Other dihydropyridines with high pH values (⬎7) exist predominantly in their charged form at physiological pH. Both stimulatory and inhibitory actions can be observed with the dihydropyridines. However, only their inhibitory action is important therapeutically. Neutral dihydropyridines bind primarily to the inactivated state of the Ca2⫹ channel. The Kd for nitrendipine block decreased from 0.73 애M at ⫺80 mV to 0.36 nM at ⫺15 mV (Bean, 1984). However, because the onset of block is rapid, there is little change in the extent of block as the duration of depolarization is increased beyond a few milliseconds. At the single channel level, dihydropyridines increase the fraction of null sweeps (Hess et al., 1984; Kawashima and Ochi, 1988). Nulls tend to occur in runs, so-called mode O gating. Mean open time is unchanged. In the activated state, block also plays an important role in the action by charged dihydropyridines.

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IX. Cardioactive Drugs

Phenylalkylamines exist predominately in the charged form at physiological pH. They access their receptor site from the cytoplasmic side of the membrane. The quaternary derivitives are inactive when applied from the extracellular side, but produce block when injected into cells (Hescheler et al., 1982). Depolarizing voltages below threshold do not increase block. Block is enhanced by brief pulses that activate the Ca2⫹ channel. At the single channel level, open channel block was confirmed by a reduction of the mean open time (McDonald et al., 1989). The fraction of null sweeps was also increased. Benzthiazepines block both inactivated and open channels. Block develops at depolarized membrane potentials and increases as the duration of depolarization is prolonged. The rate of recovery from block is intermediate between that of dihydropridines (fast, t 앑 500 msec for nitrendipine) and phenylalkylamines (slow, t 앑 15 sec for verapamil).

X. OTHER ANTIARRHYTHMIC DRUGS: ADENOSINE Adenosine, an endogenous nucleoside, is effective in terminating AV node-dependent supraventricular tachycardias and a specific subset of ventricular tachycardia (Holloway et al., 1989). Adenosine interacts with the A1 receptor on cardiac cell membranes and with the A2 receptor on endothelial and vascular smooth muscle cells. Interaction of adenosine with A1 and A2 receptors is blocked with low affinity by methylxanthines such as caffeine and aminophylline. A significant antiarrhythmic effect results from the A1 receptor interaction. Some of the side effects result from the A2 receptor interaction. The binding of adenosine to the A1 receptor releases the GTP-binding protein Gi . This protein exerts both direct and indirect effects. The direct effect results in the activation of an inward rectifying potassium channel IKACh/adenosine . The density of this channel is high in SA and AV nodes and atrial cells, but is low in ventricular and His-Purkinje cells. Activation of IKACh/adenosine slows the sinus rate and AV conduction and shortens the atrial refractory period. The slowing of AV node conduction accounts for its antiarrhythmic action in terminating supraventricular tachycardia. Gi inhibits adenyl cyclase and decreases basal and isoproterenolstimulated calcium current. This action accounts for the efficacy of adenosine in terminating epinephrinesensitive ventricular tachycardia. Adenosine action is terminated by the rapid reuptake into cells, including erythrocytes and endothelial cells. This accounts for its evanescent action. Intracellular enzymes inactivate adenosine by converting it to inosine or

by phosphorylation to adenosine monophosphate. The cellular uptake of adenosine is blocked by dipyradamole.

A. Implications of Models of Antiarrhythmic Drug Action and States Blocked The proposed models of drug interaction with the sodium channel have a number of important bearings on their clinical use. The modulated and guarded models of each class of antiarrhythmic drugs propose a single receptor. This implies that multiple drugs, including a parent compound and its metabolites, will compete for a single blocking site. In blocking regimens in which a drug has continuous access to its receptor, the blocking action of multiple drugs is usually additive. However, with ion channel-blocking drugs, the high-affinity blocking state is phasically available. A drug with rapid binding kinetics may displace a drug with slow binding kinetics. In the low-affinity (resting) state, the fast drug also dissociates more rapidly from the receptor. The net result is that at certain rates of stimulation, less block may be observed with a combination of drugs than with either drug alone. A number of case reports have described the reversal of the toxic effects of slow Na⫹ channel-blocking drugs by lidocaine (von Dach and Streuli, 1988; Whitcomb et al., 1999). It should be emphasized that the circumstances under which such reversal occurs are quite restrictive. The toxic effect of antiarrhythmic drugs is best treated by withholding the drug and providing general supportive measures. Open channel blockers are envisioned as binding within the channel pore. The permeant ion, e.g., Na⫹ or K⫹, crosses the membrane by ‘‘hopping’’ from multiple low-energy sites with the pore. The permeant ion may decrease the association of drug with its receptor and reduce block. Sodium bicarbonate and sodium lactate have been reported to reduce the blockade produced by class I drugs (Bellet et al., 1959a,b). Both Na⫹ and the accompanying alkalization contribute to this effect (Nattel et al., 1984; Pentel and Benowitz, 1984). A characteristic response of myocardial cell to injury, e.g., ischemic, is membrane depolarization. Such depolarization has significant implications for drug action. Membrane depolarization decreases the fraction of Na⫹ channel available for opening, resulting in a decrease in efficacy of open blocking channel class I drugs. Conversely, the action of inactivated state blocking drugs is enhanced, providing a mechanism for the selective blockade of conduction in injured cells.

XI. SUMMARY The increase in mortality in several long-term clinical trials of antiarrhythmic drugs has redirected the focus

48. Antiarrhythmic Drug Action

of treatment to physical methods such as radiofrequency ablation or the implantable defibrillator. However, antiarrhythmic drugs remain an important treatment modality. They retain an important place in the treatment of patients with atrial flutter/fibrillation and as adjuvant therapy in patients treated with the implantable defibrillator. The rapid advantage in membrane biophysics and molecular biology has provided a wealth of information on the structure, function, and mechanism of blockade of ion channel. The three-dimensional structure of a potassium channel from streptomyces has been reported (Doyle, 1998). We are now aware of some of the properties of antiarrhythmic drugs that are associated with a high proarrhythmic potential, e.g., sodium channel blockade with very slow dissociation kinetics. Rational drug design based on precise structural information is on the horizon. In the late 1970’s and 1980’s, the focus of drug design was an agent with a single mechanism of action. The broad therapeutic efficacy of amiodarone and its low incidence of cardiac toxicity suggest that drugs with multiple actions, e.g., ion channel and 웁-adrenergic blockade, may offer distinct therapeutic advantages.

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Zamponi, G. W., and French, R. J. (1993). Dissecting lidocaine action: Diethylamide and phenol mimic separate modes of lidocaine block of sodium channels from heart and skeletal muscle. Biophys. J. 65, 2335–2347. Zamponi, G. W., Sui, X., Codding, P. W., and French, R. J. (1993). Dual actions of procainamide on batrachotoxin-activated sodium channels: Open channel block and prevention of inactivation. Biophys. J. 65, 2324–2334. Zhang, J.-F., and Siegelbaum, S. A. (1991). Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes. J. Gen. Physiol. 98, 1065–1083.

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49 Cellular Mechanisms of Cardioprotection MASAFUMI KITAKAZE and MASATSUGU HORI Department of Internal Medicine and Therapeutics Osaka University Graduate School of Medicine Suita 565-0871, Japan

I. INTRODUCTION

ever, we do not believe that these drugs are powerful enough to treat chronic heart failure with full satisfaction. This chapter discusses how cardioprotection is achieved from these points of view in ischemic heart disease.

It is critically important to consider how cardioprotection is achieved in heart diseases because mortality and morbidity of heart diseases have increased all over the world, and the burden from the viewpoint of not only the individual patient but also social and economical aspects is increased as we begin the 21st century. To decrease this burden, it is essential to protect the heart against ischemic stress, and there are three different aspects to achieve cardioprotection in ischemic hearts. First, acquiring tolerance against ischemia and reperfusion injury before the onset of ischemia is effective for patients with coronary artery disease or coronary risk factors. For example, development of collateral circulation in advance can reduce the severity of ischemia when coronary occlusion occurs. Preventing rupture of the atheroma is another paradigm used to attenuate the incidence of acute coronary syndrome. Furthermore, if the trigger mechanisms of cardioprotection due to ischemic preconditioning are elucidated, they can be applied in high-risk patients before acute myocardial infarction. Second, it is also important to develop the tool for the treatment of ischemic and reperfusion injury. To our knowledge, we still do not have the drugs or tools to decrease either infarct size or cardiac remodeling in patients with acute myocardial infarction, except for recanalization therapy. Even when patients survive acute myocardial infarction, chronic ischemic heart failure may occur. Therefore, as the third paradigm, we need to obtain drugs to treat chronic ischemic heart failure. Angiotensin-converting enzyme (ACE) inhibitors and 웁 blockers have been proven to attenuate the mortality and morbidity of chronic heart failure; how-

Heart Physiology and Pathophysiology, Fourth Edition

II. ACQUISITION OF CARDIOPROTECTION BEFORE THE OCCURRENCE OF ISCHEMIA When the effective pretreatment to attenuate the ischemic injury is applied prior to the occurrence of ischemic heart disease in subjects with high-risk factors for acute myocardial infarction, such as hyperlipidemia, smoking, and hypertension, the pretreatment before the onset of myocardial ischemia will become an effective method for the attenuation of ischemia and reperfusion injury. This is similar to the immunization therapy for infectious diseases such as mumps.

A. Plaque Rupture Since it is well recognized that acute myocardial infarction occurs due to the abrupt plaque rupture followed by the accumulated platelet aggregation, it is critically important to prevent plaque rupture in patients with coronary artery disease. One possibility is to apply cholesterol-lowering therapy. Although pravastatin, one of the inhibitors of HMG-CoA reductase, is reported to decrease stenotic coronary artery only to a modest extent, it decreases the incidence of cardiac death in patients with coronary artery disease or in high-risk patients (1). It is suggested that pravastatin

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stabilizes the plaques and prevents the rupture of plaques. Pravastatin or simvastatin may decrease the content of cholesterol and the accumulation of macrophages at the stenotic site of the coronary artery and reduce oxidative stress and inflammatory actions. Casscells et al. (2) reported that active plaques produce heat, suggesting that inflammatory reactions are involved in active plaques, which may make the fibrous cap fragile. This inflammation may be caused by bacteria such as chlamydia or by lipid metabolites which activate monocytes. Because probucol, an antioxidant drug with a moderate cholesterol-lowering capability, is very effective in decreasing the mortality and morbidity in patients with coronary artery disease, oxidative stress may be an important factor for the onset of plaque rupture. Therefore, oxidative stress at the atheroma attacks fibrous caps, although there is no direct evidence or report of the methods to prevent plaque rupture at present.

B. Collateral Circulation Development of a collateral circulation from nonischemic myocardium to ischemic myocardium is an important strategy in attenuating the severity of myocardial ischemia when coronary arterial occlusion occurs. The fibroblast growth factor (FGF), transforming growth factor-웁 (TGF-웁), and vascular endothelial growth factor (VEGF) families are known to be important growth factors for angiogenesis (3–5). Basic FGF has been proved to be responsible for the development of collateral circulation (6). Yanagisawa et al. (6) showed clearly that basic FGF levels increase after the onset of myocardial infarction, suggesting that basic FGF may play a role in angiogenesis of collateral circulation in patients with coronary artery disease. However, heparin is also known to cause angiogenesis because heparin activates HB-EGF, which induces the migration and proliferation of smooth muscle cells (7, 8). Furthermore, VEGF is also known to cause potent angiogenesis due to the proliferation and migration of endothelial cells (9). Interestingly, adenosine is shown to increase mRNA and the protein levels of VEGF (10), suggesting an important role for the development of collateral circulation. Furthermore, adenosine increases the proliferation and migration of endothelial cells in vitro (11). However, in the in vivo condition, adenosine stimulates angiogenesis on the chick chorioallantoic membrane (Fig. 1) (12), and dipyridamole increases adenosine-induced angiogenesis. Finally, repeated administration of dipyridamole in vivo for several weeks increases the regional myocardial flow of the ischemic area compared with the control, and this effect cannot be mimicked by diltiazem (13). This result suggests that coronary vasodilation per se does not affect the development of collateral circula-

tion, but the enhancement of adenosine during the administration of dipyridamole can increase the development of collateral vessels. Taken together, either or all of the growth factors, such as VEGF, HB-EGF, and basic FGF, may induce angiogenesis in patients with coronary artery disease, which may constitute cardioprotection against ischemia and reperfusion injury. Furthermore, pharmacological interventions, such as heparin or adenosine administration, may enhance angiogenesis via the induction of growth factors. Gene transfection may lead to the development of collateral circulation (5, 14). Indeed, Losordo et al. (5) initiated a phase 1 clinical study to determine the safety and bioactivity of direct myocardial gene transfer of VEGF as the sole therapy for patients with symptomatic myocardial ischemia. In five patients who had failed conventional therapy for angina, naked plasmid DNA encoding VEGF (phVEGF165) was injected directly into the ischemic myocardium. They found that all of the patients had a significant reduction in angina and that the postoperative left ventricular ejection fraction was either unchanged or improved. Objective evidence of reduced ischemia was documented using dobutamine single-photon emission-computed tomography (SPECT) imaging in all patients. Coronary angiography showed an improved Rentrop score in all of the five patients. Therefore, gene transfection to increase collateral circulation may become one of the potential treatments of coronary artery disease. Another method used to increase ischemic tolerance is to induce cardioprotection prior to the ischemia of acute myocardial infarction, and one potential way is to induce in advance the infarct size-limiting effect of ischemic preconditioning in patients with coronary artery disease.

C. Ischemic Preconditioning Ischemic preconditioning has received much attention from both basic researchers and clinicians. This was first described by the research group of Murry et al. (15). Results to date have shown that ischemic preconditioning limits infarct size to 10–20% of the risk area in the reperfused ischemic myocardium (16–19). Liu et al. (16) have implicated endogenous adenosine as a trigger or mediator in ischemic preconditioning by demonstrating that the administration of 8-(sulphophenyl)theophylline abolishes the salutary effect of ischemic preconditioning. These investigators have hypothesized that ischemic preconditioning occurs via adenosine A1 receptor activation. Adenosine A1 receptor activation activates protein kinase C (PKC) via the activation of phospholipase C, and several investigators, including

49. Cardioprotection

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FIGURE 1 A formalin-fixed chorioallantoic membrane after removal from egg illustrating the representative response to a 3-mg adenosine pellet (ADN) and mannitol control pellet (M). The pellets are 1.2 cm apart. From Dusseuau et al. (12).

the authors, found that the activation of PKC is transiently observed after the procedure of ischemic preconditioning (18, 19). Furthermore, the inhibition of PKC blunts the infarct size-limiting effect of ischemic preconditioning (17–20). Therefore, at present, activation of protein kinase C is believed to be a common pathway in triggering cardioprotection. The next question is how protein kinase C activation triggers the infarct size-limiting effect of ischemic preconditioning. Activation of protein kinase C opens KATP channels, and the opening of KATP channels may be cardioprotective against ischemia and reperfusion injury. The opening of mitochondrial KATP channels is also activated via protein kinase C (20, 21), suggesting that it is mitochondrial KATP channels which are responsible for cardioprotection. It was shown that the activation of PKC increases ecto-5⬘-nucleotidase activity (Fig. 2) and mediates the cardioprotection via the enhancement of adenosine production in ischemic preconditioning (17, 18). Cardioprotection due to the ischemic preconditioning procedure disappears in several hours, and this

disappearance could be attributable to dephosphorylation of the cardioprotective proteins or enzymes. Interestingly, cardioprotection will reappear in 24–48 hr after the ischemic preconditioning. This is known as the second window of ischemic preconditioning (22, 23). Marbe et al. (22) reported that HSP72 induction is important in mediating the infarct size-limiting effect of ischemic preconditioning, and Kuzuya et al. (23) reported that Mn-superoxide dismutase (Mn-SOD) induction may contribute to the cardioprotection in ischemic preconditioning.

III. ACQUISITION OF CARDIOPROTECTION DURING ISCHEMIA AND REPERFUSION There are two different ways to find the method to directly decrease ischemia and reperfusion injury. One is to find mediators of the infarct size-limiting effect of ischemic preconditioning, and the other is to invent drugs that attenuate the ischemia and reperfusion injury independent of cardioprotection mechanisms of ische-

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FIGURE 2 The dose–response relation between phorbol 12-myristate 13-acetate (PMA) and ecto-5⬘-nucleotidase activity with and without either GF109203X (an inhibitor of protein kinase C) or cycloheximide (an inhibitor of protein synthesis) in rat cardiomyocytes. Ecto5⬘-nucleotidase activities in the control conditions were 6.44 ⫾ 0.89, 5.96 ⫾ 0.78, and 5.81 ⫾ 0.44 nmol/mg protein/min in PMA, PMA with GF109203X, and PMA with cycloheximide groups, respectively. From Kitakaze et al. (17).

mic preconditioning. Because the former was discussed briefly above as well as in Chapter 50, the latter strategy is mainly discussed here.

A. Factors That Cause Ischemia and Reperfusion Injury There are many factors that constitute ischemia and reperfusion injury, and these factors seem to synergistically cause cellular injury in the heart. 1. ATP Depletion Intracellular ATP levels are thought to determine the turning point between reversible and irreversible cellular injury. This is because the maintenance of intracellular homeostasis is energy dependent: Ca2⫹ pump, Ca2⫹ ATPase, and interaction of actin and myosin. A 90% decrease in ATP coincidentally results in the irreversible deterioration of the myocardium (24), leading to the idea that depletion of ATP content in reperfused myocardium may be a critical factor in the process of irreversible injury. However, when adenosine is administered throughout ischemic and reperfusion periods, a 90-fold increase of ATP synthesis is obtained in the reperfused myocardium (25). It is known that (1) adenosine stimulates glycolysis in rat hearts, (2) intracoronary

infusion of adenosine increases glucose uptake, and (3) dipyridamole enhances glucose uptake accompanied by an increase in myocardial ATP in the newborn lamb. Thus, enhanced glucose metabolism by adenosine may contribute in part to a decrease in the rate of ATP depletion during ischemia. However, one may argue that the repletion of intracellular ATP via the administration of ribose or adenosine in the reperfused myocardium after the establishment of reperfusion injury does not promote recovery of contractile function, indicating that the recovery of intracellular ATP levels does not necessarily improve contractile function. Adenosine-induced attenuation of contractile dysfunction may not be due to the recovery of ATP levels but rather to adenosine receptor activation. Therefore, there is consensus that decreases in ATP levels are important for the determination of irreversible cellular injury, but may not be important in reversible cellular injury, such as myocardial stunning or hibernation. 2. Ca2ⴙ Overload Ca2⫹ overload is thought to disrupt cellular membrane and intracellular homeostasis via the activation of calpain. If this process occurs during ischemia and reperfusion injury, Ca2⫹ overload may play an important role in ischemia and reperfusion injury. When myocardial ischemia occurs, cellular acidosis increases, which activates Na⫹ /H⫹ exchange via the accumulation of H⫹ with resulting increased intracellular Na⫹ levels (26). Accumulation of Na⫹ causes Ca2⫹ overload via Na⫹ / Ca2⫹ exchange. It was shown that ischemia increases intracellular Ca2⫹ levels in in vitro Langendorff hearts (27). Furthermore, using the integrity of microtubules, Ca2⫹-sensitive structures in the cells, as an index, we showed that Ca2⫹ overload occurs in ischemic canine hearts (28). Therefore, Ca2⫹ overload is believed to occur in ischemic and reperfused hearts. Because intracellular Na⫹ levels are increased before the rise in intracellular Ca2⫹ levels and the inhibitor of Na⫹ /H⫹ exchange attenuates the Ca2⫹ overload, the route of entry of Ca2⫹ into the cells is thought to be via Na⫹ /H⫹ and Na⫹ / Ca2⫹ exchanges. The question is how important Ca2⫹ overload is for the pathophysiology of ischemia and reperfusion injury. Transient Ca2⫹ exposure in the ferret Langendorff preparation causes myocardial dysfunction (Fig. 3) (29), and attenuation of Ca2⫹ overload attenuates the severity of myocardial dysfunction, suggesting that Ca2⫹ overload may be an important cause of myocardial stunning. However, there is no clear consensus that Ca2⫹ overload contributes to the cause of myocardial necrosis. Sustained intracellular acidosis, which inhibits Na⫹ /Ca2⫹ exchange and thus Ca2⫹ overload, is reported to attenuate myocardial ischemia (30), hinting

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FIGURE 3 Changes in developed pressure after repeated transient exposure to Ca2⫹ (10 mM) for 5 min separated by 10 min. Ca2⫹ exposure causes the reduction of developed pressure 10–20 min after exposure. From Kitakaze et al. (29).

that attenuation of Ca2⫹ overload may limit myocardial necrosis (31), although intracellular acidosis is beneficial for the ischemia and reperfusion injury aside from the attenuation of Na⫹ /Ca2⫹ exchange. 3. Free Radicals When hearts are reperfused abruptly, oxygenderived free radicals and hydroxyradicals are produced and released from leukocytes and endothelial cells. In the ischemic and reperfused condition, xanthine dehydrogenase changes to xanthine oxidase because xanthine oxidase is activated by protease sensitive to Ca2⫹ accumulation. These radicals attack the cellular membrane and cause cellular damage via the inactivation of membrane enzymes, pumps, and proteins, such as Na⫹ / K⫹-ATPase, Ca2⫹ channels, and ecto-5⬘-nucleotidase (Fig. 4) (32, 33). Gross et al. (34) reported that myocardial contractile dysfunction produced by 15 min of

ischemia and 3 hr of reperfusion is restored by superoxide dismutase (Fig. 5) (34). Furthermore, Sekili et al. reported that hydroxyradicals are produced within a minute after the onset of reperfusion and that these radicals contribute to the generation of myocardial contractile dysfunction (35). These results suggest that free radicals are important for the development of myocardial stunning; however, there is no clear consensus that free radials generated during ischemia and reperfusion may cause cellular necrosis. Indeed, the clinical trial of the TIMI study revealed that 10 mg/kg of SOD does not attenuate infarct size in 120 patients with acute myocardial infarction (36). This may be attributable to the fact that (1) oxygen-derived free radicals do not contribute to the formation of myocardial necrosis during ischemia and reperfusion or (2) the therapeutic time window of SOD may be narrow in patients with acute myocardial infarction. However, oxidative stress causes vascular remodel-

FIGURE 4 Staining of ecto-5⬘-nucleotidase in nonischemic canine endocardium (A). Ischemia following 1 min of coronary occlusion increases ecto-5⬘-nucleotidase activity (B), and enzyme activity is markedly enhanced when ischemic myocardium is exposed to SOD (C).

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FIGURE 5 Effects of superoxide dismutase and catalase on myocardial contractile function following 15 min of coronary occlusion. PO, prior to occlusion; OCC, occlusion. From Gross et al. (34).

ing, such as thickening of intima and plaque rupture. These effects may be very important in the genesis of acute myocardial infarction because drugs with antioxidant capability are reported to attenuate vascular events.

sense, norepinephrine release causes two opposite effects on vascular tone. High norepinephrine concentrations associated with severe prolonged ischemia may mask the adenosine-related cardioprotection and any favorable intramyocardial flow redistribution.

4. Catecholamines

5. Microcirculatory Disturbances

When myocardial ischemia occurs, presynaptic vesicles release norepinephrine via the accumulation of Na⫹. Increases in Na⫹ levels activate the reverse uptake of norepinephrine (37), which facilitates norepinephrine release. Norepinephrine activates both 움- and 웁-adrenoceptors: 움-adrenoceptor stimulation increases intracellular Ca2⫹ levels and causes coronary vasoconstriction and 웁-adrenoceptor stimulation increases myocardial oxygen consumption. These factors may cause deterioration of myocardial contractile and metabolic functions during ischemia and reperfusion. Indeed, many experimental and clinical studies reveal that blockers of 웁adrenoceptors are effective in the treatment of ischemic heart disease. Interestingly, the amount of norepinephrine released during ischemia may enhance adenosine production and adenosine-induced coronary vasodilation through 움1and 움2-adrenoceptor stimulations (38, 39). Furthermore, 움-adrenoceptor stimulations are reported to increase myocardial endocardial blood flow at the expense of epicardial blood flow (40). This may be attributable to differences in the sensitivity of endocardial and epicardial arteries during 움-adrenoceptor stimulation. In this

Even if ischemic myocardium is reperfused by the once occluded coronary artery in acute myocardial infarction, coronary microvasculature does not necessarily receive enough flow. Rather, myocardial perfusion becomes more heterogeneous; some areas receive enough flow, but some areas receive less flow than necessary. This is called the ‘‘no reflow phenomenon’’ (41, 42). The no reflow phenomenon is reported to predict the size of the myocardial necrosis and functional recovery in the chronic phase in patients with acute myocardial infarction (42). The no reflow phenomenon can be caused by myocardial cellular injury, platelet plugging, leukocyte adhesion, and increases in the tone of small coronary vessels. Kloner et al. (41) reported that 90 min of ischemia causes heterogeneous myocardial flow distribution, but that 40 min of ischemia does not. Furthermore, the no reflow area is evident 10–12 sec after the onset of reperfusion, suggesting that the no reflow phenomenon is observed transiently at early phases of reperfusion. Because 40 min of ischemia does not cause the no reflow phenomenon but causes myocardial necrosis, several investigators suggest that the no reflow phenomenon may not be involved in the pathophysiology

49. Cardioprotection

of reperfusion injury. However, because there are differences in the sensitivity to detect necrosis and the no reflow phenomenon and because there is clinical evidence to consider the no reflow phenomenon as a cause of myocardial injury, the no reflow phenomenon is believed to constitute reperfusion injury in ischemic heart disease. 6. Adhesion Molecule Ischemia and reperfusion activate adhesion between leukocytes and endothelial cells. Adhesion molecules in leukocytes are LFA-1, Mac-1, and the selectin family, and adhesion molecules in endothelial cells are ICAM1 and L-selectin (43). There is contradictory evidence whether attenuation of these adhesion molecules using antibodies does or does not limit infarct size. Therefore, we cannot determine the importance of the activation of adhesion molecules in the pathophysiology of ischemia and reperfusion injury. 7. Endothelin Endothelin (ET) is divided into three subtypes: ET1, ET-2, and ET-3. ET-1 causes potent vasoconstriction and it increases in patients with vasospastic angina and acute myocardial infarction. There are two endothelin receptors, ETA and ETB , and ET-1 activates ETA . Interestingly, when the ETA receptor antagonist is administered before or after the onset of myocardial ischemia, it decreases infarct size to 30–40% (44). This indicates that endothelin plays an important role in the formation of reperfusion injury. However, the mechanisms by which endothelin is deleterious to ischemic hearts, such as coronary vasoconstriction, Ca2⫹ overload, or leukocyte or platelet activation, are not clear at present. 8. Apoptosis Ischemia and reperfusion cause apoptosis, and ischemic preconditioning attenuates the extent of cellular apoptosis during ischemia and reperfusion (45). However, how much of the area of ischemic and reperfused myocardium becomes apoptotic or necrotic and the importance of apoptosis in the pathophysiology of diseased hearts have not yet been clarified.

B. Endogenous Factors That Cause Cardioprotection

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via adenosine receptors (38, 39): (1) Attenuation of the release of catecholamine, 웁-adrenoceptor-mediated myocardial hypercontraction, and Ca2⫹ overload via A1 receptors and (2) increase in coronary blood flow and inhibition of platelet and leukocyte activation via A2 receptors. Furthermore, adenosine inhibits renin release and TNF-움 production in experimental models. It has been thought that the stimulation of adenosine A2 receptors activates adenylate cyclase in the coronary arteries to produce cyclic adenosine monophosphate (cAMP) and relaxes coronary vascular smooth muscle. Increases in cAMP may increase the uptake of Ca2⫹ into the sarcoplasmic reticulum and cause vasorelaxation. Furthermore, cyclic AMP may open KATP channels and decrease Ca2⫹ movement inward into smooth muscle cells. Indeed, adenosine-induced coronary vasodilation is attenuated by glibenclamide, an inhibitor of KATP channels (46). In the ischemic heart, thromboembolism in small coronary arteries, which is believed to be one of the causes of the ‘‘no reflow phenomenon’’ of the reperfused myocardium, may worsen the severity of acute myocardial infarction. Small coronary microembolizations are caused by platelet aggregation, and stimulation of adenosine A2 receptors has been reported to inhibit the platelet aggregation induced by norepinephrine in vitro (47, 48). We have investigated whether endogenous adenosine inhibits thromboembolism secondary to platelet aggregation in in vivo ischemic hearts (Fig. 6) (47). We further examined the cellular mechanisms of platelet aggregation when adenosine receptors are inhibited (Fig. 6). The appearance of P-selectin in the platelet increased 8-(sulfophenyl)theophylline treatment, and the inhibitor of P-selectin inhibited platelet aggregation with leukocytes, and thus with endothelial cells (48). Thus, endogenous adenosine released in the ischemic myocardium inhibited the activation of platelet P-selectin and inhibited microembolization in small coronary vessels. Adenosine also inhibits leukocyte chemotaxis (49) and the production of oxygen-derived free radicals through the stimulation of adenosine A2 receptors. This decrease in the inflammatory response may also be cardioprotective. Interestingly, the activation of leukocytes decreases ecto-5⬘-nucleotidase activity (50), which may decrease adenosine production and activate leukocytes further. These vicious cycles in leukocytes may enhance the injury in ischemic hearts by the release of oxygen-derived free radicals and adhesion to endothelial cells to obstruct small coronary arteries.

