AAdvances in
EELECTROCARDIOLOGY 2 2004
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Proceedings of the 31st International Congress on Electrocardiology
Advances in
ELECTROCARDIOLOGY 2004 27 June - 1 July 2004
Kyoto, Japan
editors
Masayasu Hiraoka Tokyo Medical and Dental Universitg Tokyo
Satoshi Ogawa Keio University, Tokyo
ltsuo Kodama Nagoya Universitg Nagoya
Hiroshi lnoue Toyama Medical and Pharmaceutical UniversitL: Toyama
Hiroshi Kasanuki Tokyo Women’s Medical University, Tokyo
Takao Katoh Nippon Medical UniversitL: Tokyo
Y N E W JERSEY
LONOON
World Scientific
SINGAPORE
BElJlNG
-
SHANGHAI
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HONG KONG * TAIPEI * C H E N N A I
Published by
World Scientific Publishing Co. Re. Ltd.
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British Library Cataloguing-in-PublicationData A catalogue record for this book is available from the British Library
ADVANCES IN ELECTROCARDIOLOGY 2004 Proceedings of the 31st International Congress on Electrocardiology Copyright 0 2005 by World Scientific Publishing Co. Re. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.
For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.
ISBN 981-256-107-2
Printed in Singapore by World Scientific Printers (S)Pte Ltd
The Organizing Committee President Honorary President Honorary Vice-president Vice-president Secretary General
Masayasu Hiraoka (Tokyo) Kazuo Yamada (Nagoya) Tsuneaki Sugimoto (Tokyo), Shoji Yasui (Nagoya) Junji Toyama (Toyama) Satoshi Ogawa (Tokyo)
Committee Chairpersons Scientific Program committee ltsuo Kodama (Nagoya) Program committee Hiroshi lnoue (Toyama) Fund-Raising Committee Hiroshi Kasanuki (Tokyo) Finance Committee Yoshifusa Aizawa (Niigata) General Affairs Committee Takao Katoh (Tokyo) Public Relations and Registration Committee Tohru Ohe (Okayama) Shiro Kamakura (Osaka) Exhibition Committee Site Management and Liaison Committee Minoru Horie (Shiga)
International Society of Electrocardiology President Past President Honorarynreasurer Council Members
L. de Ambroggi (Italy) J. Liebman (U.S.A.) P.W. Macfarlane (U.K.)
C.Antzelevitch (USA) L. Bacharova (Slovak Republic) E.P. d’Alch6 (France) M. Hiraoka (Japan) H. lnoue (Japan) J. K. Jagielski (Poland) G. Kozmann (Hungary) R. MacLeod (U.S.A.) J.E. Madias (U.S.A.) J.A. Malmivuo (Finland) M. Monoach (Israel) R. Nadeau (Canada) 0. Pahlm (Sweden)
C.A. Pastore (Brazil) I. Preda (Hungary) A.J. Pullan (Newzealand) M.P. Roshchevsky (Russia) Y. Rudy (U.S.A.) R.H.S. Selvester (U.S.A.) M. Sobieszczanska (Poland) L.I. Titomir (Russia) J. Toyama (Japan) M. Tysler (Slovak Republic) A.van Oosterom (The Netherlands) W.Zareba (U.S.A.)
Supported by The International Society of Electrocardiology The Organizing Committee of the 31st International Congress on Electrocardiology The Japanese Society of Electrocardiology The Japan Heart Foundation V
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PREFACE Electrocardiology has witnessed a century of development since the introduction of Einthoven’s Galvanometer and still it stands as standard diagnostic and therapeutic means in clinical practice. With a rapid progress in the field of electrocardiology of scientific, technological and clinical aspects in recent years, electrocardiology covers wide ranges of topics from genes and molecules as electrical origin of the heart to pathogenesis, diagnostic and therapeutic measures for cardiovascular diseases. The book contains the fruits of the presentation at the 3 1st International Congress on Electrocardiology held in Kyoto, Japan, June 2004, which includes latest information and development in molecular biology, genetics and structure-function relationship of ion channels, channelopathy such as long QT syndrome, advancement in computer technology and signal processing of electrical activity, new diagnostic application of electrocardiogram, introduction of new drugs and non-pharmacological treatments including novel devices for treatment of cardiac arrhythmias, and future prospect of regeneration of heart tissue. Unresolved issues as to diagnosis, risk stratification, treatment and prevention of sudden cardiac death, are one of the main targets for exchange of opinions. Thus, the book will provide the hottest discussion and latest advancement of information in all areas of electrocardiology from basic science to clinical cardiology.
Masayasu HIRAOKA, MD, PhD.
vii
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CONTENTS Preface
V
1 Long QT Syndrome
1
1979-2004: 25 Years of the International Registry for the Long QT Syndrome. Its Impact on Knowledge and Clinical Management P. J. Schwartz
3
2 Atrial Fibrillation
19
Ion Channel Remodeling and Atrial Fibrillation: Clinical Aspects H.J.G.M. Crijns
21
Long-Term Efficacy of Antiarrhythmic Drug Therapy and its Influence to the Prognosis in Patients with Paroxysmal Atrial Fibrillation
25
K. Okumura Electrophysiology of Pulmonary Vein Myocardial Sleeves and their Role in Atrial Fibrillation H. Honjo Efficacy of Bepridil and Aprindine in Pharmacological Conversion of Long Lasting Atrial Fibrilaltion
26
34
A. Fujiki, T. Tsuneda, K. Nishida, M. Sakabe, M . Sugao, K. Mizumaki and H. Inoue Progressive Nature of Paroxysmal Atrial Fibrillation - Observations from a 14-Year follow-up Study
42
T. Kato, T. Yamashita, K. Sagara, H. Iinuma and L.-T. Fu
3 Basic Electrophysiology
47
Mechanisms of Ventricular Fibrillation: Role of Inward Rectifyer Channels
49
J. Jalife Reciprocal Regulation of RGS Proteins by Phospholipid and Ca'+/Calmodulin in Cardiomyocytes: Implication for Cholinergic Regulation of Heart Rates M. Ishii and Y. Kurachi ix
50
X
HERG Potassium Channel is Regulated by Protein Tyrosine Kinase (PTK) in Human Embryonic Kidney Cells
54
L.-M. Wu, K. Ueda, Y. Hirano, T. Furukawa and M. Hiraoka Acute Amiodarone Prolongs VT Cycle Length and Prevents Wave-Break of Spiral Type Excitations H. Nakagawa, M. Yamazaki, Y. Okuno, S. Nashimoto, T. Yamaguchi, T. Arafune, I. Sakuma, N. Shibata, H. Honjo, K. Kamiya and I. Kodama Losartan Decreases the Arrhythmogenic Activity of Pulmonary Vein Cardiomyocyte
57
58
Y.-C. Chen, Y.-J. Chen, S.-A. Chen and C.-I. Lin Enalapril Preserves Sinus Node Function in a Canine Bradycardia-Tachycardia Syndrome Model M. Sakabe, A. Fujiki, K. Nishida, M. Sugao, T. Ttsuneda, K. Mizumaki and H. Inoue Amiodarone not only Reverses Electrical Remodeling but Suppresses Matrix Metalloproteinases 2 Activity in Canine Pacing-Induced Persistent Atrial Fibrillation Model K. Ashikaga, T. Kobayashi, M. Kimura, S. Owada, T. Higuma, S. Sasaki, A. Iwasa, K. Furukawa, S. Motomura and K. Okumura Heterogenic Process of the Appearance of the Atrial Electrical Remodeling in Canine Rapid Stimulation Model
59
63
64
J. Kojima, S. Niwano, D. Sato, M. Moriguchi, Y. Wakisaka, K. Ikeda, K. Inuo, H. Hara, T. Yoshida and T. Izumi Electrophysiological Properties of the LA-PV Tissues of Xh-Gene Knockout Mice
69
C. Y. Song, Y. J. h i , Y. X . Loh, Y. C. Chen and C. I. Lin
Heterogeneous Distribution of the Muscarinic K+ Channels in Guinea-pig Atria Y. Yasuda, H. Toda, W.-G. Ding, F. Toyoda, M. Itoh, M. Horie and H. Matsuura Electrophysiology of Single Cardiomyocytes Isolated from Left Atrium and Pulmonary Veins of Myopathic versus Heathly Hamsters
73
74
Y.-X. Loh, Y.-C. Chen and C.4. Lin Development of a Dynamic Gap Junction Model Including the Ca2+Gate C. Oka, H. Matsuda, N. Sarai, S. Matsuoka and A. Noma
75
xi Role of ERK-Mediated Suppression of Gap Junction Permeability in Cardioprotection Afforded by MITO-KATPChannel Activation
81
Y. Ichikawa, T. Miura, T. Miki, J. Sakamoto, Y. Nakamura, T. Yano and K. Shimamoto Extracellular Potassium Dependent Negative Dromotropic Actions of Nicorandil: Experimental and Computational Study
82
T. Maruyama, F. Kuma, H. Ito, Y. Kaji and T. Kiyosue Opening of Cardiovascular ATP-Sensitive K+ Channels is Induced by Dimerization of Nucleotide-Binding Domains of Sulfonylurea Receptors 2A and 2B M. Yamada and Y. Kurachi
87
Platelet Activating Factor Affects Intracellular Calcium Concentration by Modulating L-Type Calcium Channel T. Kaku, H. Ozaki, S. Ishii, T. Shimizu and K. Ono
92
Long-Term Effects of Amiodarone on the Transcription of T3-Responsive Genes in Rat Hearts
97
R. Shi, J.-K. Lee, Y. Takeuchi, M.Horiba, K. Yasui, F. Kambe, Y. Murata and I. Kodama Effects of Inwardoutward Current Injection at the Early Plateau Phase on Cardiac Action Potential Durations: A Computational Study
103
Y. Hirano and M. Hiraoka Electrophysiological Effects of Palmitate on Rabbit Pulmonary Vein Myocardial Cells S. Higa, Y.-C. Chen, J. Wei, M. Shimabukuro and C.-I. Lin Rapid Atrial Pacing Upregulates Synthesis of Asymmetric Dimethylarginine in Canine AF Model T. Kobayashi, K. Ashikaga, M. Kimura, S. Owuda, T. Higuma, S. Sasaki, A. Iwasa, T. Osunai, S. Motomura and K. Okumura Combined Effects of Nifekalant and Lidocaine on Spiral-Type Reentrant Ventricular Arrhythmias in Rabbit Hearts
108
109
110
M. Amino, K. Yoshioka. K. Usui, Y. Deguchi, T. Tanabe, M. Yamazaki, H. Nakagawa, K. Yasui, H. Honjyo, K. Kamiya and I. Kodama Regional Abnormality of Restitution Properties Cause Electrical Alternans and Arrhythmia in Chronic Myocardial Infarction
Y. Hosoya, K. Yuuki, I. Kubota and M. Yumaki
111
xii Unexcited Core of Spiral Wave Reentry has a Small but Obviously Depolarized Potential - Study by Optical Mapping and Computer Graphics T. Namba, T. Yao, H. Nakagawa, M. Yamasaki, T. Ikeda, K. Nakazawa, H. Honjo, K. Kamiya, I. Kodama, T. Arafune, A. Mishima, I. Sakuma and T. Ohe Analysis of Virtual Electrode Polarization Induced Break Excitation and Capture Mechanisms of Excitation Propagation by Electrical Point Stimulus T. Arafune, Y. Takata, S. Nashimoto, T. Yamaguchi, E. Kobayashi, I. Sakuma, N. Shibata, H. Nakagawa, M. Yamazaki, H. Honjo, K. Kamiya and I. Kodama Simultaneous Detection of Wave Propagation Velocity and Direction in Optical Mapping Data of Cardiac Excitation Using Optical Flow Y. Takata, S. Nashimoto, T. Yamaguchi, T. Arafune, E. Kobayashi, I. Sakuma, N. Shibata, H. Honjo and I. Kodama A Canine Model of Brugada Syndrome Using Regional Epicardial Cooling of the Right Ventricular Outflow K. Nishida, A. Fujiki, K. Mizumaki, M. Sakabe, M. Sugao, T. Tsuneda and H. Inoue Long-Term Treatment with Glibenclamide Increases Susceptibility of Streptozotocin-Induced Diabetic Rat Heart to Reperfusion-Induced Ventricular Tachycardia N. Takahashi, T. Ooie, M. Nakagawa and T. Saikawa
4 Genetics of Arrhythmias Ion Channel Gene Profiling in a Mouse Model of Acquired QT Prolongation and Ventricular Tachyarrhythmias Secondary to Complete AV Block J.-K. Lee, D. Yuasa, M. Iwase, S. Futaki, M. Hayashi, M. Horiba, K. Yasui, Y. Hayashi and I. Kodama Ventricular Repolarization Abnormality in Japanese Carriers of G643S Single Nucleotide Polymorphism of KCNQI Gene T. Ozawa, M. Ito, S. Tamaki, T. Yao, T. Ashihara, Y. Kita, H. Ueshima and M. Horie GenotypicPhenotypic Characteristics in Japanese Patients with KCNJ2-Associated Andersen-Tawil Syndrome A. Kobori, T. Inoue, Y. Hosaka, T. Washizuka, T. Murakami, H. Yamanouchi, H. Ushinohama, Y. Nakamura, T. Ai, Y. Aizawa, T. Kita and M. Horie
112
113
114
119
124
125 127
131
134
xiii The Relationship between Underlying Heart Rhythm and Inducibility of Ventricular Fibrillation in Brugada Syndrome H. Itakura, Y. Enjoji, A, Moriyama,T. Nakae, T. Sakata, M. Nor0 and K. Sugi Characteristics of the Patients with Brugada Syndrome and Tachyarrhythmias Excluding Both Ventricular Fibrillation (VF) and Atrial Fibrillation (AF)
135
136
H. Okazaki, H. Yamaguchi, J.-C. Oh, T. Tejima, H. Sakurada, M. Nishizaki and M. Hiraoka Clinical Relevance of Pilsicainide Challenge Test for Risk Stratification of Sudden Cardiac Death in Patients with Brugada Type ECG
137
S. Sasaki, A. Iwasa, T. Higuma, S. Owada and K. Okumura Complete RBBB Pattern in ECGs in Patients with Brugada Syndrome is Associated with a Higher Incidence of Ventricular Fibrillation Events N. Tsuboi, I. Kodama, Y. Yoshida, K. Tajima, H. Hirayama, T.Itoh, J. Toyama, K. Yamada, T. Yamada and Y. Murakami Clinical and Electrophysiological Characteristics of Aborted Sudden Cardiac Death Patients without Structural Heart Disease
138
139
S.Fukamizu, S. Imai, A. Ikeda, Y. Sakai, I. Sunagawa, H. Yagi, H. Aoyama, H. Tanaka, K. Togawa, S. Yamaji, H. Takase, K. Matsudaira, N. Takahashi, K. Sugino and H. Yagi
5 Computer Simulation Shock-Induced Changes in Transmembrane Potential: What is the Asymmetry Due to? Insights from Bidomain Simulations
141 143
T. Ashihara and N. Trayanova Investigation of Electrical Defibrillation of Chaotically Fibrillating Human Ventricular Myocardium in a Computer Model I. M. Popp, G. Seemann and 0. Dossel
148
Computer Simulations on High Frequency Components of ECG Due to Micro-Necroses Y. Okamoto, N. Zenda, M. Kasama, H. Shimojima and T. Tsutsumi
152
On the Genesis of the Injury Potentials Y. Okamoto, M. Kondoh and S. Mashima
153
xiv Spatiotemporal Dynamics of Ventricular Fibrillation in an Anisotropic Human Heart Model
154
J. R. Fitz-Clarke, J. C. Clements and B. M. HoraEek Assessment of Local Repolarization Changes Using Model Based BSPM Interpretation M. Tysler, M. Turzova and S. Filipova
6 Sudden Cardiac Death Electrocardiographic Predictors of Cardiac Events in MADIT I1 Patients
158
163 165
W. Zareba Heart Rate Turbulence A. Bauer, P. Barthel and G. Schmidt
166
Short Term Measures of Heart Rate Variability E. Hodgart, E. Clark, S. Latifand P. W. MacFarlane
174
Risk Stratification for Sudden Cardiach Death Using Microvolt T-Wave Alternans in Postmyocardial Infarction Patients T. Ikeda, K. Sugi and H. Yoshino
182
Clinical Application of BRS (Baroreflex Sensitivity) for Risk Stratification of Sudden Cardiac Death and CHF R. Nohara
192
Corrected QT Dispersion is the Predictor in Coronary Microvascular Ischemia T. Ohta, S. Kodama, N. Morito, E. Yahiro, H. Mihara, K. Miyoshi, Y. Yamanouchi and H. Urata
199
Clinical Characteristics of Patients with Idiopathic Ventricular Fibrillation
200
H. Okamura, S. Kamakura, T. Noda, K. Otomo, K. Satomi, K. Suyama, W. Shimizu. T. Kurita and N. Aihara
7 New Frontier in Basic Cardiac Electrophysiology Clinical, Genetic, Molecular, and Cellular Aspects of the Brugada Syndrome
C. Antzelevitch
201 203
xv Functional Development of Ca2+Signaling Pathways in Mouse Embryonic Stem Cells during Differentiation to Cardiomyocytes
219
S. Kawano, S. Shoji, A. Kuruma, Y. Hirayama, K. Otsu, E. Yanagida, Y. Muto, F. Yoshikuwa and T. Furuichi Developmental Changes of L-Type and T-Type Ca2+Channels in Cardiac Cells
230
K. Yasui
8 Ion Channels Co-Cultured Skeletal Myocyte and Cardiomyocyte Cell-Sheets could not Establish Electrical Communication, but Caused Fibrillating Activity in Cardiomyocyte S. Miyoshi, Y. Itabashi, K. Fukuda, K. Tanimoto, T. Shimizu, Y. Hagiwara, A. Furuta, T. Tanaka, N. Nishiyama, T. Okano, H. Mitamura and S. Ogawa Remodeling of Gap Junction Connexin in Atrial and Ventricular Fibrillation I. Imanaga, L. Hai and K. Ogawa A Mathematical Model of the Proposed Fuzzy Space for Na' and Ca2+in Left Ventricle Cardiomyocytes
239 24 1
242
246
G. T. Lines, P. Grgttum, J. B. Sande, T. A. Str@rnmeand 0. M. Sejersted ATP-Sensitive K+ Channel is not Involved in the Extracellular K+ Accumulation in Ischemic Mouse Heart T. Sato, T. Saito, T. Miki, S. Seino and H. Nakaya
250
Sealing of Electrically Ruptured Pores by LA3+and Polyethyleneglycol in Rabbit Ventricular Cell Membrane R. Ochi, Y. Song and L. Fan
25 1
Comparative Effects of Insulin and Insulin-Like Growth Factor-1 on Dog Ventricular Muscles and Rabbit Cardiomyocytes
252
C. H. Hsu, C . 4 Lin, Y. X . Loh and Y. C. Chen Stimulatory Action of Angiotensin I1 on IKs Potassium Current in Guinea-pig Atrial Cells D. Zunkov, W.-G. Ding, H. Matsuura and M. Horie Molecular and Functional Properties of T-Type Ca2+Channel in Mouse Embryonic Hearts
N. Niwa, K. Yasui, T. Opthof; H. Takemura, A. Shimizu, M.Horiba, J.-K. Lee, H. Honjo, K. Kamiya and I. Kodama
257
26 1
xvi
Effects of Eicosapentaenoic Acid on the Electrophysiological Characteristics of Rabbit Left Atrial-Pulmonary Vein Cardiomyocytes I.-J. Chen, Y.-C. Chen, J. Wei and C.4.Lin
262
Two Modes of Polyamine Block Regulating the Cardiac K+ Current IK1as Revealed by a Study of the Kir2.1 Channel K. Ishihara
263
Effects of Antiarrhythmic Drugs on the Currents of Xenopus Oocytes Expressing HERG and KvLQTUminK Channels K. Ishii. K. Nakashima and M. Endoh
264
Relationship between KChIP2 and Transient Outward Current of Developing Rat Heart
268
T. Kobayashi, Y. Yamada, M. Nagashima, M. Fukao, K. Kameda, S. Seki, M. Tsutsuura, Y. Ito, I. Sakuma, H. Hamada, T. Abe and N. Tohse Deprivation of Membrane Cholesterol Depresses Camp-Dependent Enhancement of L-Type CA Current in Rabbit Ventricular Myocytes H. Tsujikawa, H. Masumiya, Y. Song, C. Jin and R. Ochi Potentiation of IKSPotassium Current in Guinea-pig Ventricular Myocytes by Sphingosine- 1-Phosphate
269
270
H. Toda, W.-G. Ding, F. Toyoda, Y. Yasuda, M. Ito, M. Horie and H. Matsuura Differential Effects of Mefenamic Acid on Cardiac 1, and the KCNQllKCNEl Channels F. Toyoda, W.-G. Ding, Z. Dimitar and H. Matsuura p,-Selective Antagonists are More Effective for the Treatment of Type 1 Long QT Syndrome K. Kawakami, T. Nagatomo, H. Abe, Y. Oginosawa, T. Tsurugi and Y. Nakashima Open-State Unblock Characterizes Acute Inhibition of ,I Potassium Current by Amiodarone in Guinea-pig Ventricular Myocytes D. Zunkov, W.-G. Ding, H. Matsuura and M.Horie
27 1
274
278
Acute Myocarditis Causes Structural and Electrical Ventricular Remodeling
- the Role of Reduction of ITo-Related Molecules in Experimental Autoimmune Myocarditis Rat Y. Wakisaka, S. Niwano, H. Niwano, J. Saito, T. Yoshida, S. Hirasawa and T. Izumi
282
xvii
A Novel Deletion Mutation of KCNQl that Causes Long QT Syndrome in a Near-Drowning Patient’s Family H. Yamazaki, K. Ohta, A. Ishizaki, N. Nakamura, T. Saito, Y. Niida and S. Koizumi Phenotypical Overlapping of Sick Sinus find Brugada Syndromes in a Family with a Novel SCN5A Mutation F. Yanagisawa, Y. Higashi, H. Shimojima, T.Tsutsumi, Y. Takeyama, N. Zenda, T. Makiyama and M. Horie KCNQl Mutation Causing Dominant-Negative Suppression due to Defective Channel Trafficking Underlies Cardiac Arrest in a Patient with Long QT Syndrome Y. Aizawa, L.-M. Wu, K. Ueda, S. Kawano, Y. Hirano, A. Kimura, Y. Aizawa and M.Hiraoka
9 Genetic Basis for Cardiac Arrhythmias
283
287
288
293
DNA Microarrays and Arrhythmias D. G. Escande
295
Molecular, Genetic and Clinical Aspects of Arrhythmia Disorders C. R. Beuina and A. A. M. Wilde
297
Allelic Variants in Cardiac Ion Channel Genes in Patients with Drug-Induced Long QT Syndrome
311
H. Kanki Genetic Basis of Cardiac NA Channelopathies N. Makita and M. Horie
312
10 Clinical Arrhythmias
319
Bepridil Regularizes Ventricular Response during Atrial Fibrillation in accordance with Prolongation of Fibrillation Cycle Length T. Tsuneda, A. Fujiki, M. Sugao, M. Sakabe, K. Nishida, K. Mizumaki and H. Inoue The Suppressive Effect of Bepridil on Atrial Flutter Organized from Persistent Atrial Fibrillation during Class Ic Antiarrhythmic Therapy M. Suzuki, M.Nishizaki, T.Arakawa, T. Ohara, A. Matsurnura, Y. Hashimoto and M.Hiraoka
321
326
xviii
Conversion and Maintenance Effects of Sinus Rhythm by Bepridil in Patients with Persistent Atrial Fibrillation Y. Nakazato, M. Yasuda, A. Sasaki, Y. lida, Y. Kawano, K. Nakazato, T. Tokano, H. Daida, Y. Mineda, M. Sumiyoshi and Y. Nakata Bulgarian General Practitioners’ Knowledge about the Atrial Fibrillation Management B. Georgiev, N. Gotcheva and I. Tomov Effect of Cardiac Resynchronization Therapy on the Incidence of Atrial Fibrillation in Patients with Poor Systolic Function J. W. Fung, Y. Zhang, A. Chan, M. Wang, P. Ho, G. Yip, C. M. Yu and J. E. Sanderson The Differences in the Bulgarian General Practitioners’ and Cardiologists’ Approach to the Supraventricular Tachycardias Management
327
330
335
336
B. Georgiev, N. Gotcheva and I. Tomov Efficacy of Nifekalant Hydrochloride on the Management of Life-Threatening Ventricular Tachyarrhythmias in Patients with Non-Ischemic Cardiomyopathy
340
T. Washizuka, M. Chinushi, H. Furushima, H. Watanabe and Y. Aizawa Efficacy and Safety of Low Dose Amiodarone
344
Y. Kawano, Y. Nakazato, A. Sasaki, Y. Iida, K. Nakazato, T. Tokano, M. Yasuda, H. Daida, Y. Mineda, M. Sumiyoshi and Y. Nakata Role of Combined Treatment of Bepridil in Patients with Implantable Cardioverter Defibrillators D. Izumi, H. Watanabe, M. Chinushi, T. Washizuka, H. Sugiura, T. Hirono, S. Komura, Y. Hosaka, Y. Tanabe, H. Furushima, S. Fujita and Y. Aizawa Prognostic Impact of Amiodarone Compared to D, L-Sotalol in Patients with Implantable Cardioverter Defibrillator
348
349
Y. Yokoyama, Y. Yamauchi, K. Kumagai, Y. Tanaka, K. Kurihara, A. Sato, A. Takahashi and K. Aonuma Repolarization, Estimation Criterion for Evolution in Cases with Myocardial Infarction and Necrosis Q Wave Disappearance
R. Grigore and C. Sutescu
350
xix Theoretical Study on the Nonlinear Nature in HR-Dependency of VPC Appearances N. Ikeda, A. Takeuchi, S.Okayarna, N. Mamorita, H. Hara and K. Takuyanagi Effect of Quinidine and Sotalol on QT Interval and Heart Rate in Miniature Swine H. Nitta, M. Kuwahara, H. Tsubone, E. Kumagai and M. Tanigawa Critical Roles of Pilsicainide at Termination of Atrial Flutter Studied by Noncontact 3D Mapping in the Canine Incision Model K. Uno, T. Iwa, I. Kato, Y. Suzuki, M. Fukuta, Y. Wakita, T. It0 and K. Shimamoto Cyclic Heart Rate Recovery Speed from the Hot Water Bathing Stress M. Ishijima
354
358
362
363
Prospective follow-up Study of QT Dispersion in Maintenance Hemodialysis Patients L. Zhao, G. Fukuda, M. Fukuda, K. Fukuda, K. Tanaka, M. Shibuya, Y. Yamamoto, T. Yarnaishi, T. Katsuki and K. Hagiwara
366
Baseline Tumor Necrosis Factor - Alpha Measurement Corraletes Coronary Collaterals, Predicts Left Ventricular Function and Ischemic Events in Patient with Acute Myocardial Infarction C. Zorkun, K. Zmudka, M. Pasowicz and W. Tracz
376
Combination of ST Segment Resolution and Baseline Troponin I Level Predicts Fatal Events at 1 Year in Acute Myocardial Infarction C. Zorkun, K. Zmudka, M. Pasowicz and W. Tracz
380
11 Clinical Electrophysiology Distinctive Electrophysiological Properties of Pulmonary Veins in Patients with Paroxysmal Atrial Fibrillation against Isoproterenol K. Motoki, H. Takui, H. Yabushita, K. Ikoma, R. Yasuoka, T. Hayashi and K. Ishikawa Quantitative Assessment of the Effects of Pulmonary Vein Isolation on the Trigger and Maintenance of Atrial Fibrillation T. Yamane, K. Inada, S. Matsuo, S. Miyanaga, T. Date, H. Miyazaki, K. Abe, K. Sugimoto and S. Mochizuki
385 387
388
xx Clinical Outcome of 4-Pulmonary Vein Isolation in Patients with Persistent Atrial Fibrillation S. Matsuo, T.Yamane, K. Inada, S. Miyanaga, T. Date, H. Miyazaki, K. Sugimoto and S. Mochizuki Clinical and Electrocardiographical Predictors of Immediate Recurrence of Atrial Fibrillation after External Cardioversion B. Gorenek, G. Kudaiberdieva, Y. Cavusoglu, 0. Goktekin, A. Birdane, N. Ata, A. Unalir and B. Timuralp Relation Between Transverse Conduction Capability and the Anatomy of the Crista Terminalis in Patients with and without Atrial Flutter
393
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Y. Okumura, I. Watanabe, K. Ohkubo, H. Sugimura, K. Hashimoto, Y. Takagi, T. Nakai, S. Saito, Y. Ozawa and K. Matsumoto Pulmonary Vein Potentials were Completely Organized by Pilsicainide Administration just before Termination of Atrial Fibrillation A. Iwasa, S. Ohwada, S. Sasaki and K. Okumura Different Response between Electrophysiological Test and Challenge Test with Sodium Channel Blocker in the Brugada Syndrome with Saddleback-Type ST Elevation M. Nishizaki, H. Sakurada, T.Furukawa, Y. Mizusawa, T. Ogawa, S. Sugawara, H. Fujii, T. Ashikaga, N. Yamawake, M. Arita, M. Isobe and M. Hiraoka Electropharmacologic Evaluation of Quinidine in the Brugada Syndrome Y. Mizusawa, H. Sakurada, T. Sakai, H. Yamaguchi, J.-C. Oh, H. Okazaki, T. Tejima, M. Nishizaki and M. Hiraoka Comparison of Clinical and Electrophysiological Characteristics between Symptomatic and Asymptomatic Brugada Syndrome N. Amaya, J.-D. Lee, A. Nakano, H. Uzui, T. Geshi, K. Toyoda, H. Shirasaki, T. Mizuguchi, M. Watanuki, S. Ikeguchi and T. Ueda Significance of Small Notch Potentials in the Late Phase of T-Waves in Brugada Syndrome
400
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408
R. Kishi, K. Nakazawa, A. Takagi, K. Osada, 0.Miyadu, Y. Watanabe, S. Nishio, H. Matsuda and F. Miyake Possibility of Medical Treatment for Brugada Syndrome
A. Takagi, K. Nakazawa, R. Kishi, K. Osada, T. Sakurai, 0. Miyazu, Y. Watanabe, S. Nishio, H. Matsuda and F. Miyake
42 1
xxi Clinical Profiles and Prognosis of Patients with Symptomatic and Asymptomatic Brugada Syndrome from a Multi-Center Study in Japan
430
N. Aihara, S. Kamakura and Brugada Syndrome Investigators in Japan Electro-Anatomical Mapping System Reduced Recurrence Rate of Isthmus Dependent Atrial Flutter during Long-Term follow-up T. Asano, Y. Kobayashi, T. Matsuyama, N. Watanabe, S. Ryuu, M. Kawamura, K. Tanno and T. Katagiri A Novel Radiofrequency Catheter Ablation Technique for Creation of Cavotricuspid Isthmus Block in Isthmus-Dependent Atrial Flutter
43 1
435
M. Maruyama, Y. Kobayashi, Y.-K. Iwasaki, Y. Miyauchi, S. Miyamoto,
T. Tadera, T. Ino, H. Atarashi, T. Katoh and T. Takano Frequent Association of Atrial Fibrillation does not Mean Increased Atrial Vulnerability in WPW Syndrome: Presence of “Patients Referral Bias”?
