Inflammatory Cardiomyopathy (DCMi) - Pathogenesis and Therapy (Progress in Inflammation Research)

Progress in Inflammation Research Series Editor Prof. Michael J. Parnham, PhD Director of Science MediMlijeko d.o.o. 1...

Author: Heinz-Peter Schultheiss | Michel Noutsias

15 downloads 749 Views 4MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Progress in Inflammation Research

Series Editor Prof. Michael J. Parnham, PhD Director of Science MediMlijeko d.o.o. 10000 Zagreb Croatia

Advisory Board G. Z. Feuerstein (Wyeth Research, Collegeville, PA, USA) M. Pairet (Boehringer Ingelheim Pharma KG, Biberach a. d. Riss, Germany) W. van Eden (Universiteit Utrecht, Utrecht, The Netherlands)

Forthcoming titles: Endothelial Dysfunction and Inflammation, A. Karsan, S. Dauphinee (Editors), 2010 Muscle, Fat and Inflammation: Sustaining a Healthy Body Composition, S.A. Stimpson, B. Han, A.N. Billin (Editors), 2011 The Inflammasomes, I. Couillin, V. Petrilli, F. Martinon (Editors), 2011 (Already published titles see last page.)

Inflammatory Cardiomyopathy (DCMi) Pathogenesis and Therapy

Heinz-Peter Schultheiss Michel Noutsias Editors

Birkhäuser

Editors Heinz-Peter Schultheiss Michel Noutsias Department of Cardiology and Pneumology Charité – Universitätsmedizin Berlin Campus Benjamin Franklin Hindenburgdamm 30 12200 Berlin Germany

Library of Congress Control Number: 2010923938

Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de

ISBN 978-3-7643-8351-0 The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use, permission of the copyright owner must be obtained. © 2010 Springer Basel AG P.O. Box 133, CH-4010 Basel, Switzerland Part of Springer Science+Business Media Printed on acid-free paper produced from chlorine-free pulp. TCF d Cover design: Markus Etterich, Basel Cover illustration: see p. 207, with friendly permission by Michel Noutsias Printed in Germany ISBN 978-3-7643-8351-0 987654321

e-ISBN 978-3-7643-8352-7 www.birkhauser.ch

Contents

List of contributors Preface

.................................................................

vii

..............................................................................

xi

Myocarditis and inflammatory cardiomyopathy – clinical management, epidemiology and prognosis Bernhard Maisch, Konstantinos Karatolios, Sabine Pankuweit and Arsen Ristic Pathogenesis, diagnosis and treatment of pericarditis . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

G. William Dec Epidemiology and prognosis of myocarditis and dilated cardiomyopathy: Predictive value of clinical parameters and biopsy findings . . . . . . . . . . . . . . . . . . . . . . .

25

Animal models of myocarditis – autoimmunity and viral infection; therapeutic interventions Christoph Berger und Urs Eriksson Autoimmune murine myocarditis and immunomodulatory interventions . . . . . . . .

37

Michel Noutsias and Peter Liu Coxsackievirus-induced murine myocarditis and immunomodulatory interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

Susanne Rutschow, Michel Noutsias and Matthias Pauschinger Myocardial proteases and matrix remodeling in acute myocarditis and inflammatory cardiomyopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71

Alterations of the immune system in human viral and inflammatory cardiomyopathy Jeffrey A. Towbin and Matteo Vatta Molecular genetics of cardiomyopathies and myocarditis . . . . . . . . . . . . . . . . . . . . . . . .

91

Contents

Roland Jahns, Valérie Boivin, Georg Ertl and Martin J. Lohse Pathogenic relevance of autoantibodies in dilated cardiomyopathy

.............

157

Jesus G. Vallejo and Douglas L. Mann The role of cytokines in inflammation-induced cardiomyopathy: Pathogenesis and therapeutic implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Diagnosis of inflammatory and viral cardiomyopathy Annalisa Angelini, Fiorella Calabrese and Gaetano Thiene Histology and immunohistology of myocarditis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Michel Noutsias, Heinz-Peter Schultheiss and Uwe Kühl Immunohistological diagnosis of inflammatory cardiomyopathy and diagnosis of cardiotropic viral infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Matthias Gutberlet Cardiac magnetic resonance imaging: A non-invasive approach for the detection of myocardial inflammation – Potentials and limitations. . . . . . . . . . . . . . . 227 Treatment strategies in inflammatory cardiomyopathy patients Kenneth L. Baughman† Clinical management of acute myocarditis and cardiomyopathy . . . . . . . . . . . . . . . . . 239 Andrea Frustaci and Cristina Chimenti Immunosuppressive treatment of inflammatory cardiomyopathy patients

......

257

Heinz-Peter Schultheiss, Michel Noutsias and Uwe Kühl Antiviral interferon-B treatment in patients with chronic viral cardiomyopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Stephan B. Felix and Alexander Staudt Immunoadsorption in dilated cardiomyopathy patients Index

vi

..........................

279

................................................................................

291

List of contributors

Annalisa Angelini, Department of Medico-diagnostic Sciences and Special Therapies, Special Pathological Anatomy, University of Padua Medical School, Via A. Gabelli, 61, 35121 Padova, Italy; e-mail: [email protected] Christoph Berger, Departments of Internal Medicine and Research, University Hospital, Petersgraben 4, 4031 Basel, Switzerland Valérie Boivin, Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany; e-mail: [email protected] Fiorella Calabrese, Department of Medico-diagnostic Sciences and Special Therapies, Special Pathological Anatomy, University of Padua Medical School, Via A. Gabelli, 61, 35121 Padova, Italy; e-mail: [email protected] Cristina Chimenti, Department of Cardiovascular and Respiratory Sciences, La Sapienza University, Viale del Policlinico 155, 00161 Rome, Italy; e-mail: [email protected] G. William Dec, Cardiology Division, Massachusetts General Hospital, Heart Failure and Transplantation Unit, Boston, MA 02114, USA; e-mail: [email protected] Urs Eriksson, GZO AG Spital Wetzikon, Spitalstr. 66, 8620 Wetzikon, Switzerland; and Universitätsspital Zürich, Rämistr. 100, 8091 Zürich, Switzerland; e-mail: urs. [email protected] Georg Ertl, Department of Internal Medicine, Medizinische Klinik und Poliklinik I, Cardiology, University Hospital of Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, Germany; e-mail: [email protected] Stephan B. Felix, Klinik für Innere Medizin B, Ernst-Moritz-Arndt-Universität, Friedrich-Loefflerstr. 23a, 17475 Greifswald, Germany; e-mail: felix@uni-greifswald. de

vii


Andrea Frustaci, Department of Cardiovascular and Respiratory Sciences, La Sapienza University, Viale del Policlinico 155, 00161 Rome, Italy; e-mail: [email protected] Matthias Gutberlet, University Leipzig/ Leipzig Heart Center, Department of Diagnostic and Interventional Radiology, Strümpellstrasse 39, 04289 Leipzig, Germany; e-mail: [email protected] Roland Jahns, Department of Internal Medicine, Medizinische Klinik und Poliklinik I, Cardiology, University Hospital of Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, and Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany; e-mail: [email protected] Konstantinos Karatolios, Department of Internal Medicine-Cardiology, University Hospital of Giessen and Marburg, Baldingerstrasse 1, 35033 Marburg, Germany; e-mail: [email protected] Uwe Kühl, Department of Cardiology and Pneumonology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; e-mail: [email protected] Peter Liu, Heart & Stroke Medicine and Physiology, Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research, Toronto General Hospital, University of Toronto, Canada; e-mail: [email protected] Martin J. Lohse, Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany; e-mail: [email protected] Bernhard Maisch, Department of Internal Medicine – Cardiology, University Hospital of Giessen and Marburg, Philipps-Universität Marburg, Baldingerstrasse 1, 35033 Marburg, Germany; e-mail: [email protected] Douglas L. Mann, Division of Cardiology, Washington University School of Medicine, St. Louis, MO, USA; e-mail: [email protected] Michel Noutsias, Department of Internal Medicine – Cardiology, University Hospital of Marburg and Giessen, Philipps-Universität Marburg, Baldinger Strasse 1, 35033 Marburg, Germany; e-mail: [email protected] Sabine Pankuweit, Department of Internal Medicine-Cardiology, University Hospital of Giessen and Marburg, Baldingerstrasse 1, 35033 Marburg, Germany; e-mail: [email protected]

viii


Matthias Pauschinger, Department of Cardiology, Medizinische Klinik 8, Klinikum Nürnberg Süd, Breslauer Straße 201, 90471 Nürnberg, Germany; e-mail: matthias. [email protected] Arsen Ristic, Department of Cardiology II, Belgrade University, Belgrade, Serbia; e-mail: [email protected] Susanne Rutschow, Department of Cardiology and Pneumonology, CharitéCentrum 11 for Cardiovascular Medicine, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; e-mail: [email protected] Heinz-Peter Schultheiss, Department of Cardiology and Pneumonology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; e-mail: [email protected] Alexander Staudt, Klinik für Innere Medizin B, Ernst-Moritz-Arndt-Universität, Friedrich-Loefflerstr. 23a, 17475 Greifswald, Germany; e-mail: staudt@uni-greifswald. de Gaetano Thiene, Department of Medico-diagnostic Sciences and Special Therapies, Special Pathological Anatomy, University of Padua Medical School, Via A. Gabelli, 61, 35121 Padova, Italy; e-mail: [email protected] Jeffrey A. Towbin, The Heart Institute, Division of Pediatric Cardiology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA; e-mail: [email protected] Jesus G. Vallejo, Section of Infectious Diseases, Department of Pediatrics and Winters Center for Heart Failure Research, Baylor College of Medicine and Texas Children’s Hospital, 6621 Fannin Street, Houston, TX 77030, USA; e-mail: jvallejo@ bcm.tmc.edu Matteo Vatta, Baylor College of Medicine, Texas Children’s Hospital, 6621 Fannin Street, MC 19345-C, Houston, TX 77030, USA