1. Adenosine

2. Nitric Oxide

Adenosine, produced not only in cardiomyocytes but also in endothelial cells, is known to be cardioprotective

Nitric oxide (NO) activates soluble guanylate cyclase and increases cyclic GMP levels. Increases in cGMP

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FIGURE 6 Hypoperfused coronary arteries without (left) and with (right) the intracoronary administration of 8-phenyltheophylline during coronary hypoperfusion (38 ⫾ 2 mm Hg). 8-Phenyltheophylline is a potent antagonist of intracoronary adenosine receptors and induced thrombosis in the small coronary arteries. Tissue was excised following in situ perfusion fixation for 3 min following the onset of ischemia. Bars in the right corner are 50 애m (hematoxylin and eosin). From Kitakaze et al. (47).

cause several cardiovascular actions. NO relaxes smooth muscles, inhibits platelet aggregation, inhibits the activation of leukocytes, attenuates myocardial contraction, attenuates the presynaptic release of norepinephrine, and inactivates the renin–angiotensin system. NO also attenuates the expression of adhesion molecules. These actions of NO are very similar to those of adenosine, although the cellular signal transduction is different. NO is believed to attenuate ischemia and reperfusion injury as does adenosine. Although NO with oxygen radicals produces peroxymitrite, and peroxymitrite is very harmful to the cells, the consensus is that NO is beneficial to ischemia and reperfusion injury as a whole because the beneficial actions of NO may overcome the deleterious pathway of peroxymitrite. 3. ANP and BNP Both ANP and BNP are released from the atrium and ventricle of the heart, which may play an important role in the homeostasis of the cardiovascular system (51). Both ANP and BNP activate particulate guanylate cyclase and increase cellular cyclic GMP levels. ANP and BNP regulate coronary vascular tone. ANP is increased in patients with chronic heart failure, chronic renal failure, systemic hypertension, and paroxysmal artial tachycardia. However, there is no clear consensus whether ANP is involved in the ischemic myocardium. Interestingly and importantly, when ANP was infused into the canine ischemic myocardium, we found that coronary blood flow increases and myocardial contractile and metabolic function recover in canine ischemic hearts. Furthermore, we found that ANP attenuates

myocardial necrosis following 90 min of ischemia and 6 hr of reperfusion in open chest dogs. BNP is also increased in the mechanically stressed heart. These data support that either ANP or BNP is cardioprotective against ischemia and reperfusion injury. Of course, ANP and BNP reduce blood volume by increasing urine output and decrease heart size, which mainly favor cardioprotection. There are no clear data whether ANP or BNP affects leukocytes or platelets to reduce myocardial damage further. 4. Acidosis Cellular acidosis is thought to be a natural defense mechanism against myocardial ischemia and reperfusion injury. H⫹ blocks Ca2⫹ channels and Na⫹ /Ca2⫹ exchange and antagonizes Ca2⫹ overload in the myocardium (30, 31). Furthermore, H⫹ increases NO and adenosine production of ischemic myocardium. Indeed, data show that transient cellular acidosis attenuates reperfusion injury, restores myocardial function, and limits cellular necrosis (30, 31). These results indicate that acidosis is a self-protecting mechanism and that the moderate enhancement of cellular acidosis may induce cardioprotection against ischemia and reperfusion injury. 5. Endothelium-Derived Hyperpolarizing Factor (EDHF) Endothelial cells produce not only nitric oxide, but also the substance that decreases the membrane potential, i.e., endothelium-derived hyperpolarizing factor.

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EDHF decreases membrane potentials and causes relaxation of the vessels. EDHF has not been identified yet, but (1) bradykinin is thought to increase EDHF levels and (2) EDHF opens Ca2⫹ activated K⫹ (KCa) channels. The inhibitor of KCa channels decreases coronary blood flow and worsens the contractile and metabolic functions of ischemic myocardium and EDHF, or the opening of KCa channels, plays an important role in the regulation of coronary blood flow in the ischemic myocardium (52). Furthermore, the opening of KCa channels induces an infarct size-limiting effect (53).

IV. HOW TO MEDIATE CARDIOPROTECTION DURING ISCHEMIA AND REPERFUSION The potential treatment of acute myocardial infarction is to reperfuse the occluded coronary artery. Either percutaneus transluminal coronary angioplasty (PTCA) or percutaneous transluminal coronary recanalization (PTCR) is recognized to be the most effective treatment of acute myocardial infarction in clinical settings. However, our impression is that either PTCA or PTCR limits ischemia and reperfusion injury to a modest extent because of the diminution of a beneficial effect by reperfusion injury, and we need to find the adjunctive therapy to treat ischemia and reperfusion injury directly. Because many factors are involved in ischemia and reperfusion injury, the idea is (1) to use many drugs that inhibit each deleterious factor or (2) to use one drug that inhibits many deleterious factors. The latter seems to be more plausible for clinical settings. The candidates are adenosine, NO, and ANP for treatment. Because adenosine also attenuates reversible and irreversible myocardial cellular injury after reperfusion in various species of animals, intracoronary infusion of adenosine results in a 75% reduction in myocardial infarct size in dogs (54) and attenuates contractile dysfunction in rats. The AMISTAD trial reveals that adenosine administration is effective for the treatment of acute myocardial infarction (55), and we are also planning an ATP administration trial for patients with acute myocardial infarction (COAT trial) (56). Adenosine-related compounds may be effective for cardioprotection against ischemia and reperfusion injury. Vesnarinone, a new inotropic agent, has been reported to inhibit adenosine uptake in immune cells (57), which was also demonstrated in myocytes, endothelial cells, and smooth muscle cells (58). Furthermore, we also observed that vesnarinone activates ecto-5⬘-nucleotidase via protein kinase C-independent mechanisms. These data suggest that vesnarinone may be effective for acute myocardial infarction. When testing this hypothesis, we observed that vesnarinone limits infarct size, an effect which is blunted by an antagonist of adenosine receptors (59).

Furthermore, both methotrexate and sulfasarazine, drugs for rheumatoid arthritis, are reported to attenuate inflammation via adenosine and ecto-5⬘-nucleotidase (60). We also tested whether a methotrexate analogue mediates cardioprotection. We found that a methotrexate analogue mediates an infarct size-limiting effect, which is completely abolished by either an antagonist of adenosine receptors or AOPCP (unpublished data). AICA riboside also increased the activity of ecto-5⬘nucleotidase, which may have merit for cardioprotection. Enhancement of nitric oxide or ANP/BNP may be another tool used to attenuate ischemia and reperfusion injury. Indeed, the V-Heft I trial reveals that nitrate, the NO donor, is effective for the treatment of chronic heart failure. Furthermore, ACE inhibitors, which are used in the treatment of ischemic or nonischemic chronic heart failure, are reported to increase cardiac NO levels via bradykinin-dependent mechanisms in experimental and clinical studies. We have proved that either cilazapril or imidaprilat increases cardiac NO levels and attenuates the severity of myocardial ischemia via a NO-dependent pathway. Nipradilol can be used for the NO donor with the additional effects of 웁-adrenoceptor blockade, which may be useful compared to ordinary NO donors or 웁-adrenoceptor blockers. ANP is available for the treatment of heart failure, although it is difficult to use ANP for the treatment of chronic heat failure because there is only an intravenous type of ANP. Candoxiatril, which inhibits the degradation of ANP, has been developed and can be used for the treatment of chronic heart failure.

V. TREATMENTS AFTER ACUTE MYOCARDIAL INFARCTION A. Pharmacological Approach Chronic heart failure, the end state of the ischemic heart, is characterized by the reduction of cardiac performance relative to the oxygen demand of the body; however, several neurohormonal factors have been reported to exacerbate the severity of chronic heart failure. Catecholamine, renin–angiotensin, and cytokines may be involved in the pathophysiology of chronic heart failure. Indeed, chronic heart failure is treated effectively by 웁-adrenoceptor antagonists and angiotensin-converting enzyme inhibitors (61, 62), and these drugs have been proven to be effective in the treatment of chronic heart failure in mass studies. Carvedilol is reported to be more effective than ordinary 웁-adrenoceptor blockers (63). Because carvedilol is characterized as a 웁-adrenoceptor-blocking agent with modest 움-adre-

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FIGURE 7 Changes in ejection fraction and oxygen consumption during the bicycle exercise test in patients with chronic heart failure treated with dipyridamole for 6 months. From Kitakaze et al. (65).

noceptor blocking capability, one may argue that the vasodilatory capability of 움1-adrenoceptor blockade may be effective in the 웁-blockaded condition, as the 웁blocker decreases cardiac output, but 움1-adrenoceptor blockade may compensate to maintain cardiac output. However, prazosin has not been proven to be effective for the treatment of chronic heart failure, suggesting that 움1-adrenoceptor blockade does not explain the beneficial effect of carvedilol. Carvedilol has an antioxidant effect. This effect may be important, as oxidative stress has been implicated in the pathophysiology of chronic heart failure. How about adenosine or NO? Adenosine and NO are reported to decrease sympathetic tone and activity of the renin–angiotensin and the cytokine systems, and NO has been proven to be effective in the treatment of chronic heart failure. Interestingly, we observed that the plasma adenosine levels increased according to NYHA classification in patients with chronic heart failure (64). Furthermore, when we administered dipyridamole or dilazep (Fig. 7 and 8) to patients with chronic heart failure for 6 months, ventricular wall motion and exercise capacity in these patients were increased (65). Therefore, adenosine may be an alternative treatment of chronic heart failure. Endothelin receptor antagonists are also effective in the treatment of chronic heart failure and have been tested in clinical trials.

FIGURE 8 Changes in plasma adenosine levels (A), the New York Heart Association functional classification (B), ejection fraction (C), and oxygen consumption during the bicycle exercise test (D) in patients with chronic heart failure treated with dilazep for 6 months. From Kitakaze et al. (65).

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FIGURE 9 Teratomas formed by human ES cell lines in SCID beige mice. Human ES cells after 4 to 5 months of culture (A) were injected into the rear leg muscles of 4-week-old male SCID beige mice (B). In B, many tissues appeared. (A) Gut-like structure (scale bar: 400 애m), (B) rosettes of neural epithelium (scale bar: 200 애m), (C) bone (scale bar: 100 애m), (D) cartilage (scale bar: 100 애m), (E) striated muscle (scale bar: 25 애m), and (F) tubules interspersed with structures resembling fetal glomeruli (scale bar: 100 애m). From Thomson et al. (66).

B. Molecular Approach

C. Mechanical Approach

Other strategies for the treatment of chronic heart failure are to compensate for the loss of myocardium and to decrease myocardial fibrosis. Genes to change mature cardiomyocytes to myoblasts with proliferative capability have not yet been identified. It is difficult to reproduce cardiomyocytes from the myoblasts in vivo at present. However, many molecular investigators are focusing on finding the master gene to regulate the proliferative capability of cardiomyocytes. In the future, the proliferation of cardiomyocytes will be one of the treatments of chronic heart failure. Another method that could potentially be used to restore the volume and numbers of cardiomyocytes is the transplantation of cardiomyocytes. Because human embryonic stem (ES) cells are available, if we find a way to change ES cells to cardiomyocytes, we can transplant the cardiomyocytes to failing hearts. Indeed, transfection of Flk genes changes ES cells to endothelial cells (9), suggesting that the transfection of cardiomoycytespecific genes may cause changes in morphogenesis to cardiomyocytes. Furthermore, when human ES cells are transplanted into leg muscles of mice, teratomas form in which human ES cells can be transformed into various human tissues and cells (66) (Fig. 9), suggesting that the analysis of teratoma may give a hint to the methods of how ES cells become cardiomyocytes. These issues need further investigation.

Another fascinating approach for the treatment of chronic heart failure is the artificial heart. Although heart transplantation is used all over the world, the number of donor hearts is not sufficient to treat all of the patients with chronic heart failure. Therefore, it is important to develop artificial hearts for the treatment of chronic heart failure. In the clinical setting, the left ventricular assist device is available to support failing hearts.

VI. FUTURE DIRECTIONS OF INVESTIGATION OF CARDIOPROTECTION Because many factors that cause deterioration of the function and metabolism of the heart are activated in ischemic hearts, it is important to recognize and inhibit the multiple deleterious factors. One strategy of pharmacological interventions is to administer corresponding drugs to attenuate the multiple deleterious factors, e.g., superoxide dismutase for oxygen-derived free radicals. However, this strategy is not realistic in the clinical setting because this strategy requires the administration of many drugs to attenuate multiple deleterious factors. Another possibility is to administer one substance that has multiplicity of action. The candidate is adenosine or nitric oxide.

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Two other directions are gene therapy and artificial organs, although these two strategies have not been investigated thoroughly. If human cardiomyocytes are cultured in the test tube and are transplanted to failing hearts, this method may become a potential treatment for the failing heart, although we need to elucidate how coronary vascular systems and extracellular matrix systems are incorporated to support myocardial cells. These three directions, i.e., pharmacological interventions, gene interventions, and artificial interventions, may synergistically mediate cardioprotection for failing hearts.

VII. SUMMARY Both prevention and attenuation of ischemia and reperfusion injury in patients with acute coronary syndromes are critically important for cardiologists. To save these patients from deleterious ischemic damages, there are three different strategies. The first strategy is to increase ischemic tolerance before the onset of myocardial ischemia. Prevention of plaque rupture comes first; HMG-CoA reductase inhibitors such as pravastatin may attenuate plaque rupture. Growth factors can induce collateral circulation to prevent or attenuate the ischemic damages. Finding the trigger mechanisms of the infarct size-limiting effect of ischemic preconditioning is important in inducing ischemic tolerance in advance. The second strategy is to attenuate ischemia and reperfusion injury when the irreversible process of myocardial cellular injury occurs. Pharmacological interventions, such as adenosine or nitric oxide, may contribute to attenuate the ischemic damages. The third strategy is to treat ischemic chronic heart failure that is caused after acute myocardial infarction. Gene therapy or the development of artificial hearts may provide a potential treatment in chronic failing hearts. Taken together, we need to investigate potential mechanisms of the cellular damages and the tools for cardioprotection before, during, and after the onset of acute myocardial infarction.

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50 Ischemic Preconditioning: Description, Mechanism, and Significance MICHAEL V. COHEN and JAMES M. DOWNEY Departments of Medicine and Physiology University of South Alabama College of Medicine Mobile, Alabama 36688

I. INTRODUCTION

60 min! Although some observers undoubtedly voiced disbelief, this phenomenon, termed ischemic preconditioning, has been duplicated in every species tested, including humans (Lawson and Downey, 1993). It was postulated that if the mechanism of this protective effect could be established, it might be possible to devise a clinical intervention that would result in the salvage of ischemic tissue.

A major complication of the sudden occlusion of a coronary artery is the attendant loss of contractile myocardium served by that artery. This condition, termed acute myocardial infarction, is a common clinical occurrence and often leaves the patient with a failing heart. There has been a search for a treatment that would reduce the amount of tissue loss in this setting, which in turn would reduce the incidence of heart failure. To date only acute reperfusion with either thrombolytic agents or catheter-based interventions such as coronary angioplasty have been proven to successfully salvage ischemic myocardium. However, reperfusion takes time. The thrombolytic agent must chemically dissolve the offending thrombus before reflow can be achieved, and procedural delays are unavoidable whenever patients must be brought to the cardiac catheterization suite. Time delays translate into infarcted myocardium. Therefore, despite the success that has attended revascularization, it is apparent that an additional therapy is needed that would preserve viability until blood flow can be restored to ischemic myocardium. In 1986 Murry and colleagues made a most improbable observation. In an anesthetized dog preparation, a 40-min occlusion of a coronary artery resulted in 29% infarction of the myocardium served by that vessel (Fig. 1). However, if that 40-min occlusion had been preceded by four cycles of 5 min occlusion/5 min reperfusion, then infarction was only 7% of the risk zone. Thus infarction was diminished by 75% even though the aggregate duration of ischemia had increased from 40 to

Heart Physiology and Pathophysiology, Fourth Edition

II. NATURAL HISTORY Ischemic preconditioning demonstrates a saturating kinetic pattern. Whereas 2 min of either coronary occlusion in rabbits (Van Winkle et al., 1991) or low-flow ischemia in pigs (Schulz et al., 1998) before a longer period of myocardial ischemia yields no protection, prolonging the short ischemic period to 5 min in rabbits and 10 min in pigs produces maximal protection. Intermediate periods of ischemia in the pig result in an intermediate degree of protection. Li et al. (1990) observed that a single 5-min coronary artery occlusion in dogs is as protective as six cycles of a 5-min occlusion, each followed by reperfusion. Miura et al. (1992) also observed that multiple preconditioning cycles in rabbits were no more effective at protecting the heart than a single cycle, although Sandhu and colleagues (1997) noted that three 5-min coronary occlusions in rabbits produced a small increment in protection over one cycle of brief occlusion and reperfusion. In rats, cardiac protection is still evident if the reperfusion period is shortened to 1 min, although there is no protection if reperfu-

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Copyright  2001 by Academic Press. All rights of reproduction in any form reserved.

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though the signal transduction pathway leading to protection in this second window phase shares many of the steps identified for early phase protection, this chapter concentrates on observations made for the first window.

III. TARGETS AND TRIGGERS OF PROTECTION

FIGURE 1 Infarct size as a percentage of risk zone is plotted on the ordinate for individual control dogs with only a 40-min coronary occlusion and for preconditioned dogs with four preceding cycles of 5 min coronary occlusion/5 min reperfusion. In the latter, infarct size averaged 7%, significantly smaller than the 29% infarction in the former (P ⬍ 0.001). Open symbols represent infarct size of individual animals, whereas closed symbols represent mean group infarction and bars indicate SEM. Adapted from Murry et al. (1986).

sion is only 30 sec. However, neither reoxygenation nor full reflow is critical. Thus hypoxia (Walsh et al., 1995) or low-flow ischemia (Koning et al., 1995) preceding the long period of myocardial ischemia can successfully precondition the heart. The anti-infarct effect of preconditioning, however, is relatively short-lived. Typically a 5-min period of ischemia followed by up to 60 min of coronary reflow before the more prolonged ischemic interval results in salvage. However, if the reperfusion interval is prolonged beyond 1–2 hr in the anesthetized rabbit (Van Winkle et al., 1991; Miura et al., 1992) or dog (Murry et al., 1991) or 2–4 hr in the conscious rabbit (Burckhartt et al., 1995), protection is no longer evident. However, protection can be reinstituted with a second cycle of preconditioning ischemia/reperfusion (Yang et al., 1993). Thus there is a definite and fairly rigid timetable for preconditioning. Somehow the myocardium ‘‘remembers’’ that it had been preconditioned by a brief ischemic period that had occurred up to several hours before the prolonged ischemia. The mechanism of this memory continues to elude investigators up to the time of this writing. While this first window or early phase of protection is fleeting, a second window of protection appears 24 hr after ischemic preconditioning (Yellon et al., 1998), and this latter antiinfarct effect is believed to last for up to 3 days (Baxter et al., 1997). The second window of protection is assumed to result from expression of a protective protein, possibly inducible nitric oxide synthase (Bolli et al., 1998). Al-

When first described by Murry et al. (1986), preconditioning and its protection were elicited by brief coronary occlusion. Soon, however, a variety of preconditioning stimuli were described, including rapid cardiac pacing, hypoxia, thermal stress, and various pharmacological agents. Additionally the specter of preconditioning’s salutary effect was broadened as protection of other targets of ischemic injury was described. Ischemic preconditioning protects against infarction in all species tested, including some avian hearts (Rischard and McKean, 1998). However, it has been difficult to demonstrate an antistunning effect of the first window or early phase of protection in dogs, rabbits, and pigs, but the second window of protection does demonstrate a strong antistunning effect, emphasizing that its mechanism, at least in part, is different from that of classical or early phase preconditioning. Of particular interest has been the rat model. The buffer-perfused, Langendorff rat heart preparation contracts isovolumetrically around a fluid-filled balloon. When the heart is subjected to global ischemia, its mechanical function is abolished within seconds. If the heart is reperfused after 20 min, a predictable degree of recovery of mechanical contractile activity will occur. The magnitude of postischemic recovery has been a popular index of ischemic injury, although residual dysfunction is clearly a mixture of infarction and stunning. For some unknown reason, the postischemic recovery of function is greatly enhanced by preconditioning in rat hearts, but not in other species, such as rabbit or dog. It has never been resolved whether this protection in the rat is related to an anti-infarct effect, an antistunning effect, or both. Ischemic preconditioning also has an antiarrhythmic effect in several species, including the rat and dog. It is difficult to demonstrate any effect against arrhythmias in pig or rabbit. It is not known whether the antiarrhythmic effect of preconditioning shares any mechanisms with the anti-infarct effect. Because of the uncertainty concerning mechanisms of protection against stunning and arrhythmias, the remaining sections of this chapter concentrate on the anti-infarct effect of ischemic preconditioning.

IV. PROTECTION IS RECEPTOR MEDIATED Preconditioning’s anti-infarct effect can be shown to be independent of recruitment of coronary collaterals

50. Ischemic Preconditioning

or stunning of the jeopardized myocardium (Murry et al., 1986, 1991). Liu and co-workers (1991) proposed that a metabolite produced by the briefly ischemic myocardium could in some fashion be affecting the tissue. Because ischemic tissue is characterized by breakdown of ATP to adenosine, they initially evaluated a possible role of the latter metabolite. A 5-min infusion of adenosine or an adenosine analogue, R(⫺) N 6-(2-phenylisopropyl)adenosine (rPIA), in anesthetized rabbits in lieu of brief ischemia protected hearts to the same extent as brief ischemia

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(Liu et al., 1991) (Fig. 2). Conversely, an adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (SPT), infused just prior to the brief ischemia completely blocked the protection (Liu et al., 1991) (Fig. 2). At least four adenosine receptor subtypes have been identified, and three have been cloned. Using receptor subtype-specific antagonists, investigators have demonstrated that A1 and A3 but not A2 adenosine receptors could initiate the protection of preconditioning (Thornton et al., 1992; Liu et al., 1994; Auchampach et al.,

FIGURE 2 In an in situ rabbit model, preconditioning with a single cycle of 5 min coronary occlusion/10 min reperfusion before a 30-min occlusion significantly decreased infarct size plotted for each heart on the ordinate to 10% of the risk zone from 39% in control animals experiencing only the 30-min occlusion (p ⬍ 0.01). That protection of preconditioning was abrogated if rabbits were also treated with the adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) (top). In isolated hearts the protection of ischemic preconditioning could be mimicked by a 5-min infusion of either adenosine or its analogue R(⫺) N6-(2-phenylisopropyl)adenosine (rPIA) in lieu of 5 min of coronary occlusion (bottom). The infusion of either agonist was discontinued 10 min before the 30-min occlusion. Open symbols represent infarct size of individual animals, whereas closed symbols represent mean group infarction and bars indicate SEM. Modified and reproduced with permission from Cohen et al. (2000).

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1997). Both A1 and A3 receptors are present on cardiac myocytes. Because A3 receptors are also found in abundance on mast cells, one intriguing hypothesis suggested that preconditioning was related to the degranulation of myocardial mast cells by adenosine release during brief ischemia (Linden, 1994). Then, during the prolonged ischemia, granule-poor mast cells in the myocardium would no longer be able to release massive amounts of toxic substances, thereby resulting in less damage. Although attractive, this proposal was later discarded when it was noted that there was dissociation between early mast cell degranulation and ultimate protection (Wang et al., 1996a).

V. G-PROTEINS AND PHOSPHOLIPASES Cell surface A1 and A3 adenosine receptors are coupled to inhibitory G-proteins, Gi. Indeed, chronic treatment with pertussis toxin, which blocks Gi, did abolish preconditioning’s protection in the rabbit model (Thornton et al., 1993). The adenosine receptor is coupled through Gi to, among other things, phospholipases C (PLC) and D (PLD). After adenosine binds to its receptor the trimeric G-protein is split into its 움 and 웁웂

parts. Both components are capable of activating the 웁 isoform of PLC in the sarcolemma. PLC catalyzes the hydrolysis of membrane inositol-containing phospholipids: phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. The latter is hydrolyzed to inositol trisphosphate (IP3) and diacylglycerol (DAG). Whereas IP3 causes release of Ca2⫹ from the sarcoplasmic reticulum, which in turn is believed to elicit positive inotropy, DAG stimulates the translocation and activation of PKC. PLD is able to degrade other membrane phospholipids, including phosphatidylcholine. The immediate degradation products are choline and phosphatidic acid. The latter is metabolized further by a phosphohydrolase to DAG. Many agonists, including norepinephrine and endothelin, can stimulate both PLC (Kaku et al., 1991) and PLD (Ye et al., 1994), although the coupling mechanism for the latter is not well understood. The onset of the PLC reaction typically is very rapid and DAG production is short-lived, peaking at 30 sec, whereas the PLD reaction is delayed but accounts for a more prolonged production of DAG, which is associated with a prolonged activation of PKC (Billah and Anthes, 1990). Thus both pathways are thought to be critical components of PKC activation.

FIGURE 3 Infarct size as a percentage of risk zone is plotted on the ordinate for control rabbits (no PC) and rabbits preconditioned with a single cycle of 5 min coronary occlusion/ 10 min reperfusion before a 30-min occlusion (PC) (left). The latter had significantly smaller infarcts (p ⬍ 0.05). Staurosporine, a PKC antagonist, effectively blocked the protection of PC (middle). In the isolated heart, 5-min infusions of either of two PKC activators, phorbol 12myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), in lieu of 5 min of coronary occlusion were just as protective as ischemic preconditioning (p ⬍ 0.05 vs control) (right). Open symbols represent infarct size of individual animals, whereas closed symbols represent mean group infarction and bars indicate SEM. Modified and reproduced with permission from Cohen et al. (1996).

50. Ischemic Preconditioning

VI. PROTEIN KINASE C Hence adenosine released by ischemic cells binds to cell surface receptors, which are linked to phospholipases mainly through Gi proteins. Because these phospholipases in turn activate PKC, it was logical to assume that adenosine was an activator of PKC and that this activation was a critical step in preconditioning’s signal transduction pathway. This assumption was supported by several studies that demonstrated adenosine could increase PKC activity (Kohl et al., 1990; Schwiebert et al., 1992), although this view was not universal. Ytrehus et al. (1994) noted in the rabbit that PKC activation by infusion of either phorbol 12-myristate 13-acetate (PMA) or the DAG analogue 1-oleoyl-2-acetyl-snglycerol in lieu of brief ischemia was equally protective as ischemic preconditioning against infarction (Fig. 3). The converse was also demonstrated. PKC blockade with staurosporine (Ytrehus et al., 1994) (Fig. 3), polymyxin B (Ytrehus et al., 1994), or chelerythrine (Liu et al., 1994) prevented brief ischemia from salvaging ischemic myocardium. Similar observations were also made at that time in the rat heart model in which recovery of function rather than infarction was the end point (Mitchell et al., 1995). These experimental data strongly implied that PKC played an important role in eliciting preconditioning’s protection. In the hearts of dogs (Przyklenk et al., 1995) and pigs (Vahlhaus et al., 1996) the involvement of PKC in preconditioning has been more controversial, in part because of an alternate signaling pathway with activation of a protein tyrosine kinase (see later).