436
K. Kuga, M. Endoh, B. Niho, A. Suzuki, M. Kanemoto. M. Enomoto, K. Yoshida and I. Yamaguchi A Case of Tachycardia-Induced Cardiomyopathy Caused by an Ectopic Atrial Tachycardia Originating from the Right Atrial Appendage T. Kubota, K. Tsuchiya, T.Hirano, K. Yamaguchi, K. Nishigaki, S. Minatoguchi, H. Fujiwara, M. Goya, A. Takahashi and Y. Iesaka Optimal Temperature and Acute Effect on Sinus Node of Ablation at Junction between Superior Vena Cava and Right Atrium; Electrophysiological and Histological Evaluation Using our Thermal Balloon Catheter K. Tanaka, H. Sohara, S. Satake, Y. Watanabe and M. Tanaka Latent Mahaim Fiber Conducted during only in Atrioventricular Reentrant Tachycardia A. Moriyama, Y. Enjoji, H. Itakura, K. Kumagai, T. Sakai, T. Nakae, N. Tezuka, T. Sakata, M. Nor0 and K. Sugi A Simple Criterion of “V-H-A Pattern by Ventricular Extrastimulus” for the Diagnosis of Atrioventricular Nodal Reentrant Tachycardia
437
442
447
448
S. Owada, T. Higuma, S. Sasaki, M. Kimura, T. Kobayashi, K. Ashikaga and K. Okumura Specific Findings of the Standard 12-Lead Electrocardiogram in Patients with Transient Left Ventricular Apical Ballooning: Comparison with Anterior AM1
R. Ogura, Y. Hiasa, T. Takahashi, T. Tomokane, Y. Ohara, H. Miyajima, T. Ogata, N. Suzuki, K. Yuba, K. Kishi and R. Otani
449
xxii Septal Q Waves in V6 Lead Disappear during Narrow QRS Supraventricular Tachycardia: A New ECG Observation M. Noda, F. Suzuki, K. Motokawa and M. Isobe Causes of Exercise-Induced ST-Segment Elevation in Old Anterior Myocardial Infarction S. Taniai, Y. Koide, S. Yusu, K. Sakata, M. Yotsukura, T. Ikeda and H. Yoshino Electrocardiographic Characteristics of Idiopathic Ventricular Tachycarrhythmia Originating from the Right Ventricular Inflow Tract
450
45 1
452
Y. Tanaka, Y. Yamauchi, A. Takahashi, K. Kumagai, Y. Yokoyama, K. Kurihara, A. Sato and K. Aonuma Morphological Evaluation of Idiopathic Left Ventricular Tachyarrhythmia from Anterior Aspects of Mitral Annulus Compared with those from Aortic Sinus Cusps K. Kumagai, A. Takahashi, Y. Yamauchi, Y. Yokoyama, K. Kurihara, Y. Tanaka. A. Satou and K.Aonuma Idiopathic Ventricular Fibrillation Initiated by Premature Extrasystoles Originating from Right Ventricular Outflow Tract T. Noda, W. Shimizu, A. Taguchi, M. Yokokawa, H. Okamura, K. Ofomo, K. Satomi, K. Suyama, T. Kurita, N. Aihara and S. Kamakura Brugada-Like ECG Fingings Associated with Non-Cardiac Diseases
453
454
455
A. Sasaki, Y. Nakazato, Y. Kawano, Y. lida, Y. Mineda, T. Tokuno, K. Nakazato, M. Yasuda, M. Sumiyoshi, Y. Nakata and H. Daida Arrhythmogenic Right Ventricular Cardiomyopathy with Right Bundle Branch Block and Right Precordial ST-Segment Elevation - A Case Report H. Kawano, N. Komiya, S. Fukae, R. Nakamizo, Y. Koide, G. Toda and K. Yano Effect of Intracoronary Acetylcholine Injection on the Electrocardiogram in Patients with Brugada Syndrome Y. Abe, K. Kadowaki, K. Terata, A. Shoji, H. Kumagai, T.Sat0 and M.Miura Influence of Acute Vagal Activity in the Patients with Brugada Syndrome
N. Yamawake, M. Nishizaki, T. Ogawa, S. Sugawara, H , Fujii, T. Ashikaga, M. Arita, H. Sakurada, M. Isobe and M. Hiraoka
459
462
466
xxiii Brugada Syndrome Showed Consistent J Wave and ST Segment Elevation in 12-Lead Electrocardiogram: Time Course of Variation on J Wave
467
T. Namiki and K. Matsumoto Inappropriately Shorter QT Interval at Slower Heart Rate can Differentiate Patients with Idiopathic Ventricular Fibllation from Asymptomatic Brugada Syndrome M. Sugao, A. Fujiki, T.Tuneda, K. Nishida, M. Sakabe, K. Mizumaki and H. Inoue Prevalence of Atrial Fibrillation Caused by Acute Atrial Dilation Decreased in Rat Diseased Heart
47 1
476
H. Suzuki, H. Otake and Y. Maruyama Long Term Efficacy of PV Isolation for Penitent Atrial Fibrillation Patients
417
K. Hashimoto, I. Watanabe, K. Kawauchi, Y. Okwnura, K. Ookubo, H. Sugimura, T. Nakai, S. Saito, Y. Ozawa and K. Matumoto P Wave Morphology of an Arrhythmogenic Focus with Paroxysmal Atrial Fibrillation Originating from SVC or Right Superior Pulmonary Vein K. Ohkubo, I. Watanabe, Y. Okumura, T. Yamada, H. Sugimura, T. Nakai, K. Hashimoto, S. Saito, Y. Ozawa and K. Matsumoto Coronary Sinus Dilatation in Patients with the Atrial Flutter R. Kato, K. Matsumoto, C. Suga, T. Tosaka and S. Nishimura Effects of Pulmonary Venous Isolation during Atrial Fibrillation: Frequency Domain Analysis of Electrograms Recorded by a Basket Catheter
478
419
483
S. Sakurai, K. Uno, D. Nagahara, K. Tsuchihashi and K. Shimamoto Autonomic Nervous Activity and QT Dispersion at Common Bile Duct Treatment during Endoscopic Retrograde Cholangiopancreaticography: Correlation with Cardiac Accidents M. Nomura, Y. Nakaya, A. Nishikado, K. Koshiba, K. Yamaguchi, T. Kawano and S. It0 Role of Autonomic Nervous System and Recurrence of Atrial Fibrillation after Successful Cardioversion E. Watanabe, T. Arakawa, M. Q. Tong, T. Uchiyama, I. Kodama and H. Hishida Effect of Breathing Rate on Heart Rate Variability
T. Princi, A. Accardo and D. Peterec
484
489
492
xxiv
12 Body Surface Mapping Remarks to the Activation Sequence Invariance of QRST Integral Maps
497 499
G. Kozmann and K. Szakolczai Analysis of the QRST Isointegral Maps in Patients with Systemic Sclerosis A. Bietous- Wilk, M. Mical-Strqk, M. Sobieszczariska, J. Jagielski, L. Rusiecki, D. Jagielski and D. Kalka Isointegral Map Extrema Variability during Initial Parts of QRS Complex in Young People
503
507
K. Kozlikovri, J. Martinka and J. Bulas Abnormal Left Atrial Depolarization Wavefront in Patients with Paroxysmal Atrial Fibrillation Assessed with Magnetocardiography R. Koskinen, M. Karvonen, V. Mantynen, H. Vaananen, M. Makijarvi, J. Nenonen, J. Montonen and L. Toivonen Fifty Years of the International Congresses on Electrocardiology; The Hungarian Contribution I. Prida and Z. Antalbczy Development of a Tri-Polar Laplacian Electrocardiogram Gram Electrode Using Nine Point Finite Difference Method
512
517
521
W. G. Besio and R. Aakula Automated Laplacian ECG Moment of Activation Determination Algorithm during Pacing W. G. Besio and A. K. Kota
525
Discriminative Role of the Extreme Values of the Isopotential Maps in RBBB versus LBBB Diagnosis L. Rusiecki, J. Jagielski, M. Sobieszczariska and D. Kalka
529
Isointegral Map Extrema Variability during QRS Complex and its Thirds in Young People
533
J. Martinka, K. Kozlikovri and J. Bulas The S I T Integral in Detection of Healed Myocardial Infarction Assessed with Body Surface Potential Mapping
P. Vesterinen, H. Hanninen, M. Karvonen, K. Lauerma, M. Holmstrom, M. Makijarvi, H. Vaananen, J. Nenonen and L. Toivonen
538
xxv
The Effect of Percutaneous Coronary Artery Intervention on Body Surface Potential Maps M. Medvegy, E. Szucs, G. Simonyi, T. Bauemfeind, G. Duray, K. Szakolczai, R. G. Kiss, R. J. Bedros, R. A. Nadeau and I. Prkda How does Sodium Channel Dysfunction Relate to Repolarization Abnormalities in the Right Ventricular Outflow Tract in Brugada Syndrome? M. Yokokawa, W. Shimizu, H. Takaki, H. Okamura, T.Noda, K. Otomo, K. Suyama, T. Kurita and S. Kamakura QRS Isointegral Maps in a follow-up of the Patients with Hypertensive Left Ventricular Hypertrophy M. Sobieszczahka, D. Kaika, J. Jagielski, L. Rusiecki and J. Bolanowski Assessment of the Diastolic Function by Body Surface Potential Maps in Ischemic Heart Disease T.Bauemfeind, I. Prkda, E. Szucs, G. Duray, G. Simonyi, K. Szakolczai, R. G. Kiss, R. J. Bedros and M. Medvegy Simulation and Measurement of Single Component versus Three Component Cardiomagnetic Fields J. Haueisen, L. di Rienzo, C. M. Arturi, M. Liehr and M. Goering Non-Invasive Assessment of Atrial Wave Length by P Wave Signal Averaged Electrocardiography N. Makino, H. Maekawa, T. Shimonagata, N.Misaki, T. Yamada, M. Asai, H. Kioka, S. Tamaki, T. Matsumoto and M. Fukunami
542
543
544
548
549
550
Electric Heart Field Changes in Patients Treated with Dosulepine 0. Kittnar, J. SlaviEek, M. MlEek, 3. Havrbnek, A. Dohnalovb, I. Paclt, E. Kitzlerovb and M. Balikova'
55 1
The Normal Variability of the QRS Autocorrelation Maps A. D. Corlan and L. De Ambroggi
557
13 ECGNCG Signal-Averaged ECG Variables in Prone Position: Comparison with those in Supine Position Y. Cho, H. Kim, J. Heo, M. Park, D. H. Yang, H. S. Park, S. C. Chae, J.-E. Jun and W.-H. Park
561 563
xxvi Parametric Modeling Analysis of Abnormal Intra-QRS Potentials in Signal-Averaged ECG C.-C. Lin, T.-F. Yang, C.-M. Chen and I.-F. Yang Screenig of Left Ventricular Function by Signal-Averaged Electrocardiogram K. Aihara, Y. Nakazato, Y. Kawano, K. Nakazato, M. Yasuda, T. Tokano and H. Daida Body Surface Potential Mapping to Identify Patients with Ejection Fraction Improvement after Biventricular Pacing N. Samesima, C. A. Pastore, N. Tobias, A. Pedrosa, L. F. Moreira, S.Nishioka, M. Martinelli F" and J. F. Ramires Noninvasive Assessment of Activation and Repolarization Dynamics by QRS and QRST Integral Maps G. Komann and K. Haraszti Body Surface Potential Mapping Electro-Temporal Study of Resynchronization in Patients with LBBB and Heart Failure: Comparison of Right and Left Ventricular Activation
567
572
574
585
590
C. A. Pastore, N. Tobias, M. M. Filho, A. Pedrosa, S. Nishioka, R. A. Douglas, L. F. Moreira and J. F. Ramires The Duration of the QT Interval and Heart Rate in Miniature Swine M. Kuwahara, M. Hashimoto, H. Tsubone, E. Kumagai and M. Tanigawa
608
Bazett's QT Correction is still not Recommended! E. Miller and P. W. MacFarlane
612
Circadian Variation of the QT Interval in Patients with Heart Failure
617
T. Uchiyama, E. Watanabe, K. Yasui, H. Takeuchi, T. Terasawa, I. Kodama and H. Hishida The Response of T-Wave Parameter during Exercise Testing in Paediatric Patients with QT Prolongation and Bifid T-Wave
62 1
T. Akaike and M. Iwamoto Analysis of Atrial Fibrillation by Autocorrelation Function A. Shirnizu, M. Esato, T. Veyama, R. Kametani, N. Inoue, M. Kanemoto,
A. Sawa and M. Matsuzaki
627
xxvii Changes in QRS Amplitude to Left Ventricular Mass Relation in Rats Treated by Antihypertensive Drugs
636
L. Bacharova, J, Kyselovic, J. Klimas and D. Kucerova Electrocardiography Fails to Diagnose Left Ventricular Hypertrophy Accurately in Women
640
H. Ochi, S.Miyata, A. Noda, M. Iwase, S. Kuroki, Y. Koike, R. Ito,
H. Yamada and M. Yokota Pictorial Representation of Atrial Depolarization on the Basis of Dipole Electrocardiotopography (DECARTO) for Diagnosis of Atrial Enlargement L. I. Titomir. V. G. Trunov, E. A. I. Aidu, T. A. Sakhnova and E. V. Blinova A Difference of Time-Frequency Power Spectrum during QRS in Q from Non-Q Wave Myocardial Infarction N. Zenda, T. Tsutsumi, D. Wakatsuki, F. Yanagisawa, H, Shimojima, Y. Higashi and Y. Takeyama Myocardial Salvage Effects of Primary Angioplasty under Distal Protection in Patients with Myocardial Infarction H. Komatsu, M. Nakamura, H. Hara and K. Sugi Age and Sex Dependent ST-T Criteria for ST Elevation Myocardial Infarction P. W. MacFarlane, B. Devine, E. Clark, E. Miller, J. Seyal, D. W. Browne and D. R. Hampton Electrocardiographic Manifestation of Local Ischemia at Right Ventricular Outflow Tract F. Asano, M. Kondo, K. Wakabayashi,T. Sato, H. Ide, T. Tsutsumi and Y. Takeyama
644
648
653
654
658
Reproducibility of the ST/HR Analysis during ECG Test in Asymptomatic Middle-Aged Women J. Viik, R. Lehtinen and J. Malmivuo
659
Non-Uniform Discrete ECG Representation Optimised for Medical Data Fidelity
660
P. Augustyniak Evaluation of the Expert 12-Lead ECG Analysis System N. Isobe, M. Kaneko, M. Takahashi, T. Iwatsuku, N. Okamoto, S. Yasui, Y. Watanabe, Y. Abo and Y. Ichihara
665
xxviii Twelve-Lead Electrocardiogram Telemonitoring D. Wei Synthesis of 12-Lead ECG from 3 EASI Leads: Investigation of Population-Specific Transformation Coefficients
670
675
X . Liu, S. H. Zhou, J. Liu, J. Chen, K. Qiu, J. W. Warren, J. R. Fitz-Clarke and B. M. HorciEek High-Frequency Spectral Analysis in Signal-Averaged ECG T.-F. Yang, C.-C. Lin, C.-M. Chen and I.-F. Yang Assessment of Signal-Averaged P-Wave as a Predictor of Postoperative Atrial Fibrillation after Coronary Artery Bypass Graft Surgery T. Nakai, Y. Kasamaki, T. Yamada, K. Okubo, K.Tokai, K. Hashimoto, A. Sezai, I. Watanabe, S. Saito and K. Matsumoto Different Manifestation of Premature Ventricular Contractions by their Origin S. Nakahara, K. Takayanagi, T. Nakata, K. Tanaka, I. Hisauti, T. Hayashi and S. Morooka
679
684
685
Prediction of Mechanisms of Ventricular Premature Contraction (PVC) by T Wave Analysis E. Ino-Oka, S. Yumita, H. Sekino, Y. Ohtaki, H. Inooka and K. Sagawa
687
Evaluation of the AV Nodal Conduction Using RR-Interval Plotting in AF-Patients: Its Relationship with Cardiac Function A. Chishaki, H. Chishaki and K. Sunagawa
69 1
Evaluation of the T-Wave during Exercise-Testing in Patients with Idiopathic Dilated Cardiomyopathy (DCM) with and without Beta-Blockades H. Hara, S. Niwano, S. Hirasawa, T. Sasaki, N. Ikeda, H. Miyahara and T. Izumi
696
Monitoring Electrocardiograms via a Mobile Network System Using Cellular Phones X. Zhu, W. Chen, S. Ding, H, Tsuchida, M. Cohen and D. Wei
700
Development of an Automatic Network Holter Electrocardiogram Analysis System
705
A. Akahori and K. Oguri Excluding Incorrect Detection on ECG Automatic Analysis Using SVM
Y. Kikawa and K. Oguri
709
xxix
Power to Detect Prior Myocardial Infarction by ECG Findings at Health Examination H. Zhang, H. Toyoshima, H. Yatsuya, K. Tamakoshi and T. Kondo
14 Autonomic Nervous Activity Exaggeration of Morning Fluctuation of Autonomic Nervous Activity in the very Elderly Healthy Subjects
713
717 719
H. Tasaki, T. Serita, C. Ueyama, K. Kitano, S. Set0 and K. Yano Gender Differences in Autonomic Modulation of Ventricular Repolarization in Humans M. Nakagawa, T. Ooie, M. Ichinose, H. Yonemochi and T. Saikawa Responses to Head-Up Tilt Test in Vasovagal Syncope after Atenolol Treatment H. Kim, J. Heo, D. Yang, H. Park, Y. Cho, S.-C. Chae, J.-E. Jun and W.-H.Park
720
725
Assessment of Home Orthostatic Self-Training in the Prevention of Neurocardiogenic Syncope H. Abe and Y. Nakashima
730
15 Pacing
735
Optimal AV Delay is not Preferred to Spontaneous AV Conduction in Patients with Pacemaker C. Suga, K. Matsumoto, R. Kato, T. Tosaka, T. Tamaki, T. Yamazaki and S. Nishimura Measurement of Intracardiac Bioimpedance in Rate Adaptive Pacemakers
737
738
A . Kuusik, R. Land, M. Min, T. Pane and G. Poola Ventricular Pacing Thresholds Following High-Energy Ventricular Defibrillation Shocks Y. Yamanouchi, S. Kodama, T. Ohta, N. Morito, E. Yahiro, K. Miyoshi and H. Urata Prevention of Atrial Fibrillation by Biatrial Pacing: The Outcome and the Electrophysiological Mechanism of Prevention Y. Enjoji, T. Sakata, M.Noro, T. Nakae, N. Tezuka, K. Kumagai, T. Sakai, H. Itakura, A. Moriyama and K. Sugi
743
744
xxx
16 Pediatric ECG Significance of QT Dispersion and Ventricular Late Potentials in Children with Mitral Valve Prolapse: A Prospective Study W. Bobkowski, J. Zuchwieja, B. Mrozinski, A. Nowak and A. Siwinska
745 747
QT Dispersion and Corrected QT Interval in Children with Chronic Renal Failure W. Bobkowski, A. Nowak, J. Zachwieja, B. Mrozinski and A. Siwinska
752
PDA-Based System for Cardiology Home Care and Pregnancy Monitoring
756
P. Augustyniak Benign Arrhythmias in Children and Youth C. Sutescu, R. Grigore and I. Stoian Drug Sensitivity and Antiarrhythmic Treatment in Children with Idiopathic Polymorphic Ventricular Tachycardia T.Yasuda, N. Kojima, D. Fukumi and M. Nagashima Efficacy of 12 Leads Holter Monitoring System in Brugada Syndrome - Multicenter Heart Screening Study in Japan N. Surnitomo, M. Nagashima, H. Ushinohama, N. Konishi, S. Sato, S. Yasukochi, Y. Nakumura, N. Izumida, M. Yoshinaga, K. Karasawa, M. Ayusawa, H. Kato and K. Harada A New Index for Assessment the Ability of Myocardium to the Fibrillation
76 1
768
769
774
V. Kobrin, I. Konovalova and M. Tverskuya Bio-Impedance FDM-Modeling Inside Heart for Application in Implanted Devices
778
R. Gordon and A. Kuusik Successful Biventricular Pacing in an Elderly Patient with Cardiac Sarcoidosis at Risk of Congestive Heart Failure
783
0. Okazaki, M. Hiroe, N. Tezuka, M.Noro, M. Kashida, N. Akatsuka and Y. Yazaki Chronic Angiotension I1 Receptor Blocker does not Alter Ventricular Defibrillation Thresholds
79 1
Y. Yamanouchi, S. Kodama, T. Ohta, N. Morito, E. Yahiro, K. Miyoshi and H. Urata Validation of Quality of Life Questionnaire for ICD Patients S. Tsunoda, H. Abe, T. Mitsuhashi and S. Ishizuka
792
xxxi Performance of the Criteria for Gender Differences on the ECG Early Ventricular Repolarization Wave Contour E. P. Silva, E. I. Oliveira, P. Marques, V. Machado, M. G. Florentim, S. Ribeiro, M. G. Lopes and J. C. Cunha Autonomic Dysfunction in Children with Chronic Renal Failure W. Bobkowski, A. Nowak, J. Zuchwieja, B. Mrozinski and A. Siwinska
17 Modeling of Cardiac Electrical Activity
796
80 1
805
Reflections on T Waves A. van Oosterom
807
Electrocardiographic Imaging (ECGI): Validation and Application in Humans Y. Rudy
816
Whole Heart Model and ECG/MCG Inverse Problem Y. Okamoto
817
Changes in Rabbit Heart Vulnerability during Phase 1A of Acute Global Ischemia
818
N. Trayanova and B. Rodriguez Simulated Epicardial Potential Maps with a Membrane-Based Bidomain Model of the Human Heart
827
M. Potse, B. Dube', E. Be'langer, J. Richer and R. M. Gulrajani Analysis of QT Interval Prolongation by Simulation of Repolarization Process Based on KCNQI and KCNEl Expression Experiment
83 1
T. Yamaguchi, K. Kamiya, T. Arafune, K. Ouchi, E. Watanabe, H. Honjo, I. Kodama, N. Shibata and I. Sakuma Mechanisms of Shock-Induced Arrhythmogenesis: Role of Tissue Discontinuity and Electroporation in the Initiation of Focal Repetitive Postshock Acitvations T. Ashihara and N. Trayanova
18 Ablation New Ablation Technologies for VT A. d'Avila
832
837 839
xxxii
Catheter Ablation of Primary VentricularFibrillation: Mapping Methods and the Mechanism of Catheter Ablation A. Nogami
841
Endocardia1 ElectroahatomicalSubstrate and Catheter Ablation in Patients with Nonischemic Cardiomyopathy and Monomorphic Ventricular TachycGdia K. Satomi
852
Author Index
855
1 Long QT Syndrome
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1979-2004 :25 YEARS OF THE INTERNATIONAL REGISTRY FOR THE LONG QT SYNDROME. ITS IMPACT ON KNOWLEDGE AND CLINICAL MANAGEMENT PETER J. SCHWARTZ Department of Cardiology IRCCS Policlinico S. Mafteo and Universiv of Pavia, Pavia, Italy Correspondence to:
Peter J. Schwartz, MD Department of Cardiology IRCCS Policlinico S. Matteo V. le Golgi, 19 27 100 Pavia, Italy Tel: +39-0382-503567 Fax +39-0382-503002 piqt@,compuserve.com
Introduction There are not many examples in medical history of a disease which has been kept outside of mainstream cardiology and regarded as an oddity for so long and then, over just a few years, with a sudden reversal has been embraced by many as a paradigm for sudden cardiac death (1). The identification at the end of March 1995 (2,3) of the first two LQTS genes followed in January 1996 by the identification of KvLQTl (4), represented a major breakthrough not only for cardiac electrophysiology but also for cardiology as a whole and paved the way for the understanding of how tight can be the relationship between molecular and clinical cardiology. Indeed, the impressive correlation between specific mutations and critical alterations in the ionic control of ventricular repolarization has made of LQTS the best example to date for the specificity and value of the correlation between genotype and phenotype (5). Until the mid-nineties most cardiologists had been unimpressed by the clinical relevance of molecular biology, but they changed their mind largely on the basis of the very rapid developments that contributed to unravel this life-threatening disorder which represents a sort of Rosetta stone for sudden cardiac death (6). Similarly, many basic science investigators who had not even 3
4
ever heard of LQTS became involved in LQTS-related research because of its obvious potential to help to elucidate key mechanisms underlying also more common and complex clinical disorders. A critically important role, initially not fully foreseen, has been played by the International Registry for LQTS initiated by Arthur Moss and myself in 1979, 25 years ago (7). This short essay aims at revisiting the origin, the development, the main contributions and the impact of the International Registry. These events will be revisited from my own perspective. The First Steps My First Patient
In 1971, a 9 year old girl with repeated syncope, mostly triggered by emotional stress, was referred to the Department of Medicine of the University of Milan where I was then working as a young physician. Her medical exam was unremarkable with the exception of a prolonged QT interval on the ECG, but those were the days when medical textbooks were maintaining that the QT interval had no clinical significance (8). Her 17 year old sister had died suddenly after a violent emotion while participating in a live television program. Both sisters had begun at age 3 to have repeated episodes of fainting, sometimes with convulsions, whenever they were frightened or stressed. The diagnosis of the surviving sister required some time and numerous consultations; eventually a pediatric cardiologist suggested me to go to the medical library and to look in “The Lancet” where years earlier a similar case, with QT interval prolongation, had been described. This turned out to be the famous editorial published in 1964 (9). As I was then involved in experimental research focused on the autonomic nervous system and on the significance of excitatory sympathetic reflexes (10), it was a natural thing to look at this girl as an example of how sympathetic activation could have lethal consequences. Hence, our initial therapeutic management involved beta-adrenergic blocking agents. After the diagnosis of LQTS was made, beta-blockers (propranolol) were thus started, but despite a significant reduction in the number of episodes syncope and cardiac arrest recurred. Moreover, episodes of T-wave alternans during stress were noted (Fig. 1). This was then regarded as a very rare phenomenon of unknown origin and by searching the literature from 1925 onward I could find only 7 such reports in patients without LQTS; by striking contrast, a significant number of the not many existing reports on LQTS contained descriptions or figures of this very unusual ECG pattern.
5
In 1966, Yanowitz et al reported that, in dogs, left stellate ganglion stimulation or right stellate ablation prolonged the QT interval (1 1). Based on these findings, I performed experiments in cats and was able to demonstrate that both QT prolongation and T wave altemans could be produced by left stellate ganglion stimulation (12). These findings suggested a major role of cardiac sympathetic innervation in LQTS and supported left cardiac sympathetic denervation as a rational approach to management. Meanwhile, the literature offered another very important piece of information. In 1969, a patient with recurrent syncope triggered by acute emotions had been referred to Arthur Moss. The diagnosis of LQTS was made, but no valid therapy seemed available at that time for this disorder. On the basis of the Yanowitz’s findings mentioned above, Moss convinced a neurosurgeon, Dr. McDonald, to carry out a left cervicothoracic sympathetic ganglionectomy in the patient to reduce the sympathetic innervation to the ventricles. The patient has remained free of syncope for the past 35 years. The publication of this therapeutic success in 1971 (13) provided an important option for the management of LQTS. The experimental data by Yanowitz et a1 (1 1) and the clinical report by Moss and McDonald (13), coupled with my experimental results made easier my task of convincing the senior people at my institution to allow to perform surgical left cardiac sympathetic denervation in my own patient in the spring of 1973. This patient has remained asymptomatic during the subsequent 30 years. The intense emotional experience of the medical challenge posed by this child and the intriguing scientific aspects highlighted by the experimental findings gave new directions to my scientific life. Shortly afterwards, probably because of a couple of presentations I had made on my first patient, I was referred a second patient - an 8 year old boy affected by LQTS. I did initiate therapy with beta-blockers and recommended the parents to never stop the therapy. Understandably, my credibility at that time was minimal and one year later - as the episodes of syncope had disappeared - a family member decided that the child did not need medication; the boy died suddenly two months later while running up the stairs to enter his home.
Looking for Answers I was then working in Italy, in a junior position, and I had to deal with a number of constraints. My most pressing questions were “what is the best way to treat these patients?’ and “what is the mechanism underlying LQTS?’. At that time Arthur Moss, to me, was just a name on an article published in the United
6
States; little did I know that he had similar questions in his mind and that later on he would have played such an important role in making possible to get answers to our questions. One thing seemed, however, immediately obvious. Namely, that neither the scanty information available in the literature nor my two patients could have provided adequate and meaningfid information. It was then that I decided, in a very amateurish way, to begin to find answers on treatment. My first point was that the literature did not offer very much. Most of those single case reports had a very brief follow-up period and while the short term results with an impressively large number of different therapies might have appeared as promising, very little or nothing was known for longer periods of follow-up. Moreover, despite my young age I was skeptical about the likelihood of clinicians writing an update to indicate that their patients had died one or two years later, if this had happened. My second point was that “once you see something, you are more likely to see it again” and I thought more likely than not that those who had reported on a first LQTS patient might have observed a few others. It was on this basis that I began, in 1972, to write to physicians around the world asking them for additional information on LQTS. I did start with those who had described the disorder and my first letters went to Dr. Romano and to Dr. Ward (of the Romano-Ward syndrome). Dr. Romano, not a cardiologist, was not following any more his initial family but Dr. Ward, on July 20, 1972, provided an interesting reply (Fig.2). Two points are interesting in his letter. Dr. Ward first indicated that his patient, alive at the time of his publication, had actually died suddenly one year later. This confirmed my suspicion that one could not rely too much on the therapeutic results present in the published articles. The second point, I think, is of historic interest. Ward refers to a meeting with Professor Durrer in Amsterdam. Durrer was a true pioneer in cardiac electrophysiology and clearly had an excellent understanding for cardiac arrhythmias. Nonetheless, “he was pessimistic about controlling the condition (LQTS) in any way”. Today, Durrer would be quite surprised to realize that the vast majority of LQTS patients can be treated very effectively and that overall mortality is around 2%. The letters that I had written, pestering everyone who had published even a single case report on LQTS, began to bear fruit. Fig. 3 is just example of the standard letters that I used to outline on my typewriter. This one was sent to Dr. Barlow (of the “mitral click”) in South Africa, who replied with proper information (Fig. 4). For all of us it is always interesting to look at what we did more than 30 years ago. Reviewing my own letter my comment now would be
7
that the style was simple and rather to the point but, in retrospect, I find amusing to note the pride with which I was informing Dr. Barlow (and the rest of the world) that I was following two of these patients! Not just one. With an almost complete lack of sense of proportions I was already considering myself in the special league of clinicians with more than one patient with LQTS! As to the issue of the mechanisms underlying LQTS, I had begun to develop the “sympathetic imbalance” hypothesis and I had contacted Professor Anton Jervell in Oslo to ask his opinion. Fig. 5 shows one of his always very kind replies. Indeed, he shows a gentle agreement on my proposal of a triggering role for the sympathetic nervous system but he also keeps his distance for the idea that autonomic abnormalities might have been the primary cause and, cleverly, suggests “some abnormality in the myocardial metabolism”, which in 1973 was as close as humanly possible to an intracardiac abnormality of genetic origin. I am always moved in reading the conclusion of this letter where he hopes that my interest for LQTS would be more than transient, and I would very much wish to let him know that, yes, 30 years later my enthusiasm and interest for LQTS have only increased.
An American Partnership In 1973 I had moved to work for two years in Buzz Brown’s laboratory in the Department of Physiology and Biophysics at the University of Galveston in Texas. In early 1974 Dr. George Burch, the Editor-in-Chief of the American Heart Journal, had invited me to write a first review article on the long QT syndrome, which was published in 1975 (14) and eventually included a report on over 200 patients with LQTS, a staggering number for those days. Moreover, a number that for the first time allowed reasonable and meaningful statements about the efficacy, or lack thereof, of the various treatments employed up to that time. This invitation by George Burch gave new impulse to my search for information and the number of outgoing letters increased exponentially. One of them went to Dr. Arthur Moss, who replied kindly. He had been involved in LQTS already since a few years. His 1971 NEJM article, describing his pioneering therapeutic approach, had had wide resonance and he was referred a number of LQTS patients. He continued to keep track of these interesting patients and to receive information on their outcome. We met personally for the first time in Houston during the ACC 1975 meeting and had lunch with Michel Mirowski. We agreed on staying in touch. We had no idea of how intertwined our htures would have become.
8
At the 1977 American College of Cardiology meeting, in Las Vegas, Moss and I were both involved in an evening Fireside Panel program on LQTS and the effects of sympathetic nerve stimulation on ventricular repolarization. I presented an update on my database of LQTS patients collected from the medical literature and personal contacts, which now totaled over 700 patients, but was limited by retrospective, non-structured information. Given our common interest in LQTS we decided to join forces with the goal of unravelling this mysterious disease and arranged to have breakfast together the next day. During that breakfast, we formulated the concept of an International LQTS Registry to prospectively collect clinical and follow-up information on LQTS patients from around the world. We discussed plans to submit a grant to the National Institutes of Health for funding; as we both were already recipients of NIH funds, the possibility of a cooperative international grant was not unrealistic. When Arthur Moss asked how long should we run this international registry I replied “for 25 years”, an unmistakable sign that we were very young. It took two years until the International LQTS Registry started up in 1979 (15,16), and now 25 years later the Registry is still active and thriving with enrolment of over 1200 LQTS families and continuous National Institutes of Health fimding for the past 20 years. In 1977, it did not escape us that this long-term project was likely to contribute to a better understanding and management of LQTS. Quite frankly, we did not anticipate the explosion of knowledge that would result from the genetic and molecular findings of the 1990s and the central role that the Registry, with its well-defined clinical phenotypes and family pedigrees, would play in uncovering the secrets of this disorder. Objectives Our primary objectives with the International LQTS Registry were to gain insight into the natural history, the clinical course, and the efficacy of current and novel therapies so that more effective therapy could be implemented to prevent the syncope and sudden death events that represent the main burden for the LQTS patients.