ix

Preface

Acute myocarditis (AMC) and its sequelae, inflammatory cardiomyopathy (DCMi), are leading entities of heart failure and of cardiac transplantation. AMC is mostly caused by various cardiotropic viruses in the Western World. The long-anticipated association of dilated cardiomyopathy with chronic inflammation and viral persistence in terms of DCMi has been substantiated by more sensitive and specific diagnostic methods for analyzing material from endomyocardial biopsies (EMBs) during the last 15 years. This development has led to the broadly acknowledged “death of the Dallas criteria” as the single conditio sine qua non for the histological diagnosis of myocarditis. Moreover, these refined diagnostic techniques have been pertinent for successful randomized immunomodulatory trials. As such, immunosuppression, antiviral interferon treatment and immunoadsorption have shown significant beneficial results in selected DCMi patients. These compelling insights will likely lead to a renunciation of the limited usage of EMBs, seen as a consequence after the first immunosuppressive trial based on the Dallas criteria. In addition, cardiac magnetic resonance (CMR) has evolved to a powerful non-invasive diagnostic approach. “Cardiac inflammation is difficult to diagnose and, even if diagnosed, can we then treat it more effectively?” This was written 1772 by Jean Baptiste Sénag, physician to Louis XV. Are we yet able to answer this simple, but decisive question? In fact, DCMi is still an enigmatic disease, with highly diverging clinical courses, and potentially fatal outcomes. More robust prognostic variables and selection criteria for appropriate DCMi candidates and the corresponding immunomodulatory treatment strategies are needed. Almost yearly new key players of the immune system and of viral entry/persistence mechanisms are unraveled as relevant pathogenic and therapeutic targets in experimental myocarditis. “It is better to know some of the questions than all of the answers.” (James Thurber; 1894–1961). This volume focuses on major advances in DCMi over the last 10 years, encompassing: (1) Epidemiology and prognosis; (2) insights from experimental myocarditis; (3) alterations of the immune system in human DCMi; (4) diagnostic concepts of DCMi

xi

Preface

in EMBs and by CMR; and (5) conventional and immunomodulatory treatment strategies in DCMi. We thank the international group of leading authors who have contributed to this book. This volume serves as a valuable source for cardiologists, cardiovascular pathologists and related researchers to update their knowledge on DCMi. DCMi is specific cardiomyopathy entity, defined in 1996 by the WHO/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies, and since then research into this entity has made major steps from bench to bedside. January 2010

xii

H.-P. Schultheiss M. Noutsias

Myocarditis and inflammatory cardiomyopathy – clinical management, epidemiology and prognosis

Pathogenesis, diagnosis and treatment of pericarditis Bernhard Maisch1, Konstantinos Karatolios1, Sabine Pankuweit 1 and Arsen Ristic 2 1

Department of Internal Medicine-Cardiology, University Hospital of Giessen and Marburg, Marburg, Germany 2 Arsen Ristic, Department of Cardiology II, Belgrade University, Belgrade, Serbia

Abstract The spectrum of inflammatory pericardial syndromes includes acute, chronic and recurrent (relapsing) pericarditis, pericardial effusion and cardiac tamponade as well as constrictive pericarditis. The etiology of pericardial diseases is extensive, including infectious, non-infectious, systemic and autoimmune causes. A substantial proportion of idiopathic pericarditis represents viral infections or autoreactive pericarditis. The diagnosis of pericardial diseases is based on clinical presentation, electrocardiogram changes and echocardiographic findings. Pericardiocentesis, pericardioscopy with pericardial biopsy and contemporary pathology, immunohistochemistry and molecular biology techniques have extended the diagnostic armamentarium and contributed to etiopathogenetic understanding and treatment in pericardial diseases. Nevertheless, in uncomplicated cases, resolving to conventional anti-inflammatory treatment such a comprehensive approach is clinically not justified. On the other side, in chronic, recurrent or resistant forms an etiological diagnosis enables specific treatment. Furthermore, intrapericardial treatment after pericardiocentesis with cytostatic agents (cisplatin or thiotepa) in malignant pericardial effusions or sclerosing agents (triamcinolone, gentamycin) in chronic recurrent autoreactive effusions may prevent recurrences. Pericardiocentesis is life saving in cardiac tamponade and indicated in large effusions (> 20 mm in echocardiography in diastole), and suspected purulent, tuberculous and neoplastic pericarditis. Pericardiectomy is the only treatment for permanent constriction.

Classification and etiology of pericardial disease Inflammatory pericardial syndromes include acute, chronic and recurrent (relapsing) pericarditis, pericardial effusion and cardiac tamponade as well as constrictive pericarditis. Their etiology comprises infectious, systemic autoimmune, metabolic, toxic, neoplastic, postmyocardial infarction syndrome, postcardiotomy syndrome and autoreactive disorders (Tab. 1) [1–4].

Inflammatory Cardiomyopathy (DCMi) – Pathogenesis and Therapy, edited by Heinz-Peter Schultheiss and Michel Noutsias © 2010 Springer Basel

3

Bernhard Maisch et al.

Table 1. Etiology, incidence and pathogenesis of pericarditis Etiology

Incidence (%)

Pathogenesis

Infectious pericarditis - Viral (Coxsackie A9, B1-4, Echo 8, mumps, EBV, CMV, Varicella, Rubella, HIV, Parvo B19...) - Bacterial (pneumo-, meningo-, gonococcosis, hemophilus, Treponema pallidum, borreliosis, chlamydia, tuberculosis...) - Fungal (candida, histoplasma...) - Parasitary (Entameba histolytica, Echinococcus, toxoplasma...)

30–50a

5–10a

Multiplication and spread of the causative agent and release of toxic substances in pericardial tissue cause serous, serofibrinous or hemorrhagic (bacterial, viral, tuberculous, fungal) or purulent inflammation (bacterial)

Rare Rare

Pericarditis in systemic autoimmune disease -

Systemic lupus erythematosus Rheumatoid arthritis Spondylitis ankylosans Systemic sclerosis Dermatomyositis Periarteritis nodosa Reiter’s syndrome Familial Mediterranean fever

30b 30b 1b > 50b Rare Rare ~2b 0.7b

Cardiac manifestations of the basic disease, often clinically mild or silent

Type 2 (auto)immune process - Rheumatic fever - Postcardiotomy syndrome - Postmyocardial infarction syndrome - Autoreactive (chronic) pericarditis

Secondary, after infection/surgery b

20–50 ~ 20b 1–5b

23.1a

Mostly in acute phase 10–14 days after surgery DDg P. epistenocardica Common form

Pericarditis and pericardial effusion in diseases of surrounding organs -

4

Acute MI (P. epistenocardica) Myocarditis Aortic aneurysm Lung infarction Pneumonia Esophageal diseases Hydropericardium in CHF Paraneoplastic pericarditis

5–20b 30b Rare Rare Rare Rare Rare Frequent

1–5 days after transmural MI Accompanying epimyocarditis Dissection: hemorrhagic PE

No direct neoplastic infiltrate

Pathogenesis, diagnosis and treatment of pericarditis

Table 1 (continued) Etiology

Incidence (%)

Pathogenesis

Pericarditis in metabolic disorders -

Renal insufficiency (uremia) Myxedema Addison’s disease Diabetic ketoacidosis Cholesterol pericarditis Pregnancy

Frequent 30b Rare Rare Very rare Rare

- Viral/toxic/autoimmune - Serous, cholesterol rich PE - Membranous leak? - Transudation of cholesterol (sterile serofibrinous PE)

Traumatic pericarditis - Direct injury (penetrating thoracic injury, esophageal perforation, foreign bodies) - Indirect injury (Non-penetrating thoracic injury, mediastinal irradiation)

Rare

Neoplastic pericardial disease

35a

- Primary tumors - Secondary metastatic tumors: Lung carcinoma Breast carcinoma Gastric and colon Other carcinoma Leukemia and lymphoma Melanoma Sarcoma Other tumors Idiopathic

Rare

Rare Frequent 40c 22c 3c 6c 15c 3c 4c 7c 3.5a, in other series > 50a

Less frequent after introduction of topical convergent irradiation

Serous or fibrinous, frequently hemorrhagic effusion Accompanying disease during the infiltration of malignant cells

Serous, fibrinous, sometimes hemorrhagic PE with suspect viral or autoimmune secondary immunopathogenesis

CHF, congestive heart failure; DDg, differential diagnosis; MI, myocardial infarction; P., pericarditis; PE, pericardial effusion a Percentage related to the population of 260 subsequent patients undergoing pericardiocentesis, pericardioscopy and epicardial biopsy (Marburg pericarditis registry 1988–2001) [1] b Percentage related to the incidence of pericarditis in the specific population of patients (e.g., with systemic lupus erythematosus) c Percentage related to the population of patients with neoplastic pericarditis With permission from the ESC-Textbook on the Management of Pericardial Disease (Maisch et al. [12])

5


Pericardial syndromes Acute pericarditis The acute inflammation of the pericardium can be dry, fibrinous or effusive. Their etiology is extensive (Tab. 1).

Clinical presentation Retrosternal or left precordial chest pain is usually pleuritic or pericarditic in nature. It varies with posture, being worse in the supine position and relieved by sitting and leaning forward. Typical pericardial pain radiates to the trapezius ridge due to irritation of the phrenic nerves, which traverse the anterior pericardium and innervate the trapezius ridge [3, 5, 6]. Pericardial pain can also simulate ischemic pain, leading to a false diagnosis of myocardial infarction. Pericardial friction rub is diagnostic of pericarditis and is present in about 85% of patients with pericarditis. This requires a careful and frequent auscultation of the left sternal edge and the cardiac borders at expiration, with the patient sitting and leaning forward, since the pericardial rub can be transient [5, 6].

Electrocardiogram An electrocardiogram (ECG) often displays a rapid heart rhythm and, classically, diffuse concave ST elevations with PR segment depressions (Fig. 1). In approximately 40% of cases, the ECG is atypical [7].

Laboratory results They may include leukocytosis, elevated C-reactive protein and erythrocyte sedimentation rate [8, 9]. Elevated troponin levels are detectable in 1/3 of cases indicating epicardial and myocardial involvement [9, 10].

Echocardiography Echocardiography detects effusion and determines the extent of the effusion, its physiological significance, signs of constriction, concomitant myocardial disease or paracardial pathology. Acute pericarditis may be a transient, often benign disease, but also the first manifestation of an underlying neoplastic, purulent or tuberculous disorder that requires prompt specific therapy. A systematic, step-wise approach to the etiological diagnosis is essential to select the patients that can be effectively treated (Tab. 2, Figs 2 and 3) [4, 8, 10, 11]. A symptomatic treatment of acute pericarditis

6


Figure 1. Typical ECG findings in acute pericarditis with ST-segment elevation in almost all leads.

is based on chest pain management and anti-inflammatory therapy. Non-steroidal anti-inflammatory agents (NSAIDs) are its mainstay. We prefer diclophenac (75 mg bid), ibuprofen (200–600 mg tid) or indomethacin (25–50 mg tid) as alternatives to aspirin [12]. Indomethacin should not be used in patients with or suspected of having coronary artery disease, since it reduces coronary flow. Colchicine (0.5 mg bid) as monotherapy or added to NSAIDs is also very effective for initial treatment and for the prevention of recurrences [13]. Systemic corticosteroids are indicated for connective tissue diseases, and autoreactive or uremic pericarditis. Ibuprofen or colchicine should be added early for tapering of corticosteroids [4, 12]. Intrapericardial application of corticosteroids (triamcinolone) is very efficient and mainly avoids systemic side effects in patients with a large autoreactive and uremic pericardial effusion [2].