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the amount of each that is produced by ischemic myocardium. In the rabbit blockade of adenosine (Liu et al., 1991), bradykinin (Goto et al., 1995), or opioid (Miki et al., 1998) receptors or interference with free radical production (Baines et al., 1997) raises the ischemic threshold for preconditioning, thus blocking protection and indicating a physiological role for that receptor in the protection of ischemic preconditioning (Fig. 4). Conversely, administration of antagonists to 움-adrenergic (Tsuchida et al., 1994), angiotensin (Liu et al., 1995), or endothelin (Wang et al., 1996b) receptors has no effect on the ability of brief ischemia to precondition the heart, indicating that those receptor agonists are not released in sufficient quantity to independently affect the endogenous triggering of protection. Of course, in different species, other agonists may assume importance because of differences in their production. For example, in the rat, opioid receptors appear to be the principal ones involved in ischemic preconditioning (Schultz et al., 1998), and it is difficult to show a role for adenosine (Li and Kloner, 1993). Furthermore, in pigs, protection after 3 min of preconditioning ischemia is related to increased interstitial bradykinin (Schulz et al., 1998). With longer periods of ischemia, adenosine release becomes more important, and this purine then becomes a major trigger. Hence it appears that multiple agonists participate in preconditioning’s protection, and presumably all recep-

VII. MULTIPLE PKC-COUPLED RECEPTORS MAKE THE TRIGGER FOR PROTECTION REDUNDANT If PKC were involved in preconditioning, then any means of activating that kinase should successfully protect the heart. The adenosine receptor is only one of many receptors on the cardiomyocyte that couple to PKC. Thus 움-adrenergic, endothelin, angiotensin, bradykinin, muscarinic, and opioid agonists can each bind receptors on the cell surface and stimulate PKC, and each, with the exception of a muscarinic agonist, is released to some extent by ischemic myocardium. Indeed a brief intracoronary infusion of any one of these agonists will put the heart into a preconditioned state. Furthermore, exposure to free radicals that activate PKC directly can also precondition the heart (Baines et al., 1997; Tritto et al., 1997). Whereas exogenous administration of each of these agonists is capable of minimizing infarction, not all have been found to play a physiological role in the protection from brief ischemia. Presumably this is a reflection of

FIGURE 4 Brief myocardial ischemia results in release of adenosine (adeno), bradykinin (brady), opioids, and free radicals from the heart. Each activates PKC and the individual contributions sum to exceed a hypothetical threshold, which results in the triggering of preconditioning and protection. Blockade of any of the receptors (e.g., SPT for adenosine or HOE140 for bradykinin) or of release of any of the components results in insufficient stimulation of PKC by the remaining triggers and resulting loss of protection. However, increasing the number of preconditioning ischemia/reperfusion cycles results in enhanced release of the remaining components and once again attainment of the PKC threshold for triggering of protection. Modified and reproduced with permission from Cohen et al. (2000).

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tor signals sum at the level of PKC. This redundancy is perhaps a built-in protective mechanism to ensure that a brief period of ischemia will always result in protection.

VIII. BIOCHEMISTRY OF PKC PKC is a family of at least 12 serine/threonine kinases, many of which are present in the rabbit heart (Ping et al., 1997). The many isozymes can be classified into three major groups: classical or conventional (cPLC), novel (nPKC), and atypical (aPKC). The classical isozymes (움, 웁I, 웁II, 웂) require DAG, phospholipid, and Ca2⫹ for activation, whereas the novel ones (웃, ␧, ␩, ␪) lack the calcium-binding region so that these subtypes are not dependent on Ca2⫹ for activation. The atypical isozymes (␨, ␫, ␭, 애) require neither cofactor. Instead, 3⬘-phosphoinositides may be the activators of aPKCs. PKC isozymes are not distributed randomly within the cell, but are rather associated with specific compartments or structures. A peculiar trait of PKC isozymes is that they physically move about within the cell when activated. For example, Mochly-Rosen (Disatnik et al., 1994; Mochly-Rosen and Gordon, 1998) has demonstrated with immunofluorescent techniques that the inactive ␧ isozyme is found in the nucleus and perinuclear region and that it translocates to cross-striated structures (possibly the contractile elements) and cell–cell contact regions following activation. Receptors for activated C kinase (RACKs) are located on various intracellular structures and form docking sites for the activated isozymes (Gray et al., 1997; Mochly-Rosen and Gordon, 1998). RACKs are isozyme specific so that, for example, the activated ␧ isozyme will bind only to its ␧ RACK and to no other. The ␧ RACK can only bind the ␧ enzyme. It has been hypothesized that similar binding sites for inactivated PKC also exist, but have yet to be identified. Ping et al. (1997) identified 11 PKC isozymes in the rabbit heart. Using specific antibodies to each isozyme they demonstrated that a series of four cycles of 4 min coronary occlusion/6 min reperfusion caused translocation (and thus activation) of only 2 novel isozymes, ␧ and ␩. They also demonstrated that in neither the cytosol nor the particulate fraction did total PKC activity change. This study clearly documented that brief ischemia would cause PKC isozyme translocation. These investigators suggested that it was one of these 2 isozymes translocated during brief ischemia that contributed to cardioprotection. Parallel studies by multiple investigators in rat heart have confirmed that specific isozyme translocation occurs in preconditioned hearts that is not evident in

nonpreconditioned tissue. Using immunohistochemical staining, Banerjee’s group showed that PKC-웃 and -␧ isozymes were translocated in ischemically preconditioned rat hearts and PKC-웃 and -␨ in rat hearts preconditioned with phenylephrine (Mitchell et al., 1995). Yoshida et al. (1997) reported that PKC-움, -웃, and -␧ were translocated to the membrane fraction in rat hearts following ischemic preconditioning and that this movement was completely blocked by the PKC inhibitor chelerythrine. In a subsequent study in rat hearts, Kawamura et al. (1998) correlated the protection of ischemic preconditioning under various conditions with the translocation of either PKC-웃 or -␧. Ashraf’s group has demonstrated that calcium preconditioning in the rat heart causes translocation of only the 움 and 웃 isozymes (Miyawaki and Ashraf, 1997). Finally, PKC-움, -웃, and -␧ translocate in isolated rat neonatal cardiomyocytes during hypoxic preconditioning (Goldberg et al., 1997; Gray et al., 1997). Interestingly, Albert and Ford (1999) demonstrated that the 움, ␧, and ␫ isozymes of PKC do not translocate after 5 min of global ischemia and 15 min of reperfusion in the isolated rat heart, but do if there is a subsequent 30-min period of global ischemia. In these hearts, translocation was associated with the phosphorylation of specific particulate-associated proteins. However, translocation of any given isozyme does not prove that that isozyme is responsible for preconditioning. Movement of a PKC isozyme from cytosol to membrane might simply be an epiphenomenon resulting from the ischemia without any consequent triggering of protection. To further explore the role of PKC, isozymespecific antagonists were needed. Accordingly, MochlyRosen (Johnson et al., 1996; Souroujon and MochlyRosen, 1998) developed small peptides identical to portions of the binding domains of classical and several novel isozymes, including 웃, ␧, and ␩. When introduced into the cell, these peptides would bind to their target RACKs and thus effectively block translocation and, therefore, activation of the specific isozyme (Fig. 5). She demonstrated in neonatal rat ventricular cells exposed to 48 hr of hypoxia that it was only the ␧ V1-2 peptide which would abort the protection of a preceding 90-min period of hypoxia (Gray et al., 1997). A similar result was observed in isolated rabbit ventricular cardiomyocytes subjected to simulated ischemia (Liu et al., 1999). The latter is produced by centrifuging isolated ventricular cells into a pellet and covering them with either mineral oil or microballoons to exclude oxygen. The cells rapidly consume the remaining oxygen in the medium and then start metabolizing ATP and producing adenosine, lactate, and other by-products as would an ischemic tissue. In fact, the responses of these pelleted cells are quite similar to those of ischemic cells

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releasing the PKC inhibitor into the cytosol. Introduction of inhibitory peptides for the classical or the novel PKC isozymes 웃 or ␩ into the cell has no effect on the protection induced by preconditioning. However, as demonstrated in Fig. 6, the ␧ inhibitor peptide V1-2 completely aborts the protective effect of preconditioning. More importantly the ␧ V1-2 peptide blocks protection from exposure to DAG, which activates all classical and novel PKC isozymes. Thus it would appear that only PKC-␧ is capable of putting the heart into a preconditioned state.

IX. 5⬘-NUCLEOTIDASE

FIGURE 5 To be activated, PKC must translocate from its location in the cytosol to docking sites or RACKs positioned on specific membranes or cytoskeletal elements. Each isozyme has a unique RACK. After translocation, the enzymatic site of the kinase is exposed and phosphorylation can proceed (top). Small peptides have been developed that bind to specific RACKs and block translocation of the PKC isozyme normally docking on those RACKs (bottom). These peptides are, therefore, highly selective and specific antagonists of their respective PKC isozymes.

of the intact heart following interruption of blood flow. Over the ensuing 120–180 min, cell aliquots are incubated in hypotonic trypan blue. As the duration of simulated ischemia is extended, the cells become increasingly osmotically fragile, as evidenced by the increasing number of stained cells with time. This progressive increase in osmotic fragility represents a loss of integrity of the cell membrane and is thought to be related to the process of ischemic cell death, which can also be shown to involve osmotic swelling (Jennings et al., 1990). Pelleting and exclusion of oxygen for 10 min followed by resuspension prior to the 120- to 180-min period of simulated ischemia serve to precondition the cells. When the preconditioned cells are repelleted, they stain much more slowly than nonpreconditioned cells. This delay in appearance and progression of osmotic fragility represents protection (Fig. 6). When the inhibitor peptides are linked to an antennapedia homeodomain peptide, cardiomyocytes internalize the peptide complex. Once inside the cell the cys–cys bond between the antennapedia and inhibitory PKC isozyme peptides is hydrolyzed, thus

One attractive theory of preconditioning’s mechanism is based on 5⬘-nucleotidase activation. 5⬘-Nucleotidase catalyzes the dephosphorylation of 5⬘-adenosine monophosphate (AMP) to adenosine. During ischemia, ATP is metabolized to AMP as oxygen-starved cells deplete their high-energy stores. The amount of adenosine produced by the cell is then limited by the activity of 5⬘-nucleotidase in the cardiomyocte. Kitakaze and co-workers (1994) have shown that 5⬘-nucleotidase is activated by PKC and have proposed that a preconditioned heart is protected because it releases more adenosine. Kitakaze hypothesized that PKC activates 5⬘-nucleotidase, which in turn causes adenosine release, and that as long as 5⬘-nucleotidase is activated the heart remains preconditioned. There are now several observations that argue against this theory. The first is that most observers find that preconditioned hearts actually release less adenosine during ischemia than nonpreconditioned hearts (Van Wylen, 1994). Second, when canine hearts are treated with an adenosine deaminase inhibitor, the adenosine level during ischemia is increased several orders of magnitude without ensuing protection (Silva et al., 1995). Finally, it was shown that protection in rabbit hearts treated with a direct activator of PKC could not be blocked by an adenosine receptor antagonist (Iliodromitis et al., 1998). However, a PKC blocker completely prevented protection from an adenosine receptor agonist (Iliodromitis et al., 1998). Thus PKC is clearly downstream of adenosine in the preconditioning pathway, rather than upstream as proposed by Kitakaze. These observations in no way diminish the importance of activation of 5⬘-nucleotidase in preconditioning. Adenosine must be released at two important times for protection to occur. Following onset of the preconditioning ischemia, adenosine is released and serves as a trigger for protection. During the subsequent prolonged ischemia, this agonist again must be released to mediate the protection (Thornton et al., 1993). However, during the second occlusion, tissue pH will not become as

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FIGURE 6 Change in osmotic fragility of pelleted isolated rabbit ventricular cardiomyocytes as determined by the inability of cell membranes to prevent cellular uptake of trypan blue. The progressive increase in osmotic fragility as the duration of pelleting and simulated ischemia was prolonged in control cardiomyocytes was diminished significantly in preconditioned cells (PC), which underwent 10 min of pelleting to simulate ischemia and 15 min of reperfusion before the longer period of pelleting. Administration of the inhibitory PKC-␧ peptide V1-2 completely negated this preconditioning effect. (A) Osmotic fragility curves and (B) the average area under the curves. Areas under control curves and curves of PC cells treated with the ␧ inhibitor peptide were significantly greater than the area under the curve for PC cells (p ⬍ 0.05). Each point represents a group average and bars represent SEM. Modified and reproduced with permission from Liu et al. (1999).

acidic. While this attenuated acidosis can be shown to be unrelated to preconditioning’s protection, the elevated pH does cause an increased adenosine kinase activity, which in turn would result in less adenosine accumulation in the interstitial space (Decking et al., 1997). Activation of 5⬘-nucleotidase opposes this effect and guarantees that a sufficient amount of adenosine will be present during the second occlusion. Thus, activation of 5⬘-nucleotidase may serve as a positive feedback system to reinforce protection.

X. PRECONDITIONING’S MEMORY IS NOT DEPENDENT ON PHOSPHORYLATION BY PKC As noted earlier, the nature of preconditioning’s memory is obscure. According to Kitakaze’s 5⬘-nucleotidase theory, the memory is believed to be related to

phosphorylation of the enzyme. As long as the activating phosphate group remains on 5⬘-nucleotidase, the heart would be protected. While the central role of 5⬘-nucleotidase in preconditioning’s signaling pathway is in doubt, it is still possible that the phosphorylation of some other protein could be involved. However, this seems unlikely. PKC should be the first kinase in the pathway. The heart can still be ischemically preconditioned by a 5min coronary occlusion in the presence of staurosporine, a PKC antagonist that blocks PKC’s ability to phosphorylate substrates, as long as staurosporine is washed out prior to the prolonged ischemic period. However, introduction of staurosporine just prior to the long ischemia is sufficient to completely abort PC’s protection against infarction (Yang et al., 1997). Thus it would appear that phosphorylation by PKC is required only during the long occlusion and that the memory itself is not related to PKC-dependent phosphorylation. Furthermore, the ‘‘memory step’’ must reside upstream of PKC’s kinase

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activity. One possibility is that PKC’s translocation may constitute the memory. Staurosporine reversibly attaches to PKC’s ATP-binding site and blocks kinase activity, but not PKC’s ability to translocate. We have proposed that an isozyme of PKC may translocate to a critical compartment in the cell, and that as long as it is there it is poised to protect the cell with the advent of the next episode of ischemia. There is neither convincing evidence for nor against the translocation hypothesis of memory at this time.

ischemic preconditioning (Vahlhaus et al., 1996, 1998). However, if they were combined, protection was completely eliminated (Vahlhaus et al., 1998). This behavior suggests the existence of another signal transduction pathway that parallels PKC and contains at least one tyrosine kinase. The rat heart may act similarly (Tanno et al., 1998; Fryer et al., 1999). Multiple cycles of preconditioning can also overcome the abrogation of protection by PKC blockers in the rabbit heart, suggesting the presence of a similar bypass pathway in that species (Sandhu et al., 1997; Miura et al., 1998). Virtually nothing is known about the bypass pathway.

XI. TYROSINE KINASES What is beyond PKC and what is the end effector of protection? These questions have fueled much investigation. There are predicted to be approximately 4000 kinases expressed in the cell, and finding the one(s) associated with ischemic preconditioning may be a difficult task. There are some clues, however. The isoflavone genistein, a relatively selective tyrosine kinase antagonist by virtue of competitive inhibition of the enzyme’s ATP-binding site, blocks the enhanced postischemic functional recovery seen in preconditioned rat hearts (Maulik et al., 1996b). Protection following ischemic preconditioning in the rabbit can also be aborted by the administration of either genistein or the more selective antagonist lavendustin A, a noncompetitive inhibitor at the ATP-binding site as well as an uncompetitive inhibitor at the substrate-binding site (Baines et al., 1998) (Fig. 7). As with staurosporine, these kinase inhibitors are effective only when given just before the long ischemia rather than at the time of the brief preconditioning ischemia. There are two major groups of tyrosine kinases: receptor tyrosine kinases, such as those associated with the various growth factor receptors, and nonreceptor/ cytosolic tyrosine kinases, such as the pp60src family of kinases (Cantley et al., 1991). Tyrosine kinases can be either upstream or downstream of PKC in kinase cascades. Receptor tyrosine kinases may stimulate PLD (Maulik et al., 1996a), which in turn would activate PKC. Alternatively, PMA can elicit PKC-dependent tyrosine phosphorylation in cells. Because both genistein and lavendustin A can block the protection induced by PMA, a direct activator of PKC, it is apparent that the involved tyrosine kinase is unlikely to be part of a surface receptor, but rather is downstream of PKC (Baines et al., 1998). In the rabbit it appears that PKC and tyrosine kinase are in series. In other species, however, tyrosine kinases may also be present in a second pathway that bypasses PKC. In the pig heart, neither antagonists of PKC nor tyrosine kinase alone could block protection from

XII. MAP KINASES We have proposed that the p38 mitogen-activated protein kinase (MAPK) pathway is important in preconditioning. There are three major MAPK cascades, which are presented diagrammatically in Fig. 8. These MAPK cascades can be activated by receptor tyrosine kinases, PLC, G-protein-coupled receptors, and diverse cellular stresses and all have been identified in the heart (Robinson and Cobb, 1997). The 42/44-kDa extracellularregulated kinase (ERK) cascade is clearly activated by growth factor and G-protein-coupled receptors, but there is no evidence that it is involved in ischemic preconditioning. The two stress-activated MAPK families, the 46/54-kDa c-Jun N-terminal kinase (JNK) and the 38-kDa p38/reactivating kinase, are activated in response to environmental stresses such as UV radiation, osmotic shock, cytokines, lipopolysaccharide, and ischemia (Raingeaud et al., 1995; Sugden and Clerk, 1998). It is somewhat uncertain how ischemia actually targets these cascades. The link to PKC is even more tenuous. The JNK family consists of at least two isoforms: the 46-kDa JNK1 and the 54-kDa JNK2, both of which are present in the heart (Clerk et al., 1998). Clerk et al. (1998) have reported that both are strongly activated upon reperfusion, but are not affected by ischemia alone. Schaper’s group has reported significant increases in JNK activity in preconditioned pig hearts (Barancik et al., 1997) and has correlated a reduction in infarction with increased JNK activity in hearts exposed to the bacterial product anisomycin, which increases both JNK and p38 MAPK activities (Barancik et al., 1999). Most attention has been focused on the p38 MAPK cascade. At least five isoforms of p38 MAPK have been identified, although only p38 움 and 웁 are expressed to any degree within the heart (Sugden and Clerk, 1998). p38 MAPK, like all of the MAP kinases, has two activation sites, which must be phosphorylated for activation: a threonine at amino acid 180 and a tyrosine at site 182.

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FIGURE 7 Infarct size as a percentage of risk zone plotted on the ordinate for control and ischemically preconditioned (PC) hearts showing the dramatic salvage in the latter (p ⬍ 0.05). When either of the tyrosine kinase blockers genistein (GEN) or lavendustin A (LAV) was infused late (L) from shortly before to the middle of the 30-min coronary occlusion in ischemically preconditioned hearts, protection was blocked. However, early (E) infusion of genistein to bracket the preconditioning ischemia had no effect on protection. Neither kinase inhibitor had any effect on infarction when infused in nonpreconditioned hearts. Open symbols represent infarct size of individual animals, whereas closed symbols represent mean group infarction and bars indicate SEM.

FIGURE 8 Schema of activation of mitogen (MAPK)- and stress (SAPK)-activated protein kinases. The three MAP kinases, p38/RK, JNK, and ERK 1/2, are highlighted. Various stresses can activate the cascades and all pathways eventually lead to gene expression. p38 MAPK also results in phosphorylation of the small heat shock protein HSP27, which controls actin filament polymerization. Modified and reproduced with permission from Cohen et al. (2000).

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This kinase is activated by a MAP kinase kinase or MEK, which itself is activated by a MAP kinase kinase kinase (Fig. 8). We have examined phosphorylation of p38 MAPK’s tyrosine-182 residue, which is required for the kinase’s activation (Weinbrenner et al., 1997). Phosphorylation increases dramatically during the prolonged ischemia of preconditioned hearts, with a peak after 20 min of ischemia (Fig. 9). This increased phosphorylation of the kinase is not observed in either nonpreconditioned hearts or preconditioned hearts in which protection has been blocked by an adenosine receptor blocker. Thus phosphorylation of the p38 MAPK activation site occurred only during ischemia, and then only when protection from preconditioning was present. It is notable that this increase in phosphorylation occurred only during the prolonged ischemia in the preconditioned heart and, therefore, coincided with the timing of PKC and tyrosine kinase activation. SB203580 is a highly specific inhibitor of p38 MAPK (Cuenda et al., 1995). In isolated rabbit cardiomyocytes this antagonist prevents the protective effect of brief simulated ischemia on osmotic fragility (Weinbrenner et al., 1997) and blocks the anti-infarct effect of brief ischemia in isolated rat hearts (Maulik et al., 1998). These data implicating participation of this stress-activated kinase are further supported by the observation that anisomycin, an activator of p38 MAPK as well as JNK, will protect the ischemic heart when administered in lieu of brief ischemia (Baines et al., 1999). Further-

FIGURE 9 Phosphorylation of tyrosine-182 of p38 MAPK during 30 min of global ischemia in control and ischemically preconditioned (PC) rabbit hearts and preconditioned hearts also treated with the adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT). Whereas there was no change in the extent of phosphorylation in control hearts, PC increased phosphorylation significantly (p ⬍ 0.05) with a peak of 2.7-fold after 20 min of ischemia. This increase was blocked by SPT. Each point represents a group average and bars indicate SEM. Modified and reproduced with permission from Weinbrenner et al. (1997).

more, anisomycin’s protective effect in cardiomyocytes is aborted by SB203580 (Weinbrenner et al., 1997), suggesting that p38 MAPK had mediated the protection. However, anisomycin also activates JNK (Zanke et al., 1996; Foltz et al., 1998), and, therefore, the latter supportive data cannot unequivocally establish participation of p38 MAPK. It must be acknowledged that not all investigators have reported that SB203580 blocks protection in preconditioned tissue. Armstrong et al. (1999) have noted that this agent promotes injury in nonpreconditioned cells, whereas some reports claim that this agent is actually protective (Nagarkatti and Sha’afi, 1998; Mackay and Mochly-Rosen, 1999). It has been documented that SB203580 may not be as selective as once believed. It can activate Raf-1, which is directly involved in the PKC cascade (Kalmes et al., 1999). Therefore, it is possible that under certain conditions the adverse antagonistic effects of SB203580 on the stress-activated kinase cascade may be counterbalanced by beneficial stimulatory effects elsewhere.

XIII. HSP 27 p38 MAP kinase in turn phosphorylates and activates MAPK-activated protein kinase 2 (MAPKAPK-2) (Freshney et al., 1994) (Fig. 8). Interestingly, the latter phosphorylates one of the chaperone heat shock proteins (HSP25/27) (Freshney et al., 1994). In its unphosphorylated state, HSP27 acts as a cytoskeletal actin end cap and blocks actin polymerization (Guay et al., 1997). However, once phosphorylated, HSP27 actually enhances actin polymerization. Therefore, this kinase cascade can influence the integrity of the cell’s cytoskeleton. Activation of the p38 MAPK/HSP27 pathway has been shown to prevent oxygen radical-induced fragmentation of actin filaments, thus preserving cell viability (Huot et al., 1996). Overexpression of HSP27 in isolated rat myocytes confers protection against simulated ischemia, whereas depletion exacerbates injury (Martin et al., 1997). Prolonged ischemia is known to cause cytoskeletal disruption (Ganote and Armstrong, 1993). The ischemic cell finds it increasingly difficult to pump out sodium and maintain an osmotic equilibrium. In the end the ischemic cell bursts from the osmotic forces. Activation of the p38 MAPK/HSP27 pathway could well be protective against swelling by maintaining the integrity of the actin cytoskeleton. There is little direct evidence, however, to support this attractive hypothesis. Nonetheless, cytochalasin D, a drug known to disrupt the cytoskeleton, did block the protective effect of preconditioning on the increasing osmotic fragility of isolated ischemic rabbit cardiomyocytes (Baines et al., 1999).

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XIV. KATP CHANNELS Many years ago it was proposed that the ATPdependent K⫹ (KATP) channel might be a prime candidate for the end effector. The KATP channel is composed of two subunits, both of which are absolutely required for channel activity. For each channel protein, which itself belongs to the inward rectifier K⫹ channel family, there is an associated sulfonylurea receptor involved in the binding of ATP and sulfonylurea compounds (Trapp and Ashcroft, 1997; Aguilar-Bryan et al., 1998). KATP channels are inhibited by ATP in the low micromolar range and open as ATP levels fall (Trapp and Ashcroft, 1997). It was reasoned that as the ATP concentration fell in the ischemic cells, these KATP channels would open, resulting in a shortening of the action potential. The latter was supposed to spare the cell’s dwindling energy supply, ultimately leading to protection (Grover, 1997). Several investigators reported that exogenous KATP channel openers such as pinacidil indeed preserved postischemic function in guinea pig myocardium (Cole et al., 1991) and reduced infarction and cell death in dogs (Grover et al., 1990) when administered before the long period of ischemia. Interestingly, subsequent evaluations of several KATP openers, including bimakalim (Yao and Gross, 1994) and cromakalim (Grover et al., 1995), revealed a dissociation between cardiac protection and shortening of the action potential. It has become apparent that there are two distinct populations of KATP channels in the myocardial cell. Sarcolemmal and mitochondrial channels have different reactivities and different properties. Whereas pinacidil will open and glibenclamide will close both at the concentrations generally employed, diazoxide will open mitochondrial channels with at least a 2000-fold greater selectivity (Garlid et al., 1996; Liu et al., 1998), and 5-hydroxydecanoate (5HD) is a potent closer of mitochondrial channels (McCullough et al., 1991; Garlid et al., 1997). Furthermore, a recently introduced compound, HMR 1883, will close surface channels but not mitochondrial KATP channels (Go¨gelein et al., 1998; Sato et al., 2000). These pharmacologic tools have permitted in-depth investigation of these channels. Diazoxide will protect ischemic rat (Garlid et al., 1997) and rabbit (Baines et al., 1999) hearts and isolated rabbit cardiomyocytes (Liu et al., 1998), whereas 5HD blocks the protection of ischemic preconditioning (Garlid et al., 1997; Liu et al., 1998; Baines et al., 1999; Sato et al., 2000). Furthermore, HMR 1883 has no effect on the infarctsparing action of preconditioning (Birincioglu et al., 1999). Therefore, it appears that the mitochondrial, not sarcolemmal, KATP channel is responsible for preconditioning’s myocardial protection. Some important observations link the KATP channels

FIGURE 10 Infarct size as a percentage of risk zone plotted on the ordinate in control and ischemically preconditioned rabbit hearts demonstrating significantly less infarction in the latter (p ⬍ 0.05). Anisomycin (aniso), which activates both p38 MAPK and JNK, administered in lieu of the initial brief ischemia also protected ischemic hearts (p ⬍ 0.05), but this protective effect was blocked by the mitochondrial KATP channel antagonist 5-hydroxydecanoate (5HD). Open symbols represent the infarct size of individual animals, whereas closed symbols represent mean group infarction and bars indicate SEM. Modified and reproduced with permission from Baines et al. (1999).

to the already established signaling cascade. For example, protection induced by PMA in isolated rabbit hearts could be nullified by 5HD (Van Winkle et al., 1995). Furthermore, the same closer of mitochondrial channels can block the protective effect of anisomycin (Baines et al., 1999) (Fig. 10). The latter report implies that the kinase cascade activated during preconditioning is upstream of the KATP channels and ultimately leads to channel opening. Opening of the KATP channel has two known effects. First, channel opening would result in the movement of water into the mitochondria, causing them to swell. Second, opening the channels would short circuit the hydrogen ion gradient across the mitochondria and uncouple them slightly. It is unknown why either of these effects would be beneficial to the ischemic cell. Of course, it is still not proven that the mitochondrial KATP channel is indeed the end effector. One must still be receptive to other suggestions. Diaz et al. (1999) proposed that opening of a chloride rather than a potassium channel is responsible for the protection.

XV. PRESERVATION OF ATP In the earliest work on preconditioning it was noted that ATP appeared to fall more slowly in preconditioned dog heart (Murry et al., 1990). That observation led to the hypothesis that improved energetics were responsible for preconditioning’s protection. However, available

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data are not completely supportive. While most studies do show a slower utilization of ATP in ischemic tissue that has previously been preconditioned, they also show that at the beginning of the prolonged ischemia the myocardium already has a large deficit of ATP, which is related to metabolism during the preconditioning ischemia itself. Furthermore, Kolocassides et al. (1996) observed that preconditioning could be demonstrated in rat hearts even when the initial deficit of ATP was so great that the energetics of the preconditioned hearts never caught up to those in nonpreconditioned counterparts.