Impact The lnternational LQTS Registry has enhanced our knowledge on an infrequently occurring cardiac disorder, and it has become a paradigm for studying such conditions. The Registry continues to offer physicians from around the world an opportunity to obtain advice on how to manage their LQTS
9
patients. On the other hand, these physicians also contributed clinical data to the Registry by willingly filling out enrolment and yearly follow-up data forms. This approach allowed us to gather information on an impressive number of patients and - of crucial importance for the subsequent genetic developments on first and second-degree family relatives. When molecular biology techniques matured to the point of making possible the identification of disease-causing genes and disease-causing mutations what became essential was the availability of numerous and well worked out clinical pedigrees providing clear separation between “affected” and “non affected” individuals. This is what the Registry was able to provide and where it played a decisive role in sharing with molecular biologists the ideal material for their analysis. In 1979 we could not fathom the explosion of knowledge that would have followed and the now clear evidence that LQTS represents indeed a paradigm for the understanding of sudden cardiac death in more common cardiac diseases. Subsequently, similar types of registries were established by interested investigators for other uncommon cardiac disorders including hypertrophic cardiomyopathy arrhythmogenic right ventricular cardiomyopathy, and Brugada syndrome. It is gratifying to know that the International LQTS Registry helped pave the way for scientific progress in the difficult field of uncommon diseases. Molecular Genetics By the early 1990s, molecular biology had made impressive progress. The new genetic techniques, especially linkage analysis and detection of DNA sequence differences, were offering a realistic potential for the identification of disease-genes and disease-causing mutations. There was but one limiting step, and not a small one. These techniques, as powerful as they were, still depended for their success on the availability of well worked out clinical pedigrees. Carefully studied large family trees with clear separation between “affected” and “non-affected” individuals were essential. This is where the Registry, with its many large families and myriad of small families together with a quantitative QTc diagnostic marker and well defined clinical phenotypes, played a decisive role in offering molecular biologists ideal material on which to carry out their analyses. It was on this background that Mark Keating and his associates made their fundamental discoveries (2-4). It seems fair to say that in modem cardiology there have been few findings that have had such fruitful consequences as the identification of the first three LQTS genes. Merit is often not disjointed from
10
good luck. The first series of genes identified with LQTS were all encoding cardiac ion channels, and it is fortunate that techniques already existed that allowed for functional evaluation of the mutant genes by cellular expression studies. These studies provided the evidence on how a specific mutation, by altering cardiac electrophysiology and the balance between inward and outward currents, was affecting the cardiac action potential - thus explaining how these mutations result in the lengthening of ventricular repolarization coded as QT prolongation. Findings from the Registry
The Registry, with its expanding number of genotyped families, has provided an opportunity to study the clinical aspects and to explore the genotype-phenotype relationships in this unique cardiovascular disorder. The first publication of findings from the Registry occurred in 1985 when we highlighted the risk factors for cardiac events in 196 LQTS patients (17). By 1991, we expanded the prospective study of the clinical course of this disorder to 1,016 affected individuals in 328 LQTS families (18). The diagnostic criteria for LQTS have been established (19). Important findings from the Registry during the past decade have included: age and sex-related differences in the clinical manifestations of LQTS (20); influence of pregnancy on the risk for cardiac events in LQTS (21); ECG T-wave patterns in genetically distinct forms of LQTS (22); clinical course of LQTS by genotype (23,24); the spectrum of mutations in LQTS genes (25); increased risk associated with mutations in the pore region of the hERG gene (26); role played by physical exercise, emotions, arousal, and redsleep as triggers and facilitators for syncope and sudden cardiac death in LQT1, LQT2, and LQT3 (27); effectiveness of beta-blocker therapy, particularly according to genotype (28,29); potential gene-specific usefulness of sodium channel blockers (mexiletine and flecainide) in the treatment of patients with the LQT3 mutations (30,31); and left cardiac sympathetic denervation in the management of high-risk LQTS patients (32). Key to the success of the International LQTS Registry was the good fortune to have an outstanding group of committed multidisciplinary investigators who have been associated with the program through several decades and an excellent staff in Rochester. The International LQTS Registry started with three enrollment centers (Rochester, NY; Milan now Pavia, Italy; and Charlottesville, VA, with Dr. Richard S. Crampton) but over the years the Registry expanded to include LQTS enrolment centers in Jerusalem, Israel (Dr. Jesaia Benhorin), Salt
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Lake City, UT (Dr. Michael Vincent), and Houston, TX (Dr. Jeffrey Towbin) and a second center in Pavia (Dr. Silvia Priori). Challenges for the Future Additional new genes and new genetic mechanisms need to be uncovered, modifier genes that explain the variable duration of ventricular repolarization andor the variable severity of clinical manifestations in individuals with the same mutation have yet to be identified, gene-specific and mutation-specific therapy is presently in its infancy. Although the Registry has been a very successful endeavor, our quest for uncovering the secrets of LQTS continues. References 1. Members of the Sicilian Gambit: The search for novel antiarrhythmic strategies. Eur Heart J 1998;19:1178-1196and Jpn Circ J 1998;62:633-648. 2. Wang Q, Shen J, Splawski I, Atkinson D, Li Z, Robinson JL, Moss AJ, Towbin JA, Keating MT: SCNSA mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 1995;80:805-811. 3. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT: A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 1995;80:795-803. 4. Wang Q, Curran ME, Splawski I, et al: Positional cloning of a novel potassium channel gene: KVLQT 1 mutations cause cardiac arrhythmias. Nat Genet 1996;12:17-23. 5 . Schwartz PJ, Priori SG: Long QT syndrome: genotype-phenotype correlations. In: CARDIAC ELECTROPHYSIOLOGY. FROM CELL TO BEDSIDE. IV EDITION (Zipes DP and Jalife J, Eds.) WE3 Saunders Co., Philadelphia, pp. 65 1-659,2004. 6. Zipes DP: The long QT interval syndrome. A Rosetta stone for sympathetic related ventricular tachyarrhythmias. Circulation 1991;84:1414-1419. 7. Moss AJ, Schwartz PJ: 25'h Anniversary of the international Long QT Syndrome Registry: an ongoing quest to uncover the secrets of LQTS. Circulation (In press). 8. Grant RP. CLINICAL ELECTROCARDIOGRAPHY. New York, McGraw-Hill, p. 63, 1957. 9. Congenital cardiac arrhythmia. (Editorial) Lancet 1964;ii:26.
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10. Malliani A, Schwartz PJ and Zanchetti A: A sympathetic reflex elicited by experimental coronary occlusion. Am J Physiol 1969;217:703-709. 11. Yanowitz F, Preston JB, Abildskov JA: Functional distribution of right and left stellate innervation to the ventricles. Production of neurogenic electrocardiographic changes by unilateral alteration of sympathetic tone. Circ Res 1966;18:416-428. 12. Schwartz PJ, Malliani A: Electrical alternation of the T-wave: clinical and experimental evidence of its relationship with the sympathetic nervous system and with the long Q-T syndrome. Am Heart J 1975;89:45-50. 13. Moss AJ, McDonald J: Unilateral cervicothoracic sympathetic ganglionectomy for the treatment of long QT interval syndrome. N Engl J Med 1971;285:903-904. 14. Schwartz PJ, Periti M, Malliani A: The long Q-T syndrome. Am Heart J 1975;89:378-90. 15. Moss AJ, Schwartz PJ: Sudden death and the idiopathic long Q-T syndrome. Am JMed 1979;66:6-7. 16. Schwartz PJ: The idiopathic long QT syndrome. The need for a registry. Eur Heart J 1983;4:529-531. 17. Moss AJ, Schwartz PJ, Crampton RS, et al: The long QT syndrome: a prospective international study. Circulation 1985;71:17-21. 18. Moss AJ, Schwartz PJ, Crampton RS, et al: The long QT syndrome. Prospective longitudinal study of 328 families. Circulation 1991;84:1136-1144. 19. Schwartz PJ, Moss AJ, Vincent GM, Crampton RS: Diagnostic criteria for the long QT syndrome: an update. Circulation 1993;88:782-784. 20. Locati EH, Zareba W, Moss AJ, et al: Age- and sex-related differences in clinical manifestations in patients with congenital long-QT syndrome: findings from the International LQTS Registry. Circulation 1998;97:2237-2244. 2 1. Rashba EJ, Zareba W, Moss AJ, et al: Influence of pregnancy on the risk for cardiac events in patients with hereditary long QT syndrome. LQTS Investigators. Circulation 1998;97:451-456. 22. Moss AJ, Zareba W, Benhorin J, et al: ECG T-wave patterns in genetically distinct forms of the hereditary long QT syndrome. Circulation 1995;92:2929-2934. 23. Zareba W, Moss AJ, Schwartz PJ, et al: Influence of genotype on the clinical course of the long-QT syndrome. International Long-QT Syndrome Registry Research Group. N Engl JMed 1998;339:960-965.
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24. Priori SG, Schwartz PJ, Napolitano C, et al: Risk stratification in the long-QT syndrome. N Engl JMed 2003;348:1866-1874. 25. Splawski I, Shen J, Timothy KW, et al: Spectrum of mutations in long-QT syndrome genes. KVLQT1, HERG, SCNSA, KCNE1, and KCNE2. Circulation 2000;102:1178-1185. 26. Moss AJ, Zareba W, Kaufman ES, et al: Increased risk of arrhythmic events in long-QT syndrome with mutations in the pore region of the human ether-a-go-go-related gene potassium channel. Circulation 2002; 105:794-799. 27. Schwartz PJ, Priori SG, Spazzolini C, et al: Genotype-phenotype correlation in the long-QT syndrome: gene-specific triggers for life-threatening arrhythmias. Circulation 200 1;103:89-95. 28. Priori SG, Napolitano C, Schwartz PJ, et al: Association of long QT syndrome loci and cardiac events among patients treated with beta-blockers. J A M 2004;292:1341-1344. 29. Moss AJ, Zareba W, Hall WJ, et al: Effectiveness and limitations of beta-blocker therapy in congenital long-QT syndrome. Circulation 2000;101:6 16-623. 30. Schwartz PJ, Priori SG, Locati EH, et al: Long QT syndrome patients with mutations of the SCNSA and HERG genes have differential responses to Na+ channel blockade and to increases in heart rate. Implications for gene-specific therapy. Circulation 1995;92:3381-3386. 31. Windle JR, Geletka RC, Moss AJ, et al: Normalization of ventricular repolarization with flecainide in long QT syndrome patients with SCNSA: AKF'Q mutation. Ann Noninvasive Electrocardiol2001;6:153-158. 32. Schwartz PJ, Priori SG, Cerrone M, et al: Left cardiac sympathetic denervation in the management of high-risk patients affected by the long-QT syndrome. Circulation 2004; 109:1826-1833.
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Figure 1
D3
A, Control conditions, QTc 0.61. B, During fright. Tracings in B are simultaneous. T wave alternans, in amplitude (Dl) and in polarity (D2 and D3), is evident.
15
FIGURE 2
For details, see text.
16
Figure 3
Dear Dr, Berlcr~t
For details, see text.
17
FIGURE 4
University of the Witwatersrand, Johannesburg Department of Medicine. Medial School, Horpad Sneer, Johannesburg, South A f i m
2 4 t h Septentber, 1973.
Or. P . J . S c h w a r t z , I s t i t u t o D i Ricerche Cardiovascolari, Oell U n i v e r s i t i O i Milano, 20122 Milano, Via F . S f o r z a , 35,
MILAN -
Dear O r . S c h w a r t z . Thank you f o r y o u r l e t t e r , d a t e d September 6 t h . c o n c e r n i n g t h e p r o l o n g e d Q-T syndrome. To o u r knowledge, a l l t h e p a t i e n t s a r e s t i l l a l i v e b u t t h e f i r s t f a m i l y are r a t h e r u n c o - o p e r a t i v e a s f a r a s f o l l o w up i s concerned. They have, i n f a c t . n e g l e c t e d t o t a k e o u r p r e s c r i b e d t r e a t m e n t and we o n l y h e a r a b o u t them i n d i r e c t l y . The o t h e r f a m i l y i s v e r y c o - o p e s i v e and a l l members a r e doing w e l l except t h a t t h e youngest c h i l d has about syncopal a t t a c k each y e a r . They are b e i n g t r e a t e d w i t h a combination o f Dig$xin and P r o p r a n o l o l . We have n o t y e t p u b l i s h e d any o t h e r p a p e r s on t h i s s u b j e c t a l t h o u g h remain i n t e r e s t e d i n i t . I s h o u l d be g r a t e f u l i f you would s e n d me a r e p r i n t of any p a p e r s which you p u b l i s h . With k i n d e s t r e g a r d s ,
For details, see text.
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FiPure 5
For details. see text.
2 Atrial Fibrillation
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ION CHANNEL REMODELING AND ATRIAL FIBRILLATION: CLINICAL ASPECTS HJGM CRIJNS Department of Cardiology, University Hospital Maastricht, The Netherlands
[email protected] Electrical and Structural Remodeling Occurs Simultaneously The progression to sustained atrial fibrillation depends on electrical remodeling, i.e. a progressive decrease of the effective refractory period. It also relates to structural remodeling. These processes go hand in hand. Electrical remodeling induced by AF is due to intracellular calcium overload. This leads to shortening of the atrial action potential and loss of action potential rate adaptation. These electrical changes are reversible once atrial fibrillation has disappeared. In the mean time however, calcium overload may have led to activation of intracellular proteases like calpains which cleave cellular proteins of the cytoskeleton, channel proteins and the contractile elements. In this way the high atrial rate during atrial fibrillation may secondarily lead to structural remodeling.
Structural Remodeling mostly Occurs while Patient is in Sinus Rhythm Although this is one mechanism, atrial structural remodeling which usually occurs during sinus rhythm, is probably more important. Stretch of the atria in the setting of hypertension or valvular disease is the main trigger for fibrosis. In some sub sets of patients amyloidosis may occur especially in elderly women with valvular heart disease.
Clinical Consequences of Atrial Remodeling Among the clinical consequence of atrial remodeling are (1) progression to permanent AF due to changes in ion channels, connexin down regulation, myolysis and fibrosis. In addition (2) reduced drug efficacy in converting atrial fibrillation to sinus rhythm or in maintaining sinus rhythm. Also (3) reversed remodeling, which occurs especially after cardioversion of persistent atrial fibrillation as a basis for different types of recurrences among which immediate recurrences and sub acute recurrences, the latter occurring between day one after cardioversion up till 2 to 4 weeks. Reversed remodeling also determines 21
22
changes in drug effects depending on the period after electrical cardioversion with some drugs preventing immediate recurrences of atrial fibrillation whereas others are active only against sub acute or late recurrences of AF. Finally (4) atrial structural remodeling is one of the mechanisms in Virchow's Triad responsible for thrombosis and embolism. Different Models, Different Ion Channel Remodeling
Ion channel remodeling may differ between one versus the other type of atrial fibrillation. Atrial Jibrillation related remodeling is mainly associated with a decrease in ICa-L and moderate decrease in Ito as well as the Ikur. On the other hand heart failure related ion channel remodeling is associated with moderate decreases in ICa-L, Iks, as well as a moderate decrease in Ito. In addition the sodium calcium / exchanger is moderately up regulated. Studies by the group of Nattel have shown clearly that down regulation of the Ica-L is the main determinant of electrical remodeling. Brundel in our lab has shown a clear correlation between the atrial effective refractory period and L-type calcium channel protein expression. In addition, she showed that down regulation of the channel together with shortening of the atrial refractory period was most marked in patients with chronic atrial fibrillation. Experimental and clinical studies have shown that the action potential shortening and shortening of the refractory period is not enough to develop persistent atrial fibrillation. In addition to these factors, conduction delay plays a role. In this respect sodium channel down regulation, connexin down regulation as well as fibrosis leading to detour conduction are important. Ausma and coworkers have shown that connexin or gap-junctional remodeling is present in long standing atrial fibrillation. She also found that after restoration of sinus rhythm gap-junctional connexins restored whereas atrial fibrillation could still be easily induced. Therefore she concluded that fibrosis or proteolysis are far more important then connexin down regulation. Brundel in our laboratory showed that calpains are responsible voor proteolysis and structural changes in human paroxysmal and persistent atrial fibrillation. Calpain activity was especially visible in the nucleus as well as at the level of the intercalated disc. Activation of proteolysis by calpains was significantly related to the type of atrial fibrillation with low protein expression of the ICa-L channel. Especially, this was seen in patients with chronic atrial fibrillation. Nattel and co-workers showed that the renin angiotensin system is a very important fibrosis pathway especially in heart failure related atrial fibrillation.
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Class I11 Drugs and Ion Channel Remodeling
One of the consequences of molecular remodeling is that class 111 drugs lose their efficacy the longer atrial fibrillation lasts. Classical class 111 drugs block Ikr. Although this channel is not down regulated in long lasting atrial fibrillation, it is not important for repolarization in the remodeled atria. In stead, channels active early during the plateau phase of the action potential (like Ito and Ikur) are more important and blocking these channels may effectively induce prolongation of the action potential. This has been investigated by Allessie and co-workers who showed that an Ikur blocker AVEO118 can cardiovert persistent atrial fibrillation by prolonging especially the very short action potentials of the remodeled atria. It may be concluded that Ikur block is more effective in prolonging effective refractory period in remodeled compared to normal atria. In addition, inhibiting both Ikur and Ikr may produce supra-additive effects on the action potential duration. These conclusions have appeared from the work of Courtemanche and Nattel and show that mathematical models may provide insight in new ionic targets for drug therapy in AF induced electrical remodeling. Reversed Remodeling and Different Types of Postcardioversion Recurrences
Reversed electrical remodeling is important to understand the different types of recurrences after cardioversion of persistent atrial fibrillation. During the sub acute phase of recurrences, i.e. the first two weeks after the shock, several ionic mechanisms may be held responsible for these sub acute recurrences: abnormal calcium handling leading to atrial ectopy; differential ERP lengthening producing dispersion of refractoriness; sinus node dysfunction; recovery of atrial function leading to stretch induced ectopy. Hoshiyama and co workers showed in the remodeled atria down regulation of calcium handling proteins like Serca and the Ryanodine receptor. Down regulation of these proteins may lead to electrical instability once calcium flows again into the cell like it occurs immediately after cardioversion. This notion is supported by the fact that verapamil in combination with class I or class 111antiarrhythmic drugs may reduce sub acute recurrences of AF; i.e. recurrences which happen during the reversed remodeling phase after cardioversion. The same holds for irbesartan in combination with amiodarone as has been showed by Madrid, De Simone and co workers. In conclusion, ion channel remodeling as well as structural remodeling has significant clinical consequences. Understanding the molecular mechanisms is
24
mandatory for understanding the clinical pathophysiology and treatment of atrial fibrillation.
I
Types of recurrences after cardiwersion
I
LONG-TERM EFFICACY OF ANTIARRHYTHMIC DRUG THERAPY AND ITS INFLUENCE TO THE PROGNOSIS IN PATIENTS WITH PAROXYSMAL ATRIAL FIBRILLATION KEN OKUMURA Second Department of Internal Medicine, Hirosaki University School of Medicine, Japan
We examined the long-term efficacy of serial antiarrhythmic drug therapy (AAT) in 290 patients with paroxysmal AF (mean age, 69 years), and studied the relationship between the response to AAT and long-term prognosis of the patients. After sinus rhythm was restored spontaneously or by cardioversion, one of the following class I drugs, disopyramide (300 mg/day), cibenzoline (300 mg/day), and aprindine (60 mg/day), was selected by an envelope method (the first drug) and was administered orally. When AF recurred, one of the following drugs, flecainide (150 mg/day), pilsicainide (150 mg/day) and bepridil (150 mg/day), was selected by an envelope method (the second drug). When AF recurred, amiodarone or class I antiarrhythmic drug that was not used before was administered (the third drug). After treatment with the fxst drug, 51%, 47% and 35% of the patients treated with disopyramide, cibenzoline and aprindine, respectively, were free from AF recurrence at one year. After the second drug, 33%, 33% and 21% of the patients treated with flecainide, pilsicainide and bepridil, respectively, were free from AF recurrence at one year. After the third drug, 43% and 18% of the patients treated with amiodarone and class I antiarrhythmic drug that was not used before, respectively, were free from AF recurrence at one year. During a mean follow-up period of 5 1h29 months, 114 patients (39%) had no AF recurrence (Group l), 113 (39%) had repeated AF recurrence (Group 2), and the remaining 63 (22%) had permanent AF despite AAT (Group 3). Survival rate without any cardiovascular deaths at 60 months was 99% in Group 1, 95% in Group 2 and 94% in Group 3 (p=NS among 3 groups). Survival rate without symptomatic ischemic stroke was 99% in Group 1, 88% in Group 2 and 76% in Group 3 ( Ba2', showing that Isoc in mES cell is particularly selective for Ca". Thus, we confirmed the existence of Ism in mES cells.
voccs In contrast, voltage-operated Ca2+channels (VOCCs) are well known to play a central role for Ca2' entry across the plasma membrane in electrically excitable cells and may contribute to differentiation or proliferation. However, in most non-excitable cells including mES cells, the functions of VOCCs are not well
222
understood. To examine whether VOCCs express and function in mES cells , the cells were depolarized by high K" external solution in Ca" imaging experiments, however, the elevation of [Ca2'Ii was not observed (n= 14), suggesting no function of VOCCs in mES cells. In the whole cell membrane currents recordings applied to potentials between -70 mV and +lo0 mV from 80 mV holding potentials, inward currents could not be observed (20/20 cells). We also examined the expression of mRNAs for several kinds of VOCCs in mES cells. None of them included L, T, P/Q type Ca2' channels could not be detected in our experiments. From above results, we concluded that VOCCs do not function in mES cells before differentiation.
Ca2+Extrusion Systems in mES Cells Plasma Membrane Cd7+Pump ATPase A membrane Ca" pump (PMCA) has been identified as a major contributor to the cell Ca2' extrusion. We tested two different types of Ca" pump blockers, namely carboxyeosin and caloxin 2A1. Application of carboxyeosin (5 pM), which is a cell-permeable fluorescein analogue and one of the most potent blocker (14), markedly increased basal [Ca2'Ii (8/8 cells). We also tested a specific PMCA blocker, caloxin 2A1, which is a synthesized peptide obtained by screening a random peptide phage display library for binding to the second extacellular domain (residues 40 1-413) sequence of PMCA and selected to bind the second putative extracellular domain to inhibit the Ca2' pump function (15). Application of these blockers induced an elevation of [Ca2'Ii (1 1/12 cells). It is known that there are four genes PMCA isoforms 1-4 in mammals (16). These isofoms are known to be expressed in a tissue-dependent manner. Our studies in the expression of genes for PMCAs isoforms by RT-PCR showed the expression of mRNAs for PMCAl and 4 in mES cells but not for PMCA2 or 3 (12).
Na+/Ca2' Exchanger Ca2+Extrusion Systems As well as PMCAs, the Na+-Ca2+exchanger (NCX) of mammalian plasma membrane assumes to play an important role in the maintenance of the intracellular Ca" homeostasis (17). Several kinds of blockers for NCX were tested on [Ca2+]i. Since Na' gradient was necessary for the activation of Na+/Ca2' exchanger, the elimination of Na" in the bath solution to block NCX induced the increase of basal [Ca2'Ii (n= 15). The application of KBR7943, a specific Na"/Ca2' exchanger blocker, also increased [Ca2'Ii (12/13 cells). These results indicate that the Na'/Ca2' exchangers operate to extrude Ca2' from
223
cytosole and contribute to maintain low level of [Ca2’Ii in mES cells. By RTPCR, mRNAs for NCX, I, I1 and I11 could be detected in mES cells (7). #2; Development of Ca2+Signaling Pathways during Differentiation into Cardiomyocytes Ca2’ signaling pathways in mES cells are almost identical to those in the other non-excitable cells. Next question is, when or how these Ca” signaling pathways change during the differentiation to cardiomyocytes.
Ca2+Releasefrom Sarcoplasmic Reticulum It is well known that Ca” release from SR is mediated via ryanodine receptors in cardiomyocytes. We examined when the function of ryanodine receptors was observed during cardiomyogenesis using caffeine by measuring [Caz’]i responses. As described above, we did not see caffeine responses in mES cells, however, caffeine induced Ca2’ transient were observed in the middle stage of differentiation or later. The numbers of cells responded to caffeine increase during differentiation to cardiomyocytes. The magnitude of [Ca2+]itransient also increases with differentiation.
Ca2+Entry through DHP Receptros In cardiac myocytes, Ca” entry through dihydropyridine receptors (DHPR) is very important for cardiac function. To study the functional development of DHPR, the depolarization induced [Ca2+Iichanges were measured in [Ca2+Ii imaging experiments. From the middle stage of differentiation, voltage-induced [Ca2+Ii increases were observed and these responses increased with differentiated processes. In cardiac myocytes, both T-type and L-type Ca” channels are known. To distinguish these channel functions, we used each channel blocker, low dose of N?’ and DHP. In the middle stage of differentiation, DHP did not block Ca2’ transient induced by K’ depolarization, but Ni2+ completely blocked this, indicating the contribution of T-type Ca2+ channel. On the other hand, the later stage of differentiation, Ca” transient was blocked by DHP but not Ni”, indicating the contribution of L-type Ca” channel. These results were confirmed by whole cell membrane currents.
224
Activities of Na'm (pump) ATPase in mES Cells and Derived Cardiomyocytes The Na'/K' ATPase (Na' pump) maintains the high internal K' and low internal Na+ concentrations in most cells (18,19) and are well known to play very important roles for cardiac functions (17,18,20). We investigated the function of Na'/K+ ATPase in undifferentiated mES cells and derived cardiomyocytes by inhibiting its activities using K+ free solution or a cardiac glycoside, ouabain. Since, it is well known that the concentration of intracellular Na' (ma'Ii) is increased by the inhibition of Na+/K+ATPase, [Na+]i was monitored by using the cell membrane-permeable Na' indicator, acetoxymethyl ester of sodiumbinding benzofuran isophthalate (SBFI-AM). Cells were incubated in the K' free solution to block Na+/K' ATPase and then the bath solution was changed from K' free to 10 mM K' solution to activate it. These experiments gave us information that undifferentiated mES cells expressed Na+/K+ATPase and the activities at various stages of differentiation in mES cells increased with a time course of differentiation into cardiomyocytes, suggesting existence of the developmental changes of Na+/K+ATPase activities. It is known that several subtypes of a-subunit are expressed developmentally or depended on cell types (19, 21). It is also reported that the ouabain sensitivity is different depending on the isoform of Na+/K+ ATPase (19). The a 1 isoform has low sensitivity to ouabain but both a 2 and a3 subunits have high sensitivities to this drug (22). Consequently, the a1 isoform can be functionally distinguished from a2 and a3 isoforms on the basis of their differential sensitivities to ouabain. Therefore, we further examined the ouabain sensitivities of undifferentiated mES cells and derived cardiomyocytes. By the application of low concentration ouabain, small changes in could be induced in undifferentiated mES cells, but big changes in were observed in 15-day derived cardiomyocyts. On the other hand, the high concentration of ouabain (3 mM) markedly increased in both undifferentiated mES cells and derived cardiomyocytes. In the pooled data of mES cells at various stages of differentiation, the effects of low dose of ouabain were significantly increased along the time course of differentiation. The effects of high dose of ouabain also increased in derived cardiomyocytes at all stages of differentiation compared with undifferentaitated mES cells (Fig. 2A). To confirm the activities of Na+/K+ATPase, we recorded Na+/K+pump currents (Ip) using whole cell patch clamp technique. The results showed that a very tiny l,, exits in mES cells and its amplitude increases during differentiation into cardiomyocytes.
225
Functional Expression of Na+/Caz' Exchangers during Cardiomyogenesis We studied the functions of NCXs in mES cells at various stages of differentiation by measuring [Ca2'Ii using a NCX blocker, KB-R7943. By the application of KB-R7943, the dynamic changes in [Ca2'Ii were observed in almost all mES cells (87/88 cells), which showed two different patterns, namely a [Ca2'Ii transient and a sustained elevation of [Ca2'Ii. More than 90 % of undifferentiated mES cells exhibited [Ca2'Ii transients (10/11 cells). A sustained elevation of [Ca2+Iiwas observed in many derived cardiomyocytes (134 7 cells), but not in undifferentiated mES cells (1/11 cells). In pooled data at various stages of differentiation, the numbers of the cells showing a [Ca2'Ii transient decreased and those showing a sustained elevation of [Ca2']i increased during the differentiation. The magnitude of the sustained [Ca2'Ii became larger in a time course of differentiation (Fig. 2B).
Na+/K+ATPase and its Functional Couplings with Na'/Caz' Exchangers during Differentiation into Cardiomyocytes It is widely accepted that the partial inhibition of Na+/K' ATPase by ouabain causes a modest increase in [Na+]i in cardiomyocytes, which in turn regulates the activities of the Na'/Ca2' exchanger (NCX), leading to a significant increase in [Ca2']i or [Ca2"]' oscillations (18, 22, 23, 24). We examined how Na'/K+ ATPase affected [Ca2'Ii during cardiomyogenesis. In undifferentiated mES cells, the application of either 30 pM or 3 mM ouabain did not affect [Ca2'Ii (9/9 cells), indicating no coupling of both transporters. At early stage of differentiation (3/44 cells), the low dose of ouabain affected the small number of the derived cardiomyocytes. In 80 % of derived cardiomyocytes at middle and late stages, ouabain induced [Ca2']i oscillations Fig. 2C). Form these results, we conclude that the functional coupling between two Nat/K+ ATPase with NCX is established at the middle stage of the differentiation.
Molecular Study in N a ' m ATPase and Na+/Caz' Exchangers The above functional studies demonstrated the developmental changes in both Na'/K' ATPase and Na'/Ca2' exchanger during differentiation from mES cells to derived cardiomyocytes. Using RT-PCR, a1 subunit mRNA could be detected in undifferentiated mES cells and derived cardiomyocytes at all stages of differentiation. In contrast, a 2 subunit, which is high sensitive to cardiac glycoside, could not be detected in undifferentiated mES cells but detected in mES cells after 4- or longer in vitro induction.
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It is known that the Na+/Ca2+exchanger is coded by NCXl gene in adult cardiomyocytes (23). We examined the kinetics of NCXl expression and found that NCXl mRNA could be detected in undifferentiated mES cells and derived cardiomyocytes at all stages of differentiation. The expression of this gene increased during differentiation. These results are consistent with physiological functional results.
Physiological Roles of Na+@ A TPase and NCX during Differentiation There are several studies to identify the fictional expression of ion channels and signal transduction pathways in mES cells during cardiogenesis (3, 4, 25). In early stages of differentiation, inward Ca2' current, transient outwrd K' current and IUTP are demonstrated. Other cardiac ion currents such as inward rectifying K' currents, Na' current and If start to function in the later stages of differentiation (longer than 10 day in vitro differentiation) (3,4). Much different from these cardiac specific ion channels, both Na+/JS+ ATPase and Na'/Ca2' exchanger express these functions before differentiation, suggesting the universal roles for cellular functions. Because the expression patterns of Na+/K+ ATPase isoforms change during cardiogenesis, we speculate that the normal ionic balance maintained via Na+/K+ ATPase and NCX might regulate the processes of differentiation in mES cells to derive cardiomyocytes. #3; Conclusion
In this study we demonstrate a unique Ca2+-signalingsystem in mES cells. (1) InsP3 induced Ca2' release from ER, (2) Ca2' entry through the store-operated Ca" channels. (3) To maintain the low level of [Ca2'Ii,, both plasma membrane Ca2' pumps and Na'/Ca2' exchangers play important roles for extrusion of Ca2+ out of the cytosol. (4) During differentiation from mES cells to cardiomyocytes, we demonstrated the functional expression of DHP receptors, ryanodine receptors, Na+/K+ ATPase and Na'/Ca2' exchanger by physiological and molecular experiments. (5) The functional coupling between Na"/K' ATPase and Na+/Ca2+exchanger to regulate [Ca2']i was recognized in the middle stages of in vitro differentiation (10-day in vitro induction).
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References 1. P.J. Donovan, J. Gearhart Nature 414 (2001) 92-97. 2. A.M. Wobus Mol. Aspects Med. 22 (2001) 149-164. 3. J. Hescheler, B.K. Fleischmann, S. Lentini, V.A. Maltsev, J. Rohwedel, A. M. Wobus, K. Addicks Cardiovasc. Res. 36 (1997) 149-162. 4. K.R. Boheler, J. Czyz, D. Tweedie, H.T. Yang, S.V. Anisimov, A.M. Wobus, Circ. Res. 9 1 (2002) 189 - 20 1. 5. P, Anversa, B. Nadal-Ginard, Nature 415 (2002) 240-243. 6. M.G. Klug, M.H. Soonpaa, G.Y. Koh, L.J. Field, J. Clin. Invest. 98 (1996) 216-224. 7. K. Otsu, A. Kuruma, E. Yanagida, S. Shoji, T. Inoue, Y. Hirayama, H.Uematsu, Y. Hara, S. Kawano. Cell Calcium 2004 (in press) 8. Berridge MJ, Lipp P, Bootman MD. Nat Rev Mol Cell Biol2000; 1:11-21. 9. Meldolesi J. Nature 1998; 392: 863-866. 10. Berridge MJ. JPhsiol 1997; 499: 291-306. 11. Elliot AC. Cell Calcium 2001; 30: 73-93. 12. E. Yanagida, S. Shoji, Y. Hirayama, F. Yoshikawa, K. Otsu, H. Uematsu, M. Hiraoka, T. Furuichi, S. Kawano. Cell Calcium 36: 135-146 13. Berridge MJ. Nature 1993; 361: 315-325. 14. Sedova M, Blatter LA. Cell Calcium 1999; 25: 333-343. 15. Jyoti C, Walia M, Am JPhysiol-Cell Ph 2001; 280: C1027- C1030. 16. Strehler EE, Zacharias DA. Phys Rev2001; 81: 21-50. 17. Blaustein MP, Lederer WJ. Physiol Rev 1999; 79: 763-854. 18. H.G.Glitsch, Physiol. Rev. 81 (2001) 1791-1826. 19. G. Blanco, R.W. Mercer, Am. J. Physiol. Renal Physiol. 275 (1998) F633650. 20. K.D. Philipson, D.A. Nicoll, Annu. Rev. Physiol. 62 (2000) 11 1-133. 21. P.A. Lucchesi, K.J. Sweadner, J. Biol.Chem. 266 (1991) 9327-9331. 22.R. Zahler, Z.T. Zhang, M. Manor, W.F. Boron, J. Gen. Physiol. 110 (1997) 201-13. 23. M.P. Blaustein, W.J. Lederer, Physiol. Rev. 79 (1999) 763-854. 24. 0. Aizman, P. Uhlen, M. Lal, H. Brismar, A. Aperia, Proc. Natl. Acad. Sci. USA. 98 (2001) 13420-13424. 25. V.A. Maltsev, A.M. Wobus, J. Rohwedel, M. Bader, J. Hescheler, Circ. Res. 75 (1994) 233-244.
228 Figure 1 A, Ca" entry through plama membrane. When the stores were depleted with the 1 p M TG and 20 pM histamine in the 0 Ca" bath solution, [Ca"]; was strikingly decreased following a large increase of [Ca2'Ii. When [Ca2'], was increased to 4 mM, [Ca2+Iigradually increased. The application of 0.1 mM LaC13 markedly decreased [Ca2']i. Pictures indicate [Ca2'Ii imagings taken by conforcal microscopy. B, Direct recording of Isoc. Ramp clamp pulses (200 ms) were applied from -120 to +60 mV at every 2 seconds. Bath solutions contained 110 mM Ca2+.Small inward currents were induced by the application of histamine, which were blocked by 0.1 mM La3'. a, The representative ramp clamp currents at 10 minutes after application of histamine (1) and after the application of 0.1 M La3+(2). b, Isoc was obtained as a ~a~+-sensitive current [(I) - (211.
changes Figure 2A, Functional development of Nu+& (pump) ATPase. (-8F/Fo) before and after application of 30 pM were measured in the derived cardiomyocytes at various stages of differentiation (n = 17 - 53 cells). Significant changes in were indicated by * compared with mES cells and 5 compared with early stage of differentiation. Both P values (* and 5 ) were < 0.01. 2B, Functional expression of Nai/Ca2' exchangers. The intensities of fluorescence 30 minutes after application of KEbR7943 were measured at various differentiated stages (n = 11-32 cells). Significant differences were indicated by * and 5 compared with mES cell and early stage of differentiation, respectively. Both P values (* and 5 ) were < 0.001. Mean values between middle stage and late stage were also significant different (P < 0.01). 2C, Ouabain effects on (Ca2'li. Cells were loaded with fluo3-AM for 30 minutes to monitor [Ca2+]i.The numbers of cells showing [Ca2'Ii oscillations by the application of ouabain were summarized. [Ca2'Ii oscillations were not observed in undifferentiated mES cells (0/63 cells), but observed in 6.81 % cells (3/44), 77.8 % cells (28/36) and 75 % cells (12/16) in the derived cardiomyocytes at early stage, middle stage and late stage, respectively.