Chronic pericarditis Chronic (> 3 month) pericarditis comprises effusive, adhesive and constrictive forms [4, 5]. Symptoms include pericardial pain and fever and are related to pericardial inflammation. The diagnostic algorithm (Tab. 2) and symptomatic treatment are as in acute pericarditis.

7


Table 2. Diagnostic algorithm in acute pericarditis [2–4, 8, 10, 11] (with permission from [12]) Diagnostic test

Characteristic findings

Step I – Tests obligatory in all patients History and physical examination

- Identification of high-risk patients (should be hospitalized)a - Pericardial rub (mono-, bi-, or triphasic)

ECGb

- Stage I: anterior and inferior concave ST segment elevation. PR segment deviations opposite to P waves - Early stage II: all ST junctions return to the baseline. PR segments deviated - Late stage II: T waves progressively flatten and invert - Stage III: generalized T wave inversions in most leads - Stage IV: ECG returns to prepericarditis state

Echocardiography

- Effusion types B-D (Horowitz) - Signs of tamponade or concomitant heart or paracardial disease

Laboratory analyses

- ESR, CRP, LDH, leukocytes, parameters of renal and hepatic function, urine analyses - Troponin Ic, CK-MB

Chest x-ray

- Ranging from normal to “water bottle” shape. - To reveal pulmonary or mediastinal pathology

In self-limiting pericarditis (within 1 week) or known associated or systemic diseases no further diagnostic procedures are necessary. In patients symptomatic for > 1 week despite NSAID treatment, screening for systemic autoimmune disease (ANA, anti-ds-DNA, RF, C3, C4, immunoglobulins, immune complexes), thyroid disease, three sputum or gastricaspirate cultures for mycobacteria, serological tests (mycoplasma, toxoplasma, borrelia, legionela), and lymph node biopsy, tumor markers, and additional imaging (abdominal sonography, CT, MR) should be done. Step II – mandatory in tamponade, in large (> 20 mm) effusions and in suspected purulent, tuberculous or neoplastic etiology, or if previous tests were inconclusive in symptomatic patients resistant to conventional treatment Pericardiocentesis/ drainage

8

- Analyses of pericardial effusion can establish the diagnosis of viral, bacterial, tuberculous, fungal, cholesterol, and malignant pericarditis: - Analyses obligatory in all patients: - Cytology, cell counts - Acid-fast bacilli staining - Mycobacterium cultures (preferably with radiometric growth detection e.g., BACTEC-460) and PCR for M. tuberculosis - Biochemical analyses: specific gravity, protein level, glucose, LDH


Table 2 (continued) Diagnostic test

Characteristic findings

Pericardiocentesis/ drainage (continued)

- In areas with high incidence of tuberculosis: - Adenosine deaminase, interferon-gamma, pericardial lysozyme - In suspected autoreactive or viral pericarditis - PCR analyses for cardiotropic viruses - In suspected bacterial or fungal pericarditis - Cultures of pericardial fluid for aerobes, anaerobes and fungi (3x) - Blood cultures (3x) - In suspected chylopericardium - Cholesterol, triglycerides

Step III – Optional or if previous tests inconclusive in symptomatic patients resistant to conventional treatment Pericardial/epicardial biopsy (preferably by pericardioscopy)

- Histology (neoplastic and tuberculous pericarditis) - PCR for cardiotropic viruses, borreliosis and tuberculosis - Immunohistochemistry (autoreactive forms)

a

Indication for hospitalization: fever > 38°C, subacute onset, immunodepression, trauma, oral anticoagulant therapy, myopericarditis, severe pericardial effusion, or cardiac tamponade. Others can be treated as outpatients. b Typical lead involvement: I, II, aVL, aVF, and V3-V6. If ECG is first recorded in stage III, pericarditis cannot be differentiated by ECG from diffuse myocardial injury, “biventricular strain”, or myocarditis. Early repolarization is similar to stage I, but does not acutely evolve and J-point elevations are usually accompanied by a slur, oscillation, or notch at the end of the QRS just before and including the J point (best seen with tall R and T waves – large in early repolarization). Pericarditis is likely if in V6 the J point is > 25% of the T wave height (using the PR segment as a baseline). c A cTnI rise was detectable in 32.2%, but > 1.5 ng/ml in 7.6%. It was not a negative prognostic marker regarding the incidence of recurrences, constrictive pericarditis, cardiac tamponade or residual LV dysfunction.

Recurrent (relapsing) pericarditis Recurrent pericarditis encompasses two clinical forms: (1) the intermittent type with symptom-free intervals without therapy, and (2) the incessant type, in which the discontinuation of anti-inflammatory therapy causes the relapse [14]. The most common reasons of relapsing pericarditis are viral and “idiopathic” pericarditis, followed by postmyocardial infarction and postcardiotomy syndromes [14, 15]. Therapy aims at symptomatic treatment of acute episodes and the prevention of

9


Figure 2. Diagnostic and therapeutic approach to pericardial effusion.

recurrences. Symptomatic treatment includes exercise restriction and the management of acute episodes as in acute pericarditis with NSAIDs [4]. Colchicine has been demonstrated to prevent relapses [13, 15]. Corticosteroids should be restricted to patients with refractory symptoms and frequent crises. Viral infection should be excluded by analyses of pericardial fluid or epicardial tissue with polymerase chain reaction (PCR) prior to administering corticosteroids to prevent augmented viral replication leading to frequent recurrences [14]. Percutaneous balloon pericardiotomy (Fig. 4) or surgical pericardiectomy is beneficial in patients with frequent and symptomatic recurrences refractory to any other treatment [12].

Pericardial effusion Pericardial effusion can develop in any of the above-mentioned pericardial diseases. Large effusions are common with neoplastic, tuberculous, cholesterol or uremic pericarditis, with myxedema and parasitoses [16]. Large effusions develop slowly and therefore can be remarkably asymptomatic. Rapidly accumulating small effu-

10


Figure 3. Diagnostic algorithms in pericarditis. (1A) Pericardioscopy of fibrinous pericarditis with little changes on the epicardium. (1B) Pericardioscopy of exudative pericarditis with small petechiae. (1C) Pericardioscopy of exudative pericarditis with increase vascular injections. (2A) Pericardioscopy in lymphocytic pericarditis. (2B) Epicardial biopsy in lymphocytic pericarditis with CD45Rho-positive infiltrate. (2C) Cytology in lymphocytic pericarditis: numerous lymphocytes are found in the pericardial fluid. (2D) Immunohistological staining of an epicardial biopsy in lymphocytic pericardial effusion.

sions may result in symptomatic cardiac tamponade. The ECG may display low QRS and T-wave voltage, PR segment depression, ST-T changes, bundle branch block and electrical alternans [3]. In chest radiography large effusions resemble a water bottle heart (Fig. 5 left). Echocardiography classifies pericardial effusion and determines its hemodynamic significance. Effusions can be graded as small (separation of the pericardial layers in diastole < 10 mm), moderate (10–20 mm), large (> 20 mm) or very large (> 20 mm with compression of the heart) [4], or classified according to Horowitz into types B–F (Fig. 6) [17]. Hemodynamic compromise,

11


Figure 4. Transcutaneous balloon pericardiotomy. Left: After insertion of the only partially inflated balloon, showing the original small entrance to the pericardial sac. Middle: Schematic drawing of the procedure. Right: Fully inflated balloon, demonstrating a new pericardial window.

Figure 5. Left: Chest radiography with a large pericardial effusion (water bottle heart). In contrast to congestive heart failure, the pulmonary arteries are small and hardly visible. Right: Constrictive pericarditis with pericardial calcifications (arrows).

12


Figure 6. Classification of pericardial effusion according to Horowitz. EN: Endocardial; EP: epicardial; P: pericardial. Type A is a normal motion of both layers of the pericardium in systole and diastole; type B is a systolic separation of the epicardial and pericardial layer; type C shows a clear cut systolic separation of the epicardial and pericardial layer; type D shows the separation of both layers in systole and diastole; type E displays thickened pericardial layers without fluid separation and type F demonstrates residual fluid between the thickened and concomitantly moving epicardial and pericardial layer.

cardiac tamponade or suspected purulent or tuberculous pericardial effusion are absolute indications for periocardiocentesis and drainage (Tab. 3, Figs 7 and 8). Pericardiocentesis is not necessary when the diagnosis can be established by other means, or if the effusions are small, or respond well to conventional anti-inflammatory treatment [4, 12].

Cardiac tamponade Any pericardial disease can cause cardiac tamponade, but neoplastic effusion is the most frequent [3, 5]. Classical clinical findings include hypotension, elevated jugular venous pressure apparent as jugular venous distension, and pulsus paradoxus (inspiratory decline of systolic blood pressure greater than 10 mm Hg). The treat-

13


Table 3. Indications for pericardiocentesis/pericardial drainage (with permission from the ESC Textbook on the Management on Pericardial Diseases [12]) Class I indications (Evidence and/or general agreement that the procedure is useful and effective) - Cardiac tamponade - Effusions > 20 mm in echocardiography (diastole). - Suspected purulent or tuberculous pericardial effusion Class II indications (Conflicting evidence and/or a divergence of opinion about the usefulness/ efficacy of a procedure or treatment) Class IIa indications (The weight of evidence or opinion is in favor of the procedure or treatment) - Effusions 10–20 mm in echocardiography in diastole for diagnostic purposes other than purulent pericarditis or tuberculosis (pericardial fluid and tissue analyses, pericardioscopy, and epicardial/ pericardial biopsy) - Suspected neoplastic pericardial effusion Class IIb indications (Usefulness/efficacy is less well established by evidence or opinion) - Effusions < 10 mm in echocardiography in diastole for diagnostic purposes other than purulent; neoplastic or tuberculous pericarditis (pericardial fluid and tissue analyses, pericardioscopy and epicardial/pericardial biopsy). In symptomatic patients diagnostic pericardial puncture should be reserved to dedicated centers Contraindications (Class III) - Aortic dissection - Relative contraindications include uncorrected coagulopathy, anticoagulant therapy, thrombocytopenia < 50,000/mm3, small, posterior and loculated effusions - Pericardiocentesis is not necessary when the diagnosis can be made otherwise or the effusions are small and resolving under anti-inflammatory treatment

LA: left atrium, LV: left ventricle, RA: right atrium, RV: right ventricle, VCI: inferior vena cava How to perform pericardiocentesis - Recent and reliable echocardiography, best immediately before the procedure. The operator has to perform the echocardiography or see the video himself. - Pericardiocentesis guided by fluoroscopy should be performed in the cardiac catheterization laboratory in local anesthesia. The SUBXIPHOID APPROACH has been used most commonly, with a 8–17-cm, long blunt-tip needle (e.g., Tuohy-17) permitting the passage of the guide-wire, directed towards the left shoulder at a 30° angle to the frontal plane. - Pericardiocentesis guided by echocardiography can be performed in the intensive care unit, or at bed-side. Echocardiography should identify the shortest route to enter the pericardium INTERCOSTALLY (usually in the sixth or seventh rib space in the anterior axillary line). The intercostal arteries should be avoided by puncturing close to the upper margin of the rip. - It is essential that the needle approach the pericardium slowly under steady manual aspiration (negative pressure). As soon as the pericardial effusion is aspirated a soft J-tip guidewire should be inserted and after dilatation exchanged for a multi-holed pigtail catheter. - Strict aseptic conditions, ECG and blood pressure monitoring have to be provided. - Direct ECG monitoring from the puncturing needle is not an adequate safeguard. - Right-heart catheterization can be performed simultaneously, allowing the assessment of tamponade, hemodynamic monitoring of pericardiocentesis, and exclusion of constriction. - In large pericardial effusions it is prudent to drain < 1 l at the time of initial procedure to avoid the acute right-ventricular dilatation. Prolonged pericardial drainage is recommended after pericardiocentesis until the volume of effusion obtained by intermittent pericardial aspiration (every 4–6 h) fall to < 25 ml/day.