XVI. DOES SALVAGE OF ISCHEMIC MYOCARDIUM TRANSLATE INTO IMPROVED MECHANICAL FUNCTION? Despite our incomplete knowledge of the signaling pathway, it is clear that preconditioning salvages ischemic myocardium and minimizes infarction. A logical corollary would be that less infarcted myocardium should then lead to improved left ventricular function. Clinically, patients with coronary artery disease and myocardial infarction without arrhythmias most commonly die of progressive left ventricular systolic dysfunction and pump failure. Therefore, preconditioning should arrest or at least attenuate this process. Ample evidence in in vitro rat hearts has demonstrated that the recovery of left ventricular developed pressure after coronary occlusion is indeed improved if the latter were preceded by a preconditioning stimulus (Asimakis et al., 1992). However, it has always been difficult to distinguish whether this improvement was the result of attenuation of stunning and/or infarction. Improved postischemic function has been much more difficult to demonstrate in in situ models of regional ischemia, probably because marked stunning of any surviving myocardium renders the entire segment akinetic for the first few hours of reperfusion. Indeed, most studies have reported that ischemic preconditioning preceding coronary occlusions of 30–60 min in the rabbit (Sandhu et al., 1993; Jenkins et al., 1995; Lasley et al., 1995), dog (Ovize et al., 1992; Shizukuda et al., 1992), or pig (Schott et al., 1990) had no salutary effect on postischemic segment shortening, wall thickening, or left ventricular systolic or end diastolic pressures when measurements were confined to the first few hours after reperfusion. Such a short reperfusion period would not have allowed stunned myocardium sufficient time to recover. The only exception is the study by Qiu et al. (1997) in the pig in which a benefit from preconditioning was seen immediately after reperfusion. However, this is not the experience in rabbits in which there is little difference

FIGURE 11 Segment shortening as measured by intramyocardial piezoelectric crystals in chronically instrumented control and ischemically preconditioned (PC) rabbits and presented as a percentage of baseline, preischemic values on the ordinate. Each point represents a group average and bars indicate SEM. In control rabbits, segment shortening declined precipitously at 0 min when the coronary artery was occluded and the monitored segment bulged (negative segment shortening) for the duration of the 30-min coronary occlusion. Following reperfusion there was negligible recovery for the first hour. During the ensuing 71 hr, minimal recovery resulted in a final segment shortening of only 14%. Preconditioned rabbits underwent an initial 5-min coronary occlusion during which the myocardial segment bulged. Recovery during the 10-min reperfusion period was not complete because of myocardial stunning. During the 30-min coronary occlusion and initial 1-hr reperfusion there were no differences between control and preconditioned hearts. However, over the next 71 hr, recovery of function was significantly better than in control hearts (p ⫽ 0.02) with final segment shortening of 44%. Modified and reproduced with permission from Cohen et al. (1999).

in myocardial systolic shortening between control and ischemically preconditioned hearts very early following reperfusion (Cohen et al., 1999). A difference begins to emerge after 24 hr and becomes increasingly apparent at 72 hr, at which time 44% of systolic shortening has returned in preconditioned hearts in contrast to only 14% return in control hearts (Fig. 11). It was also striking that infarction of only 10% of the affected segment caused a 56% deficit in segment shortening. Thus salvage of ischemic tissue should not be expected to translate to immediate clinical improvement in left ventricular function. The improvement will likely occur gradually, but will still be more complete in preconditioned hearts.

XVII. PRECONDITIONING IN HUMANS: CLINICAL ASPECTS Preconditioning is now a well-accepted laboratory phenomenon, and many of the steps in the signal transduction cascade have been established (Fig. 12). However, it has been more difficult to prove its existence in humans. As noted previously, hypoxic preconditioning

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FIGURE 12 The signal transduction pathway used in preconditioning from receptor stimulation to opening of the mitochondrial KATP channel. Many steps have not yet been fully defined (shaded area), and it is still not certain that the KATP channel is the end effector.

can indeed protect isolated human ventricular cardiomyocytes (Ikonomidis et al., 1997) and strips of atrial (Speechly-Dick et al., 1995; Cleveland et al., 1997) and ventricular (Cleveland et al., 1996) myocardium. Several approaches have been used in an attempt to prove preconditioning’s existence in the intact human heart, but each has obvious shortcomings, which leave the issue unresolved. An early study by Yellon et al. (1993) reported that short periods of aortic cross-clamping prior to induced ventricular fibrillation for the coronary anastomosis of a bypass graft resulted in greater preservation of myocardial ATP levels than in nonpreconditioned hearts. However, a follow-up study by the same group failed to confirm these observations (Jenkins et al., 1997). Although Jenkins and colleagues (1997) noted that serum troponin T was modestly higher in nonpreconditioned patients following revascularization surgery, others have observed that there is a significantly greater creatine kinase MB release following cardioplegic arrest in patients having undergone preconditioning than controls (Perrault et al., 1996). Perhaps the most popular method introduced to prove preconditioning exists in humans has been the documentation of changes in the ECG recorded by an intracoronary electrode during serial inflations of an angioplasty balloon. It has been observed repeatedly

that the extent of S-T segment elevation during the second 2-min coronary occlusion is noticeably less than during the initial balloon inflation (Deutsch et al., 1990; Cribier et al., 1992; Tomai et al., 1994). It was proposed that this change was the result of ischemic preconditioning, although opening of collateral vessels could not be discounted. Studies in coronary collateral-deficient pigs (Shattock et al., 1996) and rabbits (Birnbaum et al., 1996; Cohen et al., 1997) supported the notion that the declining magnitude of S-T segment elevation could indeed be the result of preconditioning. Importantly, however, the contribution of coronary collateral vessels to the S-T segment response in humans still cannot be discounted. Several investigators have nicely demonstrated that coronary collateral flow during coronary angioplasty can increase from the first to second or third coronary occlusion (Cribier et al., 1992; Billinger et al., 1999). Increased collateral flow to the ischemic region would diminish the consequences of coronary occlusion without the need to postulate the existence of other cardioprotective mechanisms, such as ischemic preconditioning. More recent investigations using the rabbit have demonstrated a dissociation between myocardial cardioprotection and declining maximum S-T segment voltage recorded during sequential coronary occlusions (Birincioglu et al., 1999). Whereas the infarct-sparing

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effect of preconditioning appears to be associated with the opening of mitochondrial KATP channels, it is the opening of sarcolemmal KATP channels that results in the lessening of S-T segment elevation during the second and subsequent coronary occlusions. With ischemic preconditioning, apparently both populations of channels open simultaneously. When these channels are manipulated pharmacologically, however, it cannot be assumed that modification of the S-T segment voltage implies anything about the state of protection of the heart. Finally, a third popular tool has been the use of population studies to demonstrate that those individuals with preinfarction angina have better postinfarction left ventricular function and better prognosis than those patients in whom infarction was unheralded (Hirai et al., 1992; Anzai et al., 1994; Kloner et al., 1995; Nakagawa et al., 1995; Ottani et al., 1995; Kloner et al., 1998). These retrospective analyses have at times concluded that preinfarction angina, even up to a month before the episode of infarction, is protective. Based on experimental observations, it hardly seems possible that such remote episodes of angina could have such a long-lasting preconditioning effect. Critical review of these reports reveals some other important reservations. First, in some studies the possible effect of coronary collateral development in those with recurrent angina pectoris preceding the infarction has not been considered because of the absence of cardiac catheterization data. However, coronary collaterals are more prevalent in those with preceding angina (Hirai et al., 1992, 1993), and even in this latter group, regional left ventricular function is better in those with more developed collaterals. The importance of coronary collaterals when considering preconditioning has already been discussed. Second, those individuals with preinfarction angina lyse the thrombus occluding the coronary artery and reestablish distal flow faster than those without angina (Andreotti et al., 1996). Reperfusion occurs approximately 21 min sooner after the initiation of infusion of the thrombolytic agent in those with prior angina. Reasons for this difference are uncertain, but more rapid reperfusion might result in less infarction, which could translate into an improved clinical course without any consideration of the preconditioning phenomenon. Finally, at least one study has demonstrated clinical benefits of preinfarction angina only in those younger than 65 years (Abete et al., 1997), thus limiting the general applicability of any protective mechanism. Hence, it is unclear whether these population studies have yet proved unequivocally that ischemic preconditioning exists in humans.

XVIII. SUMMARY Preconditioning is a powerful force that is clearly present in experimental animals and which is also probably evident in humans, although available tools are not yet satisfactory to prove the latter. We have learned a lot about the signal transduction pathway, but there are still multiple steps beyond PKC that have yet to be defined and the nature of the end effector has not been established. Intimate knowledge of this pathway should allow harnessing of preconditioning’s power to treat and even prevent ischemic damage of the heart. There are still many hurdles that have to be cleared. For example, to be effective, most known triggers of preconditioning must be administered before the onset of ischemia. However, patients with myocardial infarction typically present after coronary occlusion and initiation of the ischemic/necrotic process. Therefore, an effective treatment that can be administered after the onset of ischemia is required. However, there is general confidence that these difficulties can be overcome and that preconditioning of the human heart will become a routine clinical practice.

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may mediate the protection afforded by preconditioning in the isolated rabbit heart. Cardiovasc. Res. 28, 1057–1061. Liu, G. S., Thornton, J., Van Winkle, D. M., Stanley, A. W. H., Olsson, R. A., and Downey, J. M. (1991). Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 84, 350–356. Liu, Y., Cohen, M. V., and Downey, J. M. (1994). Chelerythrine, a highly selective protein kinase C inhibitor, blocks the antiinfarct effect of ischemic preconditioning in rabbit hearts. Cardiovasc. Drugs Ther. 8, 881–882. Liu, Y., Sato, T., O’Rourke, B., and Marban, E. (1998). Mitochondrial ATP-dependent potassium channels: Novel effectors of cardioprotection? Circulation 97, 2463–2469. Liu, Y., Tsuchida, A., Cohen, M. V., and Downey, J. M. (1995). Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J. Mol. Cell. Cardiol. 27, 883–892. Mackay, K., and Mochly-Rosen, D. (1999). An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia. J. Biol. Chem. 274, 6272–6279. Martin, J. L., Mestril, R., Hilal-Dandan, R., Brunton, L. L., and Dillmann, W. H. (1997). Small heat shock proteins and protection against ischemic injury in cardiac myocytes. Circulation 96, 4343– 4348. Maulik, N., Watanabe, M., Engelman, R. M., Zu, Y.-L., Huang, C.-K., Cordis, G. A., and Das, D. K. (1996a). Preconditioning triggers tyrosine kinase-phospholipase D signaling leading to activation of multiple protein kinases in heart. J. Mol. Cell. Cardiol. 28, A187. [Abstract] Maulik, N., Watanabe, M., Zu, Y.-L., Huang, C.-K., Cordis, G. A., Schley, J. A., and Das, D. K. (1996b). Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett. 396, 233–237. Maulik, N., Yoshida, T., Zu, Y.-L., Sato, M., Banerjee, A., and Das, D. K. (1998). Ischemic preconditioning triggers tyrosine kinase signaling: A potential role for MAPKAP kinase 2. Am. J. Physiol. 275, H1857–H1864. McCullough, J. R., Normandin, D. E., Conder, M. L., Sleph, P. G., Dzwonczyk, S., and Grover, G. J. (1991). Specific block of the anti-ischemic actions of cromakalim by sodium 5-hydroxydecanoate. Circ. Res. 69, 949–958. Miki, T., Cohen, M. V., and Downey, J. M. (1998). Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits. Mol. Cell. Biochem. 186, 3–12. Mitchell, M. B., Meng, X., Ao, L., Brown, J. M., Harken, A. H., and Banerjee, A. (1995). Preconditioning of isolated rat heart is mediated by protein kinase C. Circ. Res. 76, 73–81. Miura, T., Adachi, T., Ogawa, T., Iwamoto, T., Tsuchida, A., and Iimura, O. (1992). Myocardial infarct size-limiting effect of ischemic preconditioning: Its natural decay and the effect of repetitive preconditioning. Cardiovasc. Pathol. 1, 147–154. Miura, T., Miura, T., Kawamura, S., Goto, M., Sakamoto, J., Tsuchida, A., Matsuzaki, M., and Shimamoto, K. (1998). Effect of protein kinase C inhibitors on cardioprotection by ischemic preconditioning depends on the number of preconditioning episodes. Cardiovasc. Res. 37, 700–709. Miyawaki, H., and Ashraf, M. (1997). Ca2⫹ as a mediator of ischemic preconditioning. Circ. Res. 80, 790–799. Mochly-Rosen, D., and Gordon, A. S. (1998). Anchoring proteins for protein kinase C: A means for isozyme selectivity. FASEB J. 12, 35–42. Murry, C. E., Jennings, R. B., and Reimer, K. A. (1986). Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136. Murry, C. E., Richard, V. J., Jennings, R. B., and Reimer, K. A. (1991). Myocardial protection is lost before contractile function

recovers from ischemic preconditioning. Am. J. Physiol. 260, H796–H804. Murry, C. E., Richard, V. J., Reimer, K. A., and Jennings, R. B. (1990). Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ. Res. 66, 913–931. Nagarkatti, D. S., and Sha’afi, R. I. (1998). Role of p38 MAP kinase in myocardial stress. J. Mol. Cell. Cardiol. 30, 1651–1664. Nakagawa, Y., Ito, H., Kitakaze, M., Kusuoka, H., Hori, M., Kuzuya, T., Higashino, Y., Fujii, K., and Minamino, T. (1995). Effect of angina pectoris on myocardial protection in patients with reperfused anterior wall myocardial infarction: Retrospective clinical evidence of ‘‘preconditioning.’’ J. Am. Coll. Cardiol. 25, 1076– 1083. Ottani, F., Galvani, M., Ferrini, D., Sorbello, F., Limonetti, P., Pantoli, D., and Rusticali, F. (1995). Prodromal angina limits infarct size: A role for ischemic preconditioning. Circulation 91, 291–297. Ovize, M., Kloner, R. A., Hale, S. L., and Przyklenk, K. (1992). Coronary cyclic flow variations ‘‘precondition’’ ischemic myocardium. Circulation 85, 779–789. Perrault, L. P., Menasche´, P., Bel, A., de Chaumaray, T., Peynet, J., Mondry, A., Olivero, P., Emanoil-Ravier, R., and Moalic, J.-M. (1996). Ischemic preconditioning in cardiac surgery: A word of caution. J. Thorac. Cardiovasc. Surg. 112, 1378–1386. Ping, P., Zhang, J., Qiu, Y., Tang, X.-L., Manchikalapudi, S., Cao, X., and Bolli, R. (1997). Ischemic preconditioning induces selective translocation of protein kinase C isoforms ␧ and ␩ in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ. Res. 81, 404–414. Przyklenk, K., Sussman, M. A., Simkhovich, B. Z., and Kloner, R. A., (1995). Does ischemic preconditioning trigger translocation of protein kinase C in the canine model? Circulation 92, 1546–1557. Qiu, Y., Tang, X.-L., Park, S.-W., Sun, J.-Z., Kalya, A., and Bolli, R. (1997). The early and late phases of ischemic preconditioning: A comparative analysis of their effects on infarct size, myocardial stunning, and arrhythmias in conscious pigs undergoing a 40-minute coronary occlusion. Circ. Res. 80, 730–742. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995). Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem. 270, 7420–7426. Rischard, F., and McKean, T. (1998). Ischemia and ischemic preconditioning in the buffer-perfused pigeon heart. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 119, 59–65. Robinson, M. J., and Cobb, M. H. (1997). Mitogen-activated protein kinase pathways. Curr. Opin. Cell. Biol. 9, 180–186. Sandhu, R., Diaz, R. J., Mao, G. D., and Wilson, G. J. (1997). Ischemic preconditioning: Differences in protection and susceptibility to blockade with single-cycle versus multicycle transient ischemia. Circulation 96, 984–995. Sandhu, R., Diaz, R. J., and Wilson, G. J. (1993). Comparison of ischaemic preconditioning in blood perfused and buffer perfused isolated heart models. Cardiovasc. Res. 27, 602–607. Sato, T., Sasaki, N., Seharaseyon, J., O’Rourke, B., and Marba´n, E. (2000). Selective pharmacological agents implicate mitochondrial but not sarcolemmal KATP channels in ischemic cardioprotection. Circulation 101, 2418–2423. Schott, R. J., Rohmann, S., Braun, E. R., and Schaper, W. (1990). Ischemic preconditioning reduces infarct size in swine myocardium. Circ. Res. 66, 1133–1142. Schultz, J. E. J., Hsu, A. K., and Gross, G. J. (1998). Ischemic preconditioning in the intact rat heart is mediated by 웃1-but not 애- or ␬opioid receptors. Circulation 97, 1282–1289. Schulz, R., Post, H., Vahlhaus, C., and Heusch, G. (1998). Ischemic preconditioning in pigs: A graded phenomenon: Its relation to adenosine and bradykinin. Circulation 98, 1022–1029.

50. Ischemic Preconditioning Schwiebert, E. H., Karlson, K. H., Friedman, P. A., Dietl, P., Spielman, W. S., and Stanton, B. A. (1992). Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line. J. Clin. Invest. 89, 834–841. Shattock, M. J., Lawson, C. S., Hearse, D. J., and Downey, J. M. (1996). Electrophysiological characteristics of repetitive ischemic preconditioning in the pig heart. J. Mol. Cell. Cardiol. 28, 1339– 1347. Shizukuda, Y., Mallet, R. T., Lee, S.-C., and Downey, H. F. (1992). Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc. Res. 26, 534–542. Silva, P. H., Dillon, D., and Van Wylen, D. G. L. (1995). Adenosine deaminase inhibition augments interstitial adenosine but does not attenuate myocardial infarction. Cardiovasc. Res. 29, 616–623. Souroujon, M. C., and Mochly-Rosen, D. (1998). Peptide modulators of protein-protein interactions in intracellular signaling. Nature Biotechnol. 16, 919–924. Speechly-Dick, M. E., Grover, G. J., and Yellon, D. M. (1995). Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K⫹ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ. Res. 77, 1030–1035. Sugden, P. H., and Clerk, A. (1998). ‘‘Stress-responsive’’ mitogenactivated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ. Res. 83, 345–352. Tanno, M., Tsuchida, A., Hasegawa, T., Miura, T., and Shimamoto, K. (1998). Both protein kinase C (PKC) and tyrosine kinase contribute to cardioprotection by repetitive preconditioning in rats. J. Mol. Cell. Cardiol. 30, A312. [Abstract] Thornton, J. D., Liu, G. S., and Downey, J. M. (1993). Pretreatment with pertussis toxin blocks the protective effects of preconditioning: Evidence for a G-protein mechanism. J. Mol. Cell. Cardiol. 25, 311–320. Thornton, J. D., Liu, G. S., Olsson, R. A., and Downey, J. M. (1992). Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation 85, 659–665. Thornton, J. D., Thornton, C. S., and Downey, J. M. (1993). Effect of adenosine receptor blockade: Preventing protective preconditioning depends on time of initiation. Am. J. Physiol. 265, H504– H508. Tomai, F., Crea, F., Gaspardone, A., Versaci, F., De Paulis, R., Penta de Peppo, A., Chiariello, L., and Gioffre`, P. A. (1994). Ischemic preconditioning during coronary angioplasty is prevented by glibenclamide, a selective ATP-sensitive K⫹ channel blocker. Circulation 90, 700–705. Trapp, S., and Ashcroft, F. M. (1997). A metabolic sensor in action: News from the ATP-sensitive K⫹-channel. News Physiol. Sci. 12, 255–263. Tritto, I., D’Andrea, D., Eramo, N., Scognamiglio, A., De Simone, C., Violante, A, Esposito, A., Chiariello, M., and Ambrosio, G. (1997). Oxygen radicals can induce preconditioning in rabbit hearts. Circ. Res. 80, 743–748. Tsuchida, A., Liu, Y., Liu, G. S., Cohen, M. V., and Downey, J. M. (1994). 움1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ. Res. 75, 576–585. Vahlhaus, C., Schulz, R., Post, H., Onallah, R., and Heusch, G. (1996). No prevention of ischemic preconditioning by the protein kinase C inhibitor staurosporine in swine. Circ. Res. 79, 407–414.

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Vahlhaus, C., Schulz, R., Post, H., Rose, J., and Heusch, G. (1998). Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinase in pigs. J. Mol. Cell. Cardiol. 30, 197–209. Van Winkle, D. M., Kuzume, K., Dote, K., and Wolff, R. A. (1995). Infarct limitation by protein kinase C (PKC) is attenuated by blockade of ATP-sensitive potassium (KATP) channels. J. Mol. Cell. Cardiol. 27, A142. [Abstract] Van Winkle, D. M., Thornton, J. D., Downey, D. M., and Downey, J. M. (1991). The natural history of preconditioning: Cardioprotection depends on duration of transient ischemia and time to subsequent ischemia. Coron. Artery Dis. 2, 613–619. Van Wylen, D. G. L. (1994). Effect of ischemic preconditioning on interstitial purine metabolite and lactate accumulation during myocardial ischemia. Circulation 89, 2283–2289. Walsh, R. S., Borges, M., Thornton, J. D., Cohen, M. V., and Downey, J. M. (1995). Hypoxia preconditions rabbit myocardium by an adenosine receptor-mediated mechanism. Can. J. Cardiol. 11, 141–146. Wang, P., Downey, J. M., and Cohen, M. V. (1996a). Mast cell degranulation does not contribute to ischemic preconditioning in isolated rabbit hearts. Basic Res. Cardiol. 91, 458–467. Wang, P., Gallagher, K. P., Downey, J. M., and Cohen, M. V. (1996b). Pretreatment with endothelin-1 mimics ischemic preconditioning against infarction in isolated rabbit heart. J. Mol. Cell. Cardiol. 28, 579–588. Weinbrenner, C., Liu, G.-S., Cohen, M. V., and Downey, J. M. (1997). Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J. Mol. Cell. Cardiol. 29, 2383–2391. Yang, X.-M., Arnoult, S., Tsuchida, A., Cope, D., Thornton, J. D., Daly, J. F., Cohen, M. V., and Downey, J. M. (1993). The protection of ischaemic preconditioning can be reinstated in the rabbit heart after the initial protection has waned. Cardiovasc. Res. 27, 556–558. Yang, X.-M., Sato, H., Downey, J. M., and Cohen, M. V. (1997). Protection of ischemic preconditioning is dependent upon a critical timing sequence of protein kinase C activation. J. Mol. Cell. Cardiol. 29, 991–999. Yao, Z., and Gross, G. J. (1994). Effects of the KATP channel opener bimakalim on coronary blood flow, monophasic action potential duration, and infarct size in dogs. Circulation 89, 1769–1775. Ye, H., Wolf, R. A., Kurz, T., and Corr, P. B. (1994). Phosphatidic acid increases in response to noradrenaline and enodothelin-1 in adult rabbit ventricular myocytes. Cardiovasc. Res. 28, 1828–1834. Yellon, D. M., Alkhulaifi, A. M., and Pugsley, W. B. (1993). Preconditioning the human myocardium. Lancet 342, 276–277. Yellon, D. M., Baxter, G. F., Garcia-Dorado, D., Heusch, G., and Sumeray, M. S. (1998). Ischemic preconditioning: Present position and future directions. Cardiovasc. Res. 37, 21–33. Yoshida, K.-i., Kawamura, S., Mizukami, Y., and Kitakaze, M. (1997). Implication of protein kinase C-움, 웃, and ␧ isoforms in ischemic preconditioning in perfused rat hearts. J. Biochem. 122, 506–511. Ytrehus, K., Liu, Y., and Downey, J. M. (1994). Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am. J. Physiol. 266, H1145–H1152. Zanke, B. W., Rubie, E. A., Winnett, E., Chan, J., Randall, S., Parsons, M., Boudreau, K., McInnis, M., Yan, M., Templeton, D. J., and Woodgett, J. R. (1996). Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes. J. Biol. Chem. 271, 29876–29881.

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51 Cardioplegia and Surgical Ischemia D. J. CHAMBERS* and D. J. HEARSE† *Cardiac Surgical Research/Cardiothoracic Surgery Guy’s and St. Thomas’ NHS Trust and † Cardiovascular Research King’s Centre for Cardiovascular Biology and Medicine The Rayne Institute, King’s College St. Thomas’ Hospital, London SE1 7EH, England

I. INTRODUCTION

of ischemic injury and the meaning and significance of ‘‘reperfusion injury.’’

Ischemia of the human heart can occur in many forms and may persist for only a few seconds or minutes (angioplasty or angina), for hours (cardiac surgery or infarction), or for years (chronic ischemic heart disease). This chapter focuses on the global (whole heart) ischemia that is often deliberately induced during cardiac surgery (and transplantation) and the way in which the attendant myocardial injury can be limited. Emphasis will be given to cardioplegia as a means of protection; however, it should be stressed that there are alternative means by which the myocardium may be protected during surgery (such as intermittent ischemia with electrically induced fibrillation and the exploitation of endogenous adaptive defense mechanisms such as preconditioning) and these will be briefly discussed later. In order to facilitate the best surgery, cardiac surgeons require a still (nonbeating) and blood-free operating field, and this is achieved most easily by making the heart globally ischemic and transferring the task of blood circulation to a heart-lung machine. While this may be convenient for the surgeon, it is potentially damaging for the heart, and a great deal of research has been directed at rendering surgically induced ischemia less damaging. Before considering concepts of myocardial protection it is important to have an appreciation of the complex temporal, spatial, and molecular nature

Heart Physiology and Pathophysiology, Fourth Edition

II. ISCHEMIC INJURY Severe ischemia initiates a continuum of progressively severe molecular and cellular changes that, without reperfusion, will culminate in cell death. However, the metabolic and functional changes occurring during the early minutes of ischemia are reversible such that reperfusion at this time will lead to a rapid and complete recovery of cardiac function. As ischemia progresses, tissue damage becomes more severe, recovery takes longer, and, at some point, irreversible injury will occur. Beyond this point, reperfusion can have no beneficial effect. Indeed, some investigators believe that reperfusion may hasten, or even exacerbate, the development of irreversible injury in tissue that may have been only reversibly injured in the moments before reperfusion— this phenomenon has been termed ‘‘reperfusion injury’’ (see later). From the previous discussion, it is immediately evident that any cardioprotective intervention should be initiated as early in the ischemic process as possible so as to minimize any irreversible injury and maximize the rate and extent of recovery once reperfusion is achieved.

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In considering the temporal and spatial characteristics of ischemic injury, it is well established that the injury does not evolve uniformly throughout the myocardium; regional differences in metabolism and energy requirement render the endocardium most vulnerable to injury. Thus, cell death and tissue necrosis will develop first in the endocardium and spread as a ‘‘wavefront’’ toward the epicardium (1). Any strategy aimed at protecting the ischemic heart during cardiac surgery should have two principal objectives: (i) because reperfusion is an absolute requirement for the survival of the ischemic tissue, reflow should be initiated at the earliest possible opportunity, and (ii) if early reperfusion cannot be achieved, every attempt should be made to slow the rate of development of ischemic injury in order to delay the onset of irreversible injury. In this way, the maximum number of cells will be maintained in a reversible state of injury for as long as possible, thereby ensuring that as much tissue as possible is able to recover when reperfusion is eventually achieved. During cardiac surgery, the globally ischemic heart can usually be reperfused at will; however, the prolonged ischemic durations required for technically complex procedures or storage of hearts before transplantation require that injury-slowing procedures are instituted to protect the myocardium from the development of necrosis. Successfully achieving this goal of ‘‘injury delaying therapy’’ (2) requires a knowledge of (and an ability to manipulate) the many factors that influence the rate at which ischemic injury evolves. These include (i) the extent of residual, collateral, or noncoronary collateral flow delivered to the ischemic tissue, (ii) the effects of coexisting diseases such as hypertrophy, diabe-

tes, hypertension, and ischemic heart disease, (iii) heart rate, metabolic rate, and tissue temperature, (iv) differences in metabolic responses to ischemia, particularly with regard to patterns of substrate utilization, (v) the nutritional and hormonal status of the patient, (vi) the presence of cardioactive drugs, (vii) the existence of preexisting ischemia, and (viii) age, sex, and (for experimental studies) species. Inevitable differences in the relative contributions of these factors will result in highly variable rates of injury both within and between hearts—even when subjected to identical ischemic insults. Thus, contrary to popular opinion, there is no predefined time at which ischemic tissue becomes irreversibly injured. This variability is well illustrated in a coronary artery occlusion study by Schaper and colleagues (3)—a study that also demonstrates the vital importance of collateral or residual flow as a major determinant of tissue survival (Fig. 1). They demonstrated that necrosis occurs rapidly in animals with little or no collateral flow (such as the rabbit, rat, and pig, in which infarction may be complete in around 30 min), more slowly in animals with moderate levels of collateral flow (such as the dog and cat, in which some ischemic tissue may remain viable after several hours), but not at all in animals with extensive collaterals (such as the guinea pig) in which occlusion of a major coronary artery is fully compensated for by the recruitment of collateral flow from adjoining perfusion beds. It is debatable where humans fit into this scheme, but it is likely that the young human heart is ‘‘pig-like’’ in its collateral status, whereas the older human heart is more ‘‘doglike,’’ having benefited from the progressive development of coronary artery anastomoses that are often associated with the progression of ischemic heart disease.