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DEVELOPMENTAL CHANGES OF L-TYPE AND T-TYPE CA" CHANNELS IN CARDIAC CELLS KENII YASUI Department of Circulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan Hoshigaoka Clinic, Nagoya 464-0026, Japan
Regeneration therapy by using bone marrow- or ES cell-derived cells is highlighted for the treatment of failing hearts. These cells have electrophysiological properties similar to embryonic cardiac myocytes. Information available on molecular basis of Ca2' channels in embryonic hearts is still limited. We investigated the expression of L-type (Cavl.l-l.3) and T-type (Ca"3.1-3.2) CaZ+channels, by a real-time PCR and whole-cell patch clamp in mouse ventricles from an early embryonic stage to adulthood. 1) L-type: At 9.5 dpc, Ca"l.1, Ca"1.2 and CaJ.3 mRNAs were expressed (CaJ.3 and CaJ.2 were dominant subtypes). With development CaJ.2 increased, CaJ.3 decreased and Ca"l.1 became undetectable. Activation and inactivation curves at 9.5 dpc were shifted to the left, compared with those at 18 dpc and adult. 2) T-type: Cav3.2 mRNA was the predominant subtype at both 9.5 dpc and 18 dpc. Ca"3.1 mRNA increased with development, but remained low compared with Ca"3.2 mRNA. At adult, Ca"3.1 is greater than Ca"3.2. Ttype Ca" current was observed only in the embryonic period, and Ni2' sensitivity suggested that Ca"3.2 underlies the functional T-type CaZt channels. In conclusions, CaJ.3 and Ca"3.2 channels are functionally expressed in early embryonic mouse ventricular cells. Those channels might be responsible for their potent spontaneous activity.
Introduction Embryonic murine heart initiates to beat at 8.5 dpc (days post coitum) and its spontaneous activity become regular at 9.5 dpc, when the heart shape is tube-like. Cardiac ventricle isolated from embryonic heart at 9.5 dpc beats spontaneously. With development, action potential changes and loses the spontaneous firing. The changes of expression in various ion channels underlie the alteration of the action potential '). Two distinct families of voltage-gated Ca2+channels are identified in mammalian cardiac muscle: high-voltage activated L-type Ca2' channels and low-voltage activated T-type Ca2' channels. L-type Ca2' channel play important roles in excitation and contraction of the heart. L-type Ca2' channels are coded by four genes, namely Ca,l.l (a's, skeletal muscle), Ca,1.2 (ale, cardiac and smooth muscle), Ca,1.3 (alD, neuron, neurosecretory cells and heart) and Ca,1.4 (alF,retina) *). Ca,1.2 and Ca, 1.3 channels are expressed in the adult heart. Ca"1.2 Ca2' channel is the major L-type Ca2+channel in cardiac muscle. Ca2+ 230
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entry to the cytosol through this channel triggers Ca2' release from sarcoplasmic reticulum and forms Ca" transient for cardiac contraction. Cavl.3 Ca2+channel is considered to function cardiac pacing in sinoatrial (SA) node, because Cav1.3 ablation induced SA node dysfunction '). Cav1.3 Ca" channel activates more negative potential than Cavl.2 4, and contributes to diastolic depolarization of SA node cell. It remains which subtypes of 4 different L-type Ca" channel genes underlie electrical activity in immature heart. T-type Ca2' channel is observed in pacemaker cells from the sinoatrial node and Purkinje fibers. Three subtypes of gene encoding T-type Ca" channel, Cav3.1 ( a , ~ Cav3.2 ), ( a l ~and ) Cav3.3 (alI),have been identified '). Whereas Cav3.3 was mostly detected in brain 5 ) , Cav3.1 and Cav3.2 mRNAs were also detected in human, rat and mouse hearts 63 'I. The subtype distribution is also development-stage-dependent, but the issue remains controversial. Cribbs et al. have reported that only Cav3.1 underlies functional T-type Ca2' channels in midgetational fetal mouse cardiac ventricle 'I. In rat hearts at middle to late embryonic and perinatal periods, substantial participation of both Cav3.1 and Cav3.2 to the functional T-type Ca2+ channels has been reported '). In this study, we investigated developmental changes of L-type and T-type Ca2' channel in mouse cardiac ventricle (at 9.5 dpc, 18 dpc and adult) using whole-cell patch clamp, Western blotting and a real-time PCR. Our study revealed that Ca"1.3 L-type Ca2' channel and Cav3.2 T-type Ca2+channel are expressed functionally and mainly in early-embryonic stage. Methods Animals
ICR mice (9.5 dpc, 18 dpc and 10-week adult) were used for the present study. Analysis of mRNA Expression of L-type and T-type Ca2+Channels
Total RNA of cardiac ventricle was extracted with RNeasy Mini Kit (Qiagen) from 9.5 dpc mouse embryo and with Acid Guanidinium ThiocyanatePhenol-Chloroform method from 18 dpc mouse embryo and adult mouse. Single-stranded cDNA synthesis was performed with total RNA using oligo d(T) primer using reverse transcriptase after DNase treatment of total RNA. For the quantitative analysis of mRNAs of L-type Ca2' channel genes (Cavl.l, Cav1.2, Cav1.3) and T-type Ca" channel genes (Cav3.1, Cav3.2), we used a real time fluorogenic 5'-nuclease PCR assay (Perkin-Elmer ABI Prism 7700). cDNA sample (1 50 ng) was added to each PCR tube. The threshold cycle (C,) from the baseline to reach a statistically significant increase in fluorescence signal was measured. The C, value predicts the quantity of target cDNA in the sample. The
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GAPDH gene was used as an endogenous control. PCR products were subcloned using TA cloning @GEMR-TEasy, Promega, Madison, WI) and were verified by sequencing. cDNA standards were obtained by digesting plasmid by EcoR I. Five different molecules of cDNA standards for target genes (1 x lo', 1 x lo6, 1 x lo5, 1 x lo4, 1 x lo3) were amplified to determine the standard curves between C, and log starting molecule number of cDNA standards. Western Blotting The immunoblotting for Cavl.2 Ca2+channel protein was performed by membrane fraction. For Cavl.3 Ca2' channel protein, crude homogenate was used for the blotting. Protein samples (20 pg for Cavl.2, 50 pg for Cavl.3) were loaded on 7% polyacrylamide-SDS gels and transferred to PVDF membranes. Membranes were blocked with 2.5% (for Cav1.2) or 0.3 % (for Cav1.3) non-fat milk in PBS and incubated overnight at 4 "C with a rabbit polyclonal antibody solution (anti-Cavl.2 antibody: 1/500, BD Bioscience, #5507 16; anti-Cavl.3 antibody: 1/500, BD Bioscience, #SO7 12). The immunoblots were developed with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (14 5000, Sigma, A0545) for 1 hour, followed by enhanced chemi-luminescence (SuperSiognal West Dura Extentended Duration Substrate (Piearce Biotechnology, #34075). The intensity of protein bands by chemi-luminescence was quantified by a CS Saver and Analyzer (ATTO & Rise Corporation). For analyzing Cav1.2 protein expression, rat cerebrum lysate (10 pg, BD Transduction Laboratories, #6 11463) was used as control. Electrophysiological Experiments Cultured single ventricular myocytes were prepared from ventricles of 9.5 dpc and 18 dpc mouse embryonic hearts by methods previously described lo). Cardiac ventricles were dissected from the exposed embryos and single myocytes were isolated by collagenase treatment. Cardiac myocytes were cultured on collagen-coated glass coverslips in minimum essential medium including 10% fetal bovine serum and 10 pg/ml gentamycin for 18-24 hours before current recording. Fresh single adult ventricular myocytes were used for patch clamp experiments. Adult myocytes were isolated by collagenase treatment with Langendorff perfusion. L-type CaZfchannel currents Whole cell voltage clamp recording were performed from ventricular myocytes using Axopatch 200B (Axon Instruments, USA). To record L-type (ICa,~) and T-type (ICa,T)Ca2' channel currents, myocytes were superfused with a Na'-free and K'-free external solution containing (a TEA-C1 ) 140, MgClz 1, HEPES 5 [pH 7.41, Glucose 10, CaC12 5 and 30 pM tetrodotoxin. Internal
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solution contained (mM) CsOH 60, CsCl 80, 1-aspartate 40, HEPES 5 [pH 7.21, MgATP 5, Na2-phosphocreatinine 5, 10 EGTA, CaC12 0.65 [pCa 7.961. Cell capacitance was measured by the application of a ramp voltage pulse of 0.5 V/s at a potential ranging between -50 mV and +70 mV. For inactivation-curve recording of ICa,L,200 pM NiC12 was added to external solution to inhibit Ica,~. Test pulses to 0 mV were preceded by conditioning pulse ranging -100 mV to 10 mV for 1 sec from the holding potential of -50 mV. For activation-curve, conductance (g) was obtained by dividing a peak Ca2' channel current at test potential by the difference between test and reverse potential. Inactivation and activation curves were fitted by the Boltzmann equation: I/Imax= 1/{1 + where V, is the exp[(V,-Vli2)/k]} and g/gma =I/{ 1 + e~p[(V,~~-V,)/k]), membrane voltage, VIi2is the voltage at half-maximal inactivation or activation, and k is the slope factor. All electrophysiological experiments were carried out at 3 5 -3 7°C. T-type Ca2+channel currents
L-type (&)and T-type (Ica,~)Ca2' currents were separated by applying 200 ms voltage steps in 10 mV increments, with a pulse interval of 5 s, to different test potentials from holding potentials of -100 mV and -50 mV. Isolation of ICa,T was performed by subtracting the currents obtained from the same test potential taken at the different holding potentails. Current amplitude was determined as the difference between the peak inward current and the steady-state current recorded at the end of the test pulse. The current amplitude was divided by C, to obtain current density. In experiments to study the activation and inactivation properties of ICa,T, the current was recorded from the holding potential of -100 mV in the presence of nisoldipine (3 pM) to eliminate ICa,L.Inactivation and activation curves were fitted by the Boltzmann equation. The voltage-dependence of steady-state inactivation for Ica,T was determined using a double-pulse protocol. A conditioning pulse for 1 s to various voltages ranging from -90 to -50 mV was followed by a test pulse to -40 mV for 200 ms to elicit I c ~ ,Data ~ . were normalized by dividing the test current by the maximal current elicited, and fitted according to the Boltzmann equation. The recovery of ICa,Tfrom inactivation was studied by applying two test pulses to -40 mV for 50 ms from a HP of -100 mV with increasing intervals from 10 to 3000 ms. The fractional recovery was calculated as the ratio of the current during the test pulse to the maximum current during the conditioning pulse. Statistics
Data are presented as mean f SEM. Statistical analysis of data was performed using paired and non-paired Student's t-test (patch clamp data), or
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ANOVA (mRNA and protein data). Differences were considered significant at p < 0.05.
Results L-type Caz+Channel
mRNA expressions We evaluated mRNA expression quantitatively for Cavl.1, Cavl.2 and Cavl.3 genes in cardiac ventricles at the 3 different developmental stages (at 9.5 dpc, at 18 dpc and at adulthood) using a real-time PCR (Fig. 1). At 9.5 dpc, the levels of Cavl.l, Cav1.2, Cav1.3mRNA expressions are 72 f 31, 295 f 113 and 474 f 59 molecules / lo5 GAPDH, respectively (n = 4). With development, Cav1.2 mRNA increased (429 f 118 at 18 dpc, n = 4; 1104 f 154 at adult, n = 41, Cav1.3 mRNA decreased (31 f 6 at 18 dpc, n = 4; 11 f 2 at adult, n = 4) and Cavl.1 mRNA became undetectable. Thus, in early-stage embryonic cardiac ventricle, Cav1.3 was the dominant type of L-type Ca2' channel mRNA. With development, Cavl.2 became to be the main type.
N.D.
N.D.
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Western blotting
We have studied protein level of L-type Ca” channels with development. Ca, 1.2 protein expression increased with development (0.63 f 0.13 at 9.5 dpc, 1.00 f 0.17 at 18.dpc, 2.82 f 0.70 at adult, n = 6, normalized to control peptide). Ca,l .3 protein was detected at 9.5 dpc, but not at 18 dpc and at adult. Ca,l .l protein was not expressed at all stages (data not shown).
L-type Ca2+channel current
We applied the depolarization pulses for 200 msec to various potentials from the holding potential of -50 mV to elicit L-type Ca” channel currents. Figure 2A shows representative membrane currents in response to depolarizing pulses ranging from -40 mV to 0 mV in ventricular myocytes at 9.5 dpc, 18 dpc and adult. Depolarization to -30 mV elicited a substantial inward Ca” current at 9.5 dpc, minimum current at 18 dpc, but no inward current at adult. Ca2’ channel currents activated and inactivated quickly. This kinetics neglected the presence of Ca,l.l Ca” channel current. Depolarizing pulse to 0 mV induced a larger inward Ca2’ current at adult than those at 9.5 dpc and 18 dpc. Figure 2B summarizes the current-voltage relationships (I-V curves) of Ca2’ current obtained from ventricular myocytes at 9.5dpc, 18dpc and adult. The amplitude of Ca2’ current by depolarization to 10 mV was 6.6 2 0.6 pNpF at 9.5 dpc, 5.7 $ 0.6 pNpF at 18 dpc and 13.1 f 2.2pNpF. The threshold of activation of Ca2+current at 9.5 dpc is more negative potential as compared with those at 18dpc and at adult. The potential of half-maximal activation (VIl2)was 14.6 2 2.4 mV (n = 9) at 9.5 dpc, -3.7 f 1.14 mV (n = 6) at 18 dpc, and -4.8 2 2.4 mV (n = 8) at adult. The slope factor (k) was 10.5 2 1.8 at 9.5 dpc, 7.6 f 0.5 at 18dpc, and 5.6 & 0.3 at adult. Inactivation curve at 9.5 dpc (VIl2= -40.1 2 2.jmV, n = 53 was also shifted to more negative potential than those at 18 dpc (29.7 If: 1.4 mV, n = 5) and at adult (-31.0 If: 1.8 mV, n = 6). These characteristics suggested Ca,l .3 was expressed functionally at 9.5 dpc.
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6
Fig. 2 T-type Gaff Channel mRNA expressions
mRNA expressions of T-type Ca” channel genes (Cav3.1 and Cav3.2) were quantified by a real time PCR assay (Fig. 3). At 9.5 dpc, Cav3.2 mRNA was expressed abundantly (7177 f 105, n = 7), while Cav3.1 mRNA expression was minimal (40 f 3, n = 7). At 18 dpc, Cav3.1 mRNA was increased (1226 f 121, n = 7), whereas Cav3.2 mRNA was decreased (4041 f 629, n = 7) but it still remained to, be dominant subtype. In adult ventricular tissue, a substantial level of Cav3.1 mRNA expression (741 f 148, n = 7) was detected, whereas Cav3.2 mRNA expression was minimal (57 f 19, n = 7). T-type Ca2+channel current
We used two different holding potentials to record L-type and T-type Ca” current from ventricular myocytes. As T-type Ca2’ current was almost inactivated when the holding potential is -50 mV, Subtraction of currents at the holding potential of -50 mV from those at holding potential of - 100 mV showed
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-1
T
Fig. 3 low-voltage-activated T-type Ca2+ current (ICa,~). The current-voltage relationships (I-V curves) of Ca2+ current were recorded from ventricular myocytes at 9.5 dpc, 18 dpc and adult. Both in 9.5dpc and 18 dpc myocytes, the difference current (ICa,T)was activated negative to -40 mV and peaked at -20. The peak ICa,Tdensity of 18 dpc myocytes was comparable to that of 9.5 dpc myocytes. In adult myocytes, the two I-V curves from holding potentials of 100 mV and -50 mV were almost superimposed, giving rise to minimal difference current at potentials more positive than -20 mV, which indicates the absence of IcqTat adult. The voltage-dependence of activation and that of steady-state inactivation of ICa,T were also studied in the presence of nisoldipine. The activation and steady-state inactivation curves of myocytes at 9.5 dpc and 18 dpc were almost superimposed, and there were no significant differences in Vo.s and k values between the two embryonic stages for both activation and inactivation. We examined time-dependent ICa,T recovery from inactivation in the embryonic myocytes (9.5 dpc and 18 dpc). The fractional recovery expressed as the ratio of the current during the test pulse (I,,,,) to the maximum current during the conditioning pulse (Imax)was plotted as a h c t i o n of the interpulse duration (At). The relations were best fitted by a double exponential function. The curve fits provided time constants Tfast and z,lOw,and amplitudes Afastand Aslowfor fast and slowly recovering current fractions, respectively. The recovery kinetics at 9.5 dpc (zfast37.2 6.9 ms, Afa, 0.45 f 0.08,.z,~ow276 f 21 ms, Aslow0.54 f 0.07, n=6) were similar to those at 18 dpc (qast26.9 f 5.0 ms, Af,, O S O A 0.03, z,low300 f 23 ms, Aslow0.41 f 0.05, n=8). As previously reported from heterologous expression systems, the Ni2+sensitivity of Cav3.2- and Cav3.1-related current strongly differs (I& = 12 pM and >150 pM, respectively). We, therefore, examined the effects of Ni2+on Ic~,T recorded from embryonic (9.5 dpc and 18 dpc) ventricular myocytes. ICa,T was measured at a test pulse to -40 mV from a HP of -100 mV in the presence
-
238
of nisoldipine. The average dose-response curves were obtained from 9.5 dpc (n 10) and 18 dpc (n = 7) myocytes. The two curves were almost superimposed and the half inhibitory concentrations (ICSO)were 3.l f 4 pM at 9.5 dpc and 26 f 5 pM at IS dpc. Ni2+sensitivity suggested that Cav3.2 Ca2' channels functions in embryonic cardiac ventricle.
=
Conclusion In this study, we have investigated L-type and T-type Ca2' channels in mouse ventricular myocytes during development from 9.5 dpc to adulthood. L-type and T-type Ca2' channel currents were recorded from early-embryonic stage, but T-type Ca2' channel current was not obtained from adult stage. In cardiac ventricle at early-embryonic stage, Ca"1.2 and Cavl.3 L-type Ca2' channels were functionally expressed and Cav1.3 was the dominant type of Ltype Ca2+ channel. With development, Cav1.2 became the main L-type Ca2+ channel. In embryonic stage, Cav3.2 was functional subtype of T-type Ca2' channel. We concluded that Cav1.3 and Cav3.2 might play an important role in pacemaking activity of ventricular myocytes of early-embryonic mice. References Davies MP, An RH, Doevendans P et al. Circ. Res. 78, 15 (1996). Ertel EA, Campbell KP, Harpold MM, et al. Neuron 25,533 (2000). Platzer J, Engel J, Schrott-Fischer A, et al. Cell 102, 89 (2000). Koschak A, Reimer D, Huber I, et al. J. Biol. Chem. 276,22100 (2001). Lee J-H, Daud AN, Cribbs LL, et al. J. Neurosci. 19, 1912 (1999). Cribbs LL, Lee J-H, Yang J, et al. Circ. Res. 83, 103 (1998). Perez-Reyes E, Cribbs LL, Daud A, et al. Nature 391,896 (1998). Cribbs LL, Martin BL, Schroder EA, et al. Circ. Res. 88,403 (2001). Ferron L, Capuano V, Deroubaix E, Coulombe A, and Renaud JF. J. Mol. Cell. Cardiol. 34,533. (2002). 10. Liu W, Yasui K, Arai A, et al. Am. J. Physiol. (Heart Circ. Physiol.) 276, H608 (1999).
1. 2. 3. 4. 5. 6. 7. 8. 9.
8 Ion Channels
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CO-CULTURED SKELETAL MYOCYTE AND CARDIOMYOCYTE CELL-SHEETS COULD NOT ESTABLISH ELECTRICAL COMMUNICATION, BUT CAUSED FIBRILLATING ACTIVITY IN CARDIOMYOCYTE SHUNICHIRO MIYOSHI', YUJI ITABASHI', KEIICHI FUKUDA', KOJIRO TANIMOTO', TATSUYA SHIMIZU', YOKO HAGIWARA', AKIRA FURUTA', TOMOKO TANAKA', NOBUHIRO NISHIYAMA', TERUO OKANO', HIDE0 MITAMURA', SATOSHI OGAWA' 'Cardiopuhioizary Division of Keio University School of Medicine, Tokyo, Japan, %istitute of Advanced Biomedical Engineering and Science, Tokyo Wornen's Medical University, Japan
Transplantation to the patient with enzymatically isolated skeletal myocyte (SM) cell improved cardiac function and caused serious arrhythmias, despite electrical disconnection between the SM and host cardiomyocyte (CM) was shown. Previously we reported cell culture surfaces grafted with temperature-responsive polymer, from which confluent cells detach as a cell-sheet simply by reducing temperature without any enzymatic treatments. We applied the cell-sheet technology for the transplantation to establish electrical communication between SM and CM and observed arrhythmogeneity. Methods Prepared neonate rat derived SM and CM cell-sheets were co-cultured for 3 days. The action potential was observed by optical image of voltage sensitive dye to test electrical communication between the SM-sheet and CM-sheet. The spontaneous and arrhythmic contractile activity were monitored by video motion-detecting program and [Ca2+]i imaging. Results No electrical communication between SM and CM-sheet (0/20) was observed. Spontaneous and rhythmic contraction was observed in SM (24/30). Fibrillating contraction was observed in the CM-sheet, when it was co-cultured with SM-sheet. Conclusions Our cell-sheet graft technology failed to establish the electrical connection. Fibrillating contraction in CM was observed for the first time when we co-cultured with SM-sheet. Automaticity in SM might be foe for the arrhythmogenesis via activating stretch activated channel in CM.
241
REMODELING OF GAP JUNCTION CONNEXIN IN ATRIAL AND VENTRICULAR FIBRILLATION ISSEI IMANAGAt tDepartnzent of Pliysiology, School of Medicine, Fukuoka Universiw, 7-45-1 Nanakuma, Johnan-ku, Fukuoka, 814-0180 Japan
LIN HAIt KOICHI OGAWA Department of Anatomy, School of Medicine, Fukuoka Universiwj 7-45-1 Nanakuma, Johnan-ku, Fukuoka, 814-0180 Japan
1.
Introduction
In cardiac muscle, the gap junction greatly contributes to electrical cell-tocell coupling. Dysfunction of the gap junction is one of arrhythmogenic factors. The physiological function of the gap junction depends on expression and phosphorylation of connexins which compose gap junction channel. Atrial and ventricular fibrillation arc serious arrhythmias. .4conitine, a kind of alkaloid, has been used as model of cardiac fibrillation. And then, in this study, we examined how expression and phosphorylation of connexin 43 (Cx43) (dominant expression in atrial and ventricular cells) are altered during aconitine-induced atrial and ventricular fibrillation. 2.
Methods
Male adult rats and guinea-pigs were used. Isolated heart was perfused with Krebs solution on Langendorff, at constant pressure and constant flow. In invitro experiment, atrial and ventricular muscle strips were superfused with Krebs solution. Aconitine was applied at final concentration of 0.1pM in both experiments. The beginning of flutter and fibrillation was confirmed by electrogram in isolated heart and transmembrane action potential in in-vitro experiment. Expression of Cx43 was detected by immunohistochemistry using monoclonal anti Cx43 antibody and confocal microscope. Phosphorylation of Cx43 was evaluated by Western blotting using monoclonal anti Cx43 antibody. Tissue Angiotensin I1 and PKC (each isoform) were measured by Western blotting. Intensity of immunoreactivity of Cx43 on confocal micrograph and of Western blot were analyzed with NIH Image soft ware. 242
243
3. Results 3. 1. Induction of Fibrillation and Safety Factor Generally, flutter was induced about 5 minutes after an application of aconitine. And then, aconitine was washed out just after induction of flutter. Flutter activity continued without aconitine. In the normal heart, about 8 minutes later, flutter shifted to fibrillation. In the pathologic heart, such as diabetic or PMA-treated heart, time from shift of flutter to fibrillation was remarkably shorter than that in the normal heart. Despite of absence of aconitine, fibrillation continued for about one hour. Safety factor of conduction was under 1 at fibrillation stage. In atrial tissue, fibrillation was generated earlier and more easily than in ventricular tissue.
3. 2. Expression of Cx43, Immunohistochemistry At fairly early stage of fibrillation, just after shift from flutter to fibrillation, irregular and inhomogenous expression of Cx43 at the intercalated disk had already been observed. At late stage of fibrillation, 60min later, remarkably scant and sparse expression of Cx43 was found at the intercalated disk. Scant expression of Cx43 at the intercalated disk and reduction of area of immunoreactive particle and diminution of intensity of immunoreactivity were depending on progression of fibrillation. These immunohistochemistry findings are very similar to those of the diabetic or PMA-treated heart. Expression of Cx43 in atrium was more scant than that in ventricle.
3 . 3 . Phosphorylation and Quantity of Cx43, Western Blot As fibrillation was advanced, dephosphorylation and reduction of quantity of Cx43 were augmented. 3.4. PKC and Angiotensin 11
It was proved that activation of PKCE and tissue Angiotensin I1 were augmented during fibrillation. Another isoforms of PKC (PKC a ,LI 1, P 2, 6 and 0 ) were not significantly changed.
3 . 5 . Shifi from Flutter to Fibrillation The time of shift from flutter to fibrillation was significantly shorter in the diabetic or PMA-treated than in the normal heart. It was about 7-8 minutes in the normal heart, on the other hand, about 2-3 minutes in the above pathologic hearts.
244 4.
Discussion
Moderate intracellular Ca overload or intracellular acidosis induces moderate dephosphorylation of Cx43, decreased conductivity and impairs incompletely electrical cell-to-cell coupling [ 11. Dephosphorylation of Cx43 is one of factors closing the gap junction channel. At the early stage of fibrillation, in another words, at the beginning of fibrillation, dephosphorylation of Cx43 had already been found. Dephosphorylation was augmented as fibrillation was progressed. This may be caused by intracellular Ca-overload induced by aconitine (activation of Na-Ca exchange) or high frequency activity (flutter). Previously we reported that the degenerative changes of Cx43 expression in the diabetic or PMA-treated heart were caused by acceleration of proteolytic degradation of Cx43 due to an activation of PKCE[2]. Immunohistochemistry findings of Cx43 at the early stage of or during fibrillation were similar those in the diabetic or PMA-treated heart. In these pathologic hearts, scant and sparse expression of Cx43 at the intercalated disk was characteristic. It was proved in this study that PKCE and tissue Angiotensin I1 were augmented during fibrillation. Degenerative expression of Cx43 was augmented as fibrillation was advanced. It is suggested that abnormalities of Cx43 expression during fibrillation are caused by an activation of PKCE. When very low concentration of heptanoi, 0.1 rnM was appiied during flutter, flutter shifted to fibrillation promptly within several seconds. Very low of heptanol inhibits gap junction channels, not completely. Incomplete conduction block is an important factor of generation of fibrillation. Scant and sparse expression of Cx43 at the intercalated disk, and dephosphorylation of Cx43 indicate decrease in number of the gap junction channel and closing of the channels. These structural abnormalities of the gap junction induce incomplete conduction block and asynchronous electrical interaction between cells. Shift from flutter to fibrillation indicates beginning of asynchronous electrical interaction between cells and indicates susceptibility to fibrillation. This possibility was proved in the diabetic or PMA-treated heart. Results that atrial tissue is more susceptible to fibrillation than ventricular tissue can be explained by observation that expression of Cx43 is less in atrial than in ventricular tissue. It is possible idea that fibrillation itself makes substrates accelerating fibrillation.
245
5.
Conclusion
Cardiac tissue is susceptible to fibrillation when it is essentially exposed to impairment of gap junction channel, close of channel or reduction of number of channel. And fibrillation itself makes substrate accelerating fibrillation. That is, gap junction connexin is remodeled by fibrillation itself. It is also suggested that an activation of PKCE mediated by Angiotensin I1 is concerned with structural remodeling of gap junction connexin during fibrillation. References 1. I. Imanaga, N. Hirosawa, Hai Lin, Y. Sakamoto, K. Matsumura and T.
2.
Mayama, In Heart Cell Coupling and Impulse Propagation in Health and Disease, ed. by W. C. DeMello and M J. Janse, Kluwer, Academic Pub. Dordrecht, Chapt. 7, 185 (2002). I. Imanaga, Hai Lin, Y. Nakamura and K.Ogawa, J. Mol. Cell. Cardiol. 35, A17 (2003).
cx43 before fibrillation
beginning of fihrillstion (1-2inui)
late stage of fibrillation (60min)
Ventricle Fig. 1 Confocal micrographs of immunohistochemistryfor Cx43, rat ventricle
100
wm
A MATHEMATICAL MODEL OF THE PROPOSED FUZZY SPACE FOR NA+ AND CA2+ IN LEFT VENTRICLE CARDIOMYOCYTES G. T. LINES Simula Research Laboratoly, PB 134, 1325 Lysaker, Norway E-mail:
[email protected] P. GR0TTUM Medical Faculty, University of Oslo, Norway J0RN B. SANDE, TEVJE A. STRBMME AND OLE M. SEJERSTED Ullev6l University Hospital, University of Oslo, Norway The sodium-calcium exchanger increases intracellular calcium when it operates in reverse mode. In pathological conditions with elevated sodium this calcium source might contribute. sufficiently to facilitate contraction. We propose a mathematical model where the spatial and temporal distribution of sodium and calcium is taken into account. Important channels are represented as spatially discrete entities. Under certain conditions we observe in the model that release from SR can be triggered by sodium influx and thus advanced in time. However, the present results are preliminary and in order to draw quantitative conclusions several modeling uncertainties must be clarified.
1. Introduction
The standard concept of excitation-contraction coupling is that a small current from the L-type Ca” channel triggers the ryanodine receptors of the sarcoplasmic reticulum (SR) to release a large amount of Ca” into cytosol and hence initiate contraction’. However, Leblanc and Hume2 showed that in the absence of calcium entry through the voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores (SR). Their data gave credence to the hypothesis that Na+ influx via the Na’-channels raised local and that this caused Ca” entry via the NCX. Lederer et al.3 were the first to propose that there existed a subsarcolemmal space of restricted diffusion where also the different ionic concentrations would differ from the average myoplasmic values. This subspace was designated fuzzy space. In this study we propose a model for the fuzzy space where the interaction of currents and concentration can be studied in a wide range of scenarios. 246
247 2.
Methods
The mathematical model consists of diffusion equations for calcium(c) and sodium(s), coupled to the single-cell model by Winslow et a14which is used to calculate the transmembrane potential, channel conductance and other state variables(w). The full model is as follows: -= as at
k,V2s,
X€Q
Here Q is the intracellular domain, including the fuzzy space, but not the SR. It corresponds to the whole domain outside SR and T-tubule in Figure la). r is the boundary of the domain. The subscript of r coincides with the variable name on no-flux boundaries and with the corresponding channel name where there is a channel. The channels names are as follows. Na: fast sodium channel, NaK: sodium-potassium pump, NCX: sodium-calcium exchanger, Ca: L-type channel, rel: Release current from SR. The diffusion constants are k, and k , . There is a large range of reported values in the literature. Following Langer and Peskoff we use k, =1 10'6cmZ/s and k, =2 * 10-6cm2/s.The diffusion equation for calcium is more complicated and ZTWN are than for sodium due to the presence of calcium buffers. ICMDN absorption currents to the buffers calmodulin and troponin, respectively. The Winslow-model is an ODE system with rate functions F. It depends on the state variables w and also on the mean concentration of calcium in the fuzzy space (M,(c)) and in the myoplasma (Mm,,o(c))and the mean concentration of sodium in the myoplasma (M,,, (s)) . We consider a small part of the cell near the cell membrane, where a Ttubule is closely apposed to SR. In order to reduce the computational load we assumed rotationally symmetry, thus reducing the problem to two dimensions. A
248
radius of 57nm is used for the T-tubule, and the gap between SR and the Ttubule is 12nm. In the symmetry plane there are three release units from SR, placed 37nm apart. An L-type channel is placed on the line of symmetry in the fuzzy space. The fast Na channel, the NCX and the Na-K pump are all also placed on the membrane of the T-tubule, in the fuzzy space, at 33nm, 55nm and 1O O n m from the line of symmetry, respectively.
3. Results Figure la) shows the distribution of calcium 5ms after depolarization. It is markedly elevated in the fuzzy space due to calcium induced calcium release from SR. Figure lb) shows the current through the NCX. Positive values indicate Cainflux, ie. the NCX operates in reverse mode. The dashed line termed 'Low' is the current obtained using the above described model and normal resting ion concentrations and conductances. In comparison, the solid line termed 'High' shows the NCX-current when we have significantly increased the fast sodium current. In this case the sodium concentration peaks at about 40mM (not shown), compared to just over 1O M in the 'Low' case. From the solid trace we see that there is now a much stronger reverse mode due to the elevated sodium concentration. The surplus of sodium dissipates quickly into the myoplasma and the concentration is down to the base level after a few milliseconds. Nevertheless, the reverse current has facilitated calcium SR release, as is evident from the fact that the forward mode has started 2ms earlier than in the 'Low' case. The dashed-dotted line termed 'Bulk' is the current given by the standard Winslow model where the NCX, the Na-K pump and the fast Na channel are outside the fuzzy space and only interact with bulk myoplasmic ion concentrations. The consequence is a notable reduction in the forward current because the NCX is not subjected to the high hzzy space Ca-concentrations.