14


Figure 7. Pericardial access and drainage. The subxyphoidal approach is performed under fluoroscopic guidance with a long blunt-tip needle with a mandrel (Tuohy) utilizing the epicardial halo phenomenon in the left lateral angiographic view. The needle is inserted in the subxiphoid region at a 30° angle to the skin and directed towards the left shoulder. As the needle approaches the pericardium the operator intermittently injects contrast medium and attempts to aspirate fluid. After penetration of the pericardial layer and aspiration of pericardial fluid, the needle must be replaced by a soft J-guidewire. After dilatation, the guidewire is exchanged for a multi-holed pigtail catheter to drain the pericardial fluid. The lateral approach (Mayo clinic) is particularly suited, if only echocardiography is available [35].

ment of choice is urgent pericardial drainage. Medical treatment is only a temporary measure until pericardiocentesis is performed [4].

Constrictive pericarditis Constrictive pericarditis describes impaired expansion and cardiac filling by a chronically inflamed thickened pericardium. Rarely, constriction can exist in the absence of pericardial thickening with only ultrastructural changes [18]. The most frequent causes are tuberculosis, mediastinal irradiation and previous cardiac sur-

15


Figure 8. Pericardiocentesis under fluoroscopic guidance using the epicardial halo phenomenon (black vertical arrows) in the left lateral angiographic view. The left panel shows the puncturing needle approaching the pericardium (horizontal black arrow). The right panel displays contrast medium in the pericardial sac after successful pericardial puncture.

gery [4]. Patients complain about fatigue, breathlessness and abdominal swelling. In decompensated patients venous congestion, hepatomegaly, pleural effusions, ascites and a protein-loosing enteropathy can be observed [12]. The differential diagnosis between constrictive pericarditis and restrictive cardiomyopathy may create the most serious diagnostic problems [4, 12]. Physical findings, chest radiography (Fig. 5 right), echocardiography [19], CT, MRI, hemodynamics, and endomyocardial biopsy contribute to establishing the final diagnosis (Fig. 9). A recent report demonstrated that patients with constrictive pericarditis had only minimal elevated brain-natiuretic peptide (BNP) plasma levels, whereas in restrictive cardiomyopathy the BNP levels were significantly higher [20]. Pericardiectomy is the only treatment for permanent constriction (Tab. 4).

Specific forms of pericarditis Figure 10 shows the incidence of the various specific forms of pericarditis in a population of 260 patients undergoing pericardiocentesis, pericardioscopy and epicardial biopsy (Marburg pericarditis registry 1988–2001), with neoplastic pericarditis being the leading cause [1] The corresponding etiologically differentiated treatment regimen is also represented.

16


Figure 9. Physical, ECG, CT/MRI, echocardiographic and cardiac catheterization findings in constrictive pericarditis (modified from [12]). LV: left ventricle, RV: right ventricle, RA: right atrium, LA: left atrium, AV: atrioventricular, CVP: central venous pressure, VCI: vena cava inferior (modified from [12]).

Viral pericarditis Viral pericarditis is the most common infectious disease of the pericardium, resulting from direct viral attack, the immune response (antiviral or anticardiac) or both [3]. The most frequent causative agents are enterovirus, echovirus, cytomegalovirus, adenovirus, parvovirus B19, Epstein Barr virus, herpes humanus 6 virus, herpes simplex virus, influenza virus, hepatitis C virus and human immunodeficiency virus. A definite diagnosis can only be established through evaluation of pericardial fluid or epicardial/ pericardial tissue by PCR or in situ hybridization. However, in cases of suspected viral pericarditis responsive to conventional treatment with anti-inflammatory drugs, confirmation by molecular methods may not be necessary [4]. Symptomatic treatment is as in acute pericarditis. In chronic or recurrent confirmed viral pericarditis the following treatment options are currently under investigation: hyperimmunoglobulin

17


Table 4. Indications for pericardiectomy for constrictive pericarditis [4] Clinical findings supporting referal for pericardiectomy - Presence of increasing jugular venous pressure - Need for diuretic therapy - Evidence of hepatic insufficiency - Reduced exercise tolerance Contraindications for pericardiectomy - Very early constriction (asymptomatic patients, occult and functional class I) unless otherwise shown by: - Exercise testing (preferably with maximal O2 consumption) - Jugular venous pressure - Liver function tests - Transitory constriction - Extensive myocardial fibrosis and/or atrophy in CT/MRI - Severe, advanced disease (NYHA Class IV) (operative mortality 30–40% vs. 6–19%) With permission from the ESC-Textbook on Cardiovascular Medicine, chapter on the Management of Pericardial Disease (Maisch et al. [12])

Figure 10. Incidence of specific forms of pericarditis and their specific treatment in a population of 260 patients (from the Marburg Registry [1]).

18


for cytomegalovirus pericarditis, interferon alpha or beta for Coxsackie B pericarditis and immunoglobulin for adeno- and parvovirus B19 perimyocarditis [4, 21].

Bacterial (purulent) pericarditis Bacterial pericarditis is rare since the advent of antibiotic therapy, but it is always fatal if untreated. Despite treatment, mortality is about 40%, mostly due to severe complications (cardiac tamponade, toxicity and constriction) [22]. Purulent pericarditis is an absolute indication for pericardiocentesis (Tab. 3) with drainage and rinsing of the pericardial cavity, combined with high doses of systemic antibiotics [4]. Gram, acidfast and fungal staining, as well as cultures of obtained pericardial and body fluids are mandatory. Intrapericardial instillation of antibiotics (gentamycin) is useful but not sufficient. Frequent irrigation of the pericardial cavity with fibrinolytics (urokinase or streptokinase) may liquefy the purulent exudates [23], but open surgical drainage by subxiphoid pericardiotomy is preferable [4]. Indications for pericardiectomy include dense adhesions, loculated and thick purulent effusions, recurrence of tamponade, persistent infection and progression to constriction [4, 22].

Tuberculous pericarditis The presentation of tuberculous pericarditis is highly variable; mortality rates in untreated cases reaches 85% and pericardial constriction develops in 30–50% [24]. Pericarditis in a patient with proven extracardiac tuberculosis suggests tuberculous pericarditis [3]. The definite diagnosis requires the identification of Mycobacterium tuberculosis in the pericardial fluid or tissue and/or the presence of caseous granulomas in the pericardium [3, 4]. Increased adenosine deaminase activity and interferon gamma levels in pericardial fluid are also suggestive of a tuberculous etiology. The tuberculin test may show a false negative or false positive in about one third of patients [24]. Tuberculous pericarditis should be promptly treated with a combination of anti-tuberculous drugs for 9–12 months [4, 25]. The use of corticosteroids in tuberculous pericarditis remains controversial [26]. If pericardial constriction develops, pericardectomy is the only treatment.

Pericarditis in renal failure Pericardial involvement is frequent in patients with renal failure. Uremic pericarditis (occurring in 6–10% of patients) in advanced renal failure before dialysis has been instituted or shortly thereafter [3, 27] can be distinguished from dialysis-associated pericarditis, which occurs in about 13% of patients on maintenance dialysis, caused

19


by inadequate dialysis and/or fluid overload [28]. Many patients are asymptomatic, their heart rate may remain normal despite tamponade due to the autonomic dysfunction. The ECG is near normal in a significant proportion of patients, due to the lack of myocardial inflammation. Treatment includes hemo- or peritoneal dialysis (heparinfree to avoid hemopericardium) [3, 4]. Pericardiocentesis is indicated in tamponade and large refractory effusion. In large, resistant effusions the intrapericardial instillation of triamcinolone hexacetonide 50 mg every 6 h for 2–3 days is effective [4].

Autoreactive pericarditis The diagnostic criteria of autoreactive pericarditis are: (1) increased number of lymphocytes/mononuclear cells > 5000/mm3 or the presence of anti-myocardial antibodies in the pericardial fluid, (2) inflammation in epicardial/endomyocardial biopsies by r 14 cells/mm2, (3) exclusion of active viral infection in pericardial effusion and epicardial/endomyocardial biopsies (no virus isolation, negative PCR for major cardiotropic viruses, no IgM titer against cardiotropic viruses in the pericardial effusion), (4) exclusion of tuberculosis, Borrelia burgdorferi, Chlamydia pneumoniae and other bacterial infections by PCR and/or cultures, (5) absence of neoplastic infiltration in pericardial effusion and biopsy specimens, and (6) exclusion of systemic, metabolic disorders and uremia. The comprehensive invasive diagnostic approach may not be necessary if symptoms and effusion resolve under conventional inflammatory treatment. The treatment of autoreactive pericarditis with small pericardial effusions includes NSAIDs and/or colchicine. In patients with pericardial effusions sufficiently large to perform pericardiocentesis, the intrapericardial instillation of corticosteroids (triamcinolone) is highly efficient, allowing high local dose application while avoiding systemic side effects [2].

Post-cardiac injury syndrome Post-pericardiotomy syndrome The post-cardiac injury syndrome develops within days to months after cardiac or pericardial injury [3, 29], as a result of an immunopathological process. Treatment consists of NSAID or colchicine. Only rarely corticosteroids are needed.

Post-infarction pericarditis Post-infarction pericarditis can be distinguished as two clinical entities: (1) early or epistenocardiac pericarditis as a result of direct exudation, occurring in 5–20% of transmural myocardial infarction within the first 7 days. However, pericarditis epistenocardica is rarely clinically apparent, and (2) delayed pericarditis or Dressler’s

20


syndrome, which develops 1 week to months after the subendocardial or transmural myocardial infarction or due to extension of epistenocardiac pericarditis [3]. The treatment regimen includes aspirin or ibuprofen in high doses for 2–5 days [4]. Other NSAIDs may thin out the infarction zone and corticosteroids may delay infarction healing [3, 4].