FIGURE 1 Species differences in the rate of evolution of infarction, in relation to the duration of ischemia (followed by reperfusion), demonstrating the effect of differences in collateral flow. Redrawn from Schaper (3).

51. Cardioplegia and Surgical Ischemia

In the context of cardiac surgery, coronary-to-coronary anastomoses are unable to slow ischemic injury because the heart is globally ischemic. Noncoronary collateral flow, however, can deliver blood to the heart via bronchial, mediastinal, tracheal, esophageal, and diaphragmatic arteries at the pericardial reflections (4); this will vary greatly on an individual basis, being influenced by factors such as systemic pressure and various disease states. As a result, noncoronary collateral flow may vary from 3 to 10% of normal coronary flow. However, this warm nutritive flow, while offering the advantage of providing oxygen and substrates to the ischemic tissue, can have a negative effect by washing out cold cardioprotective solutions that had been infused into the heart at the onset of ischemia. This can result in the premature resumption of contractile activity, which can only be prevented by giving additional cardioprotective infusions. As will be discussed later, the nature and mode of use of cardioprotective solutions have continuously evolved since the late 1940s; in so doing, surgeons have been presented with a bewildering array of potential protective procedures.

III. BRIEF HISTORY OF THE DEVELOPMENT OF SURGICAL CARDIOPROTECTION Since the introduction and acceptance in the early 1970’s of modern surgical protection by ‘‘cardioplegia,’’ there have been a number of books and reviews devoted to this topic (4–9). These give detailed accounts of the effects of various cardioplegic solutions, details of their formulation, and the protective effects of various additives. Therefore, this chapter does not attempt to provide a comprehensive catalogue of the multitude of differing cardioplegic solutions and approaches; instead, we have attempted to provide a rational (and somewhat personal) view of the key developments and concepts of cardioplegia and cardiac surgical protection up to the present time.

A. Early Developments As mentioned previously, when the heart is exposed to extended periods of severe global ischemia (as necessarily occurs during cardiac surgery), every attempt should be made to delay the onset of irreversible injury. One of the earliest (heroic) attempts at delaying injury was to cool the whole patient or the heart so as to slow the rate of metabolism; subsequently, the chest was opened rapidly, the operation was performed rapidly, and the chest was closed within a short period of time before rewarming the patient. Thus, in 1953, Lewis and Taufic (10) conducted the first open-heart surgery

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(without the aid of cardiopulmonary bypass from a heart–lung machine!), successfully closing an atrial septal defect in a 5-year-old girl. They used whole body cooling (to 28⬚C, using refrigerated blankets) to protect the heart, brain, and other organs from the consequences of circulatory arrest. They then prevented cardiac inflow by clamping the aorta (so as to cause whole heart ischemia, cardiac arrest, and the required bloodfree operating field) for 5.5 min, during which time a 2cm-diameter defect was hastily closed. The patient was then rewarmed by placing her in water at 45⬚C such that, after 35 min, her temperature had returned to normal. The development and clinical use of the heart–lung machine by Gibbon (11) in 1954 allowed the hazards of circulatory arrest and consequent brain ischemia to be overcome and thus the scope of open-heart surgery to expand. Surgeons were able to operate on the heart with a bloodless field while pumps supported the systemic circulation. However, while the brain and other organs were perfused, the heart became ischemic with the inevitable injury and this (rather than the disease the operation was treating) often resulted in the death of the patient. For some operations and patient groups, surgical mortality was as high as 65% (12). Despite the induction of severe ischemia, the heart would continue to beat intermittently, draining it of energy and further hampering the operation. To prevent this, Melrose and colleagues (13) in 1955 introduced the concept of ‘‘elective reversible cardiac arrest’’ using an intracoronary infusion of high concentrations (ranging from 77 to 309 mmol/liter) of potassium citrate (added to blood at 37⬚C) to arrest the heart and provide a still and relaxed operating field. In a variety of experimental animals, they demonstrated that a 2.5% (77 mmol/liter) solution of potassium citrate in blood was effective in arresting the heart in diastole and that washout of the potassium citrate resulted in the restoration of normal beating. At the onset of ischemia (by occlusion of the aorta), the blood-based potassium solution (60 to 150 ml) was infused into the coronary arteries until the heart stopped beating, and this arresting solution was used clinically by a number of centers in the late 1950s (14–16) with apparently good results. However, subsequent studies (17,18) claimed that the use of potassium citrate was associated with myocardial injury, including areas of necrosis, which had been observed at autopsy in patients who had died. As a consequence the use of elevated potassium-based cardioplegia was abandoned in most countries for about 15 years. In the 1960s, surgeons responded to the demise of potassium arrest by adopting the technique of continuous coronary perfusion, avoiding the use of potassium and preventing the damage caused by ischemia. Some

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surgeons accompanied continuous perfusion with electrically induced ventricular fibrillation as a way to stop the heart beating and provide a still operating field. However, in the early 1970s, Buckberg and colleagues (19) demonstrated that fibrillation caused subendocardial necrosis and, as a consequence, ventricular fibrillation fell out of favor. To avoid fibrillation during continuous coronary perfusion, the temperature of the heart had to be maintained above 32⬚C. Although surgical mortality appeared to decrease following the movement away from potassium arrest or fibrillation, operating on a beating heart with a blood-perfused operating field was far more difficult. To address this, surgeons introduced the practice of intermittent coronary perfusion, typically occluding the aorta for 10 min to provide a blood-free and partially quiescent operating field and then reperfusing for 3 min. The assumption was that this short period of reperfusion was sufficient to repay the ‘‘oxygen debt’’ that occurred during the preceding 10 min of ischemia (4). Interestingly, at this time, ischemic preconditioning (20) had not been discovered but nonetheless surgeons may have been exploiting it—albeit inadvertently. In the late 1960s and early 1970s, while surgeons were experimenting with the just-described techniques, Shumway (21) was protecting the heart with ‘‘profound’’ (i.e., ⬍4⬚C) topical hypothermia. He immersed the heart in ice slush (or irrigated it with cold saline) and was able to achieve excellent results with safe ischemic times of up to 150 min. Unfortunately, his results were not readily reproducible by other surgeons and it was widely felt that ischemic periods of even 60 min were excessive. Surprisingly, some surgeons, especially Cooley (22), were less concerned about the hazards of unmodified normothermic ischemia. This skilled and exceptionally fast surgeon reduced the mean operating time for valve replacement to 38 min with good postischemic function, presumably because relatively little irreversible ischemic injury had occurred in this short ischemic time. However, the majority of cardiac surgeons at that time were unable to operate as quickly as Cooley and the technique was not used for long by many. It is of interest, however, that Cooley was the first to describe the ‘‘stone heart,’’ a condition in which the myocardium goes into irreversible (fatal) ischemic contracture from which no recovery is possible (23). Biochemists were challenged to find an answer to the ‘‘stone heart’’ (24) and it was subsequently demonstrated to be a direct consequence of ischemic injury (25), a factor that eventually persuaded Cooley’s group to consider the merits of and adopt cardioplegia. Throughout this uncertain period of the 1960s, the concept of chemical cardioplegia had been maintained

in Germany. In particular, Holscher (26), in Berlin, demonstrated in animal studies that it was the elevated citrate rather than potassium that had caused the necrotic lesions (17, 18) seen in patients using the Melrose procedure. Holscher suggested that a magnesium chloride plus procaine amide solution could be used as an alternative and safer form of cardioprotection. At the same time, Bretschneider and colleagues (27–29), in Go¨ttingen, developed a solution that induced cardiac standstill, not because it contained high potassium but because it was sodium poor, calcium free, and contained procaine. This ‘‘Bretschneider solution’’ was used routinely for myocardial protection in clinical practice by a number of surgeons with apparent success (30). Unfortunately, publication in the German language of the many clinical and experimental studies of Bretschneider did not help make these advances widely known until many years later (31). The same applied to the work of Kirsch (32), in Hamburg, who also developed a cardioplegic solution, based on elevated magnesium, aspartate, and procaine, which was used routinely during cardiac surgery for many years (33).

B. The Reemergence of Potassium Cardioplegia Probably unaware of most of the German studies, but anxious to do something to combat tissue injury during cardiac surgery, surgeons reintroduced potassium-based, citrate-free (‘‘extracellular-type’’) solutions in the mid-1970s. As mentioned previously, various volumes (500 to 1000 ml) of these solutions were infused into the coronary arteries (sometimes directly via the coronary ostia) for a few minutes at the onset of the induction of ischemia (by occlusion of the aorta). In the United States, Gay and Ebert (34), using a solution containing 25 mmol/liter potassium chloride, reported good protection after 1 hr of ischemic arrest in dog hearts. This was taken up by Roe and colleagues (35) who reported their clinical experience of myocardial protection in 204 patients using potassium-based cardioplegia with a mortality of ‘‘only’’ 5.4% (a figure considered ‘‘high’’ nowadays, but ‘‘low’’ in the 1970s). Tyers and co-workers (36) also adopted this procedure, reporting a series of over 100 patients using a solution containing 25 mmol/liter potassium with good myocardial protection. Meanwhile, Hearse and colleagues (37), in the United Kingdom, reported a large series of experimental studies in which many individual components of a potassium-based cardioplegic solution were systematically characterized and optimized. From these studies, the St. Thomas’ group rationalized that the three main components of effective cardioplegic protection (38)

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to enhance protection by combating specific deleterious ischemia-induced changes. The striking additive effect of individual protective components is illustrated in Fig. 2, and such studies led to the development of the St. Thomas’ Hospital cardioplegic solutions (Table I). The initial St. Thomas’ Hospital cardioplegic solution (1000 ml of which was infused at 4⬚C for 2 min at the onset of ischemia) was first introduced into clinical practice at St. Thomas’ Hospital by Braimbridge in 1975 (39). This solution was subsequently modified (Table I) to become the St. Thomas’ Hospital cardioplegic solution No. 2 (40), which was approved in the United States by the FDA and was produced commercially as Plegisol. Such was the success of this rationalized cardioplegia that, within a very short space of time, this solution (or related formulations) had been adopted for use by most major cardiac centers, almost completely replacing the use of coronary perfusion and other procedures (41). From these early findings, a wealth of clinical and experimental studies emerged in which hundreds of potential protective agents were evaluated, almost as many cardioplegic solutions were described as there were surgeons to use them and many new ‘‘concepts’’ of cardioplegia were advocated. Indeed, new and ‘‘improved’’ solutions continue to be developed even to this day, and many surgeons (especially in the United States) continue to use and advocate the use of cardioplegia as an effective, simple, and inexpensive way to protect the heart.

FIGURE 2 An illustrative example of the effect of sequential addition of several anti-ischemic agents on postischemic recovery of function (expressed as percentage of preischemic value) in isolated rat hearts subjected to a 2-min infusion of the agents before a period of ischemia (hypothermia was maintained throughout the ischemic period).* p ⬍ 0.05 compared to control. Drawn from various studies described in Hearse et al. (4).

were (a) the induction of rapid chemical arrest to conserve energy and provide a still (nonbeating) operating field, (b) the use of hypothermia to reduce the rate of metabolism, and (c) the addition of anti-ischemic agents

TABLE I Composition of Representative Examples of Clinical ‘‘Intracellular Type’’ (Bretschneider-HTK) and ‘‘Extracellular-Type’’ (St. Thomas’ Hospital) Cardioplegic Solutions

Component (mmol/liter)

Bretschneider solution (29)

Custodiol (Bretschneider-HTK) (42)

St. Thomas’ solution No. 1 (39)

Plegisol (St. Thomas’ solution No. 2) (40)

Sodium chloride Potassium chloride Magnesium chloride Calcium chloride Procaine hydrochloride Sodium bicarbonate K-ketoglutarate 움-Histidine 움-Histidine hydrochloride Tryptophan Mannitol pH Osmolarity (mOsm/kg H2O)

12 10 2 — 7.4 — — — — — 239 5.5–7.0 320

15 9 4 — — — 1 180 18 2 30 7.1 290

144 20 16 2.2 1 — — — — — — 5.5–7.0 300–320

110 16 16 1.2 — 10 — — — — — 7.8 324

The Bretschneider-HTK solution is usually delivered at a temperature of 8⬚C and a perfusion pressure of 40–50 mm Hg for a period of 7–9 min. At an estimated perfusion rate of 1 ml/min/g heart weight, this gives a total volume of 3000–4000 ml in adults and 300–500 ml in children (42). St. Thomas’ Hospital solution is delivered at a temperature of 4⬚C and a perfusion pressure of 80 mm Hg until arrest, reducing to 50 mm Hg after arrest; the initial infusion volume is 1000 ml delivered over a 1.5- to 2-min period. Subsequent infusions of 300–500 ml are infused at 50 mm Hg.

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IV. CHARACTERISTICS OF CARDIOPLEGIC PROTECTION Despite a multitude of different formulations, there are really only two types of cardioplegic solution: those based on an ‘‘intracellular-like’’ ionic formulation and those that are more ‘‘extracellular-like.’’ The former would be exemplified by the Bretschneider solutions (Table I) and the latter by the St. Thomas’ Hospital solutions (Table I). The principal differences between such solutions relate to the concentrations of potassium, sodium, and calcium, such that intracellular-type formulations are characterized by one or more of the following: high potassium concentrations similar to that of the cytoplasm (in the range of 100–140 mmol/liter), relatively low sodium concentrations (in the order of 10–15 mmol/liter) and either zero or very low calcium concentrations. In contrast, extracellular-type solutions are characterized by one or more of the following: moderately elevated potassium concentrations (ranging from 15 to 25 mmol/liter, which is the minimum concentration to ensure cardiac arrest), near extracellular sodium concentrations (in the range of 100–140 mmol/ liter) and relatively normal calcium concentrations (between 0.5 and 2.5 mmol/liter). The mechanisms of action, relative merits, and potential hazards of these solutions vary considerably. Intracellular solutions are largely represented by those that originated in Germany in the 1960s and include Bretschneider, Kirsch, and Rodewald solutions. However, only the Bretschneider solution (Table I) remains in clinical use today and it is currently known as the Bretschneider-HTK solution (so named for its principal constituents: histidine, tryptophan, and ketoglutarate). It is commercially available as Custodiol and is used principally in Germany (42). The BretschneiderHTK solution has a complicated delivery protocol, requiring high volumes (3 to 4 liters) to be infused over a relatively long period (7 to 9 min) at the onset of ischemia to enable the myocardium to reach minimum levels of oxygen consumption (42) and, usually, only a single infusion is required regardless of the length of ischemia. There have been many studies, in several species, comparing the efficacy of intracellular solutions such as that of Bretschneider with extracellular solutions such as the St. Thomas’ solution (43–46). Often, but not invariably, the Bretschneider solution has been found to be less efficacious; however, suboptimal delivery of the Bretschneider solution may have influenced these results. Certainly, improved protection with the Bretschneider-HTK solution has been observed in some animal (47) or clinical (48) studies compared to extracellulartype solutions.

Intracellular-type solutions are used predominantly for preservation during the long-term storage of hearts and other organs prior to transplantation. Most longterm preservation solutions [such as the Euro-Collins solution (49) and the University of Wisconsin solution (50)] were initially developed for the preservation of abdominal organs (kidney, liver, and pancreas) and have subsequently been adopted for use in the heart. In contrast, the Bretschneider-HTK solution was initially developed to protect the heart during cardiac surgery, but is now used extensively for the long-term preservation of the heart and abdominal organs (51–54). Extracellular-type solutions, such as the St. Thomas’ Hospital solution, Celsior solution (55), and the Buckberg solution (56) include those that are purely ‘‘crystalloid’’ (i.e., formulated in blood-free buffer) and those that have diluted blood as a vehicle for cardioplegic and protective components. Nowadays, solutions based on an extracellular-like formulation are, by far, the predominant type used by surgeons during routine cardiac surgery. For this reason, the following sections, which address the fundamental principles of cardioplegic protection, focus on extracellular-type formulations.

V. PRINCIPLES UNDERLYING THE PROTECTION OF THE HEART DURING CARDIAC SURGERY As mentioned previously, a multitude of studies of potential protective agents have been reported since the introduction into common usage of cardioplegic solutions in the 1970s (6, 8, 57). This huge body of literature presents an overwhelming and potentially confusing choice of cardioplegic solutions, assessed by a multitude of differing techniques and delivered to the heart by a vast array of procedures. Thus, simply considering the method of administration, one can choose from ‘‘cold,’’ ‘‘warm,’’ ‘‘tepid,’’ ‘‘single-dose,’’ ‘‘multidose,’’ ‘‘continuous,’’ ‘‘intermittent,’’ ‘‘antegrade,’’ ‘‘retrograde,’’ ‘‘high-infusion pressure,’’ ‘‘low-infusion pressure,’’ ‘‘hot-shot,’’ ‘‘terminal,’’ and ‘‘resuscitation’’ cardioplegia! On top of this, the solutions may be crystalloid (asanguineous) or blood-based (with a range of hematocrits) each with an overwhelming number of potentially beneficial additives, including substrates, antioxidants, ion channel inhibitors/activators, ion exchanger inhibitors/activators, ion pump inhibitors/activators, hyperosmolar agents, buffers, oxygen, and many others—often used in widely differing concentrations. Despite this vast array of variables and multitude of experimental and clinical solutions, it can probably be fairly concluded that most solutions, however delivered, provide a comparable degree of protection as long as they conform to the three fundamental principles of rapid

51. Cardioplegia and Surgical Ischemia

arrest combined with hypothermia and the use of antiischemic agents. It would be beyond the scope of this chapter to review and discuss every reported cardioplegic solution or additive in detail; instead, we will attempt to extract, from the myriad of reports, a number of fundamental concepts pertaining to cardioplegic solutions and successful protection during cardiac surgery. Central to the following sections is our belief that key elements of effective protection are (i) to arrest the heart completely and rapidly at the onset of ischemia in a way that is safe, easily reversible, and which will assist the surgeon by inducing a flaccid heart in diastolic arrest with a good field of view, (ii) to buy time for the surgeon by slowing the onset of irreversible ischemic injury by hypothermia, (iii) to minimize damaging ischemic changes with anti-ischemic agents, (iv) to optimize reperfusion conditions such that the rate and extent of recovery from ischemic injury and reperfusion injury are maximized, and (v) to recognize that effective protection of the whole heart requires protection not only of its contractile cells but also of other constituent cells, such as the vascular smooth muscle, endothelium, and conduction tissue and (vi) we believe that it is important to recognize and exploit alternative means of myocardial protection (such as using intermittent ischemia with fibrillation or inducing endogenous adaptive

893

defense mechanisms such as preconditioning) that may supplement or even replace conventional chemical cardioplegia.

A. Inducing Rapid and Complete Cardiac Arrest Cardiac arrest in a flaccid diastolic state is required to provide the surgeon with a relaxed and still operating field that will allow technically demanding or delicate manipulations to be optimally conducted. An important consequence of elective myocardial arrest (as opposed to that which occurs following the metabolic collapse seen with ischemia) is that oxygen requirement will be reduced significantly and the rate of the damaging cellular energy (ATP) depletion that characterizes ischemia will be reduced. Thus, myocardial oxygen consumption (at 37⬚C) is significantly lower in an arrested heart (앑1.0 ml/100 g/min) compared to a beating nonworking or fibrillating heart (앑6.0 ml/100 g/min). Cooling (to 22⬚C) reduces this to 앑0.3 ml/100 g/min (58). To achieve elective cardiac arrest, those arresting agents used most commonly during cardiac surgery target various points in the pathway, leading from excitation to contraction (Fig. 3). In this way, these agents induce a depolarized arrest, a ‘‘polarized’’ arrest, or an

FIGURE 3 Diagrammatic representation of excitation–contraction coupling (from the induction of the action potential to the initiation of contraction by elevated intracellular calcium) and the relevant targets within this process that are inhibited or activated by agents that induce depolarized arrest, polarized arrest, or arrest by inhibition of calcium influx and/or calcium-induced calcium release. SR, sarcoplasmic reticulum; TTX, tetrodotoxin.

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arrest by inhibiting calcium influx; these mechanisms are discussed in more detail next. 1. Depolarized Arrest a. Hyperkalemia Moderate elevation of the extracellular potassium concentration (usually within the range of 15–40 mmol/ liter) is the method used most commonly for inducing rapid diastolic arrest during cardiac surgery. This is achieved by depolarization of the myocyte transmembrane potential; using a simplified version of the Goldman–Hodgkin–Katz constant-field equation, it can be calculated that as the extracellular potassium concentration increases, the resting membrane potential becomes progressively more depolarized (59) and at each potassium concentration a new resting membrane potential is established (Fig. 4). At potassium concentrations around 10 mmol/liter, the voltage-dependent sodium channel is inactivated [since the threshold potential of this channel is approximately ⫺65 mV (60)], which will prevent the rapid sodium-induced spike of the action potential and result in arrest of the heart in diastole. Further increases in extracellular potassium will cause further depolarization of membrane potential; when the resting membrane potential reaches about ⫺40 mV (at an extracellular potassium around 30 mmol/liter or

higher), the slow calcium channel will be activated (60) and lead to calcium influx into the myocyte, promoting calcium overload. Thus, the beneficial effects of elevated extracellular potassium concentrations are restricted to a relatively narrow membrane potential window (about 10 to 30 mmol/liter), stressing the importance of undertaking dose–response studies for potassium in relation to the other ionic constituents of the cardioplegic solution, especially calcium and sodium. Measurement of postischemic recovery of function after arrest with solutions in which the extracellular potassium concentration was increased (4, 61) showed that, in the context of the St. Thomas’ Hospital cardioplegic solution, the optimal extracellular potassium concentration for myocardial protection lies between 15 and 20 mmol/liter. In this concentration range, the level of depolarization falls approximately in the middle of the beneficial window (Fig. 4). However, even at these levels of depolarization, other ionic currents remain active. It is postulated (62, 63) that the voltage-dependent activation and inactivation of the sodium channel are governed by ‘‘gates’’ that operate at different rates and lead to a sodium ‘‘window’’ current that is a noninactivating current at these membrane potentials (Fig. 5). This will tend to increase the intracellular sodium concentration and this, in turn, will increase the calcium ‘‘window’’ current (64, 65), causing contracture even in the arrested condition and contribute to calcium overload. Thus, although the use of elevated concentrations of potassium to induce the rapid depolarized arrest of the heart in diastole is by far the most widely used technique (possibly because it is the simplest to apply and to remove), it cannot be used haphazardly, has a number of disadvantages, and is not necessarily the best and most optimally protective. 2. Polarized Arrest

FIGURE 4 Depolarization of resting membrane potential (mV) with increasing concentrations of extracellular potassium (mmol/liter), showing the inactivation threshold for the sodium channel and the activation threshold for the calcium channel. Membrane potential was calculated using a simplified version of the Goldman–Hodgkin–Katz constant-field equation (59) in which only the effects of sodium and potassium ions on membrane potential are considered.

An alternative to inducing arrest by depolarization (as occurs with elevated potassium concentrations) is to maintain polarization of the membrane potential, close to the resting membrane potential. Polarized arrest should have a number of advantages; ionic movement (particularly sodium and calcium ions) should be reduced, as the threshold potential for activation of the ion channels will not be reached and ‘‘window’’ currents will not be activated. This reduction in ionic imbalance should, in turn, reduce myocardial energy utilization for ion movements and attempt to maintain ionic gradients. Polarized arrest can be achieved in a number of ways. a. Sodium Channel Blockade Sodium channel blockade is an effective means of arresting the heart and this is achieved by preventing

51. Cardioplegia and Surgical Ischemia

895

FIGURE 5 Voltage-dependent activation and inactivation of the sodium channel, showing the membrane potential range of the sodium ‘‘window’’ current. Activation and inactivation curves were drawn from data in McAllister et al. (62).

the rapid, sodium-induced depolarization of the action potential (66). Local anesthetics, such as procaine and lignocaine (lidocaine), have been widely used, either as cardioplegic agents or in combination with other agents, to induce cardiac arrest (4, 29, 33). As with other cardioplegic additives, these agents have a bell-shaped dosedependent effect and, at high concentrations (like so many agents), show a loss of protection and even a detrimental effect (57). Procaine (at a concentration of 1 mmol/liter) was also added to St. Thomas’ Hospital cardioplegic solution No. 1, but not as a means of arresting the heart. In view of its complex dose–response characteristics and the relatively small additional protection over and above that of potassium arrest, it was removed from St. Thomas’ Hospital cardioplegic solution No. 2. However, these drugs may have other beneficial effects, not related to countering ischemia; thus, in patients, procaine has been shown to control postoperative rhythm disturbances (67). Tetrodotoxin (a highly toxic but potent and rapidly reversible sodium channel blocker) has also been investigated as a cardioplegic agent and has been shown to protect isolated rat hearts exposed to 60 min of normothermic global ischemia (68). We (69) have used tetrodotoxin to induce a rapid and reversible arrest in rat hearts before long-term preservation and hypothermic storage and demonstrated significantly improved protection compared to hyperkalemic cardioplegia. In addition, electrophysiological measurements of membrane potential during ischemic storage showed that the resting membrane potential in tetrodotoxin-arrested hearts was maintained at around ⫺70 mV (polarized arrest) compared to around ⫺50 mV in hyperkalemic (depolarized) arrested hearts (Fig. 6).

b. ATP-Sensitive Potassium Channel Activation The cardiac ATP-sensitive potassium channel was described by Noma (70) in 1983 and a number of drugs have been developed that either activate (open) or inhibit (block) this channel. At high concentrations, potassium channel openers can exert cardioplegic effects, and arrest has been suggested to occur by the induction of membrane hyperpolarization (71). Under steady-state conditions, the myocardial resting membrane potential (around ⫺80 mV) is close to the equilibration potential of potassium (about ⫺94 mV) because the relative membrane conductance to potassium is much greater than the relative membrane conductance to sodium. Potassium channel opener-induced activation of ATPsensitive potassium channels increases the difference between these conductances, causing membrane potential to be shifted toward the potassium equilibration potential (hence, a hyperpolarization from the previous membrane potential). If the membrane potential remains more negative than ⫺70 mV, activation of the fast sodium channel will not occur [because the threshold potential is around ⫺65 mV (63)] and the heart will arrest in diastole. As has been shown with several other drugs, potassium channel openers have bell-shaped dose–response profiles, with high concentrations being associated with a loss of protective effect (71, 72). Although membrane potential hyperpolarization with potassium channel openers has been demonstrated in isolated porcine coronary arteries (73) and in isolated guinea pig and human ventricular myocytes (74), it only occurs if the potassium concentration in the solution remains low. It is currently unknown, however, whether any hyperpolarization will

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FIGURE 6 Resting membrane potential (in mV), recorded every 15 min throughout 300 min of hypothermic (7.5⬚C) global ischemia, in rat hearts arrested with tetrodotoxin (‘‘polarized’’ arrest) (䊏), hearts arrested with elevated potassium (depolarized arrest) (䊊), and hearts arrested with ischemia (䉭). Redrawn from Snabaitis et al. (69).