249 [Ca] in mM, t S m s
--- Low - Hlgh
nm
4
ms
b)
Figure 1. a) The calcium concentration right after calcium release from SR. The fuzzy space is between the T-tubule and the SR. b) The current through the NCX.
4. Discussion At the spatial scale we consider, the diffusion acts very rapidly. Large gradients typically dissipates within a few millisecond. Under normal conditions it seems unlikely that the NCX contributes significantly to calcium release. Under pathological conditions with increased intracellular sodium concentration the situation is not so clear. Uncertainties in diffusion properties and channel distribution need to be clarified before anything can be said quantitatively about the role of NCX in connection with contraction. However, when more data become available we believe that the proposed model will be useful to study the role of NCX in calcium release.
References 1 . A Fabiato. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am JPhysioZ., 245, C1-14 (1983). 2. N Leblanc and JR Hume. Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science, 248 372-376 (1990). 3. WJ Lederer, E Niggli, and RW Hadley. Sodium-calcium exchange in excitable cells: fuzzy space. Science, 248,283 (1990). 4. R. L. Winslow, J. Rice, S. Jafri, E. Marban, and B. O'Rourke. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, 11, model studies. Circ Res, 84, 57 1-586 (1 999). 5. GA Langer and A Peskoff. Calcium concentration and movement in the diadic cleft space of the cardiac ventricular cell. Circ Res, 70,1169-1 182 (1996).
ATP-SENSITIVE K' CHANNEL IS NOT INVOLVED IN THE EXTRACELLULAR K' ACCUMULATION IN ISCHEMIC MOUSE HEART TOSHIAKI SATO', TOMOAKI SAITO', TAKASHI MIKI~,SUSUMU SEINO*, HARUAKI NAKAYA' 'Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan, 2Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
Using mice with homozygous knockout of Kir6.2 gene (a pore-forming subunit of cardiac KATPchannel), we investigated the potential contribution of KATp channels to the extracellular K' accumulation and electrophysiological alterations during myocardial ischemia. Coronary-perfused mouse left ventricular muscles (stimulated at 5 Hz) were subjected to no-flow ischemia. Transmembrane potential and extracellular K' concentration ([K'],) were measured by using conventional and K'-selective microelectrodes, respectively. In wild-type (WT) hearts, action potential duration at 90% repolarization (APD90) was significantly decreased by 70.1*5.2% after 10 min of ischemia (W0.05).Such ischemia-induced shortening of APDgOdid not occur in Kir6.2deficient (Kir6.2 KO) hearts. Resting membrane potential in both WT and Kir6.2 KO hearts similarly decreased by 16.8&5.6 mV (P 0. 05) and 15. b1. 7 mV (P 0.05). Deactivation kinetics was not altered appreciably; measured fast and slow time constants did
259
not differ with statistical significance. ZK, stimulation was ATl-receptor related: in the presence of lpM valsartan increase was largely prevented.
3.2. Intracellular Molecules Implicated in IKsModulation Current experimental data showed involvement of the following intracellular components: G-proteins, phosholipase C, protein kinase C. ZKs enlargement was reduced extensively after pretreatment with each of the following inhibitors: 2 mM GDPPS, 100 pM compound 48/80, 10 pM H-7, and 300 pM BIS. Subsequent ZKsincrease by 100 nM sar-AT I1 was 15.85f6.97%, 32.88&9.89%, 16+9.24%, and 9.8M6.40%, respectively (p-RELATEDMOLECULES IN EXPERIMENTAL AUTOIMMUNE MYOCARDITIS RATYUKO WAKISAKA, SHINICHI NIWANO, HIROE NIWANO, J U N K 0 SAITO, TOHRU YOSHIDA, SHOJI HIRASAWA, TOHRU IZUMI The Second Department of Internal Medicine, Kitasato University School of Medicine, Sagamihara, Japan
BACKGROUND: We have reported that experimental autoimmune myocarditis (EAM) rats showed dramatic changes in ventricular action potential, but their mechanisms are unclear. To investigate the mechanism of cardiac remodeling in acute myocarditis and subsequent heart failure, physiological and molecular changes were evaluated. METHODS: On day 14, 21, 35 and 60 after immunization to Lewis rats, electrophysiological parameters and ionic changes were evaluated. Kv’ and L-Ca” channels, ion transporters and BNP expressions in the Ieft ventricle were examined by real-time RT-PCR and Western blot analysis. Results: EAM showed acute myocarditis on day 14, day 21 and chronic DCM-like phase on day 60. HW/BW, LVEDP, and dP/dt were always higher in EAM than control. ERP and nionophasic action potential duration (MAPD) were always longer in EAM, with a peak on day 21 which was parallel to PVC inducibility. mRNA levels of Kv4.2, Kv1.5, KChIP2, frequenin and SERCA, and the protein levels of Kv4.2 and Kv1.5 were reduced especially in acute phase. CONCLUSIONS: EAM showed structural and electrical remodeling in all phase, but most prominent change was documented in acute inflamatory phase. The reduction of Ito-related molecules and the prolongation of MAPD were considered to be a key mechanism of ventricular remodeling in EAM.
282
A NOVEL DELETION MUTATION OF KCNQl THAT CAUSES LONG QT SYNDROME IN A NEAR-DROWNING PATIENT’S FAMILY* HARUYUKI YAMAZAKI, KUNIO OHTA, AKIKO ISHIZAKI, NAMI NAKAMURA, TAKEKATSU SAITO, YO NIIDA, SHOICHI KOIZUMI Department of Pediatrics, Graduate School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641 Japan
Congenital long QT syndrome (LQTS), caused by mutations in ion-channel genes, KCNQl (LQTl), HERG (LQT2), or SCNSA (LQT3), is characterized by a prolonged QT interval on the electrocardiogram, syncope and fatal ventricular arrhythmias. Factors triggering cardiac events differ among the three distinct forms of LQTS. We have investigated mutations of these three genes in a near-drowning patient’s family. The proband is a 9-year-old female. DNA sequencing confirmed the presence of a 3-bp (TCT) deletion within the transmembrane domain S5 in the LQTl gene. This 3-bp deletion results in a novel in-frame deletion of a single amino acid (phenylalanine), delF275. The identical mutation was confirmed in her sister and father, both of whom had no history of syncope. This is consistent with the notion that swimming appears to be a gene-specific (KCNQl) anythmogenic trigger for LQTS. Analysis of LQTS genes will provide useful information for selection of the careers at risk. It is still unknown, however, why the clinical phenotype may vary considerably, even among the careers of the same mutation. We need to accumulate further clinical and laboratory evidences to answer this question.
Introduction
LQTS represents a unique model for the study of genotype-phenotype correlation in hereditary arrhythmogenic disorders, because all of the three genes (KCNQI, HERG, SCNSA) are responsible for LQTS and encode ionchannels involved in the control of repolarization. Although it is demonstrated that cardiac events in LQTS patients occur under specific circumstances varies in a gene-specific manner’), it is still unknown why the clinical phenotype may vary considerably, even among the careers of the same mutation.
* This work was supported by a Grand-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of Japan 283
284
Materials and Methods Identification of Probands with LQTS Two LQTS patients were diagnosed because of near-drowning. They both had prolonged QT interval (QTc; QT interval corrected for heart rate) in repeated ECG registrations obtained in the absence of any drugs known to affect the QT interval. Informed consent or assent was obtained from each individual (andor their guardian) in the study. DNA Sequence Analysis Genomic DNA was isolated from peripheral blood lymphocytes or lymphoblastoid cell lines derived from Epstein-Barr virus-transformed lymphocytes. Genetic fragments of KCNQ1, HERG and SCN5A were amplified by using primer sets described by Syrris et a12).Fragments were amplified on a Perkin and Elmer 9600 PCR thermal cycler or Applied Biosystems 9700 PCR thermal cycler. The sequences of genomic fragments were analyzed by using the dye terminator sequencing procedure and DNA sequencer (model 3 100 genetic analyzer, Applied Biosystems). In the sequence analysis, identical primers were used as in primary PCR analysis. Restriction EnZyme Analysis A DNA encoding exon 6 of the KCNQl was amplified by PCR using a primer pair described by Syrris et al. Restriction enzyme MboII (Takara Bio), which cleaves DNA at TCTTC sequences, was used for confirmation of the delF275 mutation. The PCR product was digested for 2 hours at 37°C and checked on 2.5% agarose gels, One restriction site for MboII is present, which is split into 2 fragments of 171-bp and 68-bp. This site is disrupted in the mutant allele, which remained undigested (236-bp). Subcloning of PCR Fragments To confirm a precise sequence of delF275, subcloning of PCR fragments was performed by using a TOP0 TA cloning kit (Invitrogen Corp, Carlsbad, CA, USA) according to manufacturer’s instruction. Case 1; 9 year-old female On July 14, 1995, she drowned in a swimming pool. She was discovered face down at the bottom of the pool. The submersion time was estimated to be within
285
a minute. She recovered consciousness soon after brief pulmonary resuscitation and was transported to our hospital. An ECG revealed QT prolongation, with a QTc interval of 0.48 sec'12 at rest, and QTc interval of 0.55 sec1I2at 3 minutes of recovery after exercise test. The pattern of broad-based T wave was consistent with the LQTl genotype. DNA sequencing confirmed the presence of a 3-bp (TCT) deletion within the transmembrane domain S5 in the LQTl gene. This 3-bp deletion results in a novel in-frame deletion of a single amino acid (phenylalanine), delF275. The identical mutation was confirmed in her sister and father (Figurel). This deletion was not detected in any of the 80 unrelated normal individuals. More over, alignment of the S5 domain sequences was highly conserved from xenopus to human.
Figure 1. Detection of a KCNQI gene mutation in case 1. a, The DNA sequence of KCNQI codon 272-279 is shown. The patient presents a 3-bp deletion resulting in an in-frame deletion of phenylalanine, delF275. This deletion was not detected in any of the 80 unrelated normal individuals. b, Pedigree of case 1 family with de1275 mutation. Circles indicate female; squares, male. Solid symbols indicate carriers of delF275 mutation. Restriction enzyme MboII, which cleaves DNA at TCTTC sequences, was used for confirmation. The PCR fragment corresponding to exon 6 is 239-bp long. MboII digestion of the normal allele divides it into fragments of 171-bp and 68-bp. The mutant allele abolishes the cut site for the enzyme, resulting in a single fragment of 236-bp. Each lane corresponds to the family member in the pedigree above.
Case 2; 9 year-old female On July 17, 2002, she drowned in a swimming pool. She was discovered floating face down and pulled from the water within a few minutes. She required cardiopulmonary resuscitation briefly. She was transported to our hospital soon after she had recovered consciousness. A resting ECG revealed QT prolongation, QTc of 0.46 set"*. The pattern of normal-appearing T wave was consistent with the LQTl genotype. DNA sequencing confirmed a G to A substitution at position 1772, which
286
changes Arginine 591 in Histidine, R591H. This mutation was previously reported3'. The identical mutation was confirmed in her sister and mother (Figure 2). a
b
QTcO46
A I C GGC W C CGC
QTcO16
QTcO49
CIG AAC L'G.*
Figure 2. Detection of a KCNQl missense mutation in case 2. a, Partial KCNQl sequence of a normal control (above) and proband (bottom). The proband presents a G to A substitution at position 1772, which changes Arginine 591 in Histidine, R591H. b, Pedigree of case 2 families with R591H mutation. Circles indicate female; squares, male. Solid symbols indicate carriers of R591 H mutation confirmed by direct sequence analysis.
Summary We identified two KCNQ 1 mutations in near-drowning patient's families. Among them, a 3-bp (TCT) deletion within the transmembrane domain S5 results in a novel in-frame (delF275) deletion. These cases are consistent with the notion that swimming appears to be a gene-specific (KCNQl) arrhythmogenic trigger for LQTS . Although specific mutation in KCNQ 1 (mutation hotspot) was not associated with lethal cardiac events in this study, analysis of LQTS genes will provide useful information for selection of the careers at risk. We need to accumulate further clinical and laboratory evidences. References 1. Schwartz PJ, Priori SG, Spazzolini C, et al. Circulation. 103 89 (2001). 2. Syrris P, Murray A, Carter ND, Mckenna WM, et al. JMed Genet. 38, 705 (2001). 3. Neuroud N, Tesson F, Denjoy I, et al. Nut Genet. 15, 186 (1997)
PHENOTYPICAL OVERLAPPING OF SICK SINUS AND BRUGADA SYNDROMES IN A FAMILY WITH A NOVEL SCN5A MUTATION FUMIKO YANAGISAWA', YUKEI HIGASHI', HISA SHIMOJIMA', TAKESHI TSUTSUMI', YOUICHI TAKEYAMA', NAOKO ZENDA', TAKERU MAKIYAMA~,MINORU HORIE~ 'Cardiovascular Division Showa University Fujigaoka Hospital, Kanagawa, Japan, 2Department of Cardiology Kyoto University Graduate School of Medicine, Kyoto, Japan, 3Departnient of Cardiovascular and Respirator Medicine, Shiga University of Medical Sience, Japan
Mutation in SCNSA gene, encoding alpha-subunit of cardiac Na channels, causes a disease category called Na channelopathy. It has been noted to produce overlapping phenotypes such as long QT and Brugada syndromes (BS). We experience a case having a novel SCNSA mutation that was supposed to generate the phenotypes of sick sinus (SS) and BS. Sixty-eight-year-old male who had received a permanent pacemaker because of SSS at age 57 was admitted because of unexplained syncope. He has a strong family history of SSS and sudden death. Intravenous pilsicainide infusion (0.5mgkg) unmasked typical ECG features of BS, and programmed RV outflow stimulation could produce ventricular fibrillation repeatedly. Genetic screening for SCN5A identified an abnormal conformer in exon 27 of the patient and his elderly brother. DNA sequencing revealed that both of them contained an insertion of two nucleotides, aa at 4729. Analysis of 110 normal control individuals did not identify the same mutation. This frame-shift mutation may cause non-functional Na channels because of stop codon insertion. To our knowledge this is a fiist case report of overlapping SS and BS caused by a unique SCNSA.
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KCNQl MUTATION CAUSING DOMINANT-NEGATIVE SUPPRESSION DUE TO DEFECTIVE CHANNEL TRAFFICKING UNDERLIES CARDIAC ARREST IN A PATIENT WITH LONG QT SYNDROME YOSHIYASU AIZAWA, LONG-ME1 WU, KAZUO UEDA*, SEIKO KAWANO, YUJI HIRANO, AKINORI KIMURA*, YOSHZFUSA AIZAWA**, MASAYASU HIRAOKA Department of Cardiovascular Disease and *Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. **Division of Cardiology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata-shi, Niigata 951-8510, Japan.
Long-QT syndrome is caused by mutations in 7 defined genes that mostly encode K+ and Na+ channels, and the underlying mechanisms are variable among different gene mutations. A 13-year-old girl with a history of cardiopulmonaryarrest was referred to our institute for genetic analysis. Her QT interval at rest was prolonged (QTc=OS2sec)and it further prolonged with exercise (maximum QTc=0.62sec).PCR-SSCP and DNA sequence analysis identified a novel frameshift mutation Ala178fd105 in KCNQ1, resulting in premature stop codon that eliminates the portions of S3-S6 and C-terminus of the channel. We examined electrophysiological properties using heterologous expression system in COS-7 cells. Whole-cell patch-clamp technique demonstrated that the WT-KCNQI with KCNEl produced normal Ms current, while no current was observed in cells expressing A178fs/105 mutant and KCNEI. Co-expression of WT- and A178fdl05-KCNQl along with KCNEl suppressed the current with a dominantnegative manner. Next we examined the subcellular localization of WT and/or A178fd105 mutant channel using confocal laser microscopy which revealed trafficking deficiency of A178fs/105 mutant. Co-expression of GFP-tagged WT- with A178fs/105KCNQl also induced the intracellular retention of the channel protein. We conclude that the A178fs/105 mutation causing dominant-negative suppression due to trafficking defect is an underlying mechanism for fatal arrhythmias of this patient.
1. Background LQTl is caused by mutations in KCNQl and most frequent cause of congenital LQTS. Functional analysis of mutant KCNQl channels has revealed loss of channel function in most cases. We identified a novel mutation, Ala178fs/105, missing S3-S6 and C-terminal portions of KCNQl channel.We examined electrophysiological properties and sub-cellular localization of Ala178fd105 expressed in COS-7 cells. 288
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2. Methods 2.1. Genetic Screening by PCR-SSCP Followed by DNA Sequencing WT- and mutant-KCNQ1 constructs were generated and were introduced to the pcDNA3.1(+), pEGFP-C1 and pEYFP-C1 vectors. 2.2. Cell Culture and Transient Transfection COS-7 cells were transfected by lipofectamine method.
2.3. Whole-cell Patch-clamp Method Whole-cell patch-clamp method was applied to COS-7 cells transfected with WT- and/or mutant-KCNQ 1 channel. 2.4. Confocal Laser Microscopy
GFP-tagged WT-KCNQ 1, YFP-tagged WT-KCNQ 1, and YFP-tagged mutantKCNQ1 were transfected to COS-7 cells in glass-bottomed well slide (Lab-Tek). Fortyeight hours after the transfection, cells were fixed on slide-glass with 4% parafonnaldehyde and visualized using LSM5 10 confocal laser scanning microscope (version 2.5, Carl Zeiss Co., Ltd.). Argon laser was used to excite the GFP (excitation wavelength=488nm) and YFP (excitation wavelength=514nm).
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3.
Results
B
A
Membrane potential (mv)
C
Membrane potentlal (mv)
D
Figure 1. Current-voltage relationships of expressed currents (A) Current-Voltage relationship measured at the peak current during test depolarization. (B) Current-Voltage relationship measured at the tail current upon repolarization to -50mV following test depolarization. (C) Bar graphs showing current densities obtained from the peak current at +60mV. (D) Bar graphs showing current densities obtained from the tail current upon repolarization to 50mV from +60mV test depolarization.
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Figure 2. Sub-cellular localization of WT- and mutant-KCNQ1 protein in COS-7 cells (A)Green signals indicate GFP-tagged WT-KCNQI channel expressed on the cell surface. (B) Red signals indicate YFP-tagged mutant-KCNQ1 channel mainly expressed in the cytoplasm. (C)Transmittedlight/DIC image of (A). (D)Transmittedlight/DIC image of (B).
4.
Summary
We identified a novel KCNQl mutation, Ala178fsA05, eliminating the S3-S6 and C-terminus portions of the channel in a case of LQT patient. The mutant-KCNQ 1 could not express functional channel. Co-expression of WT- and mutant-KCNQ1 produced much decreased current compared to the WT alone, suggesting a dominant-negative effect. The sub-cellular localization of the GFP- or YFP-tagged channel proteins revealed intracellular retention of the mutant protein, as well as co-expressed WT and the mutant protein.
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5. Conclusion Our findings suggest a novel mechanism for LQTl that the truncated Sl-S2 KCNQl mutant forms hetero-multimer with WT and causes a dominant-negative effect due to trafficking defect.
Acknowledgments The authors are grateful to the patient and her family who contributed to this study. This work was supported by the Grants from the Ministry of Education, Science, Culture, Sports and Technology of Japan, and the Research Grant from the Ministry of Health, Labor and Welfare of Japan.
9 Genetic Basis for Cardiac Arrhythmias
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DNA MICROARRAYS AND ARRHYTHMIAS DENIS G. ESCANDE L 'Institut du Therax INSERM, Nantes, France
Although electrophysiologicalremodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for the complete repertoire of genes encoding ion channel subunits (IonChips) were developed and exploited to investigate remodeling of ion channel transcripts. We first evaluated the effects of hypothyroidism on the adult mouse ventricle. Hypothyroidism decreased heart rate and prolonged QTc duration. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNAS, KCNB1, KCND2 and KCNK2 transcripts, whereas KCNQl and KCNEl expression is increased. Real-time RT-PCR validated these results. Consistent with microarray analysis, western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in Ito,f and IK,slow densities, and increased IKs density in hypothyroid myocytes. In addition to the effects on K+ channel transcripts, transcripts for the pace-maker channel HCN2 were decreased and those encoding the alphalC Ca2+ channel (CaCNAlC) were increased in hypothyroid animals. The expression of Na+, C1- and inwardly rectifying K+ channel subunits, in contrast, were unaffected. We then aimed to investigate whether chronic amiodarone remodels ion channel transcripts in the heart. Mice were treated with oral amiodarone at 30, 90, 180mg/Kg or with the vehicle during 6 weeks. Dosage of amiodarone and DEA in the plasma and in cardiac tissues showed clear dose-dependency. As expected, rT3 increased and T3 decreased in the treated animals. Serial ECG recordings demonstrated prolonged PR and QRS intervals, slight bradycardia and increased QTc duration. Endocardial electrophysiologic study demonstrated prolonged AH and atrioventricular Wenckebach parameters. In amiodarone treated hearts, microarrays identified under-expression of cardiac sodium channel subunits (SCNSA, SCN4A and SCNIB) and connexin 43. KCNQ1, Kv1.5 and Kv4.2 were down-regulated whereas Kvl.4 and TWIK-1 were up-regulated. Calmodulin 1, calsequestrin 2 and iodothyronine deiodinase (Type 11) mRNA were also down-regulated. All these changes reached statistical significance. Real-time RT-PCR experiments confirmed microarrays data and also demonstrated dose-dependence of amiodarone effects. Variation of ion channel transcripts could not be 295
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unequivocally related to the hypothyroid status of the animals. We concluded that chronic amiodarone remodels cardiac ion channels that are involved in cardiac conduction (Na+ channels and connexins) and repolarization (K+ channels). Remodeling is consistent with the reported class I and class I11 effects of the drug and may explain part of its pharmacological profile.
MOLECULAR, GENETIC AND CLINICAL ASPECTS OF ARRHYTHMIA DISORDERS CONNIE R. BEZZINA, ARTHUR A.M. WILDE Experimental & Molecular Cardiology Group, AMC, Amsterdam, the Netherlands
[email protected] Introduction Each heartbeat is initiated by a pulse of electrical excitation that originates in the pacemaker cells of the sinus node and subsequently spreads throughout the heart. This impulse is conducted throughout the heart, largely by means of the rapid entry of Na' ions into the cardiomyocytes. This depolarizes the cardiomyocytes, moving them away from their resting negative intracellular charge. This is followed by a prolonged phase at which the resultant positive membrane potential is maintained and decays only slowly, as a consequence of an extrusion of K' ions through different voltage-sensitive K' channels balanced by the entry of Ca2+through membrane Ca2+channels and the release of Ca2' from intracellular stores which triggers contraction. This corresponds to the plateau phase of the action potential, which lasts for a few hundred milliseconds, ensuring sufficient time for adequate contraction and pumping of blood. A subsequent further increase in outward K+ current brings about repolarization, thereby restoring the membrane potential of the myocyte back to its negative resting potential. (Figure 1) The rapid and selective flux of ions across the cardiomyocyte membrane occurs through ion channels, which consist of protein complexes with a conducting pore. Besides selectivity for the type of ion they conduct, individual ion channels are also characterized by gating properties (regulation of channel opening and closing), kinetics (rate at which channels open or close) and pharmacology. The maintenance of normal cardiac rhythm is dependent on the proper movement of ions mediating the action potential. Thus, abnormalities in ion channel function can have disastrous consequences that manifest themselves as arrhythmias. These disorders of ion channels, commonly referred to as 'cardiac channelopathies' have been brought into focus over the last decade as mutations in genes coding for specific channels were shown to underlie specific forms of heritable arrhythmogenic disorders occurring in the structurally normal heart, 297
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namely the Long QT syndrome (LQTS), Brugada syndrome (BS), catecholaminergic polymorphic ventricular tachycardia (CPVT), Andersen syndrome, and most recently the Short QT interval syndrome (SQTS). These disorders fall under the category of monogenic disorders, that is, disorders that follow a clear Mendelian pattern of inheritance and are classified as autosomal dominant, autosomal recessive or X-linked.
Figure 1. Ion channels underlie cardiac excitability. a, The key ion channels (and an electrogenic transporter)
in cardiac cells. K+channels mediate K’ efflux from the cell; Na’ channels and Ca” channels mediate Na’ and Ca” influx, respectively. The Na’/Ca2’ exchanger is electrogenic, as it transports three Na+ ions for each Ca” ion across the surface membrane. b, Ionic currents and genes underlying the cardiac action potential. Top, depolarizing currents as hnctions of time, and their corresponding genes; centre, a ventricular action potential; bottom, repolarizing currents and their corresponding genes. Reproduced with permission from Marban E, Nature lx,. : KW.0 : u 2002;415:213-218. -m
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Disorders of Repolarization
Repolarization is a delicate process depending on an intricate balance between inward currents (Na+ or Ca”) and outward currents (K’). Prolongation or shortening of the action potential by shifts in the balance between these inward and outward currents accentuates the inherent instability of cardiac repolarization, leading to abnormal, inhomogeneous repolarization which predisposes the heart to potentially lethal ventricular arrhythmias. Long QT Interval Syndromes
The Long QT interval syndrome (1) is a repolarization disorder identified by prolongation of the QT interval on the ECG. It usually manifests in children and teenagers and leads to syncopal episodes and malignant torsades de pointes tachyarrhythmias in a high proportion of untreated patients. Two variants, an autosomal dominant (Romano-Ward) type and an autosomal recessive (Jervell
299
and Lange-Nielsen syndrome, also associated with deafness) type have been clinically recognized. In this disorder, a decrease in net outward current during repolarization leads to prolongation of the repolarization process and consequently to a prolonged action potential duration, manifest as QT interval prolongation on the ECG (Figure 2). The Na" channel gene (SCN.54 may be affected whereby mutations in this gene lead to an inappropriate increased inward Na' current (gain of function mutations). Alternatively, one of the four genes encoding the Ik current (KCNQl, KCNE1, KCNH2, KCNE2) may be affected leading to loss of K' channel function, thereby decreasing outward K" current (loss of function mutations). Another gene linked to LQTS however does not code for an ion channel protein but encodes a membrane adapter protein, Ankyrin-B, the role of which is to ensure the proper insertion of ion channels into the appropriate domains of cell membranes (2). This finding highlighted the fact that cardiac ion channels do not function in isolation, but form part of complex macromolecular assemblies (consisting of pore-forming subunits, modulatory subunits, cytoskeletal and extracellular matrix elements, and associated signalling complexes) and that mutation in elements of this complex other than ion channel proteins per se, could also lead to arrhythmias. QT interval/ action potential duration
:+basal +j
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I
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QT interval / action potential duration /+basal , .
+- short + ,I
.,
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t inward depolarizing Na" current 1 ournard repolarizing K+ current toutward repolarizing K" current Figure 2. Temporal relationship between the ventricular action potential (bottom) and the ECG (top) in Long QT interval (left) and Short QT interval (right) syndromes. A shift in the balance between inward ma+) and outward (K') currents during the action potential leads to QT interval prolongation or shortening.
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Short QT Interval Syndromes The Short QT interval syndrome ( 3 ) is a recently described clinical entity that presents with a high rate of sudden death and exceptionally short QT-intervals. Contrary to the LQTS, repolarization is hastened by an enhanced outward current during repolarization. Mutation in the KCNH2 gene was first linked to the families in whom the disorder was initially described (4). Recently, our group demonstrated that like most arrhythmia syndromes the disease is genetically heterogeneous (i.e. can be caused by mutation in different genes) and can also be caused by mutation in the KCNQJ gene (5). In the case of both these K+ channel genes, short QT interval-causing mutations lead to a gain of K’ channel function (Figure 2). Brugada Syndrome Another disorder for which the key abnormality has been proposed to lie in the repolarization process is the Brugada syndrome (6,7). This is an increasingly recognized disorder for which the hallmark ECG features of ST-segment elevation and an apparent right bundle branch block in the precordial leads may not be constantly present. Although the average age of patients experiencing Brugada events is 40 years, sudden death can strike at any age, even in very young children. The SCNSA gene, which accounts for -30% of cases, is the only gene linked thus far to the disorder (8). The functional effects of Brugada syndrome-causing SCNSA mutations are opposite to those found in LQTS. In Brugada syndrome, the Na+ channels show loss-of-function, for instance by enhanced inactivation or by a decrease in the number of functional channels on the cardiomyocyte membrane. One mechanism proposed for the disorder holds that during phase 1 of the subepicardial action potential, this loss of Na’ channel function, allows the transient outward K+ current, It,, to repolarize the myocyte beyond the voltage range in which membrane Ca” channels are activated. Failure of these Ca” channels to activate, results in the loss of the action potential plateau and an abbreviation of the action potential duration in these cells. Because I,, is not present in subendocardial cells, the action potential plateau is preserved here. The resulting disparity in action potential duration across the thickness of the ventricular wall drives electrotonic current, ST-segment elevation and tacharrhythmias based on phase 2 reentry (6) (Figure 3). Another mechanism proposed for the Brugada syndrome ascribes the ECG features and arrhythmias to conduction delay in the right ventricular wall in the region of the outflow tract. The activation delay between this region and the
301
remainder of the heart causes ST-segment elevations and arrhythmias. The observation by our group that the magnitude of ST-segment elevation correlates with the extent of right ventricular conduction delay recently provided novel evidence that the ECG features of Brugada syndrome are indeed, at least in part, caused by right ventricular activation delay (9).
A
B
Figure 3. Mechanism of ECG abnormality (A) and arrhythmogenesis (B) in Brugada syndrome. Reproduced with permission from Charles Antzelevitch,
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) CPVT is an arrhythmogenic disorder caused by adrenergic induced arrhythmias in the form of bi-directional and polymorphic ventricular tachycardia, associated with syncope and a high incidence of sudden cardiac death (10). Although patients asymptomatic until their mid-thirties have been described, CPVT mainly affects children. In the majority of cases, CPVT displays an autosomal dominant mode of inheritance and is caused by mutation in the ryanodine receptor channel (RYRZ) (1 l), an intracellular Ca2' release channel on the sarcoplasmic reticulum that releases Ca2+ in response to Ca2+entry through the membrane L-type Ca2+ channels during phase 2 of the action potential, mediating excitation-contraction. coupling. A recessive form of CPVT caused by homozygous mutation in the calsequestrin (CASQZ) gene (12,13), which encodes a protein that serves as the major Ca2+reservoir within the lumen of the sarcoplasmic reticulum, has also been recognised. Symptoms are apparently more severe in CASQ2-related CPVT, including an earlier age of onset. Furthermore, diagnosis is more
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difficult due to the absence of a positive family history, due to the recessive nature of the disease. The pathophysiological mechanism proposed for both gene defects is an aberrant sarcoplasmic reticulum Ca2' release. Spontaneous Ca" release from the sarcoplasmic reticulum during diastole would cause a transient inward current, resulting in delayed after depolarisations (DADS; abnormal depolarisations in cardiomyocytes that occur after repolarization of a cardiac action potential). If this inward current is sufficient to cause a DAD with amplitude greater than the threshold potential, depolarisation will occur, and an arrhythmia can be triggered. Recent experimental data on CPVT-causing RYR2 and CASQ2 mutations - with mutation in the former leading to an increased channel activity and in the latter leading to a decreased Ca2' storing and releasing capabilities support such a mechanism (14,15). Andersen Syndrome
Another disorder leading to ventricular tachycardia is Andersen syndrome. Cardiac manifestations include mild prolongation of the QT interval, significant U-waves, ventricular ectopy, bi-directional ventricular tachycardia, and more rarely syncope, recurrent polymorphic ventricular tachycardia, and cardiac arrest. Features of the disorder that have also made it of high interest to scientists is that besides cardiac arrhythmias, patients also suffer skeletal muscle periodic paralysis and in addition exhibit developmental problems such as cleft palate, low set ears, short stature, and developmental features in the limbs (clinodactyly, syndactyly, brachydactyl) (16). Loss-of-function mutations in the KCNJ2 gene have been linked to the disorder (17). This gene encodes the inward rectifier K+ channel K i d . 1 that conducts outward K' current during the terminal repolarization (thereby contributing to the repolarization process) and diastolic phases (keeping the membrane potential close to the resting membrane potential) of the action potential. While this readily explains the co-occurrence of abnormalities in heart and muscle excitability (Kir2.1 contributes to membrane excitability in both tissues), it is still unknown how mutation in this channel also leads to the developmental abnormalities. Of note is the marked variability in phenotypic expression of the disease between affected individuals, with different individuals displaying different combinations of the disease triad (heart, muscle, dysmorphology).