Traumatic pericardial effusion Pericardial injury often leading to a hemopericardium may be iatrogenic or caused by chest trauma (penetrating or blunt). Procedures that can be complicated by a hemopericardium with or without tamponade include coronary interventions, percutaneous mitral valvuloplasty, endomyocardial biopsy and peacemaker lead implantation. Rescue pericardiocentesis should be performed in hemodynamically significant pericardial effusions [4]. A post-cardiac injury syndrome can develop within days to months after the pericardial injury.

Neoplastic pericarditis Primary tumors of the pericardium are very rare. Secondary malignancies are far more frequent (about 40 times), resulting from direct infiltration by tumors of adjacent organs, lymphatic or hematogenous dissemination [5]. Secondary malignant pericardial diseases arise from lung and breast cancer, malignant melanoma, lymphoma and leukemia. In almost two thirds of these cancer patients the pericardial effusion is not caused by the malignancy itself, but by irradiation and/or opportunistic infections [30]. The diagnosis can only be established by confirming the malignant infiltration of the pericardium by the analysis of the pericardial fluid or epicardial/pericardial biopsy [4]. Systemic anti-neoplastic treatment remains the mainstay, preventing up to 67% of recurrences [3]. Pericardiocentesis relieves symptoms in large pericardial effusions and is life-saving in tamponade. In confirmed malignant pericardial effusions, intrapericardial instillation of sclerosing, cytotoxic agents or immunomodulators may further reduce recurrence rate. Intrapericardial instillation of cisplatin is most effective in secondary lung cancer, and thiotepa more effective in secondary breast cancer metastasis [4, 31–34].

References 1 2

Maisch B, Ristic AD (2002) The classification of pericardial disease in the age of modern medicine. Curr Cardiol Rep 4(1): 13–21 Maisch B, Ristic AD, Pankuweit S (2002) Intrapericardial treatment of autoreactive

21


3 4

5 6 7 8

9 10 11 12

13

14 15 16 17

18

19

22

pericardial effusion with triamcinolone: The way to avoid side effects of systemic corticosteroid therapy. Eur Heart J 23: 1503–8 Spodick DH (1997) The Pericardium: A Comprehensive Textbook. Marcel Dekker, New York Maisch B, Seferovic PM, Ristic AD, Erbel R, Rienmüller R, Adler Y, Tomkowski WZ, Thiene G, Yacoub MH (2004) Guidelines on the diagnosis and management of pericardial diseases. Executive summary (Full text, educational slides, and pocket guidelines are available at www.escardio.org.) Eur Heart J 25(7): 587–610 Spodick DH (2001) Pericardial diseases. In: Braunwald E, Zipes DP, Libby P (eds): Heart Disease. 6th ed. Saunders, Philadelphia, 823–1866 Spodick DH (1975) Pericardial rub: Prospective, multiple observer investigation of pericardial friction in 100 patients. Am J Cardiol 35: 357–62 Bruce MA, Spodick DH (1980) Atypical electrocardiogram in acute pericarditis: Characteristics and prevalence. J Electrocardiol 13: 61–6 Imazio M, Demichelis B, Parrini I, Giuqqia M, Cecchi E, Gaschino G, Demarie D, Ghisio A, Trinchero R (2004) Day-hospital treatment of acute pericarditis: A management program for outpatient therapy. J Am Coll Cardiol 43(6): 1042–6 Lange RA, Hillis D (2004) Acute pericarditis. N Engl J Med 351: 2195–2202 Imazio M, Demichelis B, Cecchi E, Belli R, Ghisio A, Bobbio M, Trinchero R (2003) Cardiac troponin I in acute pericarditis. J Am Coll Cardiol 42(12): 2144–8 Permanyer-Miralda G (2004) Acute pericardial disease: Approach to the aetiologic diagnosis. Heart 90(3): 252–4 Maisch B, Soler-Soler J, Hatle L, Ristic A (2006) Pericardial diseases. In: Serruys PW, Camm AJ, Lüscher TF (eds): The ESC Textbook of Cardiovascular Medicine. Blackwell, London, 517–34 Adler Y, Finkelstein Y, Guindo J, Rodriguez de la Serna A, Shoenfeld Y, Bayes-Genis A, Saquie A, Bayes de Luna A, Spodick DH (1998) Colchicine treatment for recurrent pericarditis: A decade of experience. Circulation 97: 2183–5 Maisch B (2005) Recurrent pericarditis: Mysterious or not so mysterious. Eur Heart J 26: 631–633 Soler-Soler J, Sagristà-Sauleda J, Permanyer-Miralda G (2004) Relapsing pericarditis. Heart 90(11): 1364–8 Sagrista-Sauleda J, Merce J, Permanyer-Miralda G, Soler-Soler J (2000) Clinical clues to the causes of large pericardial effusions. Am J Med 109(2): 95–101 Horowitz MS, Schultz CS, Stinson EB, Harrison DC, Popp RL (1974) Sensitivity and specificity of echocardiographic diagnosis of pericardial effusion. Circulation 50(2): 239–47 Talreja DR, Edwards WD, Danielson GK, Schaff HF, Tajik AI, Tazelaar HD, Breen JF, Oh JK (2003) Constrictive pericarditis in 26 patients with histologically normal pericardial thickness. Circulation 108: 1852–7 Seferovic PM, Spodick DH, Maisch B (eds) (2000) Pericardiology. Contemporary answers to continuing challenges. Science, Belgrade


20

21

22

23 24

25

26

27 28 29

30

31

32

33

34

Leya FS, Arab D, Joyal D, Shioura KM, Lewis BM, Steen LH, Cho L (2005) The efficacy of brain natriuretic peptide levels in differentiating constrictive pericarditis from restrictive cardiomyopathy. J Am Coll Cardiol 45: 1900–1902 Maisch B, Ristic AD, Seferovic PM (2000) New directions in diagnosis and treatment of pericardial disease: An update by the Taskforce on pericardial disease of the World Heart Federation. Herz 25(8): 769–80 Sagrista-Sauleda J, Barrabes JA, Permanyer-Miralda G, Soler-Soler J (1993) Purulent pericarditis: Review of a 20-year experience in a general hospital. J Am Coll Cardiol 22: 1661–5 Ustunsoy H, Celkan MA, Sivrikoz MC, Kazaz H, Kilinc M (2002) Intrapericardial fibrinolytic therapy in purulent pericarditis. Eur J Cardiothorac Surg 22(3): 373–6 Sagrista-Sauleda J, Permanyer-Miralda G, Soler-Soler J (1988) Tuberculous pericarditis: Ten year experience with a prospective protocol for diagnosis and treatment. J Am Coll Cardiol 11(4): 724–8 Strang JI, Nunn AJ, Johnson DA, Casbard A, Gibson DG, Girlinq DJ (2004) Management of tuberculous constrictive pericarditis and tuberculous pericardial effusion in Transkei: Results at 10 years follow-up. QJM 97(8): 525–35 Ntsekhe M, Wiysonge C, Volmink JA, Commerford PJ, Mayosi BM (2003) Adjuvant corticosteroids for tuberculous pericarditis: Promising, but not proven. Q J Med 96: 593–9 Rostand SG, Rutsky EA (1990) Pericarditis in end-stage renal disease. Cardiol Clin 8: 701–6 Lundin AP (1990) Recurrent uremic pericarditis: A marker of inadequate dialysis. Semin Dial 3: 5–9 Maisch B, Berg PA, Kochsiek K (1979) Clinical significance of immunopathological findings in patients with post-pericardiotomy syndrome. I. Relevance of antibody pattern. Clin Exp Immunol 38: 189–97 Porte HL, Janecki-Delebecq TJ, Finzi L, Metois DG, Millaire A, Wurtz AJ (1999) Pericardioscopy for primary management of pericardial effusion in cancer patients. Eur J Cardiothorac Surg 16(3): 287–91 Martinoni A, Cipolla CM, Cardinale D, Civelli M, Lamantia G, Colleoni M, Fiorentini C (2004) Long-term results of intrapericardial chemotherapeutic treatment of malignant pericardial effusions with thiotepa. Chest 126(5): 1412–6 Bishiniotis TS, Antoniadou S, Katseas G, Mouratidou D, Litos AG, Balamoutsos N (2000) Malignant cardiac tamponade in women with breast cancer treated by pericardiocentesis and intrapericardial administration of triethylenethiophosphoramide (thiotepa). Am J Cardiol 86(3): 362–4 Maisch B, Ristic AD, Pankuweit S, Neubauer A, Moll R (2002) Neoplastic pericardial effusion: Efficacy and safety of intrapericardial treatment with cisplatin. Eur Heart J 23: 1625–31 Tomkowski WZ, Wisniewska J, Szturmowicz M, Kuca P, Burakowski J, Kober J, Fijalkowska A (2004) Evaluation of intrapericardial cisplatin administration in cases

23


35

24

with recurrent malignant pericardial effusion and cardiac tamponade. Support Care Cancer 12(1): 53–7 Spodick DH (2003) Acute cardiac tamponade. N Engl J Med 349(7): 684–90

Epidemiology and prognosis of myocarditis and dilated cardiomyopathy: Predictive value of clinical parameters and biopsy findings G. William Dec Cardiology Division, Massachusetts General Hospital, Heart Failure and Transplantation Unit, Boston, MA 02114, USA

Abstract The true incidence of myocarditis is difficult to assess due to the extreme diversity of its clinical manifestations. Myocarditis has been implicated in sudden death of young adults in 8–12% of cases and has been identified by endomyocardial biopsy as a cause of unexplained dilated cardiomyopathy in 10–12% of cases. Among cases of biopsy-proven myocarditis, the pathological distribution is typically lymphocytic 55%, borderline 22%, granulomatous 15%, and eosinophilic approximately 5%. Newer immunohistochemical techniques are better able to characterize specific inflammatory cellular infiltrates and have increased the sensitivity of biopsy for diagnosing myocarditis. The spectrum of viral genome detected by biopsy has shifted from Coxsackie virus B to adenovirus in the late 1990s and, more recently, to parvovirus B19 and other viruses. Clinical manifestations range from asymptomatic electrocardiographic abnormalities during viral pandemics to frank cardiogenic shock. Although endomyocardial biopsy remains the “gold standard” for establishing the diagnosis, cardiac magnetic resonance imaging with gadolinium enhancement has rapidly emerged as the screening procedure of choice. The natural history of myocarditis varies based on its initial clinical presentation. Myocarditis masquerading as acute myocardial infarction almost always results in full recovery of cardiovascular function and rapid improvement in segmental or global wall motion abnormalities. Acute dilated cardiomyopathy is the most frequent, clinically relevant presentation. Patients with mildly impaired left ventricular function typically improve within weeks or months, while patients with more marked ventricular dilatation or more longstanding symptoms may have substantial residual cardiac dysfunction. Fulminant myocarditis appears to have an excellent, long-term prognosis with event-free survival rate exceeding 90% at 10 years if aggressive hemodynamic support is provided during the acute phase of the illness. Mortality for biopsy-verified lymphocytic myocarditis averages 20% at 1 year and over 50% at 4 years. The survival rates are similar to observational data for patients with idiopathic dilated cardiomyopathy. Survival with giant cell myocarditis is substantially lower with fewer than 20% of patients surviving 5 years. Important predictors of adverse prognosis with biopsy-proven myocarditis include giant cell histopathology, persistent viral genome on repeat endomyocardial biopsy, elevated myocardial or circulatory Fas and Fas ligand levels, extent of left ventricular enlargement and sphericity, pulmonary hypertension and bundle branch block. Treatment remains supportive as no specific treatment for biopsy-proven myocarditis has yet been shown to be effective in randomized controlled trails. Inflammatory Cardiomyopathy (DCMi) – Pathogenesis and Therapy, edited by Heinz-Peter Schultheiss and Michel Noutsias © 2010 Springer Basel