be maintained during ischemia in whole hearts (due to ischemia-induced potassium efflux from the cell); measurement of the membrane potential after arrest with potassium channel openers and subsequent ischemia is required to establish these characteristics. Potassium channel openers have also been used as additives to hyperkalemic cardioplegic solutions and shown to enhance postischemic recovery of function (75–77); however, we have observed that this protective effect was lost when added to the St. Thomas’ Hospital cardioplegia (78). When added to hyperkalemic cardioplegia, a potential protective mechanism of these drugs is to reduce the potassium-induced influx of calcium (79). c. Adenosine Adenosine (at a concentration of 50 애mol/liter) has been shown to induce complete arrest and a hyperpolarization of ⫺12 mV in isolated rabbit SA node pacemaker cells (80). Subsequently, this cardioplegic effect was compared to that of hyperkalemia on myocardial protection in isolated rat hearts (81, 82). High adenosine concentrations (1 or 10 mmol/liter), either alone or in combination with elevated potassium, reduced the time to myocardial arrest and improved postischemic recovery of function compared to hyperkalemia (20

mmol/liter potassium) alone. Electrophysiological measurements (82) showed an initial transient hyperpolarization before depolarization; this hyperpolarization was thought to arrest SA node conduction before myocyte contractile arrest, leading to a more rapid arrest and having beneficial effects on the ischemic myocardium. In a model of cardiopulmonary bypass in baboons (83), adenosine cardioplegia (10 mmol/liter adenosine in a bicarbonate buffer) was compared to St. Thomas’ Hospital cardioplegic solution No. 2 and shown to be equally effective at protecting the heart. As with many of these compounds, adenosine has been used primarily as an additive to cardioplegic solutions to enhance myocardial protection; more details of this are provided in Section V,C,5,b. d. Acetylcholine During the early years of cardiac surgery (1955– 1960), acetylcholine was used as a cardioplegic agent by a number of surgeons (12, 84). It is likely that acetylcholine arrests the heart in a similar manner to that of adenosine by suppressing sinus node automaticity and blocking sinoatrial conduction (85); it is not known, however, whether it induces hyperpolarization, although a more negative potential is thought to be in-

51. Cardioplegia and Surgical Ischemia

897

FIGURE 7 Measurement of contractile response of an isolated rat myocyte, showing the immediate abolition of shortening (arrest) when the myocyte was subjected to a calcium-free extracellular solution. The cell is stimulated continuously at 0.2 Hz, and arrows indicate stimulations in calcium-free solution. Extracellular calcium ([Ca]o) was changed by a rapid switching device between stimuli [redrawn from Rich et al. (89)].

duced. The use of acetylcholine during cardiac surgery was short-lived, as a number of studies demonstrated that high concentrations were required to maintain arrest and that, after longer arrest periods, recovery of function was severely depressed (86, 87). Thus, the concept of maintaining arrest by the induction of polarization (or hyperpolarization) compared to depolarization (induced by hyperkalemia) may have significant advantages. However, considerable more characterization is required before this could be considered for use in cardiac surgical patients. 3. Inhibition of Calcium Influx

(through the cardiac sodium channel) at the initiation of the action potential, which would tend to maintain the membrane potential close to the resting potential, again leading to diastolic arrest. Jynge and colleagues (43, 91) compared the myocardial protective effects of the Bretschneider solution and the Kirsch solution (both calcium and sodium free) to the normocalcemic St. Thomas’ Hospital solution in an isolated working rat heart preparation (Fig. 8). The Kirsch solution was poorly protective against both normothermic or hypothermic ischemia, whereas the Bretschneider solution was protective only with hypothermic ischemia; in contrast, the St. Thomas’ solution

a. Hypocalcemia In the absence of extracellular calcium, the heart arrests in diastole, a fact that has been known since Ringer’s studies in 1883 (88). A number of cardioplegic solutions have, therefore, been developed that are characterized by zero (or very low) calcium concentrations, usually in association with a reduced sodium concentration. Thus, the original Bretschneider intracellular-type solution (29) had approximately 12 mmol/liter sodium, zero calcium, and 7.4 mmol/liter procaine (see Table I). Sodium-poor, calcium-free cardioplegic solutions have the potential to arrest the heart by a number of mechanisms. Absence of calcium from the extracellular solution prevents calcium entry during the second phase of the action potential and abolishes calcium-induced calcium release; this leads to rapid arrest (Fig. 7) by the inhibition of excitation–contraction coupling (89). In addition, potassium efflux will increase extracellular potassium and lead to cellular membrane depolarization (90). At the same time, the reduction in the calciumdriving force will cause calcium efflux and sodium influx via the sodium/calcium exchanger and calcium will continue to leak from the sarcoplasmic reticulum until depleted. However, these effects might be slowed by the low extracellular sodium attenuating the sodium current

FIGURE 8 Postischemic recovery of aortic flow after 30 min of reperfusion in rat hearts subjected to a 2-min preischemic infusion of Kirsch cardioplegic solution, Bretschneider cardioplegic solution, or St. Thomas’ Hospital cardioplegic solution followed by 70 min of global hypothermic (28⬚C) ischemia. Recovery is expressed as a percentage of the preischemic (control) value. *p ⬍ 0.05 compared to St. Thomas’ Hospital solution. Graph drawn from data in Jynge et al. (43).

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X. Pathophysiology

provided good myocardial protection regardless of the temperature during the ischemic period. From these studies, the potential danger of calcium-free solutions was realized—namely that they have the potential to induce a lethal condition called the ‘‘calcium paradox’’ [contracture and massive ultrastructural injury caused when hearts, perfused with totally calcium-free solutions, are subsequently perfused with calcium-containing solutions (92, 93)]. In reality, this rarely occurs but this is only fortuitous with protection against the paradox occurring because these calcium-free solutions (i) often contain traces of contaminant calcium, which is of a sufficiently high concentration to prevent its occurrence, (ii) are used cold and hypothermia is known to protect against the calcium paradox, and (iii) contain low sodium and/or high magnesium, which also confers protection. Some of the more recently developed intracellular-type solutions, such as the University of Wisconsin solution (which is used predominantly for the long-term preservation of donor hearts prior to transplantation), have little or no calcium (94), and studies have demonstrated that the efficacy of these solutions is very sensitive to infusion and storage temperature (95) and benefit from the addition of calcium and magnesium (96), confirming the findings of earlier studies. Rapid diastolic arrest can be achieved by the depletion of calcium; however, the concentrations of calcium and sodium are inextricably linked via the sodium/ calcium exchanger, and correct stoichiometric relationships (which are extremely complex) require that both should be reduced to achieve relatively safe myocardial protection. Instead of reducing calcium to dangerously low levels, an alternative approach to blocking calciummediated contractile activity is to use drugs that influence calcium movements. b. Calcium Antagonists Calcium antagonists depress cardiac function and reduce calcium influx through slow calcium channels. At sufficiently high concentrations, calcium-induced calcium release can be prevented and the heart arrested by the inhibition of excitation–contraction coupling. Calcium antagonists (such as verapamil, nifedipine, and diltiazem) have been suggested as cardioplegic agents per se (97–100), potentially exerting comparable protection to potassium arrest in terms of recovery of function and high-energy phosphates. One disadvantage of the use of calcium antagonists is the delayed reversal of activity (possibly due to membrane binding) of these drugs, resulting in a slower recovery than that seen with potassium cardioplegia. It is possible, however, that this might confer certain benefits (such as resistance to washout by noncoronary collateral flow and potential reduction of calcium overload during reperfusion).

As discussed later, the use of calcium antagonists has been primarily to enhance protection when used as an additive to potassium cardioplegia rather than as a cardioplegic agent per se. Dose–response studies (101) have demonstrated the importance of establishing the optimal concentration of these drugs, as high concentrations may be detrimental and also prolong electromechanical arrest (102, 103). In addition, their protection is thought to be temperature dependent, with little or no protective effect under hypothermic conditions (101). Thus, although calcium antagonists have a number of antiischemic properties, any benefits that may be obtained appear to be outweighed by the disadvantages relating to their dose-dependent, temperature-dependent, and time-related effects. c. Hypermagnesemia Elevated extracellular magnesium can arrest the heart, but it is less effective than potassium and higher concentrations are needed to induce arrest (4). It is thought that the negative inotropic and cardioplegic effects of magnesium are achieved by the displacement of calcium from the rapidly exchangeable sarcolemmalbinding sites involved in excitation–contraction coupling (104). These effects are species dependent, with the rat being very sensitive and the rabbit is relatively insensitive; it is thought that the sensitivity of the human myocardium to elevated magnesium is intermediate; the differences probably reflect the sensitivity of transsarcolemmal calcium influx to magnesium (104). Magnesium was used in the Kirsch solution (33) at a concentration of 160 mmol/liter, but this was in combination with 11.0 mmol/liter procaine (see Section V,A,5,b). As with calcium antagonists, magnesium has been employed more frequently as an additive protective agent rather than as a cardiac arresting agent per se. Hearse and colleagues (105) showed it to be an exceptionally powerful protective agent that, in the rat, exhibits a bellshaped dose–response profile (Fig. 9), with an optimal protective concentration at 16 mmol/liter (irrespective of whether hearts were subjected to normothermic or hypothermic ischemia). Since this finding, magnesium has become a standard component of St. Thomas’ Hospital solutions. The addition of magnesium (at 16 mmol/ liter) to calcium-containing hyperkalemic cardioplegia has been shown to protect against calcium-induced hypercontracture during arrest (106), and similar calcium– magnesium protective interactions have been confirmed in other studies (107–110). Thus, although magnesium can act as a cardioplegic agent, it is used more as an additive component of hyperkalemic cardioplegic solutions to enhance protection, and in this respect it has been shown to be particularly effective.

51. Cardioplegia and Surgical Ischemia

899

and above that of hyperkalemic cardiac arrest (Fig. 10). The benefits of hypothermia on the metabolic requirements of the heart have also been demonstrated by Buckberg and co-workers (58), who showed that, at 37⬚C, a beating empty heart, a fibrillating heart, and an

FIGURE 9 Postischemic recovery of aortic flow after 30 min of reperfusion in rat hearts subjected to a 2-min infusion of St. Thomas’ Hospital cardioplegic solution, containing magnesium concentrations ranging between 0 and 50 mmol/liter, followed by 30 min of normothermic (37⬚C) global ischemia. Recovery is expressed as a percentage of the preischemic (control) value. *p ⬍ 0.05 compared to 0 mmol/ liter magnesium; #p ⬍ 0.05 compared to 15 mmol/liter magnesium. Redrawn from Hearse et al. (105).

B. Slowing the Onset of Irreversible Injury by Hypothermia Having achieved rapid but reversible contractile arrest of the heart, it is now necessary to slow the evolution of ischemic injury and thus delay (for as long as possible) the onset of irreversible injury. This approach allows the surgeon sufficient time to complete his task, rather than the often hurried surgery that was required before cardioplegia because of the race against the ‘‘ischemic clock.’’ This can be achieved by hypothermia. Hypothermia has been a major (and sometimes only) component of myocardial protection since the start of cardiac surgery (see Section III, A) and it is also effective in protecting other organs, such as the brain (111– 113). The effectiveness of hypothermia is dependent on its slowing effect on metabolism as a consequence of the temperature dependency of enzymes. Generally, enzyme activity is reduced by 50% for every 10⬚C drop in temperature—thus, cooling from 37 to 7⬚C would be expected to reduce activity to 12.5% of normal (114) (indeed, hypothermia can be used as a way of inducing cardiac arrest). In this way, cooling to as low as 4⬚C slows metabolism and reduces deleterious ischemia-induced changes greatly. Using the isolated rat heart, and in dog hearts in vivo, we (61, 115, 116) have shown that hypothermia confers an additive protective effect over

FIGURE 10 Additive protection of hypothermia in combination with cardioplegia. (A) Postischemic recovery of aortic flow after 30 min reperfusion in rat hearts subjected to 2-min infusions (at 0 and 60 min) of noncardioplegic solution (hypothermia alone) or St. Thomas’ Hospital cardioplegic solution (hypothermia ⫹ cardioplegia) and 120 min of global hypothermia (20⬚C). Recovery is expressed as a percentage of the preischemic (control) value. *p ⬍ 0.05 compared to hypothermia alone. Redrawn from Hearse et al. (115). (B) Cardiac output in the in situ dog heart before bypass (control) and 15 min postbypass after 120 min of global hypothermic (20⬚C) ischemia (with 2-min infusions (at 0 and 60 min) of noncardioplegic solution (hypothermia alone) or St. Thomas’ Hospital cardioplegic solution (hypothermia ⫹ cardioplegia). *p ⬍ 0.05 compared to control. Redrawn from Rosenfeldt et al. (116).

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arrested heart had a myocardial oxygen consumption of 5.6 ⫾ 2.0, 6.5 ⫾ 1.6, and 1.1 ⫾ 0.4 ml/100 g/min, respectively, whereas at 22⬚C these values were reduced to 2.9 ⫾ 0.9, 2.0 ⫾ 0.9, and 0.3 ⫾ 0.1, respectively. Although hypothermia offers considerable protection against extended myocardial ischemia, it also has some deleterious effects. The induction of rapid myocardial hypothermia (to temperatures of 0–4⬚C) can lead to the release of calcium from intracellular stores (117), causing contracture if temperature is maintained below 5⬚C (118). Hypothermic cardiac perfusion has been associated with increased vascular constriction (119, 120), with diastolic abnormalities associated with increased intracellular calcium (121, 122) and with deleterious effects on function in acutely injured myocardium (123). During ischemia, sodium pump activity is inhibited by energy depletion, but is also thought to be inhibited rapidly by hypothermia (124, 125) and, because the pump is the primary route of sodium efflux during the cardiac action potential, inhibition by hypothermia will lead to intracellular sodium and chloride accumulation down the concentration gradient; as the sodium pump will no longer be effective in making sodium an impermeant, the colloidal osmotic pressure from intracellular proteins and other impermeable anions will lead to cell swelling as a result of water accumulation (114). In rat, rabbit, and human myocytes (126, 127), cell swelling during hypothermic cardioplegia with the St. Thomas’ Hospital solution could be prevented by partial chloride substitution with an impermeant anion (such as methanesulfonate). The optimal temperature during hypothermic cardioplegic arrest has also been debated. It is necessary to establish a balance between cellular energy conservation (maintenance of high-energy phosphates), which improves with increasing degrees of hypothermia, versus the maintenance of cellular ionic homeostasis (ionic redistribution, cell swelling, and cold-induced injury), which deteriorates as temperatures are lowered. Early temperature studies by the authors (37) demonstrated significant increases in protection between 37 and 24⬚C, whereas further cooling did not make a major difference to the protection achieved (Fig. 11). This was later confirmed in experimental (128) and clinical (129) studies, which suggested that temperatures around 10–15⬚C were optimal. During routine cardiac surgery, these temperatures can be achieved by infusion of cardioplegia at temperatures around 4⬚C, thereby cooling the myocardium to 10–15⬚C (130, 131). This remains the situation for crystalloid cardioplegia; however, for blood cardioplegia (see following sections) there is evidence that these profoundly hypothermic temperatures may not be necessary. Thus, hypothermia can exert an immensely powerful

FIGURE 11 The relationship between myocardial protection and the degree of hypothermia. Postischemic recovery of aortic flow after 15 min of reperfusion in rat hearts subjected to arrest with St. Thomas’ Hospital cardioplegic solution and 60 min of global ischemia at varying temperatures. Recovery is expressed as a percentage of the preischemic (control) value. Redrawn from Hearse et al. (37).

protective effect but it is not without hazard. Good cardioplegia, however, may make hypothermia much better tolerated (especially in relation to the control of adverse ion movements).

C. Minimizing Damaging Ischemic Changes with Anti-ischemic Agents As described earlier, a veritable cornucopia of additives has been suggested to enhance the protective properties of cardioplegic solutions. A detailed discussion of all of these is beyond the scope of this chapter; however, we have attempted to discuss those that we feel are the most beneficial, have been best characterized, or have been most widely adopted for use during cardiac surgery.

1. Blood as an Additive or a Vehicle for Cardioplegia The earliest blood cardioplegic solution was that of Melrose and colleagues (13), who used high concentrations of potassium citrate added to blood to induce cardiac arrest during open heart surgery. Although adopted by many surgeons, it was soon claimed to induce myocardial injury, and the use of cardioplegic solutions with or without blood was largely abandoned for 15 years. It was not until the late 1970s that blood cardioplegia (encouraged by the reemergence of interest and success with crystalloid cardioplegia) was again advo-

51. Cardioplegia and Surgical Ischemia

cated, particularly by Buckberg’s group, which was the first to study the potential of blood cardioplegia in detail (132). Studies in dogs demonstrated that a cold (22⬚C) blood cardioplegic solution (made up from blood taken from the heart–lung machine and modified such that the calcium was reduced to 0.6 mmol/liter, the pH was adjusted to 7.8, and the potassium was increased to a final concentration of 30 mmol/liter) produced a recovery of compliance to around 80% after 2 hr of ischemic arrest. This compared to only 40% with a continuous perfusion procedure and only 17% with intermittent ischemia. Clinically, blood cardioplegia appeared to produce similarly good results and Buckberg (133) went on to propose a number of principles to apply to blood cardioplegia, some of which were very similar to those defined by Hearse (4, 38). These were (i) immediate arrest to lower energy demands and avoid energy depletion, (ii) hypothermia to reduce energy demands further, (iii) substrate provision for anaerobic or aerobic energy production, (iv) buffering to prevent acidosis, (v) hyperosmolarity to reduce edema, (vi) membrane stabilization by exogenous additives to prevent excessive hypocalcemia, and (vii) adequate experimental testing to avoid iatrogenic effects of constituents. Many experimental and clinical studies have attempted to compare the efficacy of crystalloid versus blood cardioplegia, with most concluding that blood cardioplegia provided marginally superior protection. However, it can be argued that the majority of these studies used conditions or crystalloid solutions that were suboptimal and, as such, tended to favor a good outcome for blood cardioplegia. These included ischemic arrest at relatively high (27⬚C) temperatures (134, 135), despite the knowledge that crystalloid cardioplegia was more efficacious at lower temperatures (136, 137), and the absence of calcium in crystalloid solution studies (134, 135) [despite the fact that the addition of calcium was shown to provide similar protection to blood cardioplegia (138)]. In fact, many crystalloid solutions used in these studies were crude high potassium solutions, highlighting again the importance of conducting rigorous comparative studies in which only one variable is changed. In general, although blood cardioplegia was shown to be an acceptable means of protection, comparative clinical studies were less conclusive than experimental studies in proving any real advantage (139–141). Few investigators attempted to assess the relative importance of the various components of blood cardioplegic solutions, nor did they attempt to examine the effect of the blood component (such as osmotic or oncotic properties or oxygen-carrying capacity differences) when added to a well-established crystalloid cardioplegic solution (142). A true evaluation of blood versus

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crystalloid cardioplegia in the clinical setting requires the two solutions to be formulated as closely as possible with only the hematocrit as the principal variable. Such a study was carried out by the authors (143), who compared crystalloid St. Thomas’ Hospital solution No. 1 to a blood-based version of the same solution. Although the left ventricular stroke work index was depressed postoperatively in both groups, function at 2 hr recovered significantly more rapidly in the group treated with blood-based St. Thomas’ Hospital solution compared to the group exposed to crystalloid St. Thomas’ Hospital solution. Function in the latter group recovered to the same extent as in the former but only after 24 hr, indicating improved myocardial protection with blood cardioplegia. Even this study could be criticized as the St. Thomas’ Hospital cardioplegic solution used was the No. 1 solution rather than the more efficacious St. Thomas’ Hospital cardioplegic No. 2 solution (144). Other clinical comparative studies were, however, unable to detect differences in myocardial function and metabolism between crystalloid and blood cardioplegia, either immediately postoperatively (145) or at 1 and 5 months postoperatively (146). Despite the possibility that blood cardioplegia affords better protection, a number of concerns have been expressed over its use. These include the fact that the preparation of blood cardioplegia is more complex (it has to be made up in the operating room at the time of surgery using the patient’s own blood) and it cannot be prepared to a standardized formulation (unlike crystalloid cardioplegia, which is prepared beforehand to exacting specifications and can be used ‘‘off the shelf’’); specialized delivery systems have helped overcome some of the problems but have added to the cost of the procedure. The high hematocrit (4 : 1 ratio of blood:crystalloid) and low temperature of blood cardioplegia may induce a sludging effect by rouleau formation of the red blood cells, thereby impairing reperfusion (133), possibly by the reduced recovery of capillary perfusion (147). Although the operating field is somewhat less clear with blood cardioplegia than with crystalloid cardioplegia, the use of intermittent infusion will circumvent any problem. Another potential limitation with blood cardioplegia is that there is less scope for the inclusion of potentially beneficial additives, as they may interact adversely with components of the blood.

2. Oxygenation of Cardioplegia The introduction and promulgation of blood as the vehicle for cardioplegic solutions were partly based on the hypothesis that oxygen provision played a significant role in its beneficial effects. This contrasts with crys-

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talloid cardioplegic solutions, most of which, particularly those used clinically, are not oxygenated, although they could be. Many studies, using both crystalloid and blood cardioplegia, have demonstrated the benefits of oxygenation to cardioplegic protection. The oxygen requirement of the potassium-arrested heart at 22⬚C is 0.30 ml/100 g/min (58), and a further 10⬚C decrease in the myocardial temperature (to levels that are reached routinely during surgery) reduces this requirement to 0.14 ml/100 g/min (7). Blood has a high oxygen-binding capacity, but the temperature dependency of hemoglobin oxygen dissociation characteristics means that less than 50% of the available oxygen can be released at temperatures of 10–15⬚C. In contrast, crystalloid solutions have a low oxygen-binding capacity (when oxygenated) but all the oxygen that is present is available for release regardless of temperature (148). Thus, oxygen-saturated crystalloid solutions can deliver and release as much oxygen as cold blood. Oxygen available from the intermittent infusion of these cold cardioplegic solutions may be sufficient to meet the demands of the cold ischemic myocardium, especially during multiple infusions (149, 150); nonetheless, it remains unclear as to whether any additional protection provided by blood cardioplegia is associated with the oxygen per se or whether some other component of blood (such as the buffering capacity) is more important (134, 151, 152). Beneficial effects of oxygenation of crystalloid cardioplegic solutions have also been demonstrated (153– 157); however, oxygenation of crystalloid solutions (such as St. Thomas’ Hospital cardioplegia, which has little buffering capacity) requires adequate control of the carbon dioxide content as well as oxygen to prevent alkalosis, which can influence myocardial recovery (158, 159). Thus, the intermittent provision of oxygen during cold ischemic arrest (with either crystalloid or blood cardioplegic solutions) may be sufficient to meet the reduced demands of the myocardium at the temperatures reached during surgery and thereby enhance myocardial protection. a. ‘‘Continuous’’ Oxygenated Cardioplegia and the Potential Prevention of Ischemia Once the use of intermittent cold blood cardioplegia had been successfully adopted by some surgeons, it was realized that its uses could be extended. Thus, it was suggested that an initial arrest with a continuous (around 5 min) infusion of warm (37⬚C) blood cardioplegia (before maintenance of arrest with intermittent cold infusions) allowed severely injured hearts a period of ‘‘resuscitation’’ (160, 161); in addition to this, reperfusion with warm (37⬚C) blood cardioplegia could provide

a quiescent period for myocardial metabolic recovery without the energy consumption of contraction (162). This led some investigators to question whether ischemia and hypothermia, with their attendant detrimental effects, were a necessary component of effective cardioplegic cardioprotection during cardiac surgery. Lichtenstein and colleagues (163, 164) claimed that ‘‘continuous warm’’ (37⬚C) blood cardioplegia provided similar protection to the myocardium as that of intermittent cold blood cardioplegia. Subsequent experimental and clinical studies (9, 165–169) have confirmed the efficacy of this procedure. It was argued that this technique might be more beneficial for high-risk patients whose hearts might be compromised by additional periods of ischemia. When strictly applied, continuous warm blood cardioplegia can maintain the heart in an aerobic state (170); however, for most surgeons, ‘‘continuous warm’’ blood cardioplegia is not really continuous because periods of intermittent ischemia are usually a necessary component of the procedure (when the surgeon constructs the distal anastomoses of the bypass grafts— this is necessary because continuous flow would obstruct the field of view). Although these transient interruptions in flow during normothermic cardioplegia appear to have metabolic implications (such as lactate production) (166, 167, 171), intermittent warm blood cardioplegia has been shown (172–175) to be an effective technique for myocardial protection, providing results that were similar to the more conventional intermittent cold blood cardioplegia. It has been established, however, that the intermittent ischemic periods should be less than 10–15 min in duration (176–178). A further compromise to ‘‘continuous warm’’ blood cardioplegia has developed with the tendency of surgeons to allow the temperature of the cardioplegic solution to drift down to around 29–32⬚C, leading to the term ‘‘tepid’’ cardioplegia. Perhaps not surprisingly, this has further improved myocardial protection (probably because the moderate hypothermia affords protection during the potentially dangerous intermittent ischemic periods) (179–182). Thus, current techniques of blood cardioplegia are, in many ways, mimicking the myocardial protective techniques of the 1960s by reverting to continuous or intermittent perfusion. The major difference, however, between then and now is that hearts are arrested with moderately elevated potassium that minimizes myocardial oxygen consumption. Also, the intermittent ischemic durations are much shorter than was previously used (see Section III,A) making the ratio of ischemia to perfusion (reperfusion) durations favor that of reperfusion (see also Section V,F,2). Although the prevention of additional ischemia with continuous cardioplegic infusion is an attractive theoretical

51. Cardioplegia and Surgical Ischemia

aim, in practice the technique is quite difficult to achieve. 3. Agents That Influence Buffering and pH Cellular acidosis is one of the major consequences of ischemia and therefore is a target for beneficial manipulation; the use of buffers in an attempt to prevent major pH changes during ischemia is complex and dependent on many potentially interacting factors. When assessing the buffering requirements of a cardioplegic solution, it is necessary to consider basic concepts of pH and temperature. The dissociation of water into hydrogen ions and hydroxyl ions is reduced as temperature decreases, and the point of neutrality of water shifts in an alkaline direction by approximately 0.015 of a pH unit per degree C fall in temperature (183). This shift has been addressed in the formulation of St. Thomas’ Hospital cardioplegic solution No. 2 (Table I), which is adjusted to pH 7.8 by the addition of bicarbonate so that, on cooling, the pH falls closer to the physiological range (40, 184). A similar adjustment has been made with the Buckberg blood cardioplegic solution, which has a pH of approximately 7.6 and uses Tris (hydroxymethyl) aminomethane as the buffer (56, 133). The concept of slightly alkalotic cardioplegic solutions has, however, been challenged, and a number of reports suggest that neutral or slightly acidotic pH may provide optimal protection, possibly by inhibiting metabolism (185– 187). In attempting to resolve this issue, it is important to remember that it is the buffering capacity rather than the pH that is most important in cellular pH control and cardioplegic protection. Indeed, many cardioplegic solutions [such as St. Thomas’ Hospital cardioplegic solution No. 1 (see Table I)] are not buffered and the pH of the solution is largely influenced by the pH and dissolved carbon dioxide content of the water used to prepare the solution. This often results in pH values of 5.0 or less, but this does not represent a problem as, once infused, the solution will equilibrate to the pH of the heart. As most crystalloid cardioplegic solutions have little or only poor buffering capacity, it is likely that the buffering capacity contributes little to the protective properties of the solution. However, in solutions that do have a strong buffering capacity [such as the histidinecontaining Bretschneider-HTK solution (42) and blood cardioplegia] the buffering capacity is likely to contribute significantly to the overall extent of myocardial protection. 4. Calcium Antagonists Calcium antagonists have already been discussed as components of cardioplegic solutions in the context of

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inducing diastolic arrest (Section V,A,3,b). Calcium antagonists achieve a depression of contractile function (by reducing calcium influx through the slow inward Ltype calcium channels) and thereby have intrinsic antiischemic properties. Thus, because an important component of injury during ischemia and reperfusion is thought to involve intracellular calcium overload, calcium antagonists have been considered as protective adjuncts to hyperkalemic cardioplegia. Optimal doses of diltiazem, verapamil, or nifedipine have been shown to be effective in improving the protective properties of the St. Thomas’ Hospital cardioplegic solution and the consequent recovery of contractile function when used under conditions of normothermic ischemia (101); however, it is of interest that these agents appear to confer no protective effect when used during hypothermic (20⬚C) ischemia. It has been claimed that the potentially detrimental heterogeneous delivery of cardioplegic solutions can be improved by the addition of diltiazem to a cardioplegic solution (188), but it was again shown to be ineffective when used during hypothermia (189). In surgical patients, calcium antagonists appear to be unable to confer any significant benefit to clinical outcome (102, 190, 191) and some deleterious side effects have been reported; there are also problems of light instability with some of these compounds. Overall, the advantages of calcium antagonists as additives to cardioplegic solutions appear to be outweighed by the disadvantages and few are employed as surgical protective agents, although diltiazem continues to be added to the conventional Buckberg blood cardioplegia solution (56). 5. Antioxidants and Inhibitors of Free Oxygen Radical Production The surge of interest in the 1980s in free oxygen radicals and oxidant stress as mediators of injury during ischemia and reperfusion naturally led to the suggestion that free radical scavengers or inhibitors of their formation may have a place in preventing tissue injury during cardiac surgery. Oxygen-derived free radicals, such as the superoxide anion, the hydroxyl radical, and reactive oxygen intermediates such as hydrogen peroxide are generated during normal metabolism and particularly during ischemia and reperfusion. Fortunately, endogenous antioxidant systems, such as superoxide dismutase and catalase, normally eliminate these potentially toxic intermediates, thereby preventing the occurrence of oxidative tissue injury in nonischemic tissue. Unfortunately, in ischemic tissue, antioxidant defenses are reduced (192) and, in the face of increased radical production, tissue injury may occur. Both superoxide dismutase and catalase have been examined as additives

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to cardioplegic solutions and have been shown to improve postischemic recovery in a number of animal studies (193–196). Similarly, inhibition by allopurinol (either as a pretreatment or as an additive to cardioplegia) of the radical-generating enzyme, xanthine oxidase, was found to be effective (195, 197–199). However, the claim that xanthine oxidase is undetectable in the human myocardium (200) has reduced interest in this agent during cardiac surgery and, although used by some surgeons, its clinical value remains controversial (201–205). Similarly, despite impressive results in animal studies, superoxide dismutase and catalase have not been adopted for use by surgeons, possibly because of the high cost of these enzymes and the potential problems associated with the administration of large protein molecules to patients. Metal ions (such as iron and copper) play an important mechanistic role in catalyzing the generation of oxygen radicals (206), and use of metal ion chelators (such as deferoxamine) has been suggested as a novel approach to prevent free radical-induced injury during cardiac surgery, especially when iron may be released from red cells by hemolysis. In a variety of experimental studies, deferoxamine, added to cardioplegic solutions, has been shown to improve postischemic function (195, 207) and inhibit neutrophil activation (208). It has also been shown to reduce free radical generation in clinical studies (209, 210), but again few surgeons have adopted this approach to enhancing cardioprotection. The same applies to supplementing cardioplegic solutions with organic antioxidants (such as vitamin E, vitamin C, methionine, or reduced glutathione); again, these have been shown experimentally to enhance the postischemic recovery of function (211, 212), but few, if any, have been adopted for use as cardioplegic additives. This may be because most radical production is thought to occur during reperfusion and, as such, these additives may be more appropriate as components of reperfusion solutions (see later).

a. Exogenous High-Energy Phosphates Surprisingly, exogenous ATP (which should not be able to gain access to the cell) has been shown to be an effective additive to cardioplegic solutions, enhancing myocardial protection at concentrations ranging from 0.1 (214) to 10 mmol/liter (37). Synergistic effects with phosphocreatine have also been observed (Fig. 12), with an optimal phosphocreatine concentration of 10 mmol/ liter (215). In surgical studies, beneficial effects of direct supplementation of cardioplegic solutions with highenergy phosphates have been championed in Russia (216) and subsequently confirmed by the authors (217, 218). The mechanism for this protection is unclear; it is generally assumed that these compounds are unable to cross the normal cell membrane (but possibly may be able to access the cell as a consequence of ischemiainduced membrane injury) and, for phosphocreatine, the intact molecule is required for protection (215). It could well be that they exert their protective effects at an extracellular site or that they act by some nonenergydependent mechanism such as chelating calcium (219, 220). In this regard, reducing the calcium concentration in the St. Thomas’ Hospital cardioplegic solution was shown to give similar protection (221); however, titration of calcium to normal concentrations (in the presence of phosphocreatine) did not diminish the enhanced protection (222). Other potential protective mechanisms of high-energy phosphate compounds include reducing oxidative stress (222, 223).