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Clinical Implications of Identification of Genetic Defects in These Disorders The identification of the genetic substrate underlying these inherited arrhythmia syndromes has in the last decade yielded remarkable insights into the molecular basis of cardiac electrophysiology. However, the recognition of fundamental defects in channelopathies did not only broaden our insight into the hnction of the various ion channels but has also provided insight on arrhythmia mechanisms, provided opportunities for gene-specific therapy, and importantly the availability of a genetic diagnostic test has added an important diagnostic tool for these disorders. The classification of the Long QT syndromes into the various genetic classes has provided insight into the specific triggers for arrhythmias in this disorder. LQTl patients, with mutation in the KCNQl gene seem to be characterized by exercise and stress-related events that are adrenergically stimulated. Especially, triggers such as diving and swimming are almost exclusive to LQTl patients (18,19). Patients with LQT3, with mutation in the SCNSA gene, are at particularly high risk at rest or during sleep because their QT interval is prolonged excessively at slow heart rates (20). This clear distinction between LQT 1 and LQT3 with respect to exercise-related triggers does not hold for LQT2 patients, affected by mutation in KCNH2, who tend to suffer events both at rest and during exercise. However, events provoked by auditory stimuli, such as an alarm clock or a ringing telephone, occur almost exclusively in patients with LQT2 (21). The age of onset seems also to depend on the gene involved. At age 10 almost 40% of LQTl children has become symptomatic, whereas only 10% of LQT2 and hardly any LQT3 patients has suffered from symptoms. These gene-specific features has tailored therapeutic management for genotyped LQT patients. While P-blockers are efficient in LQTl and LQT2 patients (22), LQT3 patients might be treated using Na' channel blockers (23). Pacing is also important in this latter patient group to avoid tachyarrhythmic events induced by bradycardia (24). For both these treatment strategies however, failures have been described, such that an ICD as a backup therapy seems as yet pertinent. In the presence of certain disease genes, certain modifications in lifestyle to avoid specific triggers (see above) become possible. And also, of clinical importance, the age at which treatment should be installed is genespecific with the earliest start of treatment in LQTl patients (well before 5 years of age). Furthermore, the identification of the molecular basis for these diseases
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opens new avenues of research into new forms of therapy for the future (eg. gene therapy). Importantly, the identification of the mutation within an affected family allows diagnosis in other family members independently from the electrocardiographic features and the arrhythmic manifestations. As for other Mendelian disorders, mutations leading to primary arrhythmias are most often associated with reduced penetrance (not all mutation carriers are clinically affected by the disorder), variable expression (variable severity), and pleiotropy (diverse phenotypic effects). Thus, extensive phenotypic variability is observed among family members carrying an identical mutation in a single ion channel gene, with far-reaching implications for diagnosis and therapy. Within the same kindred, some individuals carrying the mutation may exhibit overt ECG abnormalities or suffer fatal arrhythmias, while others carrying the same primary genetic mutation might not have the ECG changes or may never develop any arrhythmias during their entire lifespan. Most data pointing at this fact comes from patients with the Long QT syndrome and Brugada syndrome. In these disorders, screening of family members of a genotyped proband uncovers an unexpectedly large number of carriers among relatives that were considered unaffected based on clinical evaluation. In a study by Priori et al. (25), on family members of 5 probands with LQTS (subtypes 1 or 2, mutation in KCNQl and KCNH2 respectively), a total of 15 family members, who on clinical grounds were classified as unaffected, were found to be mutation carriers, implying a penetrance of 25%. In another study by the same group (26), 20 out of 44 electrocardiographically and clinically unaffected family members of 4 probands with Brugada syndrome were found to be carrying the (SCNSA) mutation. The identification of such silent mutation carriers is important. Data from LQTS patients shows that silent mutation carriers are susceptible to develop arrhythmias, even if their QT interval falls in the normal range (27). Moreover, these individuals have a 50% chance of transmitting the genetic defect to their offspring, who in turn might be symptomatic at an earlier age. Pleiotropy is notably evident for SCNSA mutations, leading to what nowadays are commonly referred to as 'overlap syndromes' of cardiac Na' channelopathy. Such pleiotropy was first reported by our group (28) in a multigenerational family with the SCNSA 1795insD mutation, and was subsequently observed by others (29,30). This family presented with an extremely malignant phenotype, with a high incidence of nocturnal sudden cardiac death. Clinical features included bradyarrhythmias, intrinsic sinus node dysfunction, bradycardia-dependent QT-prolongation, and ST-segment
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elevation in V 1-V3 (28,24). Furthermore, generalized conduction abnormalities and bradycardia were also observed among mutation carriers. Overlap of these clinical features occurred in some individuals, while in others they occurred separately. This diversity of clinical phenotypes is at least partly attributable to the biophysical properties of the multidysknctional mutant Na' channel since the SCNSA 1795insD mutation responsible for the disease in this family is associated with biophysical alterations that can contribute to all the observed features; (i) a sustained inward Na' current leading to QT interval prolongation, (ii) an enhanced slow inactivation leading to Brugada syndrome features and conduction disease, and (iii) a combination of sustained inward Na' current and negative shift in voltage dependence of inactivation leading to bradycardia (3 1,32). However, this pleiotropy also provides strong evidence that genetic modifiers play an important role in determining the ultimate phenotype and severity of disease in cardiac channelopathies. Thus, the ultimate clinical presentation, not only depends on the mutation involved but also on the genetic background on which it occurs. This means that the ultimate clinical presentation should be considered as a complex phenotype, which is not only the product of the ion channel mutation alone, but also of genetic variation in genes encoding other (protein) players in that particular biological pathway. This genetic variation - in the form of single nucleotide polymorphisms (SNPs) occurring in a number of different genes - is expected to contribute to the final clinical presentation. Such genetic modifiers of cardiac electrical phenotypes are largely uncharacterised, and very few examples exist in the literature. In an interesting study by Viswanathan et al. (33), the effect of an SCNSA mutation identified in a patient with second degree AV block, appears to be modified by an SNP in the same gene. This SNP actually occurred in cis with the mutation on the same allele. Comparison of the (in vitro) biophysical properties of the channel harbouring the mutation (T512I) with that of the channel harbouring both the mutation and the polymorphism (H558R) revealed that the polymorphism actually attenuates the deleterious effects of the mutation. We have recently reported on another such potential genetic modifier in a family with the rare arrhythmia familial atrial standstill (34). This modifier consisted of two closely linked SNPs in the promoter region of the gene for the gap junction protein, connexin 40 (Cx40), potentially giving rise to reduced expression of Cx40 levels. Various members of the family were found to be carriers of an SCN5A mutation (D1275N), the primary genetic defect . However, only those carriers who were also homozygous for the rare Cx40 promoter SNPs
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actually displayed the disease. Because Cx40 is only expressed in atria an "atrial phenotype" results. An important parallel to be drawn here is with that genetic variation constituting susceptibility to common 'acquired' arrhythmias such as in ischemic heart disease, hypertrophy, heart failure or during the use of (QT intervalprolonging) medications, with the difference however, that in these latter disorders polymorphisms are expected to influence the susceptibility to arrhythmias in the absence of a disease causing mutation. Evidence for such an effect by a polymorphism has been provided by Splawski et al. (35). These investigators identified a variant, an SNP leading to an amino acid substitution (S 1 102Y) found primarily in individuals of African descent (and absent in Caucasians), which was far more prevalent in individuals with drug-induced QT prolongation than in the general population, suggesting that it increases risk. The identification of such modifiers of primary electrical disease and susceptibility genes for acquired arrhythmias is however still in its infancy and actually constitutes a major challenging next step in our understanding of the genetics of arrhythmias. The number of genes expected to contribute to susceptibility is unknown but likely to be multiple, necessitating the construction of large databases of patients phenotyped in a very standardized fashion to ensure adequate power for association studies. Genes encoding cardiac ion channels form very plausible candidates in which to search for such variation, but then the multitude of other players within the diverse pathways leading to arrhythmia, such as cytoskeleton proteins, structural proteins (such as those linked to sudden death in familial hypertrophic cardiomyopathy) and proteins involved in processes such as fibrosis, inflammation, cell-to-cell communication and electrical and structural remodeling, are also of interest. With recent advances in genotyping technology, comprehensive screening of multiple genes in different pathways is now feasible. This together with the availability of a high-resolution genome-wide map of SNPs and the technology to perform large-scale genome-wide screens in a high-throughput system (unbiased approach), expected to become available in the short term, shall in the coming decade undoubtedly increase considerably our insight into the genetics of common acquired arrhythmias. This will provide novel tools for risk stratification and open new opportunities for prevention of lethal arrhythmias in the common pathologies.
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Acknowledgements Work from the authork laboratory is funded by Netherlands Heart Foundation grants: 2000B059,2003B195 and 2003T302.
References 1. Kass RS, Moss AJ. Long QT syndrome: novel insights into the mechanisms of cardiac arrhythmias. J Clin Invest 2003;112:810-5. 2. Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, Bennett V. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 2003;421:634-9. 3. Gussak I, Brugada P, Brugada J, et al. Idiopathic short QT interval: a new clinical syndrome? Cardiology. 2000; 94: 99-1 02. 4. Brugada R, Hong K, Dumaine R et al. Sudden death associated with shortQT syndrome linked to mutations in HERG. Circulation 2004;109:r151r156. 5 . Bellocq C, van Ginneken ACG, Bezzina CR, Alders M, Escande D, Mannens MAM, Bar6 I, Wilde AAM. Mutation in the KCNQl gene leading to the Short QT Interval Syndrome. Circulation, in press. 6. Antzelevitch C, Brugada P, Brugada J, Brugada R, Shimizu W, Gussak I, Perez Riera AR. Brugada syndrome: a decade of progress. Circ Res 2002;91:1114-8. 7. Alings M, Wilde A. "Brugada" syndrome: clinical data and suggested pathophysiological mechanism. Circulation 1999;99:666-73. 8. Bezzina CR, Rook M.B., Wilde A.A. Cardiac sodium channel and inherited arrhythmia syndromes. Cardiovasc Res 2001;49:257-27 1 9. Tukkie R, Sogaard P, Vleugels J, de Groot IK, Wilde AA, Tan HL. Delay in right ventricular activation contributes to Brugada syndrome. Circulation 2004;109:1272-7. 10. Coumel P, Fidelle J, Lucet V. Catecholamine-induced severe ventricular anhyhmias with Adams-Stokes syndrome in children: report of four cases. Br Heart J 1978;40:23-37. 11. Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation 200 1;103:485-90.
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12. Lahat H, Eldar M, Levy-Nissenbaum E, Bahan T, Friedman E, Khoury A, Lorber A, Kastner DL, Goldman B, Pras E. Autosomal recessive catecholamine- or exercise-induced polymorphic ventricular tachycardia: clinical features and assignment of the disease gene to chromosome lp132 1. Circulation 2001;103:2822-7. 13. Postma AV, Denjoy I, Hoomtje TM, Lupoglazoff JM, Da Costa A, Sebillon P, Mannens MM, Wilde AA, Guicheney P. Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res 2002;91 :e2 1-6. 14. Wehrens XH, Marks AR. Altered function and regulation of cardiac ryanodine receptors in cardiac disease. Trends Biochem Sci 2003;28:67 1-8. 15. Viatchenko-Karpinski S, Terentyev D, Gyorke I, Terentyeva R, Volpe P, Priori SG, Napolitano C, Nori A, Williams SC, Gyorke S. Abnormal calcium signaling and sudden cardiac death associated with mutation of calsequestrin. Circ Res 2004;94:47 1-7. 16. Andersen ED, Krasilnikoff PA, Overvad H. Intermittent muscular weakness, extrasystoles, and multiple developmental anomalies. A new syndrome? Acta Paediatr Scand 1971;60:559-64. 17. Plaster NM, Tawil R, Tristani-Firouzi M, Canun S, Bendahhou S, Tsunoda A, Donaldson MR, Iannaccone ST, Brunt E, Barohn R, Clark J, Deymeer F, George AL Jr, Fish FA, Hahn A, Nitu A, Ozdemir C, Serdaroglu P, Subramony SH, Wolfe G, Fu YH, Ptacek LJ. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. CelZ2001;105:511-9. 18. Schwartz PJ, Priori SG, Spazzolini C, Moss AJ, Vincent GM, Napolitano C, Denjoy I, Guicheney P, Breithardt G, Keating MT, Towbin JA, Beggs AH, Brink P, Wilde AA, Toivonen L, Zareba W, Robinson JL, Timothy KW, Corfield V, Wattanasirichaigoon D, Corbett C, Haverkamp W, Schulze-Bahr E, Lehmann MH, Schwartz K, Coumel P, Bloise R. Genotype-phenotype correlation in the long-QT syndrome: gene-specific triggers for life-threatening arrhythmias. Circulation 200 1;103:89-95. 19. Moss AJ, Robinson JL, Gessman L, Gillespie R, Zareba W, Schwartz PJ, Vincent GM, Benhorin J, Heilbron EL, Towbin JA, Priori SG, Napolitano C, Zhang L, Medina A, Andrews ML, Timothy K. Comparison of clinical and genetic variables of cardiac events associated with loud noise versus swimming among subjects with the long QT syndrome. Am J Cardiol 1999;84:876-9.
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20. Schwartz P, Locati E, Napolitano C, Priori S. The Long QT syndrome. In: Zipes D, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. 2"d ed. Philadelpha, WB Saunders; 1995:778-811. 21. Wilde AA, Jongbloed RJ, Doevendans PA, Duren DR, Hauer RN, van Langen IM, van Tintelen JP, Smeets HJ, Meyer H, Geelen JL. Auditory stimuli as a trigger for arrhythmic events differentiate HERG-related (LQTS2) patients from KVLQTl-related patients (LQTS1). J Am Coll Cardiol 1999;33:321-32. 22. Moss AJ, Zareba W, Hall WJ, Schwartz PJ, Crampton RS, Benhorin J, Vincent GM, Locati EH, Priori SG, Napolitano C, Medina A, Zhang L, Robinson JL, Timothy K, Towbin JA, Andrews ML. Effectiveness and limitations of beta-blocker therapy in congenital long-QT syndrome. Circulation 2000;101:616-23. 23. Benhorin J, Taub R, Goldmit M, Kcrem B, Kass RS, Windman I, Medina A. Effects of flecainide in patients with new SCN5A mutation: mutationspecific therapy for long-QT syndrome? Circulation 2000; 101:1698-706. 24. van den Berg MP, Wilde AAM; Viersma JW, Brouwer J, Haaksma J, van der Hout AH, Stolte-Dijkstra I, Bezzina CR, van Langen IM, BeaufortKrol GCM, Cornel JH, Crijns H.J.G.M. Possible Bradycardic Mode of Death and Successful Pacemaker Treatment in a Large Family with Features of Long QT Syndrome Type 3 and Brugada Syndrome. J Cardiovasc Electrophysiol2001;12:630-6. 25. Prion SG, Napolitano C, Schwartz PJ. Low penetrance in the Long QT syndrome. Circulation. 1999;99:529-533. 26. Prion SG, Napolitano C, Gasparini M, Pappone C, Della Bella P, Brignole M, Giordano U, Giovannini T, Menozzi C, Bloise R, Crotti L, Terreni L, Schwartz PJ. Clinical and Genetic Heterogeneity of Right Bundle Branch Block and ST-Segment Elevation Syndrome : A Prospective Evaluation of 52 Families. Circulation 2000;102:2509-15. 27. Priori SG, Schwartz PJ, Napolitano C, Bloise R, Ronchetti E, Grillo M, Vicentini A, Spazzolini C, Nastoli J, Bottelli G, Folli R, Cappelletti D. Risk stratification in the long-QT syndrome. N Engl J Med.2003;348: 1866-74. 28. Bezzina C, Veldkamp MW, van Den Berg MP, Postma AV, Rook MB, Viersma JW, van Langen IM, Tan-Sindhunata G, Bink-Boelkens MT, van Der Hout AH, Mannens MM, Wilde AA A Single Na(+) Channel Mutation Causing Both Long-QT and Brugada Syndromes. Circ Res 1999;85:1206-13
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29. Kyndt F, Probst V, Potet F, Demolombe S, Chevallier JC, Bar0 I, Moisan JP, Boisseau P, Schott JJ, Escande D, Le Marec H. Novel SCNSA mutation leading either to isolated cardiac conduction defect or Brugada syndrome in a large French family. Circulation. 200 1;104:3081-6. 30. Grant AO, Carboni MP, Neplioueva V, Starmer CF, Memmi M, Napolitano C, Priori S. Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation. JClin Invest. 2002;110:1201-9. 31. Veldkamp MW, Viswanathan PC, Bezzina CR, Baartscheer A, Wilde AAM, Balser JR. Two distinct congenital arrhythmias evoked by a multidysfunctional Na' channel. Circ Res 2000;86:e9 1-e97. 32. Veldkamp MW, Wilders R, Baartscheer A, Zegers JG, Bezzina CR, Wilde AAM. Contribution of Sodium Channel Mutations to Bradycardia and Sinus Node Dysfunction in LQT3 Families. Circ Res 2003;92:976-83. 33. Viswanathan PC, Benson DW, Balser JR. A common SCNSA polymorphism modulates the biophysical effects of an SCNSA mutation. J Clin Invest 2003;111:341-6. 34. Groenewegen WA, Firouzi M, Bezzina CR, Vliex S, van Langen IM, Sandkuijl L, Smits JP,Hulsbeek M, Rook MB, Jongsma HJ, Wilde AA. A cardiac sodium channel mutation cosegregates with a rare connexin40 genotype in familial atrial standstill. Circ Res 2003;92: 14-22. 35. Splawski I, Timothy KW, Tateyama M, Clancy CE, Malhotra A, Beggs AH, Cappuccio FP, Sagnella GA, Kass RS, Keating MT. Variant of SCNSA sodium channel implicated in risk of cardiac arrhythmia. Science 2002;297; 1333-1336.
ALLELIC VARIANTS IN CARDIAC ION CHANNEL GENES IN PATIENTS WITH DRUG-INDUCED LONG QT SYNDROME HIDEAKI KANKI Cardio-Pulmonary Division, Department of Medicine, Keio University School of Medicine, Japan
Marked QT interval prolongation and the morphologically distinctive ventricular tachycardia Torsades de Pointes (TdP) develop in 1-8%of patients receiving QT prolonging antiarrhythmic drugs, such as quinidine, sotalol, ibutilide, and dofetilide. Drug-associated QT prolongation and TdP are also well recognized during therapy with non-cardiovascular agents. DNA variants appearing to predispose to drug-associated long QT syndrome (aLQT) have been reported to occur in congenital LQTS disease genes. However, the incidence of such genetic risk factors has not been systematically evaluated in a large set of patients with aLQT. We used PCR-SSCP to screen the entire coding regions of the genes encoding the pore-forming channel proteins KCNQ1, HERG, and SCN5A in aLQT and controls. We evaluated the frequency of three common nonsynonymous coding region polymorphism, 1355SR (SCNSA), Q 1027R (SCNSA) and K897T (HERG). Missense mutations (absent in controls) were identified in 5/92 patients: 3 in SCNSA, 1 in KCNQl (R583C), and 1 in HERG (R784W). We screened extended kindred of the aLQT patient with R583C in KCNQ1. 9/18 relatives were mutation carriers, but the longest QTc was 435 msec and C583 penetrance is - 0% in this kindred. Before the mutation had been identified, the proband's two children (ages 41 and 46) participated in a study of ibutilide and QTc rose markedly only in the affected son. Although the SCN5A variants did not alter INain vitro, arguing that they played no role in the aLQT phenotype, the KCNQl and HERG mutations reduced K' currents, consistent with the idea that they augment risk for aLQT.
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GENETIC BASIS OF CARDIAC NA CHANNELOPATHIES NAOMASA MAKITA* Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Sapporo 060-8638, Japan
MINORU HOME * Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Shiga, Japan 520-2192, Japan Mutations of cardiac Na channel gene SCNSA are responsible for lethal arrhythmic syndromes such as type 3 congenital long QT syndrome and Brugada syndrome. Recent genetic studies have revealed that SCNSA mutations are also responsible for conduction disturbance including atrial standstill, and some form of acquired LQTS, constituting a spectrum of disease entity termed cardiac Na channelopathies.
1. Cardiac Na Channelopathies Voltage-gated Na channels are responsible for the rapid membrane depolarization that characterizes the initial phase of the action potential. The primary Na channel subunit expressed in the heart is Navl.5 encoded by a gene SCNSA. More than 100 SCNSA mutations have been reported, which are known to evoke multiple life-threatening arrhythrmc syndromes including long QT syndrome (LQTS), Brugada syndrome, cardiac conduction disturbance (CCD), sudden infant death syndrome (SIDS), constituting a disease entity termed cardiac Na channelopathies.
2. Congenital LQTS Congenital LQTS is an inherited disorder characterized by prolonged QT interval and a predisposition for syncope or torsades de pointes (TdP). There are 7 distinct responsible genes have been reported, and one subtype of this disorder,
* This work is supported in part by the research grants 15090711 and 14370225 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the research grants for cardiovascular diseases (13A-1) (16B-3) from the Ministry of Health, Labour and Welfare, Japan, and Japan Research Foundation for Clinical Pharmacology. 312
313 A D356N
NH2
R1623Q
/
AKPQ
L’I825PfJ COOH
Figure 1 . Membrane topology of Navl.5 and the location of the SCN5A mutations. Boxes represent the transmembrane segments Sl-S6. AKPQ is located at the cytoplasmic loop connecting domain (D) 3 and 4, a putative inactivation gate.
LQT3, is caused by mutations in SCNSA. The first LQT3 mutation AKPQ was an in-frame deletion mutation located at the putative inactivation gate (Fig 1). Despite the location of the mutation, AKPQ expressed in Xenopus oocytes showed current decay similar to wild type (WT). But a small persistent Na current was evident only in AKPQ (Fig 2). Single channel recordings showed that WT channels open briefly once or very few times during depolarization, whereas AKPQ showed similar frequency of initial openings, but exhibited intermittent reopening and bursting behavior during the late phase of a depolarizing stimulus. The mean open time was nearly identical between two channels. Computer simulation successfully simulated the late current without slow current decay by setting two inactivation modes; one normal inactivation and the other impaired inactivation such that the channel fluctuating between two modes. Therefore, small late Na current due to modal gating is sufficient to prolong the action potential duration without slowing current decay’, and it was speculated that severer Na channel dysfunction as is observed in some skeletal muscle Na channelopathiesmay be incompatible with life. In 1998, we found a novel de novo SCNSA mutation R1623Q in a Japanese baby girl who showed 2: 1 AV block and TdP since she was born2. The mutation was located at the positive residue located at the putative activation gate at the domain 4 (D4). Macroscopic current of the R1623Q was significantly slower than that WT or AKPQ, indicating severer inactivation defect. R1623Q channel showed not only slow current decay but also persistent current, a hallmark of LQT3 mutations. Furthermore, inactivation properties are less voltage-sensitive, suggesting that the coupling between inactivation and
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activation is impaired. Single channel recording showed bursting behavior as was observed in AKPQ, and significantly prolonged mean open time (2.8 times longer than WT). These results suggest that defective coupling between activation-inactivationresults in delaying open to inactivation transition which in turn prolongs the mean open time and delay the current decay, demonstrating a novel biophysical mechanism underlying QT prolongation. This baby with LQT3 was successfully treated with mexiletine and a pacemaker, but she would otherwise have been a case with sudden infant death syndrome (SIDS).
m
pq
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Figure 2. Whole-cell current recordings (upper) and the inside-out patch clamp recordings (lower) of the WT and AKPQ channels expressed in Xenopus oocytes.
3. Brugada Syndrome Brugada syndrome is one form of idiopathic ventricular fibrillation without structural heart diseases, characterized by unique ECG findings of ST elevation in leads V1 through V3, and aborted cardiac death. Brugada syndrome is most prevalent in Southeast Asia, and has male preponderance. Molecular basis of Brugada syndrome is decrease of cardiac Na current due to mutations in SCNSA
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attributable to several mechanisms including 1) non-functional channels, 2) gating modulation such as enhanced inactivation and 3) defect of channel protein trafficking to the plasma membrane. Loss-of-function of Na channel during the initial phase of the action potential leaves transient outward K current (Ito) unopposed in the phase 1, and results in the large transmural voltage gradient predominantly observed in the right ventricle, which in turn leads to ST elevation in the right precordial leads. Despite the fact that the mutations have thus far only been identified in SCNSA, considerable clinical heterogeneity has been recognized; ST elevation is occasionally observed in leads other than right precordial leads. Moreover, SCNSA mutations have been linked to multiple arrhythrmc syndromes, but the vast majority of the clinical phenotypes of these diseases are ventricular arrhythmias. We experienced two cases of Brugada syndrome with distinct clinical manifestations despite having non-functional SCNSA mutations.
Case 1. A 62 year-old man with syncope showed coved-type ST elevation in V1 and V2. Ventricular fibrillation (VF) was induced at programmed electrical stimulation (PES), and he received an ICD. A novel missense mutation D356N was found at the S5-S6 loop of the D1, a part of the putative pore structure (Fig 1). Mutant channel expressed in tsA-201 cells showed no current, probably the mutant residue is within a critical structure required for Na ion permeation. Functional consequence of this mutation is estimated to be 50% reduction of Na current in myocardial cells. Case 2. A 37 year-old female recurrent syncope showed coved-type ST segment elevation in V2 and J waves are in the inferior leads3. A few hours after admission, show experienced two episodes of spontaneous VF, which was preceded by a long pause due to atrial standstill. Atrial standstill persisted after defibrillation. Provocation test using procainamide markedly widened the QRS complexes, augmented the J-wave elevation in the inferior leads, and induced idioventricular rhythm with atrial standstill. However ST elevation was not induced by procainamide. PES failed to induce VF. We found a missense mutation R367H at the D1 pore region (Fig 1). Since both mutations of D356N and R367H are nonfunctional, their functional consequence appears to equivalent: 50% reduction of cardiac Na current, which in turn results in ST elevation. Nevertheless, probands of D356N and R367H showed distinct electrophysiological manifestations. D356N resulted in spontaneous and druginduced ST elevation in V1.3, whereas R367H exhibited J wave elevation in the inferior leads, and spontaneous ST elevation in V2 that was not provoked by
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drug. Furthermore, this case showed atrial standstill that was provoked by procainamide. It is intriguing that the clinical or electrophysiological features may vary between affected individuals carrying electrophysiologically equivalent SCNSA mutations, suggesting that existence of some unknown modifier genes that may be a critical determinant of the clinical manifestations of Na channelopathies. These may include subclinical mutations or polymorphisms of Na channel genes or other genes such as connexins. 4. Acquired Long QT Syndrome
Congenital long QT syndrome is a relatively rare inherited arrhythmia. In contrast, acquired LQTS is a more prevalent disorder and is often caused by a number of drugs that preferentially block rapid component of delayed rectifier K current (I&). However, drug-induced LQTS does not occur in every patient treated with these drugs, but most likely occurs in a subset of susceptible individuals. Recent studies indicate that some drug-induced torsade de pointes are associated with polymorphisms or subclinical mutations in K channel genes responsible for congenital LQTS. It is therefore suggested that a subset of individuals with normal or borderline QT interval may carry subclinical mutations in LQTS disease genes and are susceptible to life-threatening arrhythmias upon drug exposure. Here we present the first SCNSA mutation demonstrated in an acquired drug-induced LQTS4. A 70-year-old woman was admitted to the hospital because of recurrent syncope. QT interval of her baseline ECG was normal. Soon after the prescription of cisapride, a gastrointestinal medicine with side effects of IJSr blocking activities, she showed severe QT prolongation, bradycardia, and TdP. QT interval was normalized after terminating cisapride and the cardiac pacing. Genetic screening revealed a novel missese mutation L1825P located within the C-terminal region of Na channel. The proband’s grandfather had sudden death of unknown cause, but the rest of her family members are asymptomatic. Despite the normal baseline QT interval of the patient, the recombinant L1825P channel showed profound biophysical abnormalities. Heterologously expressed L1825P channel expressed in mammalian cells showed a robust persistent late current. Current decay was significantly slower than WT. These electrophysiological properties are characteristic for the mutation R16234, an infantile congenital LQT3 mutation I showed in the previous slide. In addition to the gain-offunction, L1825P channel showed distinct loss-of-function properties characteristic for Brugada syndrome. Steady-state inactivation curve was shifted in a negative direction, and activation curve was shifted in a positive direction.
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Moreover, closed-state inactivation was also enhanced. These severe loss-offunction properties are compatible with the decreased peak Na current of the mutant channels. In order to determine whether cisapride directly affected the functions of the mutant channel and manifested QT prolongation, 1 pM cisapride was applied to the bath solution. However, cisapride failed to change the channel kinetics or the amplitude of the persistent current. These results indicate that mutant channel L1825P exhlbited overlapping biophysical properties of both LQT3 and Brugada syndrome. It is possible that the gain-of-function abnormalities might have been offset by the concomitant loss-of-function properties, which masked the functional abnormality and QT prolongation at base line. Furthermore, although the functional abnormalities of L1825P were clinically unmasked by cisapride exposure, QT prolongation was probably mediated through a mechanism other than direct effects of cisapride on mutant channels. Inactivationdefect L1825P
Block by Cisapride
\
ICa
Tolerated by Repolarization reserve
1
Tolerated by Repolarization reserve
Compensation exhausted
I QT prolongation I
Figure 3. Hypothesis for the cisapnde-induced QT prolongation in the proband with L1825P mutation X indicates dysfunction of cardiac ion channels provoked by mutations or drugs.
Figure 3 illustrates our hypothesis for the molecular basis of cisaprideinduced LQTS. Outward currents of myocardial cells consist of multiple distinct K currents such as Ito,IK,, or IK,. Minor insults on one of these K currents could be potentially compensated by other K currents, and maintain normal repolarization (left). This mechanism is called “repolarization reserve”. To extrapolate this hypothesis, it is assumed that repolarization reserve allows the
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dysfunction of the L1825P mutation to be tolerated (middle). However, administration of cisapride blocked IK, and exhausted the repolarization reserve, leading to manifest the QT prolongation and TdP (right). In conclusion, mutations of SCNSA underlie not only LQT3 and Brugada syndrome, but also conduction disturbance including atrial standstill, and some forms of acquired LQTS, constituting a spectrum of disease entity termed cardiac Na channelopathies.
Acknowledgments The authors thank Naohumi Takehara, Yuichiro Kawamura, and Takeru Makiyama for providing clinical data and the genomic samples of the patients, and helpful discussion.
References 1. Bennett, P.B., Yazawa, K., Makita, N.George, A.L., Jr. Molecular mechanism for an inherited cardiac arrhythrma. Nature 376,683-5 (1995). 2. Makita, N., Shirai, N., Nagashima, M., Matsuoka, R., Yamada, Y., Tohse, N. Kitabatake, A. A de novo missense mutation of human cardiac Na' channel exhibiting novel molecular mechanisms of long QT syndrome. FEBS Lett 423,5-9 (1998). 3. Takehara, N., Makita, N., Kawabe, J., Sato, N., Kawamura, Y., Kitabatake, A. Kikuchi, K. A cardiac sodium channel mutation identified in Brugada syndrome associated with atrial standstill. J Intern Med 255, 137-42 (2004). 4. Makita, N., Horie, M., Nakamura, T., Ai, T., Sasaki, K., Yokoi, H., Sakurai, M., Sakuma, I., Otani, H., Sawa, H. Kitabatake, A. Drug-induced long-QT syndrome associated with a subclinical SCN5A mutation. Circulation 106, 1269-1274 (2002).
10 Clinical Arrhythmias
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BEPRIDIL REGULARIZES VENTRICULAR RESPONSE DURING ATRIAL FIBRILLATION IN ACCORDANCE WITH PROLONGATION OF FIBRILLATION CYCLE LENGTH TAKAYUKI TSUNEDA, AKIRA FUJIKI, MASATAKA SUGAO, MASAO SAKABE, KLJNIHIRO NISHIDA, KOICHI MIZUMAKI, HIROSHI INOUE
The Second Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194, Japan
Bepridil is effective not only in converting long-lasting atrial fibrillation (AF) but also in improving symptoms despite persistence of AF. The aim of this study was to investigate modification of ventricular response during AF by bepridil. Methods: For pharmacological conversion 35 consecutive patients (mean age of 60i10 years) with AF lasting more than one month received bepridil (200mdday) for 4 weeks. Before and 7 days after bepridil administration, fast-Fourier transform analysis of fibrillation waves was performed and simultaneously the maximum, the minimum, the mean and coefficient of variation (CV) of RR intervals within 90 sec period were determined. Results: Bepridil converted AF to sinus rhythm in 24 patients. In converters bepridil prolonged both the mean (from 0.77*0.14 to 0.84*0.14 sec, p7 days.) Ablation Before the procedure, computerized tomography scan of heart with 3demensional reconstruction was performed in all patients to define the anatomy of PVs. All patients provided written, informed consent. PV isolation was performed during sinus rhythm, coronary sinus pacing or ongoing AF rhahm by delivering radiofrequency (RF) energy at its ostial site that had the earliest bipolar potential or polarity reversal of PV potential guided by duodecapolar catheter with a distal ring configuration (Lasso catheter, Biosense Webster). RF energy was delivered at the PV ostium with target temperature of 50 degree and maximum power output of 30-35watts for 60-90 seconds, with the endpoint of bidirectional conduction block between PV and left atrium. Analysis of Clinical Outcomes and Follow-up All patients were given a periodical follow-up in an outpatient clinic and recurrence of AF was evaluated by symptoms, ECG recordings and 24-hour ambulatory monitoring (1, 3, 6, 9, 12 months after the procedure). The mean duration of follow-up was 12.1k5.2 months. No patients were lost to follow-up. For the purpose of categorizing the clinical outcomes after PV isolation, success was defined as no recurrence of AF without antiarrhythmic drug (AAD). Effective was defined as no recurrence of AF under AAD. Unsuccess was defined as recurrence of AF under AAD.When sustained AF recurred after the early unstable period, AADs, which was previously non-effective, were restarted either temporarily or continuously. If patients were regarded as unsuccess at 3-6months of follow-up, a repeat ablation procedure was recommended. Continuous variables are expressed as mean -+ SD. Continuous variables were compared by Student’s t test. Categorical variables were compared by x2 analysis. P 2mm at its peak in one or more of the precordial leads (Vl-V3) spontaneously or during intravenous administration of class I anti-arrhythmic agents. 2) Polymorphic ventricular tachycardia (VT) or VF induced by ventricular extrastimuli. 3) No structural heart disease.
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Patient Classification Twelve patients having a history of survived sudden cardiac death, syncope and pre-syncope with blackout before recruitment were classified as symptomatic Brugada syndrome patients (10 men, 2 women, mean 49+/-17 years; group A) and the another 7 patients were classified as asymptomatic (7 men, mean 49+/-9 years; group B). Then, ICD was provided in all subjects after the sufficient informed consent and followed up prospectively (mean 29.8 months, range 4 to 91 months).
3. Results
Protocol of VTNF Induction VTNF could be induced by a couple ventricular extra stimuli in one patient in group A (8%) and 3 in group B (43%). A triple ventricular extra stimulus was needed to induce VTNF in remained 11 patients in group A (92%) and 4 in group B (57%). However, these frequencies were not different between both groups. We didn’t use the coupling interval less than 180 ms shortening.