25

G. William Dec

Pathological features Myocarditis is clinically and pathologically defined as inflammation of the myocardium [1]. The diagnosis may be established by endomyocardial biopsy, cardiac explantation during transplantation, or at autopsy. The more routine use of the endomyocardial biopsy during the past two decades has helped in achieving a better definition of the natural history of this disorder, clarifying its clinical and pathological correlates and defining its outcomes. Clinical presentations of the disease range from nonspecific systemic symptoms (such as fever, mylagias, palpitations, or exertional dyspnea) to fulminant, hemodynamic collapse and sudden death [2]. The extreme diversity of clinical manifestations has made the true incidence of myocarditis difficult to ascertain precisely. Recent prospective postmortem studies have implicated myocarditis in the sudden cardiac death of young adults at rates ranging from 8.6% to 12% [2, 3]. Myocarditis has been identified as the cause of dilated cardiomyopathy in approximately 10% of cases of unexplained dilated cardiomyopathy in a large prospective series from Johns Hopkins Medical Center [4]. The Dallas pathological criteria have served for over two decades as standardized guidelines for the histopathological diagnosis of myocarditis [5]. In this classification, active myocarditis is characterized by an inflammatory cellular infiltrate and evidence for myocyte necrosis; borderline myocarditis demonstrates inflammatory cellular infiltrates without evidence for myocyte injury. The amount of inflammation may be mild, moderate, or severe and its distribution may be focal, confluent, or diffuse, respectively. A retrospective study of 112 consecutive patients with biopsy-proven myocarditis from our institution has demonstrated the following histopathological distribution: lymphocytic 55%, borderline 22%, granulomatous 10%, giant cell 6%, and eosinophilic 6%. Sampling error remains a significant limitation to the diagnostic accuracy of endomyocardial biopsy. Although 4 to 6 biopsy samples are routinely obtained, a careful postmortem analysis of proven myocarditis cases demonstrated that over 17 samples were necessary to correctly diagnose myocarditis in over 80% of the cases [6]. Substantial interobserver variability further limits these diagnostic criteria [2]. Newer immunochemical methods using antibodies to characterize the type of inflammatory infiltrate more precisely have increased the sensitivity of biopsy for diagnosing myocarditis. Similarly, induction of major histocompatibility antigens in the microvasculature and on the surface of myocytes indicates active myocardium inflammation and is becoming increasingly useful in establishing the diagnosis of myocarditis and/or inflammatory cardiomyopathy [7].

26

Epidemiology and prognosis of myocarditis and dilated cardiomyopathy

Clinical presentations Clinical manifestations of myocarditis range from asymptomatic electrocardiographic abnormalities to cardiogenic shock [1, 2, 8, 9]. Transient repolarization abnormalities on ECG suggest myocardial involvement, which commonly occurs during community viral pandemics; most patients remain entirely asymptomatic. The incidence of a reported viral prodrome is highly variable, ranging from 10% to 80% of the patients with documented myocarditis [1, 2, 10]. Laboratory testing may show mild leukocytosis, an elevated erythrocyte sedimentation rate, or elevation of creatine kinase or troponin. Acute dilated cardiomyopathy is one of the most dramatic and clinically relevant presentations of acute lymphocytic myocarditis [2]. The causal link between myocarditis and acute dilated cardiomyopathy is most convincingly provided by endomyocardial biopsy findings. The two largest biopsy series using the Dallas pathological criteria have confirmed myocarditis in 9–16% of cases of new onset dilated cardiomyopathy [2, 4]. Further, the Giant Cell Myocarditis Study Group identified heart failure and cardiomyopathy as the primary presentation in over 70% of patients with giant cell myocarditis on biopsy [11]. Neither symptoms nor clinical course have been shown to correlate with histopathological features and outcome (see below). Myocarditis may occasionally masquerade as an acute coronary syndrome [12]. Dec and colleagues evaluated 34 patients who presented with a syndrome suggesting acute myocardial infarction but lacking coronary artery disease at catheterization. Endomyocardial biopsy revealed lymphocytic myocarditis in 27%, including several who presented with cardiogenic shock requiring intraaortic balloon counterpulsation. ECG changes suggesting acute myocardial ischemia typically include ST segment elevation in > 2 contiguous leads (54%), T-wave inversions (27%), widespread ST-segment depression (18%), and pathological Q waves (18–27%) [12, 13]. Segmental or global echocardiographic wall motion abnormalities are frequently evident despite angiographically normal coronary anatomy. Recent data by Mahrholdt et al. [14] suggest that such patients typically have myocardial damage due to parvovirus B (PVB) 19 infections involving the lateral wall of the left ventricle. Rapid recovery is the rule rather than the exception. Clinicians should consider acute myocarditis in younger patients (< 50 years of age) who present with acute coronary syndromes when coronary risk factors are absent, electrocardiographic abnormalities extend beyond a single coronary artery territory, or global rather than segmental left ventricular dysfunction is evident by left ventriculography or echocardiography. Myocarditis may also produce variable effects on the cardiac conduction system. Ventricular tachycardia is an uncommon initial manifestation of lymphocytic myocarditis but may develop during long-term follow-up [1, 2]. The Giant Cell Myocarditis Study Group has reported an initial incidence of ventricular tachycardia

27

G. William Dec

below 5% in their multicenter cohort [11]. However, ventricular tachyarrhythmia and high-grade conduction defects requiring implantable cardioverter defibrillator or pacemaker placement frequently complicate long-term management.

Natural history The natural history of myocarditis varies based on its initial clinical presentation. Myocarditis masquerading as myocardial infarction almost universally results in a full recovery of cardiovascular status in previously healthy adults [2, 12, 13]. Segmental wall motion abnormalities or global left ventricular dysfunction resolve within days to several months and recurrences are extraordinarily uncommon. Acute dilated cardiomyopathy is the most frequently observed clinical presentation. Patients who present with heart failure symptoms may have mildly compromised left ventricular function/ejection fraction (LVEF) of 40–50% and typically improve within weeks to months. Ventricular function typically normalizes but mild degrees of residual systolic dysfunction may persist. Chronic left ventricular dilatation may also persist, even after resolution of impaired systolic function. Alternatively, a smaller cohort of patients will present with more advanced left ventricular dysfunction (LVEF < 35%, left-ventricular end-diastolic dimension > 60 mm) [1, 2]. Among this group, approximately 50% of patients will develop chronic left ventricular dysfunction, and 25% of patients will progress ultimately to transplantation or death; however, the remaining 25% of patients will have spontaneous improvement in ventricular function with excellent long-term outcome [2]. Somewhat surprisingly, fulminant myocarditis has been described by McCarthy and colleagues as having the best long-term prognosis with an event-free survival rate exceeding 90% at 10 years [9]. These patients typically become acutely ill after a distinct viral prodrome, display severe cardiovascular collapse, and have histological evidence for multiple foci of active lymphocytic myocarditis [9, 13]. Patients with fulminant myocarditis typically have symptoms for less than 1 week, are febrile at presentation, and have echocardiographic features that include lack of left ventricular dilatation, increased septal wall thickness, and markedly depressed ventricular contractility [9]. Transient high-dose vasopressor or even mechanical circulatory support is often required. In view of their excellent long-term survival and subsequent rapid normalization of ventricular function over the course of days to several weeks, every effort should be made to hemodynamically support this population until ventricular recovery occurs. The Myocarditis Treatment Trial reported mortality rates for biopsy-verified myocarditis of 20% and 56% at 1 year and 4.3 years, respectively [15]. These survival rates are similar to observational data from the Mayo Clinic, which show a 5-year-survival rates of ~50% [16]. In that series, patients with biopsy-verified lymphocytic myocarditis had 1- and 5-year-survival rates that were identical to

28


Figure 1. Kaplan-Meier survival curves for patients with biopsy-proven giant cell myocarditis (n = 38) in the Giant Cell Myocarditis registry, and lymphocytic myocarditis (n = 111) enrolled in the Myocarditis Treatment trial. Duration refers to time from biopsy diagnosis. p < 0.001 by logrank test. Reproduced with permission from [11].

biopsy-negative patients with idiopathic dilated cardiomyopathy. Survival with giant cell myocarditis is substantially lower, with fewer than 20% of patients surviving 5 years (Fig. 1) [11].

Predictors of outcome Predicting prognosis for the individual patient with newly diagnosed cardiomyopathy due to biopsy-proven myocarditis remains problematic. Nonetheless, a number of important variables have been identified that may help guide the clinician (Tab. 1). Fuse et al. [17] evaluated clinical, hemodynamic, and laboratory variables in patients with acute lymphocytic myocarditis. No clinical variable was able to accurately predict either intermediate or long-term survival. Importantly, serum levels of soluble Fas and soluble Fas ligand were significantly higher among patients with fatal myocarditis, suggesting that the extent of cytokine activation and cellular

29

G. William Dec

Table 1. Predictors of clinical outcome in biopsy-verified myocarditis Predictors of adverse outcome Giant cell pathology Persistent viral genome on repeat endomyocardial biopsy Elevated circulating soluble Fas and Fas ligand levels Left ventricular enlargement and sphericity Left ventricular ejection fraction < 45% Pulmonary hypertension Bundle branch block Predictors of favorable outcome Fulminant clinical presentation Short duration of symptoms Myocardial infarction mimicry presentation Left ventricular ejection fraction >45% “Borderline” myocarditis histopathology Resolution of myocarditis on repeat endomyocardial biopsy Cardiac uptake on anti-myosin scintigraphy

apoptosis may provide important prognostic information [17]. Magnani et al. [10] used a multivariate predictive model and identified presentation with syncope, presence of bundle branch block, or the presence of LVEF below 40% as significant predictors of increased risk of death or need for transplantation. Some series have indicated that advanced heart failure symptoms (NYHA class III or IV) and elevated left ventricular filling pressures may also predict worse prognosis [2]. Pulmonary hypertension has also been shown to predict increased mortality in heart failure populations in general, and this relationship is especially important in patients with myocarditis [18]. Histological findings are highly predictive. Patients with lymphocytic myocarditis experience substantially better outcome than those with giant cell myocarditis [11]. At our institution, we have demonstrated the survival advantage for patients diagnosed with borderline myocarditis compared to the lymphocytic myocarditis, but other centers have not found such a clear-cut relationship between this histopathology and outcome [9]. Finally, histological resolution of active myocarditis on repeat endomyocardial biopsy has also been shown to predict a more favorable clinical outcome [2]. Noninvasive findings may also be of prognostic use. Cardiac uptake of antibodies directed against heavy-chain myosin has been shown to predict a high rate of spontaneous improvement in ventricular function, both among patients with biopsy-proven myocarditis and those with acute dilated cardiomyopathy and a negative biopsy for myocarditis [2, 13]. Lauer et al. [19] have reported that circulating anti-