6. Manipulation of Metabolism and Substrate Utilization A major consequence of ischemia is the rapid depletion of myocardial ATP; it has been suggested that there may be a threshold of ATP below which the heart will fail to recover any mechanical function upon reperfusion (213). Cold cardioplegia induces rapid arrest and reduces the rate of depletion of high-energy phosphates; despite this, ATP continues to be depleted by residual energy-requiring cellular reactions. For this reason, various metabolic maneuvers have been investigated in an attempt to reduce further high-energy phosphate loss or to enhance anaerobic ATP production or availability.

FIGURE 12 The protective effect of optimal concentrations of ATP (0.1 mmol/liter), phosphocreatine (CP) (10 mmol/liter), or a combination of ATP and CP when added to St. Thomas’ Hospital cardioplegic solution (STH). Postischemic recovery of aortic flow after 35 min of reperfusion in rat hearts subjected to 270 min of hypothermic (20⬚C) global ischemia. Recovery expressed as a percentage of the preischemic (control) value. *p ⬍ 0.05 compared to STH, #p ⬍ 0.05 compared to STH⫹ATP and STH⫹CP. Redrawn from Robinson et al. (214).

51. Cardioplegia and Surgical Ischemia

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b. Adenosine and ATP Catabolites

d. Glucose and Glycolytic Intermediates

Catabolites of ATP have also been proposed as effective additives to cardioplegic solutions, with most recent studies concentrating on the protective effects of adenosine. As indicated earlier (Section V, A, 5, C), adenosine, at high concentrations, has been shown to be able to induce arrest (81); however, it is used more generally as an additive to hyperkalemic solutions to enhance protection (82, 224, 225). Adenosine has concentrationdependent properties; high levels were proposed to act by beneficially influencing the repletion of adenine nucleotide stores (224), whereas lower concentrations exploit its potent vasoactive effects to enhance coronary flow during reperfusion (225). The efficacy of adenosine, however, has been shown to be reduced at lower temperatures (226). Interestingly, the addition of adenosine to hyperkalemic solutions may reduce the rate of membrane depolarization (227), thereby reducing the calcium overload associated with hyperkalemic depolarization (228). Clinical trials of adenosine as an additive to blood cardioplegia have, however, been disappointing with only minimal or no beneficial effects on clinical outcome (229–231). In addition to adenosine, compounds that enhance tissue adenosine content during ischemia (such as AICAriboside) have been shown to improve postischemic recovery in a number of animal models (232–234); however, clinical trials have again been inconclusive with regard to efficacy of AICAriboside (235). Inosine, another catabolite of ATP, has also been shown to exert protective effects when used as an additive to cardioplegic solutions (236, 237).

For decades, glucose (often in combination with insulin and low doses of potassium) has been promoted as an effective way to protect the regionally ischemic heart during evolving myocardial infarction. Although clinical results have been far from exciting, strong interest in the concept remains—driven predominantly by the fact that glucose can promote glycolytic anaerobic ATP production in the absence of oxygen. It is not surprising, therefore, that surgeons elected to study glucose as an obvious cardioplegic additive. Some early cardioplegic solutions [e.g., Roe’s solution (35) and Lolley’s solution (243)] used massive (278 mmol/liter) concentrations of glucose, whereas others [such as Conti’s Birmingham solution (244) or Craver’s solution (245)] have employed concentrations nearer the physiological range. However, the St. Thomas’ group believes that the use of glucose (with or without insulin) should be considered with caution, especially when coronary flow is zero or very low (246). In this situation, glucose metabolism will lead to increased intracellular acidosis and lactate production, causing cellular injury and possible inhibition of other, potentially beneficial metabolic pathways. Mannitol and other nonmetabolizable sugars (such as sorbitol) have also been included in cardioplegic solutions, predominantly for their osmotic properties; however, the usefulness of these compounds remains unclear with conflicting results being obtained (246, 247). Other glycolytic intermediates (such as fructose diphosphate, phosphoenolpyruvate, and pyruvate) have also been shown to exert beneficial effects when used as additives to cardioplegic solutions (248–254). Although the predominant effect of metabolic manipulation by the addition of substrates to cardioplegic solutions is to enhance the postischemic recovery of function, a cautious approach should be taken when using compounds that might enhance the accumulation of potentially toxic intermediates. It is important to fully characterize dose–response effects and interactions with other components of the solutions. Any significant beneficial effects of substrate additives to cardioplegic solutions during clinical cardiac surgery have, however, yet to be fully established.

c. Amino Acids Enhancement of cardioplegic solutions with amino acids has been advocated as an effective means of improving postischemic function. The most commonly used amino acids are glutamate and aspartate, both of which participate in energy-yielding reactions involved in the tricarboxylic acid cycle and the malate–aspartate shuttle resulting in substrate-level ATP production (238, 239). During prolonged ischemia, the addition of glutamate to blood cardioplegia has been shown to enhance myocardial protection in energy-depleted hearts, improving myocardial oxygen consumption and anaerobic metabolism (240); warm (37⬚C) induction of arrest (for 5 min) with additional aspartate supplementation further improved protection in these energy-depleted hearts and led to the term ‘‘active resuscitation’’ (241). The clinical importance of glutamate and aspartate as additives to cardioplegic solutions has not been evaluated systematically, but some clinical studies are consistent with the experimental studies and claim that these amino acids improve postischemic hemodynamics and enhance metabolism (161, 242).

D. Optimizing Reperfusion to Maximize Postischemic Recovery As mentioned previously, early reperfusion is an absolute requirement to prevent ischemic injury continuing to cell death and tissue necrosis. However, reperfusion itself is thought to induce injury over and above that sustained during the ischemic period, and this has led to the concept of ‘‘reperfusion injury’’ (255–257).

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It follows from this that if reperfusion is carried out differently, it should be possible to improve the rate of recovery and possibly even the extent of recovery. While there is ample evidence that reperfusion can exacerbate the extent of reversible injury and thereby slow the rate of recovery during reperfusion, it is controversial whether reperfusion can induce irreversible injury (i.e., increase the number of cells that die) in tissue that was still in a state of reversible injury in the moments before reperfusion. If this form of lethal or irreversible reperfusion injury were to occur then combating reperfusion injury might be expected to increase the extent as well as the rate of recovery. Because of the controversy over the existence of lethal reperfusion injury, we have divided reperfusion injury into (i) reversible and (ii) irreversible forms (256, 257). Examples of the former would be myocardial stunning (a condition familiar to most surgeons who have to contend with a slowly recovering, weakly performing heart for some time after reperfusion) and reperfusion arrhythmias (again frequently seen by surgeons). Stunning, although unfavorable, is spontaneously reversible (although several hours may be required for full reversibility to be accomplished) and reperfusion arrhythmias often self-terminate or can be electrically converted to a normal sinus rhythm. Although both of these types of reperfusion injury are reversible, they can be considered as undesirable and thus represent potential therapeutic targets (258–260). However, it is worth repeating that successful interventions against reversible reperfusion injury can only accelerate the rate of recovery and not the extent of recovery (261). Irreversible reperfusion injury, if it exists, would be manifest as the induction or extension of infarction by the act of reperfusion. Clearly, if such a phenomenon were to be quantitatively significant, it would be very undesirable and would represent an important target for intervention, especially as success would result in an increased extent of tissue salvage (i.e., limitation of infarct size). However, at this time there is little or no conclusive evidence that this form of reperfusion injury exists. In the context of developing procedures to minimize the impact of reperfusion injury, cardiac surgery presents a unique situation in that the time of onset and duration of ischemia are known and can be controlled and, by virtue of the surgical exposure, the timing, nature, and composition of reperfusion can be controlled. Cardiac surgery, therefore, represents the ideal test bed to study reperfusion injury and also to develop, administer, and evaluate interventions designed to prevent reperfusion injury and enhance recovery. Surgical studies from Buckberg’s group (262) have demonstrated that the depression of postischemic myocardial function (stunning) seen after a period of ischemic cardiac arrest

can be attenuated by transiently reperfusing the heart with blood at 37⬚C in which either (i) extracellular calcium is lowered (by citrate), pH is elevated to pH 7.8 by the addition of buffer, and arrest is maintained by increasing potassium or (ii) osmolarity is increased by the addition of mannitol. A combination of these conditions resulted (262) in an almost complete recovery following 60 min of topical hypothermic ischemic arrest (Fig. 13). This study provides compelling evidence for the potential value of developing reperfusion solutions to complement, follow, and sustain the protection afforded by cardioplegia. As a result, numerous studies have focused on components of reperfusion, modification of which might be expected to substantially improve recovery. We believe that reperfusion injury has two main interconnecting mechanisms: oxidative stress and ionic disturbances. Ionic homeostasis is disturbed at the end of ischemia, with increased intracellular sodium and calcium, and these ionic changes can be exacerbated at the beginning of reperfusion (due to oxygen-derived free radical effects, correction of intracellular acidosis, and activation of the sodium/hydrogen exchanger and by cell swelling). Thus, the best approach to avoiding reperfusion injury is to (i) control ionic disturbances during reperfusion, (ii) combat free radical production and oxidative stress, and (iii) optimize the recovery of energy metabolism.

FIGURE 13 Effect of modified blood reperfusion (low calcium (0.47 mmol/liter), pH adjusted to 7.8, hyperkalemia (32 mmol/liter), and hyperosmolarity (350 mOsmol/liter) compared to unmodified blood reperfusion on left ventricular function (systolic pressure) at increasing diastolic volume in dogs subjected to 60 min of topical hypothermic (16⬚C) arrest and ischemia. Control (nonischemic) function (䊐), unmodified reperfusion (䊊), and modified reperfusion (䉱). Redrawn from Follette et al. (262).

51. Cardioplegia and Surgical Ischemia

1. Control Ionic Disturbances during Reperfusion The simplest and most obvious approach to the development of reperfusion solutions that maintain mechanical arrest is to start by giving conventional hyperkalemic cardioplegic solutions (which may have various other modifications) at the beginning of reperfusion in addition to that given at the beginning of ischemia. In this way, the maintenance of mechanical arrest (at 37⬚C) during the initial minutes of reperfusion, using cardioplegia, to dissociate excitation–contraction coupling from that of oxidative phosphorylation seems a logical extension to ischemic arrest with cardioplegia. As energy supplies are gradually restored during early reperfusion, the newly generated ATP can be directed initially toward repair processes and restoring ionic homeostasis rather than be consumed by the immediate return of contractile function (which, at that time, is not necessary as the patient remains on cardiopulmonary bypass) (263). a. Hyperkalemia A short infusion (500–750 ml delivered over a 10min period) of hyperkalemic blood cardioplegia at 37⬚C (the ‘‘hot-shot’’ technique), at the onset of reperfusion (in combination with supportive bypass), significantly enhanced postischemic recovery of contractile function (264). However, it should be noted that, in this particular study, the cardioplegic reperfusion solution also had reduced calcium, additional buffer, and was substrate enhanced. Thus, although testifying to the power of reperfusion solutions, this study did not allow the protective effects of maintained hyperkalemic arrest to be dissected from the other components of the reperfusion solution. Asanguineous cardioplegic reperfusion solutions have also been shown (265, 266) to exert beneficial effects; experimental and clinical studies demonstrated an improved rate and extent of postischemic recovery when 1 liter of crystalloid cardioplegic solution is infused over 3–5 min at the beginning of reperfusion. This particular solution contained normal calcium concentrations but had additional substrate (glutamate), it was buffered to pH 7.6, and was hyperosmolar. Again, the identification of the relative contributions of the individual components was impossible to determine. The authors concluded that crystalloid reperfusion solutions were preferable to blood-based solutions because (i) potentially toxic metabolites washed out during reperfusion could be discarded instead of being returned to the circuit, (ii) the reduced coronary vascular resistance and lower reperfusion pressure reduce the risk of edema formation associated with crystalloid solutions, and (iii) crystalloid solutions allow better quality control, ease of storage, and handling. Similar beneficial

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effects of manipulating reperfusion were observed when 500 ml of St. Thomas’ Hospital cardioplegic solution No. 2 was administered at 4⬚C to dog hearts as a transient (2–3 min) reperfusion solution (267). Clinical studies involving emergency surgical revascularization for acute coronary occlusion (failed angioplasty) have shown significantly improved recovery of myocardial function when controlled blood-based cardioplegic reperfusion is used (268, 269); these studies also highlighted the fact that older and sicker patients undergoing cardiac surgery require more attention to the protection of their myocardium, especially during the early reperfusion period. b. Hypocalcemia Reperfusion is thought to trigger a transient increase in calcium uptake, the severity of which is related to the severity and duration of the preceding ischemia (270). This, on top of the calcium and sodium overload that has likely occurred during ischemia, is deemed to be undesirable. Certainly, after cardiac surgery, there is an acute left ventricular dysfunction and a pattern of recovery that is consistent with stunning-like reperfusion injury (271). In a nuclear magnetic resonance (NMR) spectroscopy study (272), functional impairment was suggested to be related to increased intracellular calcium during both ischemia and reperfusion; however, the detrimental effects on recovery of function could be ameliorated by the initial reperfusion with low calcium. Reperfusion under conditions of transient hypocalcemia has been shown to reduce reperfusioninduced cellular calcium uptake (273) and improve postischemic recovery (274); associated with this hypocalcemia, increased potassium or magnesium (at concentrations sufficient to induce arrest) further enhanced recovery of function. These findings were confirmed (275) when transient (20 min) hypocalcemic (150– 250 애mol/liter) reperfusion with a modified blood cardioplegic reperfusate (buffered to pH 7.6 and containing additional substrates) significantly reduced infarct size after regional ischemia in the dog heart; in addition, postischemic recovery of systolic shortening was improved. The addition of the calcium antagonist diltiazem (300 애g/kg) to this hypocalcemic reperfusate further limited infarct size and enhanced recovery of function. c. Reduction of Reperfusion-Induced Sodium Overload Lazdunski and colleagues (276) hypothesized that increases in intracellular sodium during ischemia occur as a result of sodium pump inhibition; subsequent reperfusion (with continued sodium pump inhibition) activates the sodium/proton exchanger to reduce intracellular acidosis and this increases intracellular sodium

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further, which promotes reverse mode sodium/calcium exchange activity, leading to calcium accumulation and the harmful effects of calcium overload. Support for this hypothesis was demonstrated by Tani and Neely (277), who showed increasing intracellular sodium during ischemia, which was decreased rapidly during reperfusion at the same time as intracellular calcium increased; this uptake of sodium could be ameliorated by an inhibitor of the sodium/proton exchanger (277, 278). More recently, NMR measurements of sodium (279) have demonstrated that, after relatively short (20 min) periods of global ischemia, the sodium pump is immediately activated, which rapidly decreases intracellular sodium; however, inhibition of the sodium pump reveals a continued increase in intracellular sodium, indicating sodium/proton exchanger-mediated activity. The recent development and use of specific sodium/proton exchange inhibitors has demonstrated that inhibition of this mechanism of intracellular sodium influx, either during ischemia or during reperfusion, improves postischemic recovery (280–285) and might represent a promising therapeutic modality to ameliorate postoperative stunning after cardiac surgery. There seems to be good evidence that ionic manipulation during reperfusion (particularly using maintained arrest with hyperkalemia as well as hypocalcemia) exerts beneficial effects on reperfusion injury. However, ionic manipulation during early reperfusion is currently little used during cardiac surgery, although Buckberg’s blood cardioplegic reperfusate attempts to reduce calcium overload. New therapeutic drugs (which control specific ion exchangers) may offer some advances in this area but clinical studies have yet to be conducted.

peroxidase] have been used as additives to the cardioplegic solution and the initial reperfusion solution. Superoxide dismutase, in combination with catalase, added to the initial reperfusion solution given to dog hearts after hypothermic ischemic arrest was shown (289) to improve postischemic cardiac performance and reduce lipid peroxidation. We (196) have demonstrated similar results in rat hearts, with improved postischemic recovery of function when a combination of superoxide dismutase and catalase was added either during ischemia and reperfusion or reperfusion alone (Fig. 14). Additional studies from our laboratory have demonstrated that these antioxidant enzymes also abolish reperfusioninduced arrhythmias (287). More recently, overexpression of superoxide dismutase transgene has been shown to improve myocardial protection in mouse hearts (290). The addition of coenzyme Q10 to a hyperkalemic substrate-enriched blood cardioplegic reperfusion solution has also been shown (291) to reduce infarct size and to improve contractility after regional ischemia in dog hearts. Similar results were obtained when a blood cardioplegic reperfusion solution was supplemented with a cocktail of endogenous-free radical scavengers (superoxide dismutase, catalase, coenzyme Q10, and glutathione peroxidase) (292). b. Pharmacological Inhibition of Radical Production Pharmacological inhibition (with agents such as allopurinol or oxypurinol) of radical generation by xanthine

2. Combat Free Radical Production and Oxidative Stress Oxygen-derived free radicals (such as superoxide, hydrogen peroxide, and the hydroxyl radical) have been implicated as major mediators of reperfusion injury (193, 196, 258, 260, 261, 286, 287). Electron spin resonance studies have demonstrated that a burst of free radicals occurs immediately after reperfusion of ischemic myocardium (288) and that this is dependent on reoxygenation and not reflow. Antioxidants [either present during the ischemic period (see Section V, C, 5) or given before or at the moment of reperfusion, but not after] attenuate myocardial stunning and limit the extent of radical production (261). There are a number of different types of antioxidant mechanisms that have been used in an attempt to reduce reperfusion injury. a. Supplementation of Antioxidant Enzymes Antioxidant enzymes [such as superoxide dismutase, catalase, coenzyme Q10 (ubiquinone), and glutathione

FIGURE 14 Effect of antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) as additives to St. Thomas’ Hospital cardioplegic solution and/or initial reperfusion solution. Postischemic recovery of aortic flow after 35 min of reperfusion in rat hearts following 30 min of global normothermic (37⬚C) ischemia; recovery expressed as a percentage of the preischemic value. *p ⬍ 0.05 compared to control. Redrawn from Chambers et al. (196).

51. Cardioplegia and Surgical Ischemia

oxidase has been shown to ameliorate free radical-induced myocardial injury in both experimental (195, 197, 198, 287) and clinical (201–203, 205) studies. As described earlier (Section V, C, 5), metal ions (such as iron and copper) are involved in the generation of oxygenderived free radicals (206). Chelators of metal ions (such as deferoxamine) have, in addition to being added to cardioplegic solutions, been used as additives to reperfusion solutions; significant improvements in postischemic recovery of function have been observed when added to the reperfusate (293), whereas others (294) have observed protection only when used as a pretreatment, with addition to the reperfusate having no effect. c. Supplementation of Organic Antioxidants A number of organic antioxidants have been used as additives, either of cardioplegic solutions or of reperfusion solutions, to prevent free radical-induced injury. We (287, 295, 296) have shown that compounds, such as methionine, glutathione, and ascorbate (vitamin C), or free radical spin traps (scavenge free radicals and form relatively stable adducts for electron spin resonance) reduce reperfusion-induced arrhythmias. Vitamin E, a major membrane antioxidant, decreases during early reperfusion after cardiac surgery (297); however, although supplementation with vitamin E ameliorated this decrease, there was no apparent reduction in myocardial injury (298). Thus, antioxidant therapy, given either immediately before or at the initiation of reperfusion, appears to have some therapeutic benefit; despite this, however, surgeons have been reluctant to use transient modification of reperfusion solutions in which antioxidant therapy might be of benefit. One reason for this may be that a major source of free radical generation is from activated neutrophils and, in this area, surgeons have been active in attempting to reduce reperfusion injury.

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stages of adhesion that culminate in migration across the vessel wall (305). The use of monoclonal antibodies (306–308) or drugs (309–312) that prevent the interaction between the neutrophil and the endothelium have demonstrated beneficial effects in the ischemic-reperfused myocardium. Thus, although neutrophils are essential for normal circulatory function, there appears to be a strong argument for temporarily ablating their activity during early reperfusion. We (313) have shown, in the transplanted and isolated blood perfused rat heart, that reperfusion (for 1 hr) with neutrophil-depleted blood significantly improves the postischemic recovery of function, but that this benefit is lost on continuation of reperfusion for 4 or 24 hr; in addition, the minimum duration of neutrophil depletion for benefit to be obtained was only 10 min (2 min of reperfusion with neutrophil-depleted blood had no effect) (Fig. 15). Similarly, transient (20 min) reperfusion with neutrophil-depleted blood in sheep hearts previously subjected to LAD occlusion for 90 minutes reduced the extent of stunning, the no reflow phenomenon, and infarct (314). In a similar study in pigs (315), neutrophil depletion by filtration (for 6 hr) prevented a period of transient dysfunction [commonly observed in patients undergoing cardiac surgery (271)]

d. Antineutrophil Therapy Activated neutrophils constitute a source of oxygenderived free radicals during reperfusion; in addition, they can cause other detrimental effects during reperfusion (299). Thus, cardiopulmonary bypass is associated with an inflammatory response (probably due to contact between blood and the foreign surfaces of the bypass circuit), which causes the activation of neutrophils (300, 301) and leads to neutrophil infiltration of the heart (302). Neutrophils are also activated by ischemia and reperfusion (303) and by complement activation (304) and these neutrophils interact with the vascular endothelium at sites of inflammation. This neutrophil– endothelium interaction is a multistep process in which families of cell adhesion molecules (integrins, selectins, and the immunoglobulin superfamily) regulate different

FIGURE 15 Effect of varying durations of leukopenic reperfusion on postischemic recovery of left ventricular developed pressure (LVDP) in blood-perfused isolated rat hearts subjected to 8 hr of global hypothermic (4⬚C) ischemia and 60 min of reperfusion with blood. *p ⬍ 0.05 compared to hearts reperfused with leukopenic blood for 10, 30, or 60 min. Redrawn from Galin˜anes et al. (313).

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X. Pathophysiology

and reduced ventricular stiffness, suggesting the involvement of activated neutrophils in this effect. Neutrophils, activated by cardiopulmonary bypass, have been shown to be sequestered and degranulated in the ischemic and reperfused myocardium of patients undergoing cardiac surgery (316); these effects are likely to be involved in reperfusion injury and postischemic dysfunction. In the laboratory, there are many ways to attenuate neutrophils [including chemicals such as mustine (313), monoclonal antibodies to neutrophil adhesion molecules (317), or neutrophil filters (315)]. There have also been cardiac surgical studies in which neutrophils have been depleted during cardiopulmonary bypass, including the early reperfusion period, resulting in beneficial effects on both the heart and the lung (318–320). There is also increasing evidence that neutrophil depletion of transfused blood during cardiac surgery can be beneficial (321). Thus, there is good evidence that activated neutrophils are involved in the ischemia and reperfusion injury associated with cardiopulmonary bypass and cardiac surgery. Studies in which neutrophils have been depleted during cardiopulmonary bypass, myocardial reperfusion, and transfusion have shown promising effects, as have techniques that prevent neutrophil adhesion. Further work in this field is warranted and therapeutic benefits would seem likely. 3. Optimizing the Recovery of Energy Metabolism Optimizing metabolic recovery during reperfusion has already been discussed in part (in Section V, D, 1), as prevention of contraction by potassium arrest during early reperfusion favors ionic and energetic recovery. However, another approach, which has been much studied, is selective substrate provision during reperfusion. The addition of amino acids, such as glutamate and aspartate, to an initial reperfusion solution has been shown to improve postischemic recovery after global ischemia and cardiac surgery (264); associated with this was an increased oxygen uptake and an increased energy production. It is thought that these amino acids replenish citric acid cycle intermediates and that they can enter the malate–aspartate shuttle (238). Subsequent studies (265, 322, 323) have also demonstrated the beneficial effects of amino acid supplementation of reperfusion solutions. More recently, however, there have been studies that have questioned the usefulness of these additives (324, 325). The addition of exogenous adenosine (225, 326) or drugs that maintain endogenous adenosine concentrations (233, 234, 327) during the initial reperfusion period after global ischemia improves postischemic recovery of function; endogenous adenosine has also been shown to influence neutrophil adhesion

(328). There is also evidence that drugs that enhance glucose oxidation during reperfusion (such as dichloroacetate) are beneficial (329) and may involve enhanced activity of pyruvate dehydrogenase (330). The addition of pyruvate (2 mmol/liter), in combination with glucose, to the reperfusion solution was shown to improve postischemic recovery (251), possibly by preventing free radical generation. Despite good evidence that the modification of reperfusion, using a transient infusion of warm blood-based cardioplegic solutions either with or without selective substrate additives, ameliorates reperfusion injury and enhances the rate (and maybe the extent) of recovery, surgeons have generally failed to adopt this procedure (41). Whether this is because it adds yet one more element of complexity to an already complex operation or whether it reflects surgeons, general satisfaction with the protective effects of cardioplegia used just during ischemia is open to speculation.