Requirement of Pharmacological Modification for VTNF Induction If VTNF was not induced on basal state, we administrated drugs, such as edrophonium or isoproterenol intravenously. Intravenous administration of edrophonium (1Omg) was needed in 3 patients in group A (25%) and 1 in group B patient (14%). Isoproterenol administration was needed in only 1 patients in group A patients (8%)for induced VTNF.
Drug effect of Class I Anti-arrhythmic Agents We used class I anti-arrhythmia drugs (mainly flecainide) for 9 examples from group A and 5 from group B. ST-segment elevation was observed 6 patients in group A (66%),and 3 patients in group B (60%).
Other Characteristics Complication of paroxysmal atrial fibrillations (PAF) was observed 8 patients in group A (67%) and 3 patients in group B (43%). HV interval prolongation during sinus rhythm were identified 2 patients in group A (17%), none of group B. Family history of unexpected death (55"C had the approximately 30% damaged area (Figure 2).
Discussion Generally, irreversible electrophysiologic change in myocytes had been shown to occur at temperatures >50"C (3). Moreover, Kok et a1 (4). stated that the ablative target temperature for canine pulmonary vein isolation should be limited to 55°C because the critical temperature for heat-induced contraction of pulmonary vein is likely to be between 60°C and 65°C. Thus, it would be justified that 10 pigs had been classified into the two group on the basis of the condition with the initial temperature either at 1 had a greater specificity (100%) and overall accuracy (91%) than either criteria. Conclusion: The standard 12-lead ECG on admission can accurately distinguish Transient left ventricular apical ballooning from anterior AM1.
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SEPTAL Q WAVES IN V6 LEAD DISAPPEAR DURING NARROW QRS SUPRAVENTRICULAR TACHYCARDIA: A NEW ECG OBSERVATION MAKOTO NODA’, FUMIO SUZUKI’, KATSUHIKO MOTOKAWA3, MITSUAKI ISOBE3 ’The Department of Cardiology, Haibara General Hospital, Shizuoka, Japan, 2Department of Cardiology, Fukujuji Hospital, Kiyose, Japan, ’Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Background: Q waves in left precordial leads in normal hearts are generated by left-to-right activation of the ventricular septum. We present that Q waves in V6 disappear during narrow QRS supraventricular tachycardia (SVT), compared with the QRS in sinus rhythm (SR). Methods: Eighteen SVT patients showing Q waves in V6 during SR were enrolled. Q-wave depth in V6 was measured during SR versus SVT.Atrial extrastimulus (SlS2)testing was performed in 5 pts to see whether the Q-wave depth in V6 could be changed in response to premature (S2) stimulation. Results and Conclusions: During SR, the magnitudes of Q waves in V6 were 12t0.6111111. During SVT (cycle length, 338*38), those were decreased to zero. With atrial extrastimulus testing, Q-wave completely disappeared at V1V2 intervals of approximately 400 msec. The onset of R wave gradually shifted to earlier timing relatively to septal Q activation, suggesting, “Q-wave masking” by the R wave.
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CAUSES OF EXERCISE-INDUCEDST-SEGMENT ELEVATION IN OLD ANTERIOR MYOCARDIAL INFARCTION SEIICHI TANIAI', YASUSHI KOIDE*, SATORU YUSU', KONOMI SAKATA', MASAYUKI YOTSUKURA', TAKANORI IKEDA', HIDEAKI YOSHINO' 'The Second Department of Internal Medicine, Kyorin University School of Medicine, Tokyo, Japan, 'Kugayanza Hospital Cardiology, Japan
Objectives The purpose of this study was to distinguish the causes of exerciseinduced ST-segment elevation in infarct-related leads, such as exercise-induced wall motion abnormality or transient residual myocardial ischemia (RI), using ST-Heart rate(ST-HR) loop. Methods Fifty-Eight patients having healed singlevessel Q-wave anterior wall myocardial infarction with exercise-induced STsegment elevation of 1 mm or more in lead V2. were enrolled in this study. They experienced The ST-HR loop in lead V2 was obtained and compared with diagnostic information for transient RI provided by dobutamine stress echocardiography. Results Among the 58 patients, 26 (45%)exhibited a counter clockwise rotation (CCWR) of the ST-HR loop. The incidence of RI in patients with a CCWR was significantly higher than those with a clockwise rotation (CWR) of the ST-HR loop (60% vs 6%, pl in lead I1 and R/S ratio : _ I
-~. ...
;;.,
.......
4 -"1/.~
........ , ....
. .-
. .
.
. ~ . .... ... ......
....
, -7
I
(
.~ ..
. . . . .
.
i
~
.
I
- . ~ :!- ~ : ~ .
.....
~~
.
,. .
. ~
......
.. . . . ..
.
...... . , . . . . . .
Figure 1. 12-leads ECG recorded before and after right coronary artery injection of Ach
"
465
Figure 2. Right coronav angiogram after the injection of Ach showed complete occlusion (left panel). After the injection of ISDN, there was no significant organic stenosis (right panel).
4. Discussion Experimental studies suggested that depression or loss of the action potential dome in right ventricular epicardium created a transmural voltage gradient, resulting the ST segment elevation of the right precordial leads in the Brugada syndrome. The dome of the action potential is diminished by the increase of outward currents or the decrease of inward currents. In canine myocardium, the differential response of epicardium and endocardium to Ach was observed due to the presence of transient outward current-mediated spike and dome morphology in the epicardial action potential. These findings suggest that decrease of Ca inward currents by vagal stimulation and increase of transient outward currents in right ventricular epicardium with Ach result augmentation of the ST segment elevation of the right precordial leads. It is considered that the Brugada syndrome and vasospastic angina are independent disorders. However, there might be genetic relationship because both diseases are prevalent in Asian people.
5.
Conclusion
1. Augmentation of the ST segment elevation in the right precordial leads was induced in 6 (37.5%) of 16 patients with the Brugada syndrome by the right coronary injection of Ach. Decrease of Ca inward currents by vagal stimulation and increase of transient outward currents in right ventricular epicardium with Ach result augmentation of the ST segment elevation of the right precordial leads. 2. C o r o n w artery spasm was induced in 8 (50%) of 16 patients with the Brugada syndrome. Coronary artery spasm provocation test should be recommended to assess the clinical episodes because vasospastic angina also causes malignant arrhythmias and sudden cardiac death.
INFLUENCE OF ACUTE VAGAL ACTIVITY IN THE PATIENTS WITH BRUGADA SYNDROME NORIYOSHI YAMAWAKE', MITSUHIRO NISHIZAKI', TOHRU OGAWA', SHINJI SUGAWARA', HIROYUKI FUJII', TAKASHI ASHIKAGA', MASATAKA ARITA', HARUMIZU SAKURADA', MITSUAKI ISOBE3, MASAYASW HIRAOKA4 'The Department of Cardiology, Yokohania Minami Kyousai Hospital, Kanagawa, Japan, 'The Departrnent of Cardiology, Tokyo Metropolital Hiroo Hospital, Tokyo, Japan, 'The Department of Cardiology, Tokyo Medical arid Dental University, Tokyo, Japan, 4The Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Background: It is not known whether ST segment elevation (STE) is accentuated by acute vagal stimulation in the Brugada syndrome (BS). Furthermore, some patients with BS are combined with other diseases associated with vagal activity. Methods: To assess influence of acute vagal activity on STE in BS, we examined STE in lead V1-3 on ECG during administration of Na-channel blocker, valsalva maneuvers and intracoronary administration of acetylcholine, and assessed combination of neurally mediated syncope (NMS) in 6 patients with BS. Results: STE augmentation and morphologic changes from saddleback to coved type STE were observed after Na-channel blocker in 6 and 5 patients, respectively. Moreover, STE augmentation and morphologic changes of STE were caused by valsalva maneuvers in 4 and 3 patients, respectively. Acetylcholine study was performed in 3 patients, in whom vasospasm was induced in one patient, and STE augmentation without presence of vasospasm was recorded in another patient. Two of the 6 patients'had episodes of syncope, whch were produced by NMS. Conclusion: The patients with BS had frequently STE changes during acute vagal stimulation and combination of vasospastic angina or NMS, whlch suggested that acute or latent autonomic imbalance may be associated with the pathogenesis of BS.
466
BRUGADA SYNDROME SHOWED CONSISTENT J WAVE AND ST SEGMENT ELEVATION IN 12-LEAD ELECTROCARDIOGRAM: TIME COURSE OF VARIATION ON J WAVE TAKA0 NAMIKI Chiba Prefecture Hospital of Togane, Chiba and the 2"dDepartment of Internal Medicine, Saitama Medical UniversiQ, Saitama, Japan KAZUO MATSUMOTO The 2"dDepartment of Internal Medicine, Saitama Medical Universiw, Saitama, Japan
Recently variant type of Brugada syndrome, of which electrocardiograms showed J wave and ST segment elevation not in the right precordial leads, but in the inferior leads has been reported. Despite the absent of structural heart disease, a 25-year-old male had the attack of ventricular fibrillation (VF) at the time showing prominent J wave with ST segment elevation in all 12-leads except aVR lead that were not associated with hypothermia, serum electrolyte disturbance, or myocardial ischemia. He showed natural VF attack and VF was induced by EPS. After implantable cardioverter defibrillator (ICD) was implanted, the number of VF attack was disappeared and at same time J wave in extremity-eads also diminished. Moreover during no episode of VF all J wave in 12-leads were completely disappeared, but typical ST segment elevation in V1.3 leads of Brugada syndrome was observed and low grade of ST segment elevation in the other leads except aVR was remained. In this case the prevalence of J wave in 12-leads might indicate the warning marker of VF attack.
Background Brugada syndrome, first described as a new clinical entity by Pedro and Josep Brugada in 1992, has attracted great interest because of its association with high risk for sudden death. This syndrome is characterized by marked ST-segment elevation in the right precordial ECG leads without any of ischemia, long QT syndrome, electrolyte abnormalities, or structural heart disease Although this syndrome is observed worldwide, it is more common in Asia countries. It is leading cause of death among young men in the northeastern region of Thailand, second only to automobile accidents4'. Recently variant type of Brugada syndrome, of which electrocardiograms showed J wave and ST segment elevation not in the right precordial leads, but in the inferior leads has been reported. We encountered a patient with Brugada syndrome showed prominent J wave and ST segment elevation in all 12-leads except except aVR. However, the 467
468
mechanisms responsible for not only ST-segment elevation but J wave, and the genesis of ventricular tachycardia / ventricular fibrillation (VTNF) in this syndrome remain unknown. Case Report
A 25-year-old man was admitted to our hospital with chest pain and loss of consciousness. At physical examination, the patient seemed in good health. Breath sound was normal and bilaterally equal. The heart rate was regular with a normal S, and Sz. No murmur and no extrasound was found. The abdominal examination was unremarkable. His neurological examinations including brain Computed Tomography was normal. There was no family history of sudden death. The electrocardiograph (ECG) showed incomplete RBBB and ST elevation of coved type in leads of V1 and V2 without QTc prolongation. Moreover Prominent J wave was observed in all 12-leads except aVR (Figure 1) Chest radiography was normal, as well as laboratory tests including a normal blood count and normal serum electro1ytes.A cardiac echocardiogram was performed, which was normal, including the right ventricle. Exercise testing was normal. Electrophysiologic study showed no abnormality and no VT was induced by Isoproterenol. However flecainide induced elevation of ST segment in V1-3leads, but no significant change of J wave. Coronary angiography was normal and no coronary spasm was present by acetylcholine. Moreover the patient experienced natural VF, occurring in early morning hours during sleep . After having informed consent with the patient, an implantable cardioverter defibrillator (ICD) was implanted. Two-year after implantation of ICD, small J wave in extremity leads and reduction of its amplitude in all precordial leads were observed (Figure 5A). Two and half yeas after implantation of ICD, all J wave disappeared (Figure 5B). Three-years after implantation only one episode of cardioversion for VF was recorded in ICD. The 12-lead ECG at this ICD clinic almost 0.2-0.3 mV amplitude of J wave with ST segment elevation in V3-6 leads were observed again (Figure 5C). Conclusion
The unique ECG appearance of Brugada syndrome is caused by failure of the dome of the action potential to develop. It occurs when the outward currents overwhelm the inward currents at the end of phase 1 of the action potential. The influence of the Ito on J wave in patients with Brugada syndrome were reported by some papers. We reported the case of a patient with Brugada syndrome who showed the variation on J wave in according with VF episode. Prominent J
469
waves and ST segment elevation may serve as an important diagnostic sign to detect high-risk individuals with a history of unexplained syncope. The influence of the mechanisms of arrhythmogenesis in Brugada syndrome by J wave warrants further investigation.
References 1. Brugada P,Brugada J. J A m Coll Cardiol. 1992; 20 :1391-1396 2. Miyazaki T, Mitamura H, Miyoshi S, Soejima K, Aizawa Y, Ogawa J Am Coll Curdiologv.1996; 27 :1061- 1070 3. Kasanuki H, Ohnishi S, Ohtsuka M, Matsuda N, Nirei T, Isogai R, Shoda M, Toyoshima Y, Hosoda S, Circulation. 1997 ;95 :2277-2285 4. Nademanee K. Am J Cardiology. 1997; 79:lO-11 5. Brugada J, Brugada R, Brugada P. Circulation. 1998; 97 : 457-460 6. Brugada J, Brugada P. J Cardiovascular Electrophisiol. 1997; 8 : 325-33 1
figure: f
OLP
INAPPROPRIATELY SHORTER QT INTERVAL AT SLOWER HEART RATE CAN DIFFERENTIATE PATIENTS WITH IDIOPATHIC VENTRICULAR FIBLLATION FROM ASYMPTOMATIC BRUGADA SYNDROME MASATAKA SUGAO, AKIRA FUJIKI, TAKAWKI TUNEDA, KUMIHIRO NISHIDA, MASAO SAKABE, KOUICHI MIZUMAKI, HIROSHI INOUE Second Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, Toyama Prefecture 930-0194, Japan
The electrocardiographicparameters predicting occurrence of ventricular fibrillation (VF) episode in asymptomatic subjects with Brugada-like ECG is still unknown. The aim of this study was to clarify the characteristics of QT-RR relation in idiopathic VF in comparison with asymptomatic subjects having Brugada-like ECG. Methods: The study group consisted of 9 males (age 47 k 1Oyo) with IVF (6 Brugada type and 3 non-Brugada type) who had had recent episodes of VF and 9 asymptomatic healthy males with Brugada-like ECG (age 49 +- 15yo). The relation between QT and RR interval was analyzed from 24-hour Holter ECG using automatic measurement system. From QT-RR linear regression lines, both QT intervals extrapolated to RR of 0.6 sec (QT(0.6)) and 1.5 sec (QT(1.5)) were determined. Results: The slope of QT-RR regression line was lower in IVF than in asymptomatic subjects with Brugada-like ECG (0.09 k0.02 vs. 0.13 +0.03, pO. 11s were excluded. The 24-hour ECG signals were digitized and processed off-line using a personal computer equipped with software for automated measurement of QT intervals. The software determines the QTpeakinterval as the time between the QRS onset and the peak of positive T-wave. The QTpeakmeasurement was done only when the T-wave amplitude was >0.1 mV and negative T-wave was excluded. The QTendinterval was defined as the time interval between the QRS onset and the point at which the isoelectric line intersected a tangential line drawn at the maximal downslope of the positive T-wave. In complex T-waves, the second derivative was also used to detect discontinuities after the peak, and the software excluded U-waves. Results We analyzed 108 patients (age, 66 f 12, mean f SD) with worsening heart failure. Patients were more likely to be male (68%) and 60% of the patients had non-ischemic cause of HF. After HF treatment NYHA class and left ventricular ejection fraction were improved significantly. Brain natriuretic peptide was reduced but no significant changes in the serum sodium and potassium values at admission and discharge from hospital were observed. Total ventricular extrasystoles were decreased from 1152 beatslday to 254 beatdday. Isolated ventricular ectopies also decreased before discharge. RR intervals exhibited a significant circadian rhythm in controls. RR intervals were longest from 0:OO to 4:OO am, falling sharply between 5:OO to 8:OO am. The circadian rhythm of the RR interval was blunted in patients with HF at admission, but significant circadian rhythm restored at discharge from hospital. The mean RR interval of control was 890 f 75 ms, and the corresponding value
619
of heart failure at admission was 722 f 19 ms and at discharge was 919 f 50 ms. Circadian variation of the QT interval showed similar change as RR intervals. However, QTc interval showed blunted circadian variation in these 3 groups. QTc interval in patients with heart failure at admission was longest (459 f 5 ms) compared to control (434 f 4 ms) and HF at discharge (448 f 3 ms). QT/RR slope in control and heart failure at discharge exhibited significant circadian rhythm. The 24-hour mean slope at admission was steeper than control and heart failure at admission. There was no significant difference in QT/RR slope between control and heart failure at discharge. Discussion
In the present study, patients with worsening HF had frequent ventricular extrasystoles and blunted circadian variation of the RR, QT and QT/RR slope. Improvement of HF was associated with the normalization of circadian variation of the RR, QT and QT/RR slope and decrease in the ventricular extrasystoles. HF treatment normalized the circadian variation of the RR, QT, and QT/RR slope probably through the reduction of triggers of ventricular arrhythmias (neurohumoral factor, inflammatory cytokines etc.). However, arrhythmogenic substrate in HF patients (e.g. QT prolongation) remained. Blunted circadian variations of RR and QT intervals have been shown to characterize the sudden cardiac death in post-myocardial infarction patienk2 Also, higher QT/RR slopes associates with higher mortality in patients with ischemic heart disease3,but not in idiopathic ventricular fibrillation patients! Further study is required to clarify the prognostic relevance of the circadian variation of QT interval in patients with HF. References
1. Tomaselli GF, Beuckelmann DJ, Calkins HG, Berger RD, Kessler PD, Lawrence JH, Kass D, Feldman AM, Marban E. Sudden cardiac death in heart failure. The role of abnormal repolarization. Circulation. 1994;90:2534-9. 2. Yi G, Guo XH, Reardon M, Gallagher MM, Hnatkova K, Camm AJ, Malik M. Circadian variation of the QT interval in patients with sudden cardiac death after myocardial infarction. Am J Cardiol. 1998;81:950-6. 3. Extramiana F, Neyroud N, Huikuri HV, Koistinen MJ, Coumel P, MaisonBlanche P. QT interval and arrhythmic risk assessment after myocardial infarction. Am J Cardiol. 1999;83:266-9.
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4. Tavernier R, Jordaens L, Haerynck F, Derycke E, Clement DL. Changes in the QT interval and its adaptation to rate, assessed with continuous electrocardiographic recordings in patients with ventricular fibrillation, as compared to normal individuals without arrhythmias. Eur Heart J. 1997;18:994-999.
THE RESPONSE OF T-WAVE PARAMETER DURING EXERCISE TESTING IN PAEDIATRIC PATIENTS WITH QT PROLONGATION AND BIFID T-WAVE TORU A K A I K E
Cardiovascular Center, Yokohama City University, Yokohama, Kanagawa, Japan MAR1 IWAMOTO
Cardiovascular Center, Yokohama Ciry University, Yokohama, Kanagawa, Japan
We determined ECG parameter during exercise in 1 lpatients with bifid T-wave and QT prolongation and 9 healthy children. In this study, the heart rate adaptation of QT interval during exercise was good in patients with bifid T-wave and QT prolongation. Not the T1 but the T2 component is responsible for the shoaening of QT interval during exercise.
Background Long QT syndrome (LQTS) is a congenital disease with prolonged QT interval and characteristic T-wave form. Especially, most of patients with LQT2 show type of bifid T-wave patterns. However, there were few reports about studying the change of bifid T-wave patterns during exercise. The objective of study is to evaluate the characteristics of T-wave form and repolarization parameter during exercise testing in patients with bifid T-wave and QT prolongation.
Methods
Patient Population There are 43 patients under 20 years old with LQTS or QT prolongation, who had been followed up in the Pediatric division of Yokohama City University. The study population consisted of two groups: Group 1; Patients with obvious bifid T-wave (n=11). Group 2; Healthy children (n=9). We defined patients with QT prolongation as that with QTc interval over 480rns and asymptomatic and not identified gene mutation. ECG of the obvious bifid T-wave shows bifid T-wave in at least 7 leads (Figure 1). 621
622
v1
v2
v3
v4
v5
Figure 1. A Case of 12 leads ECG in Obvious Bifid T-wave Group
ECG Measurements
12 leads ECG showing the average of 15-sec period were recorded with Marquette Case 16 under conditions of 50 d s e c and 20mdmV. Exercise stress testing was performed according to the standard Bruce protocol. Reporalization parameter was measured with Digitizer. The QT was measured as the time interval between QRS onset and the point at which the isoelectric line intersected a tangential line drawn at the maximal downslope of the positive T-wave. V5 lead was used for measurement because it is unipolar lead that reflects the potential from the free wall of the left ventricle. The QTlpeak was defined as the time interval between QRS onset and the point at the peak of the former T-wave. The QT2peak was defined as the time interval between QRS onset and the point at the peak of the latter T-wave. Tpe was then obtained by calculating as QT minus QT2p. Reporalization parameter was corrected to heart rate according to Bazett‘s formula: corrected QT (QTc; QTmR’”) and corrected QTp (QTpc; QTp/RR’”) and corrected Tpe (Tpec; Tpe/RR’/*). During exercise tests, the QT ad QTp and Tpe were measured at 6 to 12 sampling points and plotted against the corresponding the RR interval. The QT/RR and QTp/RR and Tpe/RR were calculated in each exercise test by fitting raw data to simple linear regression analysis.
623
Statistical Analysis
*
Data are presented as mean SD. Mann-Whitney’s U test was used to compare each parameter between 2 groups. A probability value < 0.05 was considered significant. Results Comparison of Clinical Characteristics
In the obvious bifid T-wave group, 5 patients had experienced emotionalrelated syncope, and 4 patients were identified HERG mutation. The age and male; female ratio did not significantly differ between the two group (Table 1). Table 1. Comparison of Clinical Characteristics
N Age, yrs
Group 1 Obvious bifid 11 13.1f4.4
Syncope
Emotional 5
RR, ms QTc, ms QTlpc, ms QT2pc, ms Tpec, ms
Group 1 Obvious bifid 783k110 521 *69 311k32 434 f 46 1 0 2 k 17
Group 2 Control 9 13.0f4.3
Group 2 Control 7 6 9 2 143 415f19
NS P 0.05), compared to VT patients.
VT
(%)
90.8%8.6" 30.3%7.443.4*26.0"
932i12.9' 32.7%12.3: 35.4% 19.9
96.3% 7.8 36.3%7.5 22.1 % 9.6
68.2 69.8 77.7
452%35.9" 62.6i45.4" 65.9%37.4105.5i62.5"
40.8%40.9" 47.6%31.7" 60.4k39.4" 91.8%58.0"
23.7h15.5 25.1i12.3 40.7%23.8 55.7*262
66.0 79.3 70.2 75.4
*: p= C a , y , K ( X , , X ) + b (3) X dESY
Typical kernel functions are the Gaussian kernel K ( X , X ' ) = exp(- IIX - X'IIZ >, o2
(4)
and others. In this research. The Gaussian kernel is used as a kernel function.
3.
Proposed Method
This recognizing method by SVM is proposed in this research. This method recognizes whether the detected place is correct as R wave by R wave detection. By this recognition, if non-R wave position is detected, it can exclude these incorrect detections. In short, it can reduce incorrect detections. This method can be applied to ECG analyzing systems which perform R wave detection, and with significant results. Also, there are often a plural number of leads in the Holter ECG, generally two. In this research, the number of incorrect detections is reduced by the use of two channels. Here we use logical addition. And by doing so, if a detected place was recognized as an R wave in either of the two
711
channels, the place is left. And if a detected place wasn't recognized as an R wave in either of the two channels, the place is excluded. An example of ch. 1 Learned S V M I
output I
Length of a waveform An example of ch. 2 Learned SVM by examples of ch. 2
1:Correctdetection class Ohcorrect detction class
Figure 1. Proposed method: First, detected places are recognized by SVM at waves of each channel. Next, those output go through OR gate.
4.
Experiment
ECG waveform data were used as learning data and estimating data for SVM in this research. The data made use of clinical Holter ECG waveform data. The number of the data is one hundred, and the length of the data is about 24hr. The data's sampling frequency is 125Hz. The part of leads are CM5 as CH1 and NASA as CH2. SVM has the ability to recognize two classes. So, the data needs to be categorized into two classes. One of the classes is correct detection class. And, the other class is incorrect detection class. Correct detection class is a group of ECG waveforms which were detected by both clinical technologists and an automatic analysis system. Incorrect detection class is a group of ECG waveforms which were only detected by automatic analysis system. This experiment was performed learning by changing the number of examples in one class N, the dimension of examples d, the positive constant C and parameter of the Gaussian kernel c 2 ,for the SVM of two channels. The conditions for each parameters are as follows:
N : 100,200 d : 15, 19, ... ,71, 75 c : 1,10,100,1000,10000 o2 : 0.01,0,1, 1, 10, 100 N pieces of examples were extracted from each class at random. The example dimensions are the converted values between 0.12 second and 0.6 second. 0.12 second is the normal width of the QRS complex in the ECG. 0.6 second is the
712
normal width of the R-R interval in ECG. Learning and estimating were performed 5 times with all combinations of all conditions. Estimated examples were all examples belonging to the classes. For the estimation of this method, a combination of parameters which was the highest result of each channel’s evaluation was used. 5.
Results and Conclusion
Figure 2 is the best-case rates of recognition in this results. In incorrect detection class, about 94% examples were excluded as incorrect detection examples correctly. It is improvement in accuracy on Holter ECG automatic analysis. However, about 3% examples were also excluded as incorrect detection examples in correct detection class. They are about 320,000 examples. This is loss, and should be improved. So as future work, this loss is going to be reduced as low as possible.
Exclusion: Advantage
Correct detection class
Incorrect detection class
Figure 2. The best-case rates of recognition in this results: The number of examples is about 9,700,000 in correct detection class, about 490,000 in incorrect detection class.
References
1. Nello Cristianini, John Shawe-Taylor: An Introduction to Support Vector Machines. Cambridge University Press, United Kingdom (2000)
POWER TO DETECT PRIOR MYOCARDIAL INFARCTION BY ECG FINDINGS AT HEALTH EXAMINATION HUIMING ZHANG Department of Public Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan HIDEAKI TOYOSHIMA, HIROSHI YATSUYA, KOJI TAMAKOSHI, TAKAAKI KONDO Department of Public Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
ECG findings at health examination in a working population were tested their accuracy to detect prior myocardial infarction (PMI). 5888 workers (aged 40-59years) taken ECG in 1997 consented to join the study. PMIs judged by Minnesota code-based criteria (MCC) and by automated diagnostic system (ADS) with doctors' correction were respectively compared with the self-reported history. 39 workers had positive history, while 87 were judged to have PMI by the MCC, and 48,by the ADS. Among 39 with positive history, only 16 and 13 were judged to have PMI by respective criteria Even when both ECG criteria were combined, 21 (54%) out of 39 were judged to have no PMI. The rate of overlooking PMI, 54%, was higher than the nonQ myocardial infarction rate of 25% reported before the advent of PCI. Among 5849 workers with negative history, 69 and 34 were judged to have PMI by respective criteria, and 15 sufficed the both ECG criteria. The 15 subjects who sufficed both criteria may have had asymptomatic myocardial infarction, cardiomyopathy, or misreported in the questionnaire. In conclusion, reliability of ECG to detect PMI seems seriously affected by wide use of PCI against acute coronary occlusion.
Objectives To test the reliability of ECG findings in detecting prior myocardial infarction (MI).
Method In 1997, a survey was conducted among civil servants who gave informed consent: 5190 males and 1079 females aged 40-59y. The survey included a selfreporting questionnaire concerning medical history and taking the findings of ECG recorded at annual health examination. All ECGs had a printed output of diagnosis judged by automated diagnostic system (ADS) with doctor's correction. Independent of this diagnosis, they were 713
714
assigned Q and ST-T codes according to Minnesota code through doublechecking by two doctors of this study group [ 13. Prior MI judged by these two kinds of criteria were respectively compared with the self-reported disease history. All participants were asked about the history of MI and the answers were used as the gold standard of the prior MI because of the following reason. Seven participants answered to have developed MI in the past to the questionnaire done in 2002. MI was confirmed in 6 and was not denied in one out of above 7 by the medical records in the hospital suggesting preciseness of the answer about MI history.
Results Among 5888 participants with full available data, 39 reported a positive history of MI. 87 participants were judged to have prior MI by ADS while 48 were judged so by Minnesota code-based criteria (MCC). Among 39 participants who reported positive history, only 16 were judged to have prior MI by MCC and 13 by ADS. Even when both ECG criteria were combined, 21 (54%) out of 39 were judged to have no prior MI. (Tables 1 and 3) Among 5849 participants who reported negative history on MI, 34 were judged to have prior MI by ADS and 69, by MCC. 15 participants were judged to have prior MI by either criteria. (Tables 2 and 4)
Discussion The rate of overlooking prior MI in this study, i.e. 54% was higher than the non-Q MI rate of 25%, which had been reported in 1986 when percutaneous coronary intervention (PCI) was not introduced in Japan. [2] For the reason that an abnormal Q-wave did not remain in the electrocardiogram of the participants with positive history of MI, the following seems possible: 1) Q wave disappeared after reperfusion by PCI or similar treatment in the acute phase, 2) Non-Q MI such as subendocardial or posterior one had occurred, and 3) history of MI was erroneous, though this is unlikely when preciseness of recollection of MI is considered. Since reperfusion therapy of the coronary artery has been commonly done in the acute phase of MI nowadays, it is quite likely that the prevalence of MI without Q-wave should increase. Among 5849 participants with negative MI history, 69 and 34 were judged to have MI by respective criteria, and 15 sufficed the both ECG criteria. The 15
715 Table 1. ECG judgments of prior MI among participants who reported positive history of MI .
ADS
Minnesota code-based criteria No Yes
All
Yes
11
2
13
No
5
2 1(54%)
26
All
16
23
39( 100%)
ADS: Automated Diagnostic System Table 2. ECG judgments of prior MI among participants who reported negative history of MI . Minnesota code-based criteria
ADS
Yes
NO
All
Yes
15
19
34
No
54
5761
5815
All
69
5780
5849
Table 3. The ECG findings of 21 participants who reported positive history of MI but were judged to have no prior MI by either criteria. dinnesota code
Number of subjects
DS
Number of subjects
-3-4 + 5-4
1
-3-4
1
1-3
1
40 Q or ST-T abnormality
1
Normal
11
3therq
h
Table 4 The ECG findings of 15 participants who reported negative history but were judged to have prior MI by ECG. Minnesota code
Number of subjects
ADS
Number of subjects
1-1
5
Anterior MI
2
I-I+ST-T
4
Inferior MI
9
1-2
5
Posterior MI
1
1-2+ST-T
1
Lateral MI
2
Infero-lateral MI
1
716
subjects may have had asymptomatic MI or cardiomyopathy, or misreported in the questionnaire, and need close examination.
Conclusion Reliability of ECG taken in health examination to detect prior MI seems seriously affected by wide use of PCI against acute coronary occlusion.
References 1. Tunstall-Pedoe H, et al: Myocardial infarction and coronary deaths in the World Health Organization MONICA Project. Registration procedures, event rates, and case-fatality rates in 38 populations from 21 countries in four continents. Circulation. 1994 Ju1;90( 1):583-612. 2. Ogawa H, et al: Comparison of clinical features of non-Q-wave and Q wave myocardial infarction. Am heart J 111:5 13-5 18, 1986
14 Autonomic Nervous Activity
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EXAGGERATION OF MORNING FLUCTUATION OF AUTONOMIC NERVOUS ACTIVITY IN THE VERY ELDERLY HEALTHY SUBJECTS HIROFUMI TASAKI, TAKUMI SERITA, CHIAKI UEYAMA, KOUEI KITANO, SHINJI SETO, KATSUSUKE YANO CardiovascularMedicine, Graduate School of Biomedical Sciences, Nagasaki University,Japan
To assess the age-related changes in autonomic nervous activities (ANAs) in the morning in the elderly, we conducted Holter monitoring twice at an interval of 15 years in 15 healthy elderly subjects (female 10, age:70.0*4.1 y.0.; at 1st monitoring) and calculated the 24-hour mean and hourly heart rate variabilities (HRVs: MeanNN[sec], HF[msec2], LF[msec2],LF/HF). Then, we chose (A) the maximum hourly value in the morning (AM4-noon) and (B) the minimum hourly value, and defined the amplitude of HRV in the morning as (B/A-1)*100. As a result, regardless of the significant decreases in the 24-mean HRVs except for HF, all the amplitude rates of HRVs in the morning increased in the very elderly, suggesting that the exaggerated ANA's fluctuation was involved in increasing cardiovascular events in the morning in the elderly.
Results
1st monitoring
15 yrs later
P
MeanNN(sec)
0.976i0.115
0.903i0.117
C0.005
HF(msec2)
221.2i138.89
3 10.78*296.73
0.1102
LF/HF
1.681i0.73 1
0.962i0.442
200msec, Smales, mean age 72* l0years). Patients were divided to patients with mildly-prolonged PR interval (group I: PR250msec, n=6). AVD was adjusted to L-AVD for the first 6months and 0AVD for the next6 months. Echocardiographic measurements and NYHA classification were compared between each setting in group I and 11. No differences were found on LVEDD (group I: 47.6*7.2mm vs 47.7&8.3mm, group 11: 425.5mm vs 45.24.4mm), LVESD (group I: 30.7h6.3mm vs 31.%8.6mm, group 11: 25.8*3.7mm vs 26.5%3.lmm), LVEF (group I: 63.5*9.5% vs 63*13.3%, group 11: 70.3*3.7% vs 71.7+3.8%) and NYHA classification (group I: 1.3k0.5 vs 1.3h0.5, group 11: 1.2h0.4 vs 1.B0.4).Conclusions: There was no significant difference on heart function between L-AVD and 0-AVD regardless of spontaneous PR interval. It is suggested that 0-AVD does not provide significant benefit in patients with spontaneous AV conduction.