30


myosin antibodies, present and 52% of their population at baseline, were associated with worst left ventricular systolic dysfunction among patients with chronic myocarditis. However, Frustaci and colleagues [20] have reported that patients with circulating cardiac autoantibodies were more likely to improve during immunosuppressive therapy for active lymphocytic myocarditis. Thus, the predictive value may depend upon the type of antibody detected and the treatment strategy employed. Genomic analysis of biopsy specimens has also produced somewhat conflicting prognostic information. Recent studies have suggested that multiple viral genomes may be found in the myocardium of patients with myocarditis or idiopathic dilated cardiomyopathy [21]. An early single center study by Figulla et al. reported significantly better 4-year event-free survival for enterovirus-positive (Coxsackie B3 viral genome) patients compared with enterovirus-negative patients (9% versus 55% survival) [22]. Furthermore, LVEF increased significantly from 35 ± 13% to 43 ± 9% (p < 0.05) in the virus-positive group, but remained unchanged in the virusnegative group. In distinct contrast, Why et al. detected enteroviral RNA in 34% of 120 consecutive patients with unexplained dilated cardiomyopathy. Enteroviral RNA presence was found to be an independent predictor of adverse outcome in this population. Actuarial survival at 2 years for genome-negative patients was substantially better than those who were positive (92% versus 68%; p = 0.02) [23]. Recently, Frustaci et al. [20] identified “non-responders” to immunosuppressive therapy as having a higher likelihood of viral genome on biopsy. In perhaps the most important observational study, Kühl et al. [24] evaluated the prognostic significance of viral persistence in the myocardium. Clearance of viral genome occurred in 37% of patients and was associated with a significant improvement in LVEF. In contrast, LVEF declined among patients with persistent viral genome. This difference was most pronounced when baseline LVEF was below 45%. In this group, LVEF increased by approximately 15 (EF units) among patients in whom virus was cleared, but fell 12 (EF units) among those with persistent virus. These most recent data seem to support the concept that viral genomic clearance may be more important than its initial detection on biopsy.

Indications for endomyocardial biopsy The role of endomyocardial biopsy remains controversial, given its invasive nature, the low rate of detection of myocarditis, and the high probability of sampling error. Biopsies performed within weeks of symptom onset have a higher potential for detecting acute myocarditis than those undertaken when symptoms are more longstanding. However, biopsies performed in chronic cardiomyopathy may be useful in detecting an active inflammatory component when new techniques such as HLA immunostaining or PCR for viral genomic detection are employed. Current American College of cardiology/American Heart Association Guidelines for the Treatment

31

G. William Dec

of Heart Failure describe biopsy as class IIB recommendation [25]. Biopsy is generally reserved for patients with rapidly progressive cardiomyopathy refractory to conventional therapeutic management, or an unexplained cardiomyopathy that is associated with progressive conduction system disease or life-threatening ventricular arrhythmias in whom the probability of giant cell myocarditis is particularly high and must be promptly differentiated from lymphocytic myocarditis with its better prognosis. It should also be considered when cardiovascular signs or symptoms develop among patients with systemic disease known to cause left ventricular dysfunction (Tab. 2). Table 2. Current indications for endomyocardial biopsy Exclusion of potential common etiologies of dilated cardiomyopathy (familial, alcoholrelated, ischemic, peripartum, cardiotoxin exposure) and one of the following features: - Subacute or acute symptoms of heart failure refractory to standard management - Substantial worsening of left ventricular function despite optimized pharmacological therapy - Development of hemodynamically significant arrhythmias, particularly ventricular tachycardia or progressive heart block - Heart failure with concurrent rash, fever, or peripheral eosinophilia - History of collagen vascular disease, such as systemic lupus erythematosus, scleroderma, or polyarteritis nodosum - New onset cardiomyopathy in the presence of known sarcoidosis, hemochromatosis, or amyloidosis - High clinical suspicion of giant cell myocarditis (young age, new subacute heart failure, or substantial arrhythmias without apparent etiology) Adapted with permission from [26].

Conclusions Myocarditis is responsible for approximately 10% of cases of acute dilated cardiomyopathy. The natural history of lymphocytic myocarditis is similar to that of idiopathic dilated cardiomyopathy. Unfortunately, clinical signs, electrocardiographic findings, and echo features do not possess sufficient sensitivity or specificity to differentiate lymphocytic myocarditis from giant cell disease. At present, biopsy is largely restricted to cases in which giant cell needs to be differentiated from lymphocytic myocarditis or to detect systemic diseases affecting the myocardium. Noninvasive methods, particularly the use of gadolinium-enhanced magnetic resonance imaging (CMRI), may help in selecting patients for biopsy and guiding the specific sites to be sampled. This imaging technique may further improve the sensitivity

32


and specificity of biopsy as a diagnostic tool. With the increased recognition of the pathogenesis of inflammatory cardiomyopathies, biopsy may ultimately prove to be useful for assessing prognosis and for guiding therapeutic strategies.

References 1 2 3 4

5

6

7 8 9

10

11

12 13 14

15

Feldman AM, McNamara D (2000) Myocarditis. N Engl J Med 343: 1388–98 Magnani JW, Dec GW (2006) Myocarditis: Current trends in diagnosis and treatment. Circulation 113: 876–90 Doolan A, Langlois N, Semsarian C (2004) Causes of sudden cardiac death in young Australians. Med J Aust 180: 110–2 Felker GM, Hu W, Hare JM, Hruban RH, Baughman KL, Kasper EK (1999) The spectrum of dilated cardiomyopathy. The Johns Hopkins experience with 1,278 patients. Medicine (Baltimore) 78: 270–83 Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon JT, Fenoglio JJ, Olsen EG, Schoen FJ (1987) Myocarditis. A histopathologic definition and classification. Am J Cardiovasc Pathol 1: 3–14 Hauck AJ, Kearney DL, Edwards WD (1989) Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: Implications for role of sampling error. Mayo Clin Proc 64: 1235–45 Narula N, McNamara DM (2005) Endomyocardial biopsy and natural history of myocarditis. Heart Fail Clin 1: 391–406 Reyes MP, Lerner AM (1985) Coxsackievirus myocarditis – with special reference to acute and chronic effects. Prog Cardiovasc Dis 27: 373–94 McCarthy RE 3rd, Boehmer JP, Hruban RH, Hutchins GM, Kasper EK, Hare JM, Baughman KL (2000) Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocarditis. N Engl J Med 342: 690–5 Magnani JW, Danik HJ, Dec GW Jr, DiSalvo TG (2006) Survival in biopsy-proven myocarditis: A long-term retrospective analysis of the histopathologic, clinical, and hemodynamic predictors. Am Heart J 151: 463–70 Cooper LT Jr, Berry GJ, Shabetai R (1997) Idiopathic giant-cell myocarditis – natural history and treatment. Multicenter Giant Cell Myocarditis Study Group Investigators. N Engl J Med 336: 1860–6 Dec GW Jr, Waldman H, Southern J, Fallon JT, Hutter AM Jr, Palacios I (1992) Viral myocarditis mimicking acute myocardial infarction. J Am Coll Cardiol 20: 85–9 Baughman KL (2005) Clinical presentations of myocarditis. Heart Fail Clin 1: 363–76 Mahrholdt H, Wagner A, Deluigi CC, Kispert E, Hager S, Meinhardt G, Vogelsberg H, Fritz P, Dippon J, Bock CT et al (2006) Presentation, patterns of myocardial damage, and clinical course of viral myocarditis. Circulation 114: 1581–90 Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME,

33

G. William Dec

16

17

18

19

20

21

22

23

24

25

26

34

Moon TE (1995) A clinical trial of immunosuppressive therapy for myocarditis. The Myocarditis Treatment Trial Investigators. N Engl J Med 333: 269–75 Grogan M, Redfield MM, Bailey KR, Reeder GS, Gersh BJ, Edwards WD, Rodeheffer RJ (1995) Long-term outcome of patients with biopsy-proved myocarditis: Comparison with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 26: 80–4 Fuse K, Kodama M, Okura Y, Ito M, Hirono S, Kato K, Hanawa H, Aizawa Y (2000) Predictors of disease course in patients with acute myocarditis. Circulation 102: 2829– 35 Cappola TP, Felker GM, Kao WH, Hare JM, Baughman KL, Kasper EK (2002) Pulmonary hypertension and risk of death in cardiomyopathy: Patients with myocarditis are at higher risk. Circulation 105: 1663–8 Lauer B, Schannwell M, Kühl U, Strauer BE, Schultheiss HP (2000) Antimyosin autoantibodies are associated with deterioration of systolic and diastolic left ventricular function in patients with chronic myocarditis. J Am Coll Cardiol 35: 11–8 Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri A (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107: 857–63 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111: 887–93 Figulla HR, Stille-Siegener M, Mall G, Heim A, Kreuzer H (1995) Myocardial enterovirus infection with left ventricular dysfunction: a benign disease compared with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 25: 1170–5 Why HJ, Meany BT, Richardson PJ, Olsen EG, Bowles NE, Cunningham L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89: 2582–9 Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112: 1965–70 Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG, Jessup M, Konstam MA, Mancini DM, Michl K et al (2005) ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation 112: e154–235 Wu LA, Lapeyre AC 3rd, Cooper LT (2001) Current role of endomyocardial biopsy in the management of dilated cardiomyopathy and myocarditis. Mayo Clin Proc 76: 1030–8

Animal models of myocarditis – autoimmunity and viral infection; therapeutic interventions

Autoimmune murine myocarditis and immunomodulatory interventions Christoph Berger and Urs Eriksson Departments of Internal Medicine and Research, University Hospital, Basel, Switzerland

Abstract Murine models of autoimmune myocarditis help in the study of immunological aspects of inflammatory dilated cardiomyopathy (DCMi). Whereas transgenic mice are preferred for delineating key molecules for drug targeting, rats are mainly used for addressing functional effects of specific immunomodulatory interventions. Promising interventions must suppress the expansion of heartspecific autoreactive T cells, abrogate the accumulation of monocytes and granulocytes within the myocardium, and prevent pathological remodeling of the chronically inflamed heart. Given their critical role in the progression of experimental autoimmune myocarditis, TNF-A, IL-1B, IL-6, IL-23, IL-17, MCP-1, and MIP-1A appear as suitable candidates for cytokine targeting. Key molecules of innate activation pathways, such as PPARG, represent other potential drug targets. A more preventive approach includes the development of vaccination strategies suppressing the expansion of autoreactive T cells or blocking pro-inflammatory cytokines. Taken together, we expect that insights from this model will soon result in clinically successful therapeutic options for DCMi.