E. Effective Cardioprotection Should Not Ignore Vascular and Conducting Tissue Optimal cardioprotection needs to consider all the cellular components of the heart and not just the myocyte. Vascular smooth muscle, endothelium, and conducting tissue are just as likely as the myocyte to be injured during ischemia and reperfusion and hence the protection of these tissues may be extremely important. Unfortunately, solutions that may exert a considerable protective effect on the myocyte may be ineffective and possibly even hazardous to other constituent cells of the heart; this is especially likely for the endothelium that is directly exposed to these often, very nonphysiological solutions. However, in comparison to the myocyte, there have been relatively few studies that have considered this aspect of cardioprotection. 1. Effect of Hyperkalemia on the Endothelium Cardioprotection studies have traditionally concentrated on the myocyte and its function, but the influence of ischemia and reperfusion on the vasculature is becoming increasingly important; however, relatively few studies have been carried out to determine the effect of existing cardioprotective strategies on the microcirculation or to develop ones specifically targeted at this highly vulnerable and important tissue. As with the myocyte, ischemic and reperfusion injury can adversely affect structural and functional alterations in the vasculature; most evidence suggests that the endothelium, rather than the vascular smooth muscle, is more vulnerable to injury and, in particular, to reperfusion injury. It is unclear whether this vascular injury occurs before or after

51. Cardioplegia and Surgical Ischemia

that of the myocyte, although it is now thought that endothelial dysfunction is a relatively early event (331), such that the ‘‘no reflow’’ phenomenon (which can be defined as an inability to perfuse previously ischemic myocardium even when blood flow is restored to the arteries supplying the tissue) can occur and potentially be additive to the overall detrimental effects of ischemia and reperfusion on survival of the myocytes (332). Specific effects of cardioplegic solutions, particularly their high potassium content, on the vasculature can significantly influence the outcome of global ischemia and reperfusion after cardiac surgery. The suggestion that crystalloid cardioplegia may be cytotoxic to the endothelium was first made in 1981 on the basis of a comparative study of 11 different cardioplegic solutions on cultured endothelial cells (333). Subsequent studies (334, 335) indicated that the addition of blood or albumin reduced the toxic effects, and it was concluded that the endothelium appeared to be more susceptible to injury during ischemia and reperfusion than myocytes. Hyperkalemia has been shown to induce vascular damage and endothelial dysfunction and impair the vasodilatory response to 5-hydroxytryptamine (336, 337), and these effects of potassium were concentration dependent. Perfusion of the hamster cremaster muscle with hyperkalemic crystalloid cardioplegia caused injury associated with decreased microvascular blood flow and increased neutrophil accumulation, which could be attenuated by adenosine-induced vasodilation (338). Multidose infusions of crystalloid cardioplegia (in crystalloid-perfused isolated rat hearts) were shown to be more protective of the endothelium and vascular smooth muscle than single-dose cardioplegia (339) and suggested that the endothelium was more susceptible to injury than smooth muscle. This was confirmed in a study on crystalloid-perfused isolated rat mesentery (to dissociate the effects of ischemia from cardioplegia and effects on vasculature from that on myocytes) (340). However, in studies in which blood-perfused hearts were used (341, 342), both the endothelium and smooth muscle were equally protected regardless of single or multiple cardioplegic infusions, suggesting that the preischemic crystalloid perfusion was influencing the effect of the cardioplegic solution on the endothelium. Bloodbased cardioplegia appears to protect the endothelium during ischemia, but endothelial reperfusion injury and no reflow persist during reperfusion (343–346). Reperfusion of the myocardium with oxygenated solutions leads to microvascular incompetence (331) and this is thought to arise as a consequence of oxygenderived free radical generation during ischemia and reperfusion (347, 348) with resulting inactivation of the nitric oxide pathway (349). Antioxidants (particularly superoxide dismutase) have been shown (347, 350) to

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attenuate endothelial dysfunction and preserve endothelial nitric oxide release. Attenuation of endothelial injury during reperfusion can also be achieved by a nitric oxide-mediated mechanism, and nitric oxide donors have been shown to improve postischemic endothelial function (351–353). Increasing evidence suggests a role for the upregulation of adhesion molecules on neutrophils and endothelium and a neutrophil–endothelium interaction being responsible for endothelial dysfunction and no reflow (303). Prevention of these interactions by various inhibitory mediators attenuates vascular dysfunction and improves postischemic myocardial recovery (354–359). It has also been suggested that ‘‘polarizing’’ cardioplegic solutions may have beneficial advantages for the endothelium (360–362) as well as for the myocardium (see Section V, A, 2). The microcirculation is increasingly being recognized as an important component within the heart that requires adequate protection during cardiac surgery. Injury to the vasculature can influence the protection of the myocytes such that further damage occurs as a result of no reflow. As our understanding of the inflammatory response affecting the microvasculature during cardiac surgery (and particularly cardiopulmonary bypass) increases, so attention will focus on therapeutic treatment to prevent endothelial injury. 2. Effects of Hyperkalemia on Conducting Tissue It is generally assumed that the conducting tissue of the heart is more tolerant to ischemia than the myocyte (363); however, there have been relatively few studies on the effects of cardioplegic solutions on myocardial conducting tissue. The increasing use of hyperkalemic cardioplegia was associated with an increase in the incidence of conduction abnormalities during reperfusion, such that prolonged heart block and supraventricular tachyarrhythmias became relatively frequent after cardiac surgery. These problems result mainly from inadequate preservation of the atrial tissue, leading to ischemic injury of the supraventricular conduction system during cardioplegic arrest (364, 365). Supraventricular arrhythmias can be attenuated by topical hypothermia of the atrium, indicating the AV node as the main site of conduction delay (366), and do not appear to be related to hyperkalemia (367). Low-amplitude electrical activity has been detected in the lower atrial septum, the AV node–His bundle complex, and the ventricular myocardium following cardioplegic arrest, even at tissue temperatures below 15⬚C (368). The site of origin of this low-amplitude activity was localized to the atrial septum containing the AV nodal tissue (369, 370); the addition of calcium channel blockers has been shown to prevent low-amplitude activity, suggesting involve-

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X. Pathophysiology

ment of calcium-mediated activation of specialized conduction tissue. Blood cardioplegia has been suggested to result in a greater incidence of perioperative and postoperative conduction disturbances than seen with crystalloid cardioplegia, possibly suggesting that blood cardioplegia should be avoided in patients with preexisting conduction problems (371); however, these findings have not been confirmed by others (372). Normothermic cardioplegia has been associated with fewer conduction abnormalities than cold cardioplegia, suggesting that hypothermia may be involved in conduction disturbances (373); however, it is likely that these are relatively short-lived. Few studies involving cardioplegia have examined the effects on conduction tissue; this may reflect the fact that surgeons do not consider that cardioplegia, with the attendant ischemia and reperfusion, causes problems that cannot be controlled (using temporary pacing). However, overall good protection should try to avoid exacerbating any conduction problems with inappropriately formulated cardioplegic solutions.

F. Alternative Approaches to Limiting Tissue Injury during Cardiac Surgery Although not the brief of this chapter, it is important to recognize that the use of cardioplegic solutions is not the only way by which the heart, undergoing cardiac surgery, can be protected. As mentioned earlier, a number of surgeons use short periods of intermittent ischemia with fibrillation, in preference to cardioplegia, when conducting coronary artery bypass surgery. There is also the possibility of exploiting endogenous adaptive defense mechanisms and the potential for the development of pharmacological mimetics. 1. Preconditioning, an Endogenous Adaptive Mechanism of Cardioprotection Ischemic preconditioning is an endogenous adaptive mechanism shown to be inducible in all species studied, including humans. Here, short periods of ischemia, hypoxia, rapid pacing, or exposure to receptor agonists confer protection when the myocardium is subjected to a subsequent prolonged period of ischemia (374). Ischemic preconditioning was originally shown to delay the onset of irreversible ischemia and thereby limit infarct size after extended periods of regional ischemia and reperfusion (20). Subsequent studies have identified a potential mechanism of action for this protection involving intracellular signaling pathways leading to the activation of protein kinase C, which is subsequently thought to phosphorylate an as yet unknown ‘‘end effector’’ [although evidence shows that the ATP-

sensitive potassium channel may be involved (375)]. Although most studies have demonstrated protection against regional ischemia and the development of infarction, ischemic preconditioning has also been shown to be protective against arrhythmias (376) and to improve contractile function after ischemia (377). We have also shown, in the setting of global ischemia, that ischemic preconditioning provides similar protection to that achieved with cardioplegic solutions (378) and that ischemic preconditioning, in combination with cardioplegia, provides additional protection (379–381); however, this conclusion is somewhat controversial (378, 382, 383). Interestingly, pharmacological preconditioning, with drugs that activate the ATP-sensitive potassium channel, has been suggested as a potential means of enhancing cardioplegic protection during cardiac surgery (384, 385). In the human, ischemic preconditioning has been shown to reduce the rate of ATP depletion (386) and reduce troponin T release (387). However, improvement in postischemic function has not been demonstrated (388). Although preconditioning has been shown to be a powerful protective mechanism, in the surgical setting the evidence that it can enhance protection achieved with cardioplegia is limited. It is possible that pharmacological preconditioning may be of benefit, but considerably more studies into this mechanism need to be done before it can be recommended. 2. Intermittent Ischemia with Fibrillation Intermittent ischemia with fibrillation (‘‘intermittent cross-clamp fibrillation’’) involves inducing global ischemia (at temperatures of around 32–34⬚C) by clamping the aorta (to obtain a bloodless field) and inducing electrical fibrillation (to obtain a nonbeating heart) for durations ranging from 6 to 13 min while the distal anastomosis of a coronary graft is constructed. The heart is then reperfused for a similar duration while the proximal anastomosis on the aorta is constructed. Since the first coronary artery bypass operations (389), this technique has continued to be favored by some surgeons, in both low and high-risk patients (390). Intermittent ischemia with fibrillation has been claimed to be a safe technique for myocardial protection during coronary artery bypass surgery (391–395), producing similar levels of protection to that of hyperkalemic cardioplegia (crystalloid or blood) in terms of markers of ischemic injury (troponin T, troponin I, or the myocardial-specific isoenzyme of creatine kinase) or mortality. However, the ischemic duration during intermittent ischemia with fibrillation is generally significantly shorter than that of cardioplegic arrest. In an isolated rat heart study, we have demonstrated that identical ischemic durations for

51. Cardioplegia and Surgical Ischemia

intermittent ischemia with fibrillation and multidose cardioplegia provide similar levels of protection (396), suggesting that intermittent ischemia with fibrillation is inherently protective. Renewed interest in this technique has arisen because it has been suggested that the technique may be inducing a ‘‘preconditioning’’ protection (see earlier discussion). Thus, intermittent ischemia with fibrillation may confer benefits on the myocardium, leading to additional protection during surgery; we (397) have demonstrated these beneficial effects in experimental studies. The current technique of intermittent ischemia with fibrillation is used successfully and safely for myocardial protection during cardiac surgery. It may enhance endogenous protective mechanisms and be particularly relevant when considering the changes occurring in the population of patients coming to surgery, who increasingly tend to be more elderly with more diffuse and severe disease, which requires enhanced protection.

4.

5. 6. 7.

8. 9.

10.

11. 12.

VI. SUMMARY 13.

This chapter provided a brief historical overview of the development and use of cardioplegic solutions during cardiac surgery. We have also attempted to rationalize, from the multitude of studies in this area relating to the protective effects of cardioplegic solutions, some of the mechanisms involved in the induction of arrest, slowing the onset of irreversible ischemic injury, and blocking specific ischemic changes and minimizing reperfusion-induced injury. In addition, we have looked briefly at other tissues of the heart and not just the myocytes. At the present time, myocardial protection during cardiac surgery is predominantly achieved by the use of hyperkalemic solutions, albeit in a great variety of forms. This principle has stood the test of time and has been one of the major influences on the development and success of modern cardiac surgery. Alternative means of arresting and protecting the heart are still being developed and investigated; wheteher, in time, any of these techniques will usurp the predominance of elevated potassium remains to be seen.

14.

15. 16.

17.

18.

19.

20.

21.

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52 Apoptosis ARMIN HAUNSTETTER

SEIGO IZUMO

Department of Cardiology University of Heidelberg Heidelberg, Germany

Cardiovascular Division Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts 02215

I. INTRODUCTION

ence results in different morphological, biochemical, and diagnostic features (Table I). A cell can undergo apoptosis in reaction to several different stimuli. Although these noxious stimuli may be as diverse as ligands for death receptors at the cell membrane, DNA-damaging agents, prooxidants, and growth factor depletion, the cell death signaling converges on a cell autonomous death pathway (Haunstetter and Izumo, 1998). Characteristically, the common final mechanism involves the activation of a characteristic subgroup of proteases, called caspases (Thornberry and Lazebnik, 1998). These proteases, once activated, cleave a plethora of cellular proteins, leading to characteristic morphological alterations of the dying cell. It is only at this advanced stage of apoptotic cell death that morphological alterations become apparent, whereas the early initiation and propagation phases display changes that are only detected by biochemical techniques. In typical apoptosis, the nuclear chromatin condenses, and nuclei shrink and break up in small, subnuclear fragments (Majno and Joris, 1995). In addition, genomic DNA is cleaved in between nucleosomes where the DNA is normally wrapped around complexes formed by histones (Wyllie, 1980). Advanced apoptosis results in volume loss of the whole cell and formation of membrane blebs, which may contain cell organelles (e.g., mitochondria in cardiac myocytes) and finally break away from the cell to form cellular remnants (apoptotic bodies). Although during the whole process the integrity of the cell membrane remains preserved, subtle membrane alterations can be detected. For example, the phospholipid phosphatidylserine, which is re-

Apoptosis can be defined as cellular suicide involving specialized initiation and execution mechanisms within the cell. Literally, the term denotes the drop of leaves from trees in the fall season, reflecting the sporadic and gradual loss of cells in tissues. In recent years, apoptosis attracted increasing interest in the cardiology research community, essentially for two major reasons. First of all, in pathologic and experimental studies, apoptosis emerged as a widespread feature in several cardiac diseases, including ischemic heart disease and congestive heart failure. Second, apoptosis is a regulated form of cell death that may provide novel approaches for therapeutic intervention to prevent the loss of cardiac myocytes and thus prevent or slow the progression of cardiac disease. It is the scope of this chapter to summarize current knowledge of the morphological and molecular features of apoptosis in general. In addition, important aspects of apoptosis in cardiac disease will be addressed.

II. APOPTOSIS: A DISTINCT TYPE OF CELL DEATH Based on cellular pathophysiology, classification of cell death has increasingly focused on two major entities: necrosis (sometimes termed oncosis) and apoptosis (Majno and Joris, 1995). The basic pathogenic difference between the two forms of cell death can be summarized as destruction of the cell by an external stimulus in necrosis versus self-destruction in response to unfavorable conditions in the case of apoptosis. This differ-

Heart Physiology and Pathophysiology, Fourth Edition

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TABLE I Features of Apoptotic and Necrotic Cell Death Apoptosis

Necrosis

Cell morphology

Cell shrinkage Cell fragmentation

Cell swelling

Nuclear morphology

Chromatin condensation Nuclear fragmentation

Nuclear swelling

Membrane alterations

Intact membrane Phosphatidylserine exposure

Membrane rupture

DNA fragmentation

Internucleosomal or large fragments (50–300 kbp)

Random

Caspase activation

Yes

No

Energy dependence

Yes

No

stricted to the inner leaflet of the cell membrane through an active process in living cells, is exposed to the outside of the apoptotic cell (Fadok et al., 1992; Bennett et al., 1995; Zwaal and Schroit, 1997). Furthermore, several lines of evidence indicate that apoptosis is an ATPdependent process and thus requires some residual metabolic activity of the cell (Leist et al., 1997; Li et al., 1997). In contrast, necrotic (oncotic) cell death seems to be triggered by energy depletion. The maintenance of cellular integrity, especially the maintenance of ionic concentration gradients across the cell membrane, requires ATP to fuel transport systems such as the Na⫹ / K⫹-ATPase or Ca2⫹-ATPases. Deregulated ion transport induces water shifts that lead to the swelling of the cellular cytoplasm and intracellular organelles, such as mitochondria and the nucleus. Membrane rupture is the irreversible consequence that kills the cells (Majno and Joris, 1995). Although necrotic cell death is associated with the activation of proteolytic enzymes, caspases typically do not seem to play a major role. Furthermore, degradation of protein is not limited to caspase substrate proteins, but involves a nonspecific proteolysis of all cellular protein constituents. Likewise, the degradation of genomic DNA is random and therefore does not show the characteristic DNA fragments suggestive of internucleosomal DNA cleavage.

III. TECHNIQUES TO DETECT APOPTOTIC CELL DEATH As outlined in the previous section, apoptotic cell death is associated with several distinct biochemical and

morphological features that help distinguish cells dying by apoptosis from cells undergoing necrotic cell death. Based on these differences, several techniques have been developed to detect and quantify apoptosis in cultured cells and tissue sections (Table II). Cellular shrinkage and fragmentation can be visualized by light microscopy. Electron microscopy is helpful in delineating the alterations of subcellular structures. This is especially true for nuclear alterations, where chromatin clumping and margination may ultimately result in a horseshoe or half-moon appearance of the condensed nuclear chromatin prior to nuclear fragmentation (Kerr et al., 1972; Deliliers and Caneva, 1999). In order for light microscropy to visualize nuclear alterations, fluorescent staining with the membrane-permeable dye Hoechst 33258 (bisbenzimide) is required. In contrast to Hoechst 33258, propidium iodide does not permeate intact membranes and failure to stain nuclear DNA provides indirect proof for the integrity of the cell membrane in the dying cell. When propidium iodide stains the nucleus, the mechanism is considered necrotic rather than apoptotic. Early exposure of phosphatidylserine on the outer leaflet of the cell membrane during apoptosis allows for cell labeling with fluorescent-labeled annexin V, a protein that requires phosphatidylserine to bind to cellular membranes (Martin et al., 1995). Biochemical evidence for apoptosis can be provided by the demonstration of caspase activation. Caspases are proteolytic enzymes with specific cellular substrate proteins. Caspase activation can thus be verified by in vitro enzymatic tests. Incubation of artificial substrates, such as the oligopeptide aspartate–glutamate–valine– aspartate–AMC (DEVD-AMC) with apoptotic cell ly-

TABLE II Assays to Identify Apoptotic Cells Cytological/biochemical alteration

Assay method

Cell morphology

Light microscopy Electron microscopy

Nuclear morphology

Electron microscopy Hoechst 33258 staining

Membrane alterations

Propidium iodine staining Annexin V staining

Caspase activation

In vitro enzyme assay Immunoblotting (cleavage of caspase or capsase substrates) Sensitivity to caspase inhibitor

DNA fragmentation

Agarose gel electrophoresis Pulsed field gel electrophoresis TUNEL (TdT) Taq-mediated double-strand DNA ligation

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sates will yield free AMC that can be determined by fluorometry. Alternatively, immunoblotting of cellular lysates will detect cleavage of cellular target proteins or the active subunits of caspases as caspase activation requires prior cleavage of the caspase proform into proteolytically active subunits (Thornberry and Lazebnik, 1998). Indirect evidence for caspase-mediated cell and tissue alterations can be provided by the inhibition of caspase activity (e.g., with the modified tetrapeptide DEVD-CHO). Most evidence for the occurrence of apoptosis in cardiovascular tissues is based on the demonstration of DNA fragmentation (Wyllie, 1980). Separation of genomic DNA by agarose gel electrophoresis will show DNA fragments with multiples of 180–200 bp in length. Sensitivity can be increased by radioactive labeling of DNA fragments prior to electrophoresis and exposure of the gels to X-ray films. However, evidence suggests that under certain circumstances, DNA degradation does not proceed to the stage of internucleosomal cleavage, but only results in large DNA fragments of 50–300 kbp of length, which requires specialized pulsed field gel electrophoresis for verification (Brown et al., 1993; Oberhammer et al., 1993; Susin et al., 1999). Staining of individual apoptotic nuclei in cell culture or tissue sections is made possible by enzymatically adding fluorescent-labeled nucleotides to the ends of fragmented DNA. In contrast to DNA electrophoresis, TUNEL (TdT-UTP nick end labeling) staining allows for the detection of individual apoptotic nuclei and their quantification (Gavrieli et al., 1992). Combinations of propidium iodine, annexin V, and TUNEL staining are used to quantitate both apoptotic cells (TUNEL or annexin V positive) and necrotic cells (propidium iodide positive) by fluorescence microscopy or fluorescentactivated cell sorting (FACS) analysis. While the commonly used TUNEL technique uses the enzyme terminal deoxynucleotidyl transferase (TdT) to transfer nucleotides to the ends of DNA fragments, another technique utilizes the Taq DNA polymerase to ligate labeled double-stranded fragments to cleaved DNA (Gavrieli et al., 1992; Didenko and Hornsby, 1996). The latter technique is claimed to be more specific for double-strand DNA breaks that occur during apoptosis. In contrast, TdT attaches nucleotides to single-strand breaks, potentially reducing its specificity for apoptotic DNA degradation. In fact, some concerns have been raised regarding the specificity of TUNEL staining in verifying apoptosis, as positive DNA labeling has been associated with markers of DNA repair or with morphological alterations suggestive for oncotic, and not apoptotic, cell death in cardiac myocytes (Ohno et al., 1998; Kanoh et al., 1999).

IV. A WORM LEADS THE PATH The discovery of a distinct morphology of cells dying in the context of tissue and organ development or in hormonally regulated cell turnover (e.g., in the ovary) was an interesting observation, which has been known for decades without a major impact (Kerr et al., 1972; Majno and Joris, 1995). Only the understanding of the molecular mechanisms involved in the execution of apoptosis attracted the interest of many researchers in different fields of biomedical research. The basis for these insights was established by the work of Robert Horvitz’ laboratory at the Massachusetts Institute of Technology during the 1980s (Ellis and Horvitz, 1986; Metzstein et al., 1998). They found that during the development of the nematode worm Caenorrhabditis elegans, 131 cells out of a total of 1090 somatic cells undergo programmed cell death, more than 75% of which are of neuronal lineage. Some mutant worms display abnormalities in cell number, either exhibiting excessive cell loss or inappropriate survival of cells that are normally bound to die. Meticulous genetic analysis defined more than 10 genetic loci that regulate the elimination of cells during the development of C. elegans. As is obvious from Table III, which lists the most important apoptosisrelated genes in C. elegans and known mammalian counterparts, more than one gene exists in the mammalian genome for most of the cell death genes in the C. elegans genome. This observation reflects the higher complexity of mammalian organisms and mammalian genomes (with approximately 100 ⫻ 103 genes) compared to the nematode genome (containing approximately 20 ⫻ 103 genes). Because each functional step in apoptotic cell death in C. elegans is served by a single gene locus, C. elegans provides a better tool for functional genetic analysis. In contrast, a certain degree of redundancy of overlapping function in the mammalian genome makes functional genetic analysis much more difficult, if not impossible. Functionally, the cell death genes in C. elegans can be roughly categorized into two groups of genes. One group of genes (including ced-1, -2, -5, -6, -7, and ced-10) mediates the removal of the cell corpse by phagocytosis (Liu and Hengartner, 1998; Metzstein et al., 1998; Wu and Horvitz, 1998a,b). Current evidence suggests that these genes are mostly expressed in the phagocytosing cell, and loss of function leads to an accumulation of dead cell corpses in tissues of the developing nematode. The second group of genes (including ced-3, ced-4, ced-9, and egl-1) regulates and mediates cell death in the dying cell (Yuan and Horvitz, 1992; Yuan et al., 1993; Hengartner and Horvitz, 1994; Conradt and Horvitz, 1998). For example, loss of function of the gene ced-3 inhibits apoptosis in all cells bound to undergo

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TABLE III C. elegans Cell Death Genes (ced) and Their Mammalian Homologues Gene

Function

Mammalian homologue

Ced-1

Cell body removal

Ced-2

Cell body removal

?

Ced-3

Caspase (apoptotic effector protease)

Caspases 1–14

Ced-4

Proapopototic regulator

Apaf-1, FLASH, nod1

Ced-5

Cell body removal

DOCK180

Ced-6

Cell body removal

SH3 adaptor protein

Ced-7

Cell body removal

ABC transporter proteins

Ced-8

?

?

Ced-9

Antiapoptotic regulator

Antiapoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xL)

Ced-10

Cell body removal

?

Egl-1

Proapoptotic regulator

Proapoptotic BH3-domain only proteins (e.g., Bid, Bik)

apoptosis, irrespective of mutations affecting the other genes, thus defining ced-3 as a proapoptotic gene that lies most downstream in the apoptotic cascade (Metzstein et al., 1998). Gain of function mutations of ced-4 induce cell death in cells that normally survive. However, the proapoptotic activity of ced-4 depends on functional ced-3, indicating that ced-4 is upstream of ced-3. Additional analysis of single and double mutant worms led to the definition of a cascade of apoptotic events as depicted in Fig. 1. The hierarchy of control goes from egl-1 to ced-9 and ced-4 and finally to ced-3, with ced-9 being antiapoptotic, whereas the other three genes are proapoptotic (Metzstein et al., 1998). Genetic analysis of apoptosis in C. elegans proved to be instrumental in defining the central pathway and regulation of apoptosis in mammalian cells. However, the current understanding of apoptosis in C. elegans is strictly limited to apoptosis in the context of development. In contrast, apoptosis in cardiovascular disease is generally not initiated by a genetic developmental program, but by noxious stimuli acting on differentiated cells of adult tissues. The initiation process involves metabolic, toxic, and receptor-mediated mechanisms for

?

which C. elegans has not yet been shown to represent an adequate model.

V. CASPASES Caspases play a central role in the initiation and execution of apoptotic cell death (Nicholson and Thornberry, 1997; Thornberry and Lazebnik, 1998). Contrary to C. elegans, in which only a single caspase is known (ced-3), at least 14 caspases have been identified in the mammalian genome so far (Table IV). Caspases constitute a unique class of intracellular proteases that characteristically cleave substrate proteins behind aspartate amino acids. However, substrate recognition requires three additional amino acids preceding the aspartate residue. A tetrapeptide recognition sequence allows for the specificity of cleavage sites and potential target proteins (Thornberry et al., 1997). In addition, different caspases exhibit substrate specificity regarding the tetrapeptide sequence. For example, caspase-3 efficiently recognizes and cleaves proteins at aspartate– glutamate–valine–aspartate (D-E-V-D) sequences, whereas caspases-8 and -9 preferably cleave target pro-

FIGURE 1 Schematic diagram showing the cell death pathway in C. elegans. Representative mammalian homologues are indicated in parentheses.

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TABLE IV Caspasesa Caspase

Group

Molecular mass (kDa)

Prodomain

DED

CARD

1 2 3 4 5 6 7 8 9 10 11 12 13 14

INF REG DOWN INF INF DOWN DOWN REG REG REG INF INF INF INF

45 46 32 43 48 34 35 55 46 55 42 47 43 30

Long Long Short Long Long Short Short Long Long Long Long Long Long Short

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹

⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺

⫺

⫺

a

Caspases are grouped into inflammatory caspases (INF), upstream regulatory caspases of apoptosis (REG), and downstream executioner caspases of apoptosis (DOWN). Information on the presence of DED (death effector domain) and CARD (caspase recruitment domain) is included where available.

teins in the context of a (valine/leucine)–glutamate– (threonine/histidine)–aspartate sequence motif (V/LE-T/H-D) (Thornberry et al., 1997). Common to all known caspases is a five amino acid sequence motif containing a central cysteine residue, which proves to be crucial for proteolytic activity. Caspases can be classified into three main functional groups: inflammation (INF), upstream regulatory (REG), and downstream executioner (DOWN) caspases (Table IV) (Thornberry and Lazebnik, 1998). The first class (inflammatory caspases) containing the prototypical mammalian caspase ICE (caspase-1) is required for the proteolytic activation of the precursor for the inflammatory cytokine interleukin-1웁 (Kuida et al., 1995). Knockout studies in mice showed that the generation of mature interleukin-1웁 is inhibited in caspase-1and caspase-11-deficient mice (Table V) (Kuida et al., 1995; Wang et al., 1998b). However, apoptosis is not substantially affected in these mice and cardiovascular

abnormalities are not observed. Based on sequence similarities, caspases-4, -5, -12, -13, and -14 appear to belong to the same subgroup of caspases, although their functional role is not well characterized. The second group of caspases (upstream regulatory caspases) encompasses caspases-2, -8, -9, and -10, which are activated early during apoptosis. Their substrate specificity seems to be restricted to the cleavage and thus activation of downstream, executioner caspases (Fernandes-Alnemri et al., 1996; Li et al., 1997). Caspase-9 is believed to mediate mitochondria-dependent caspase activation, whereas caspases-2 and -8 are involved in the initiation of apoptosis through death receptors (Boldin et al., 1996; Muzio et al., 1996; Duan and Dixit, 19