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MEASUREMENT OF INTRACARDIAC BIOIMPEDANCE IN RATE ADAPTIVE PACEMAKERS ALAR KUUSIK Department of Electronics, Tallinn University of Technology, Ehitajate tee 5 Tallinn, 19086, Estonia RAUL LAND Department of Electronics, Tallinn University of Technology, Ehitajate tee 5 Tallinn, 19086, Estonia MART MIN Department of Electronics, Tallinn University of Technology, Ehitajate tee 5 Tallinn, 19086, Estonia TOOMAS PARVE Department of Electronics, Tallinn Universiv of Technology, Ehitajate tee 5 Tallinn, 19086, Estonia GUSTAV POOLA Department of Electronics, Tallinn University of Technology, Ehitajate tee 5 Tallinn, 19086, Estonia
Using of intracardiac electrical bioimpedance (EBI) for pacing rate control requires trustable measurements. Usually, the short ( 4 m s ) and low level (10pA) excitation pulses are used to get the response characterizing the impedance. Unfortunately, the response is weak and spread over the frequency range, and it is difficult to interpret the measurement results. In our novel approach, the excitation energy is concentrated at the frequency of interest, and reliable determination of both, the real (Re) and imaginary (Im) parts of the impedance is achieved at selected frequencies. Different bipolar pulse waveforms are used for excitation and for lock-in demodulator. Obtained EBI-based information is trustable for determination of the beat-to-beat stroke volume and duration of systolic and diastolic intervals, used for adaptive adjustment of the pacing rate, and for maintaining required myocardium’s energy supply level.
1. Introduction
Pacing rate control, based on information extracted from the measurement of intracardiac electrical bioimpedance (EBI) is safe only when the measurement results are trustable [I]. This is not an easy task, particularly in case of 738
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implantable pacemakers, which have to operate for years without any service. Usually, therefore the simplest methods of EBI measurement are exploited, which work properly for determining the parameters of breathing activity and the cardiac activity [l]. In general, the EBI comprises more information, e.g., on the status of cardiac muscle (myocard) [l, 21. This information can not be easily obtained from the results of pulse based EBI measurement, as it usually is based on analysis of the transient response processes. Typically, a short ( 4 m s ) and low level (1OpA) excitation pulses (Fig.la) are used to get the response, which is used to determine the impedance [l]. Unfortunately, the response to a short pulse is spread over a wide frequency range and reflection of the certain components of the equivalent circuit in the response signal is weak. So it is difficult to interpret the measurement results, even if the simplest three-element equivalent circuit is used [3]. For three-element equivalent circuit, both the transient response and the frequency response measurement can be used. But in the case of real EBI, which in fact has a much more complicated equivalent circuit, it is quite complicated or even impossible to perform, because only limited computing resources are available in the implanted devices. 2.
Method
As the pulse form signals are very suitable for the implantable devices, it is of the interest to obtain the EBI measurement method, where the pulse form signals are used, though the term of complex impedance has been defined for the sine wave signals. But it is still possible to measure directly only the active and reactive components R andX of the complex impedance Z=R+JX (or G and B of the complex admittance Y= G +jB), which are mutually in quadrature [3,4,51. To avoid excessive mathematical conversion errors, R and X (or G and B ) must be measured with required uncertainty, which is hardly achievable in the implantable devices.
a) Conventionalsolution
I
Figure 1 . Conventional solution of pulse wave signals (a) and novel solution of pulse wave signals for lockin signal conversion (b), used for EBI measurement.
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In our novel approach, Measurement channel the pulse waves are successfully used for high precision EBI measurements thanks to using of the lock-in approach, where the excitation energy as well as measurement sensitivity are I I concentrated at the Reference channel frequency of interest, and reliable determination of Figure 2. Block diagram of the lock-in EBI measurement both, the real (Re) and system based on application of the novel pulse form signals. imaginary (Im) parts of the impedance is achieved at selected frequencies [6]. The block diagram of the lock-in EBI measurement system based on application of the novel pulse waveform signals in Fig. 2, where traditional lock-in system is modified without introducing significant complexity. Essential is to reduce the higher odd harmonic content of the excitation signal, and to decrease the sensitivity of the switching-type synchronous detectors to the lower order of harmonics of the excitation signal. The simplest appropriate approximation of the sine wave is shortening of the rectangular signal pulses and introducing zero-level intervals, yielding spectrum, given by: f ( N ) =-
1
cos5b . sin a- + sm 3.1-4- s m 5 s + ... = 3 5
(1) where - a is the magnitude value of the pulse signal, b characterises the shortening of pulses , and is equal to the duration of the signal's zero value segment within half period ( b = 0.. .n/2). According to Eq.( l), from all of the easy-to-generate waveform pairs with maximally different harmonic content, the best one is consisting of waveforms having 30" (d6) and 18" (d10) pulse shortening, which removes the harmonics 3(2n+ 1) and 5(2n+ 1) correspondingly from the signal spectra. As different bipolar pulse waveforms are used for excitation, and for lock-
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in demodulation (Fig. 1 b), the errors caused by higher odd harmonics are reduced significantly [6]. In the case of. typical three-element equivalent circuit the systematic error of determining the frequency response of impedance is reduced to a level not exceeding 0.3% (against 10% in the case Of using common rectangular Figure 3. Block diagram of the synchronous detector (SD) with shortened pulse. waveforms). In Fig. 3 a block diagram of the modified synchronous detector is shown, which is modified in comparison with the conventional switching type SD. The operating mode with shortened pulses is achieved by introducing the third, zero-gain phase of the synchronous detector [7].
3.
Conclusions
Obviously, the lock-in conversion technique on the basis of rectangular waveforms with shortened pulses is sufficiently simple and power efficient to be used in implantable biomedical devices. Despite its simplicity, it ensures acceptable estimates of the real (Re) and imaginary (Im) parts of the electrical bioimpedance. Obtained estimations are trustable for determination of the beatto-beat stroke volume and duration of systolic and diastolic intervals, playing an important role in the adaptive adjustment of pacing rate and maintaining the required myocardium's energy supply level. Acknowledgments
This work was supported by Estonian Science Foundation grants 5892, 5897, 5902 and by Japan Society for the Promotion Science (JSPS) 2003 postdoctoral fellowship program. References
1. J.G. Webster (Ed.), Design of Cardiac Pacemakers. IEEE Press, Piscataway, NJ, 1995. 2. International patents PCT WO 0057953 and PCT WOOOh7954, 2000, M. Min, A. Kink and T. Parve. 3. S. Grimnes and O.G. Martinsen, Bioimpedance and Bioelectricity Basics. Academic Press, San Diego, 2000.
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4.
M. Min, 0.Martens and T. Parve, Measurement. 27, no.l,21 (2000). M. Min, T. Parve, V. Kukk and A. Kuhlberg, IEEE Trans. Instrum. h Meas., 51,674 (2002). 6. A.Kuusik, R.Land, M.Min and T.Parve. Internat. Journ. of BioElectroMagnetism, 5, 1,23 (2003). 7. International patent application PCT/EE03/00006, filed 28.1 1.2003, M. Min, A. Kink, R. Land and T. Parve. 5.
VENTRICULAR PACING THRESHOLDS FOLLOWING HIGHENERGY VENTRICULAR DEFIBRILLATION SHOCKS YOSHIO YAMANOUCHI, SUNAO KODAMA, TAKEAKI OHTA, NATSUMI MORITO, EIJI YAHIRO, KEI MIYOSHI, HIDENORI URATA Department of Cardiology, Fukuoka University Clzikushi Hospital, Fukuoka, Japan
Increased ventricular pacing thresholds have been observed following monophasic waveform shocks in implantable cardioverter defibrillators (ICDs). This study aimed to examine such changes following high-energy biphasic shocks in ICDs. Method: Ten episodes of VF were induced every 10 minutes in 10 pigs (23.1k3.0 kg). After 10 seconds of VF a 40J biphasic shock (total 10 shocks) was delivered for successful defibrillation in the true-bipolar sensing lead system of the ICD. Ventricular bipolar pacing thresholds before and after these shocks were evaluated at one-minute intervals. Results: The mean pacing threshold before shock delivered was 0.066%0.059uJ. Those of the first,second and third minutes after the first shock were 0.052;t0.061 uJ,0.044&0.039 uJ,respectively; showing that pacing thresholds gradually decreased. Conclusion: It may not be necessary to pace at a high-voltage output after biphasic shocks in ICDs.
i
Number of ICD shocks
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PREVENTION OF ATRIAL FIBRILLATION BY BIATRIAL PACING: THE OUTCOME AND THE ELECTROPHYSIOLOGICAL MECHANISM OF PREVENTION YOSHIHISA ENJOJI, TAKA0 SAKATA, MAHITO NORO, TAKESHI NAKAE, NAOKI TEZUKA, KENTA KUMAGAI, TSUYOSHI SAKAI, HIDETOSHI ITAKURA, AKIYOSHI MORIYAMA, KAORU SUGI Division of Cardiovascular Medicine, Toho University, Ohashi Hospital, Japan
We previously reported the superiority in AF prevention by biatrial pacemaker (BAP) for a short term period. The purpose of this study is comparing the preventive effect of AF for a long time period and assessing the mechanism of prevention by BAP. Twelve patients with paroxysmal AF received BAP with 2 leads in the right atrium and coronary sinus. A crossover trial between BAP and right atrial pacing (RAP) was performed every 3 month. Non-invasive EP study for measuring the effective refractory period (ERP) of both atria and inducing AF was done after every 3 month of pacing. Results: The mean follow-up period was 33+7 months. Although AF episodes tended to decrease even by the RAP, they were more prominent by BAP (p 10 % and & 5% (Fig.2B). The linear increase of a leads to the same change of a level of fibrillation. The saltatory transitions was not observed, thus the dependence was
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characterized large dispersion. Also have been found unexpected effects, namely, non-monotonous dependence sites of level of fibrillation on average threshold of excitation. Numerous experiments have revealed possibility of target control setting of model parameters to develop strategy of shortest exit from the fibrillation mode.
References 1. Kobrin V.I. Myocardial heterogeneity and cardiac arrhythmiasllUsp. Fiziol.Nauk (USSR), 1993 Oct-Dec; 24(4): 47-59 2. Konovalova I.N., Kobrin V.I., Konovalov M.G.11 Computer model for simulating the process of excitation propagation in cardiac tissue. In book: Eclectrocardiology’99,ed.M.P.Roshchevsky, Syktyvkar, 2000, p. 121- 126
BIO-IMPEDANCE FDM-MODELING INSIDE HEART FOR APPLICATION IN IMPLANTED DEVICES M U N O GORDON, ALAR KUUSIK Department of Electronics, TTU, Ehitajate tee 5 I9086 Tallinn, Estonia, E-mail: rauno@,elin.ttu.ee
In this work segmented MFU images are used to construct 3D geometry model of the heart and its close surroundings. Then a 3D mesh of nodes is applied with connections between nodes representing frequency dependent characteristics of tissue of that area. Several tissue types are used with their corresponding frequency-characteristics. An electrical signal then is applied to the nodes of supposed cardiac-pacemaker electrodes and a distribution of potentials is calculated with FDM. As the admittance of tissues varies with frequency, the 3D-potential field inside the heart is also frequency dependant. Animations of different fields with frequency sweep were generated to gain insight on how to use multifrequency impedance measurement for improved adaptive cardiac pacing. Frequency sweep animations were made containing information from potential distribution and gradient fields with separated real and imaginary parts.
1. Introduction Intracardiac bioimpedance measurement is relevant to implanted devices such as a cardiac pacemaker [I]. A cardiac pacemaker needs to give the heart the right pulse and be rate-adaptive to different loads on the heart. One way to gain information about cardiac muscle condition and stroke volume is to measure bioimpedance between various electrodes inside the heart or in the thorax. In this work an impedance measurement is simulated in a situation, where the pacemaker electrodes are inserted to the left side of the heart. One electrode is placed close to the apex in the center of the left ventricle and the other is placed in the left atrium. With an advanced simulation system and different size models precise virtual experiments could be made with the electrodes in any desired position in the region of interest.
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Heart exterior
Figure 1.
A) 3D heart model from segmented MRI slices. Front view. B) Antialiasing of slices and admittance values.
When we have 3D models of the beat-to-beat dynamic heart geometry, then we can simulate time-varying impedance signal, as it is measured in the heart. One of the main future goals of this simulation is to develop a model for strokevolume estimation based on dynamic impedance signals measured at different frequencies and perhaps in different locations of the heart. The 3D model of the heart is made of segmented MRI images (Fig. 1A). In this simulation three tissue types have been used: blood, heart muscle and lung. In order to increase the accuracy of the simulation, antialiasing of the images is performed during the down-scaling procedure (Fig. IB). Anisotropic heart muscle properties are also used to estimate their influence on simulation accuracy. 2.
Tissue Models and Simulation Mesh
For the FDM mesh node connections, tissues are modeled with their electrical equivalent circuits. The blood and lung tissue have their own specific frequency characteristics and heart muscle has two different frequency characteristics parallel to the direction of the muscle fibres and transversal to the muscle fibres (Fig. 2 ) [ 2 ] .
Figure 2. Admittance characteristics of tissues and their respective models.
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A hexahedron mesh is applied to the geometric model of heart tissues (Fig. 3). Fibres on heart muscle are considered to circle on horizontal planes around the ventricles to make modeling easier. A map of angles is made for each plane of the model, where an angle of the muscle fibre is specified for every node (Fig. 4). Admittance between nodes is calculated as an average of the nodes it connects. Fibre direction decides the appropriate muscle admittance value for muscle nodes. For the hexahedron mesh the anisotropic properties are not distributed homogeneously and it affects the accuracy of modeling (Fig. 4). The admittance matrix is made of complex admittance values of the connections between nodes. For each frequency new complex admittance values have to be taken from tissue equivalent circuit formulas to compose the admittance matrix at that frequency.
Figure 3. Antialiased slices and the FDM mesh of equivalent circuits.
Figure 4.Map of muscle fibre angles on one plane (left). Distribution of anisotropic sensitivity (right). Only bright areas can be modeled anisotropically.
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3.
Simulation Results
3D current flow lines are calculated from potential gradient field and plotted separately for real and imaginary parts in Fig. 5. The electrodes are positioned inside the left atrium and the left ventricle of the heart. This electrode positioning can be realized in practice as a cardiac pacemaker lead with two electrodes is inserted into the left side of the heart. The current flow tubes shown here are calculated with the system of 36960 nodes on the 32*33*35 modeling box size [3, 41. We can see (Fig. 5A) that those flow-lines that penetrate the muscle, tend to do that perpendicularly to the simplified fibre structure. As the admittance of the muscle parallel to the fibre is almost two times better than transversal to the fibre, roughly half the muscle nodes (that are close to 45 degree angle) allow a lot more current to pass through the muscle, than they should. Therefore it is not clear at this point, whether this kind of limited anisotropic modeling increases the accuracy or vice versa. A different type of mesh is needed for reliable anisotropic modeling.
Isotropic muscle (parallel fibre properties)
1024 k~~
Anisotropic muscle
Figure 5 . Simulation results. A) Current flow lines at 32 !&z and 1024 !&z frequency with real and imaginary parts. B) Nyquist plot (Cole-Cole plot) of voltage drop between electrodes on complex plane. Current was fixed at 1 OpA.
The differences in the outcome of the simulation (Fig. 5B) are very small between isotropic and anisotropic modeling mostly because electrodes are surrounded with blood and far from the muscle (Fig. 1A). Most of the current is passing through the blood (isotropic). Current efforts of this study are directed towards acquiring a dynamic model of the heart (heartbeat) for modeling the dynamics of generated fields on a number of frequencies. It is also planned to improve the modeling mesh and anisotropics and to find an iterative solver good enough to solve a large system of complex linear equations.
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References 1 . J. G. Webster (Ed.), Design of Cardiac Pacemakers, New York, IEEE Press, 1995. 2. An Internet resource for the calculation of the DIELECTRIC PROPERTIES OF BODY TISSUES. ITALIAN NATIONAL RESEARCH COUNCIL, Institute for Applied Physics, “Nello Carrara“ - Florence (Italy). httr,://niremf.ifac.cnr.it/tisssrod. 3. G. H. Golub, C. F. van Loan, Matrix Calculations, The Johns Hopkins University Press Ltd., London, 1996. ISBN 0-8018-5413-X. 4. D. Day, M. A. Heroux, “Solving complex-valued linear systems via equivalent real formulations”. SIAM Journal of Scientific Computation, Vol. 23, No. 2, pp. 480-498. Society of Industrial and Applied Mathematics 2001. Acknowledgments
This work has been fimded by EITSA (The Estonian Information Technology Foundation) and Grants no. 5892 and 5897 of Estonian Science Foundation.
SUCCESSFUL BIVENTRICULAR PACING IN AN ELDERLY PATIENT WITH CARDIAC SARCOIDOSIS AT RISK OF CONGESTIVE HEART FAILURE OSAMU OKAZAKI, MICHIAKI HIROE, NAOKI TEZUKA, MAHITO NORO, MITSUO KASHIDA, NOBUHARU AKATSUKA, YOSHIO YAZAKI Division of Cardiology, International Medical Center of Japan, Japan KEYWORDS Biventricular pacing Cardiac ResynchronizationTherapy Cardiac sarcoidosis Paroxysmal atrial fibrillation Brain Natriuretic Peptide Corresponding address: 1-21-1 Toyama, Shinjuku-ku, 162-8655 Tokyo, Japan E mail address: ookazaki0 irnci.hwn.ro.jp
Abstract We report on a successful caridiac resynchronization therapy (CRT) in a patient with congestive heart failure (CHF‘) caused by cardiac sarcoidosis with mitral regurgitation (MR) and paroxysmal atrial flutto-fibrillation (PAF/AFL). A 78 year-old woman was transferred because of CHF. She was diagnosed with uveitis and cardiac sarcoidosis by echo cardiography, CAG and LVG in 1996. VDD pacemaker was implanted to treat the advanced AV block in 1997. She had been given amiodarone for ventricular tachycardia since 1999. In 2003, she was admitted because of progressive CHF with pulmonary congestion and severely impaired left ventricular function with 19% of ejection fraction (EF‘) due to PAF/AFL. To prevent from CHF, biventricular pacing was undertaken on Mar, 2003. The left ventricular pacing lead was connected with the previously implanted generater with Y connector. Her functional class improved from NYHA III to II. The QRS duration decreased from 209 to 160msec a year later. Plasma brain natriuretic peptide levels decreased from 691 to 240pg/ml. Left ventricular end-diastolic dimension decreased 75 to 70mm, EF increased from 19 to 41% and the grade of MR improved from III to II. Bi-ventricular pacing may be a successful tool in an elderly patient with CHF at risk for dyssynchrony of cardiac sarcoidosis besides during PAF/ALF.
Background Biventricular pacing (BVP) or cardiac resynchronization therapy (CRT)” for severe heart failure has been available in Japan since April 2004 with the national health insurance. Previous studies have suggested that CRT achieved through atrial synchronized BVP produces clinical benefits in patients with CHF who have an intraventricular conduction delay as shown in MIRACLE2’ during sinus rhythm. MUSTIC3’ trial included chronic AF with a wide QRS complex 783
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who required a permanent pacemaker because of a slow ventricular rate. The duration of the QRS complex may not be the optimal criterion for selecting patients who will benefit from CRT. The effects of CRT have also been evaluated in patients with CHF who have observed atrial fibrillation. However the validation for CRT in patients with cardiac sarcoidosis during VDD pacing and PAFIAFL is not clear. We report on a successful BVP treatment of CHF caused by cardiac sarcoidosis with MR and PAFIAFL. Case Report A 78-year-old female was admitted with dyspnea of chief complaint on Feb, 2003. She was diagnosed with uveitis and cardiac sarcoidosis by echo cardiography. Coronary angiography (CAG) demonstrated normal coronary arteries and left ventricular angiography (LVG) revealed diffuse hypokinesis with 29% of EF and ventricular aneurysm as a cause of ventricular tachycardia (VT) in 1996 as shown in figure 1. We performed myocardial biopsy, but unfortunately the non-epithelioid granuloma lesion was not found. VDD pacemaker was implanted to treat the advanced AV block in 1997. She had been given amiodarone for VT since 1999. Figure 2 demonstrated the septal thinning on CT scan, and myocardial aneurysm on dual SPECT. She was admitted because of progressive CHF with pulmonary congestion in 2003. On physical examination, her vital signs were as follows: BP 110178mmHg; and heart rate 117 irregular beatslmin. Jugular veneous distention was present. Cardiac auscultation revealed a distant S2 and a grade 316 mid systolic murmurs at apex. Lung fields were fine cracle to auscultation bilaterally. Extremities were without clubbing, and edema was present. An ECG was interpreted as VVI pacing rhythm on representing PAFIAFL and chest radiograph showed pulmonary congestion at the emergency room as shown in Figure 3-A. For the purpose of preventing CHF, after the informed concent CRT was undertaken on Mar, 2003 during PAF as shown in Figure 3-B. According to venography and measurement of pacing threshold via coronary sinus, the left ventricular pacing lead into the great cardiac vein was connected with the previously implanted generator with Y connector in Figure 4. AV delay was set at the sinus rhythm as 100msec. Figure 5 showed the QRS duration decreased from 209 to 160msec a year later. Plasma brain natriuretic peptide levels decreased from 691 to 240pdml as shown in Figure 6. UCG demonstrated that left ventricular end-diastolic dimension decreased 75 to 70mm, EF increased from 19 to 41% and the grade of MR improved from I11 to I1 in Figure 7. Her functional class improved from NYHA I11 to 11. As a result, her clinical course was summarized in table 1.
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Discussion CRT2’ is a miracle therapy to improve left ventricle dyssynchrony in patients with severe heart failure with left bundle branch block and NYHA functional class I11 or IV. LVEF is equal to or less than 35%, and QRS duration is more than 130msec. QRS duration indicates ventricular contraction synchronism. However there are two groups of responder and non-responder for CRT. A difference is not clear between these groups with a state of CHF, QRS duration, and AV optimization. In general, CRT indicated the CHF patients due to ischemic heart disease and idiopathic dilated cardiomyopathy. However it is not yet clear about the indication for secondary cardiomyopathy such as this elderly cardiac sarcoidosis case4).This 78 years-old female has been uveitis and cardiac sarcoidosis with repeated congestive heart failure due to involvement of intracardiac conduction disturbance and arrhythrmas due to ventricular aneurysm. Echocardiogram indicated a dilated cardiomyopathy-like movement with a diffuse hypokinesis. CAG showed non-ischemic heart disease without a significant stenosis, and LVG revealed severely reduced LVEF with an anterior aneurysm as a focus of VT. SPECT showed a blood flow and a metabolic disorder on the same legion. VDD pacemaker for advanced AV block was already implanted, and ECG showed CLBBB with 209msec of the QRS duration. Some medications including amiodarone had reached a limitation, and we decided the introduction of CRT. It was thought that the activity of sarcoidosis was low, but low-dose steroid 5mg has been given. Because there was left ventricular aneurysm and pacing threshold was high on the anterior wall, the optimal pacing site have to put it on a lateral wall into the great cardiac vein. In addition an AV delay was set in lOOmsec due to paroxysmal atrial fibrillation prevention. Recently amiodarone was changed to sotalol due to hypothyroidism. As for CRT, future development is expected to the new therapy for CHF with the involvement of cardiac sarcoidosis. VDD pacing will be change to DDD andor CRTD after the ablation to AFL as an option for the next step. We reported a successful CRT in an elderly patient with severe CHF due to cardiac sarcoidosis besides during PAFIAFL at risk for dyssynchrony.
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References 1. Leon AR, Greenberg JM, Kanuru N, Baker CM, Mera FV, Smith AL, Langberg JJ, DeLurgio DB: Cardiac resynchronization in patients with congestive heart failure and chronic atrial fibrillation: effect of upgrading to biventricular pacing after chronic right ventricular pacing. J Am Coll Cardiol, 39:1258-1263,2002 2. St John Sutton MG, Plappert T, Abraham WT, Smith AL, DeLurgio DB, Leon AR, Loh E, Kocovic DZ, Fisher WG, Ellestad M, Messenger J, Kruger K, Hilpisch KE, Hill MR; Multicenter InSync Randomized Clinical Evaluation (MIRACLE) Study Group.: Effect of cardiac resynchronization therapy on left ventricular size and function in chronic heart failure. Circulation, 107:1985-90,2003 3. Cazeau S, Leclercq C, Lavergne T, Walker S, Varma C, Linde C, Garrigue S, Kappenberger L, Haywood GA, Santini M, Baailleul C, Daubert JC: Multisite Stimulation in Cardiomyopathies (MUSTIC) Study Investigators : Effects of multisite biventricular pacing in patients with heart failure and intraventricular conduction delay. N Engl J Med 344:873-880,200 1 4. Sat0 A, Ohara T, Algowhary M, Suzuki M, Matsumura A, Hashimoto Y , Isobe M: Beneficial biventricular pacing in a patient with cardiac sarcoidosis and refractory heart failure: a case report, J Cardiol 42:22 1-226, 2003
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Figure 2
CT scan demonstrated antero-septa1thinning and aneurysm on the anterior wall. Dual SPECT using 201-T1 and 123-1 BMIPP revealed perfusion and metabolic disturbance on the aneurysm area as a focus of ventricular tachycardia.
ECG @ama.siixilAn) Feb. 24" 2003 Feb. 14u17 2003
A-P
dew
(CTR68%) Congestion(f )
Figure 3-A ECG showed atrial flutto-fibrillation and CLBBB pattern due to VVI pacing on the mode switch at the emergency room. Chest Xp demonstrated pulmonary congestion and cardiomegaly with 68% of CTR on A-P view.
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Figure 3-8 ECG showed atrial fibrillation and BVVI pacing on the mode switch. Chest Xp demonstrated bi-ventricular pacing and 63% of CTR without pulmonary congestion.
1, V endocardial lead implantation following with the CS venography for BVP
Figure 4 LV lead using Y connector was implanted into the grate cardiac vein with the CS venography and pacing threshold examination.
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BVP 011 QRS duration
Ks
c)= 209111scc
llIXrx
Figure 5 ECG showed wide QRS duration with 209msec by RV pacing only, and narrow QRS with 160msec by BVP in limb leads.
e line of BN
igure 6 Time line of BNP, QRS dulation and EF demonstrated improvements of conditions.
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Figure 7 UCG demonstrated the improvement of MRIII to MR I1 degree, and 19% to 4 1% of LVEF a year later.
Table I
CHRONIC ANGIOTENSION I1 RECEPTOR BLOCKER DOES NOT ALTER VENTRICULAR DEFIBRILLATION THRESHOLDS YOSHIO YAMANOUCHI, SUNAO KODAMA, TAKEAKI OHTA, NATSUMI MORITO, EIJI YAHIRO, KEI MIYOSHI, HIDENORI URATA The Department of Cardiology, Fukuoka University Chikushi Hospital, Japan
Angiotensin I1 receptor blockers (ARBS) are efficacious in the treatment of cardiovascular diseases, and are potentially useful in patients with implantable cardioverter defibrillators (ICD) who are suffering from heart failure. The purpose was to determine the long-term effects of ARB, candesartan, administration on the defibrillation thresholds (DFT). Methods: DFTs were evaluated using a hot can defibrillation lead system in six dogs (ARB Group, 13.4iZ4.1kg). Candesartan was fed orally at a dose of 10 mg/kg/day for one year. A further six dogs were given no candersartan (Control Group, 17.8i~7.9kg). Results: The DFT energy values of both groups at each period are shown in the table. Conclusions: Chronic ARB therapy did not result in increased DFT energy levels and therefore might not decrease the margin of safety in ICD patients.
1
Before
i treatment
1
.................................................
'
i
3-months 6-months 12-months after I after after treatment i:.......................................................... treatment treatment .&............................. ,
~
~
i
~
Control/ 16.1211.3 113.7+8.6115.2&9.5j 16.0k9.7 Group j ARB 17.0k9.1 15.6+-7.9:15.5+-7.9/ 12.9f9.1 Group
\
I
P=n.s. before vs. each period in both groups.
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VALIDATION OF QUALITY OF LIFE QUESTIONNAIRE FOR ICD PATIENTS SOICHI TSUNODA Medtronic Japan Co., Ltd, Kawasaki, Japan HARUHIKO ABE’, TAKESHI MITSUHASHI’, SATOSHI ISHIZUKA3 University of Occupational and Environmental Health, Kitakyushu, Japan, ’Jichi Medical School, Minamikawachi-machi, Japan, ’Medtronic Japan Co., Ltd., Kawasaki, Japan
’
Background and Objective: Considering the uniqueness of ICD patients’ anxiety and environment, we developed a Quality of Life Questionnaire by focus-group method. The aim of this study was to assess the correlation between the outcome of developed QOL questionnaire for ICD patients and that of already validated QOL questionnaire. Method: As a validated QOL questionnaire, Japanese version of WHO-QOL26 which consists of 26 questions was used. Questions in both QOL questionnaires were grouped in 5 domains. Twenty six ICD patients were enrolled and were given both WHO-QOL26 and the ICD questionnaire to answer. Averaged scores for both QOL questionnaires were calculated and tested for correlation. Results: Correlation was calculated for each domains and average of all. All domains showed statistical significant correlation between ICD questionnaire and WHO questionnaire. Conclusion: Newly developed QOL questionnaire for ICD patients for Japanese population showed good correlation with WHO-QOL26 and it was proved to be reliable.
1. Background Implantable Cardioverter Defibrillator (ICD) therapy gains its popularity in recent years in Japan. Quantitative measurement of Quality of Life (QOL) of those patients becomes more important parameter in order to provide better post-implant patient care. ICD therapy apparently reduces the risk of sudden cardiac death, however occasionally patient’s QOL is deterioratedl. Although there are several QOL questionnaires which were used in various studies for ICD patientsv, it may not be appropriate to adopt a QOL tool from western society because of cultural differences and patient’s lifestyle in Japan. Therefore it is needed to develop a Japan specific QOL measurement tool to aid healthcare professionals to assess QOL in ICD patients and their family members. 2.
Objective
The objective of this study was to develop a QOL questionnaire for ICD patients and validate it by comparing with an already validated questionnaire. The QOL 792
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questionnaire will be ICD specific in order to identi@ the source of QOL deterioration, if any, and it will be used conjunction with WHO-QOL26 (Japanese version) which is well validated QOL questionnaire developed by WHO (World Health Organization) 4
3. Method The process of developing and validating the questionnaire is depicted below.
IVZ" I Fignrel. Developing and validation flow for QOL questionnaire for ICD patients.
The major determinants of ICD patients' QOL deterioration were various kinds of anxieties. The assessment of anxiety was done by; Literature search Analysis of inquiries from Patient Hot-line Service at Medtronic Japan Patient Focus Groups (F.G.) A list of anxieties of ICD patients was obtained by these activities and was sorted out to eliminate generic anxieties which would be measured by WHOQOL26. Two focus groups were held in Tokyo (highly populated area) and Sapporo (less populated local area), and total of 13 patients (male 9, age 19-74, mean 60 years old) and 5 family members participated in the focus groups. F.Gs. were conducted by group interview method with a professional interviewer. After the interview, patients were informed that the information gathered during the session will be used for the questionnaire development with secure identity protection and they agreed on that. The alpha-version of anxiety questionnaire was designed from the list. It was tested for usability in 34 healthy subjects and time to complete the questionnaire and understanding of questions were assessed. The questionnaire was modified according to the usability test results and beta-version was completed. Questions in newly developed QOL questionnaire for ICD patients (QOLQ-ICD) and WHO-QOL26 were grouped in 5 domains.
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4.
Results
4.1. Patients Hot-line Service
Medtronic Japan provides toll-fi-ee Patients Telephone Service which often reveals patients’ anxiety about the implant procedure, device, and post-implant daily life. Recent inquiries list as follows as example; (Nov. 2002 -Jan. 2003) 1was informed my cardiac disease was Brugada Syndrome. What is that? Anxieties about home appliances such as rice cooker, electric blanket, snow blower, and IH (Inductive Heating) cooker. Pain at pocket site. How ICD works. What is ATP? Driving restriction. 4.2. Summary List
The following anxiety list was obtained as the result. Item 1, 2 were added to measure general acceptance of the ICD therapy. And they were categorized in five groups where applicable. (A=Physical, B=Psychological, C=Level of independency, D=Social relationship, E=Environment) 1. Total satisfaction in ICD therapy 2. Anxiety in ICD reliability 3. Pain and physical effect by shock therapy (A) 4. Body image change by device implant (B) 5. Pain and scar at pocket site (A) 6. Economical disadvantage (E) 7. Driving restriction (C) 8. EMI(E) 9. Anxiety in genetic nature of disease (D) 10. Mental stress of family members (D) 1 1. Negative effects on surroundings when shock occurs (E) 12. Reliability of work place, school, and society (D) 13. Embarrassment when shock occurs in public (B) 14. Lack of information about ICD and/or therapy (B) 15. (as a family member) Mental care for the patient (B) 16. (as a family member) Effects to oneself when the patient gets shock (A) 4.3. Usability Test
The alpha-version was tested with 34 healthy subjects (17 males, mean age 33), and the mean time to complete the questionnaire was 4 minutes and 35 seconds. Some expressions in the questionnaire were clarified for the beta-version.
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4.4. Validation
In order to validate the beta version of the questionnaire, total of 26 (1 1 male, mean age 6 3 ) ICD patients in two centers were enrolled and were given both WHO-QOL26 and the QOLQ-ICD at one time to answer them. Averaged scores for both questionnaires were calculated and tested for correlation by Pearson’s correlation coefficient. Table 1. Corrclation between QOL questionaire for ICD patients and WHO-QOL 26. Domain Physical Psychological Social Relationship Environment Total QOL Average
r 0.39 0.43 0.41 0.49 0.45 0.69
p (n=26) C0.05