The murine autoimmune myocarditis models Inflammatory dilated cardiomyopathy (DCMi) represents up to 50% of all cases of dilated cardiomyopathy, the most common cause of heart failure in young patients. Development of DCMi often follows viral myocarditis [1–3]. So far, several lines of clinical evidence suggest that autoimmune mechanisms play an important role in the progression of DCMi [4–6]. To study immunological aspects of DCMi pathogenesis in the absence of infection, murine models of experimental autoimmune myocarditis (EAM) have been developed. The EAM models also offer a helpful in vivo tool to design novel immunomodulatory treatment strategies. Neu et al. [7] developed a mouse model of autoimmune myocarditis by injection of isolated cardiac myosin from mouse hearts together with a strong adjuvant. Similarly, Kodama and Matsumoto [8] induced autoimmune myocarditis in rats by immunization with porcine myosin and Complete Freund’s adjuvant (CFA).


37

Christoph Berger and Urs Eriksson

For rats and mice, several pathogenic peptides representing the immunodominant epitopes of A-myosin were identified [9–11]. Immunization with these peptides usually results in a higher disease score and prevalence compared to whole myosin immunization [12, 13]. Furthermore, injection of activated dendritic cells loaded with myosin peptides induces autoimmune myocarditis in susceptible mice [14]. Importantly, the genetic background of the animals strongly determines disease severity and prevalence [7, 13, 15]. Myocarditis severity peaks 3 weeks after the first immunization with A-myosin/CFA. At this time point, inflammatory infiltrates mainly consist of CD45+CD11b+ monocytes, representing up to 70% of the cells. Lymphocytes, representing 10–20% of the infiltrating cells, are predominately of a CD3+CD4–CD8– or CD3+CD4+ phenotype and to a lesser extend CD3+CD8+ cells. The relative percentages of heart-infiltrating T cells increase further later on, peaking around 50 days post immunization in BALB/c mice [16]. This progression of myocarditis is associated with ventricular dilation and heart failure [14, 16, 17]. Although autoimmune myocarditis can be induced in mice as well as rats, histology differs substantially. Compared with mice, inflammation is generally more marked in susceptible rats, and includes giant cells [8, 18]. Compared to mice, rats are particularly suitable for echocardiography, and for studies addressing hemodynamic aspects of the inflamed hearts. The availability of transgenic mice lacking, or overexpressing, candidate genes makes the mouse model particularly favorable to study molecular disease mechanisms in vivo.

Targeting the recruitment of inflammatory cells in murine autoimmune myocarditis Activation of heart-resident interstitial, and bone marrow-derived cells constitutively expressing cardiac self antigens precedes the development of cardiac infiltrates in autoimmune myocarditis [13, 19]. Targeting the activation mechanisms of these cells might therefore ameliorate or abrogate autoimmune myocarditis. TNF-A, for example, represents a key cytokine mediating the activation of heart-resident interstitial cells in EAM [13]. Furthermore, TNF-A promotes the apoptosis of cardiomyocytes, impairs cardiac contractility [20] and promotes the development of an inflammatory cardiomyopathy phenotype in TNF-A-overexpressing transgenic mice [21]. It is therefore not surprising that mice lacking the TNFRp55 are protected from EAM [22]. Another approach to reduce disease is the prevention of infiltrating mononuclear cell accumulation. In fact, blocking macrophage migration inhibitory factor (MIF) in rats ameliorates autoimmune myocarditis [23]. Furthermore, mice lacking the chemokine receptors CCR2 or CCR5 for the mononuclear cell attractors MCP-1 or MIP-1A are protected from myocarditis [24]. Suppression of leukocyte chemotaxis

38

Autoimmune murine myocarditis and immunomodulatory interventions

might also explain the disease ameliorating effects of the anti-oxidative and antiinflammatory redox-regulatory protein thioredoxin-1 in immunized BALB/c mice [25].

Targeting T cells in autoimmune murine myocarditis In most mouse and rat strains, autoimmune myocarditis is a CD4+ T cell-mediated disease. Accordingly, injection of CD4+, but not CD8+, T cells isolated from diseased mice results in myocarditis in LPS-pretreated wild-type mice [13] or SCID (severe combined immunodeficiency) mice [26]. Humoral immune responses, on the other hand, are not essential for autoimmune myocarditis development in most mouse strains. In fact, B cell-deficient mice still develop disease after immunization with A-myosin [26, 27]. Nevertheless, autoantibodies might contribute to, and exacerbate, cardiac dysfunction in T cell-mediated cardiac inflammations. Anti-troponin autoantibodies for example, promote the development of severe dilated cardiomyopathy in mice lacking the immunoregulatory PD-1 receptor [28, 29]. Given the crucial role of CD4+ T cells in myocarditis development, T cells appear as promising target for immunomodulatory strategies. In fact, depletion of CD4+ T cells protects from myocarditis after subsequent myosin immunization [26, 30]. Whereas mice lacking CD8+ T cells develop exacerbated disease, myocarditis severity is markedly reduced in CD4–/– mice [30]. Furthermore, blocking costimulatory signals of T cell activation, such as CD40L-CD40, CD28-B7, and inducible costimulator (ICOS) [31, 32] as well as cytokine receptor CCR1 [33], reduced EAM severity in rats. Finally, immunoglobulin treatment reduced the activity of self-reactive lymphocytes in vitro, and in vivo after adoptive transfer of T cells from rats with EAM into SCID mice [34].

Modulating different T helper cell subsets in murine autoimmune myocarditis Several subsets of CD4+ T cells have been described, according to their cytokine production profile. Historically, CD4+ T cell responses were subdivided in Th1 and Th2 responses, which are cross-regulated and probably evolved to protect mammals more efficiently from infections with intracellular pathogens (Th1) and nematodes (Th2), respectively. Th1 cells are characterized by IFN-G production, whereas Th2 cells produce by definition IL-4 [35]. The Th1-Th2 concept has been used to explain the regulation of organ-specific autoimmune diseases. In the context of EAM, it has been suggested that autoimmune myocarditis in mice is a Th2-mediated disease. Indeed, anti-IL-4 antibody treatment of A/J mice, which exhibit characteristics of a Th2 response, markedly reduced disease severity

39


[36]. IL-4 receptor-deficient mice on the BALB/c background, however, are still susceptible to autoimmune myocarditis, albeit at a lower disease prevalence and showing an earlier peak of cardiac inflammation [37]. Th1 type immune responses, on the other hand, have long been considered as essential for the expansion and effector function of autoreactive CD4+ T cells in most autoimmune disease models. From this point of view, it was surprising that mice lacking the key Th1 cytokine IFN-G or its receptor developed increased myocarditis severity with a high mortality [37, 38]. Furthermore, mice lacking T-bet–/–, a T-box transcription factor required for Th1 cell differentiation and IFN-G production, show higher disease scores compared to wild-type controls [39]. These findings in mice contrast data from studies in rats showing that the administration of IL-12p70, a key factor for Th1 development, exacerbates EAM [40]. IL-12p70 is a heterodimer consisting of IL-12p40 and IL-12p35 subunits, which signals by binding to the IL-12 receptor (IL-12R) consisting of the IL-12RB1 and IL-12RB2 heterodimers [41]. Mice lacking IL-12p40, IL-12RB1, or STAT4, but not IL-12p35–/– mice, are completely protected from EAM [37, 42]. These observations argue against a critical role for IL-12p70 in the development of autoimmune myocarditis in mice. In fact, IL-23, a novel cytokine of the expanding IL-12-like cytokine family, is composed of a unique p19 subunit and a shared p40 subunit common with IL-12. Moreover, the IL-23 receptor shares the B1 chain with the IL-12R together with another transmembrane protein [43]. Thus, IL-23 rather than IL-12 appears to be responsible for autoimmune myocarditis development, and current levels of evidence suggest that IL-23 promotes the expansion of another specific CD4+ T cell subset characterized by IL-17 production [39, 44]. The idea that Th17 T cells are critical for EAM development also fits the observation that IL-6-deficient mice are protected from myocarditis [12]. Together with TGF-B, IL-6 is critical for the induction, whereas IL-23 promotes the expansion of Th17 T cells. So far, however, it is not known whether IL-17 plays a direct pathogenic role in EAM, or whether it just characterizes a specific self-pathogenic T helper cell subset.

Pharmacological interventions in murine autoimmune myocarditis Various immunosuppressive regimens have been tested in murine models of autoimmune myocarditis. Cyclosporine and mycophenolat mofetil (MMF) effectively prevented myocarditis in rats, whereas prednisolone and acetyl salicylic acid were ineffective [45]. Rapamycin ameliorated disease severity in rats [46] and in a recent study, FTY720, a pharmacological analogue of sphingosine binding to sphingosine1-phosphate receptors, inhibited the lymphocyte egress from secondary lymphoid organs and the thymus and prevented the development of EAM [47]. Several commonly used cardiovascular drugs also exhibit immunomodulatory properties, and the EAM model represents a good tool for evaluating these drugs for

40


their potential therapeutic use in DCMi. Findings in rats and mice, for example suggest anti-inflammatory and protective effects for the betablocking agent Carvedilol, but not for other betablockers [48]. Furthermore, the anti-arrythmic drug Amiodarone [49], ACE inhibitors like Captopril or Temocapril [50, 51], and angiotensin II receptor antagonists [52, 53] all suppressed autoimmune myocarditis. Anti-inflammatory effects of HMG-CoA reductase inhibitors have been described recently. In the EAM model, treatment with the HMG-CoA reductase inhibitors atorvastatin or fluvastatin improved cardiac function and reduced inflammation in myosin/CFA-immunized Lewis rats. Treated animals showed down-regulation of MHC class II antigen expression, and inactivation of nuclear factor-KB. Of note, these effects were independent of cholesterol reduction [54, 55]. Table 1 summarizes specific interventions in murine EAM.

Conclusions Murine autoimmune myocarditis has become a useful tool to develop immunomodulatory treatment strategies, and some of the experimentally defined treatment approaches have already been transferred to selected patients. A study on patients with chronically impaired cardiac function and biopsy-proven active lymphocytic myocarditis, for example, suggested a favorable effect of immunosuppression in individuals with no detectable viral genome on heart biopsy samples but elevated titers of cardiac autoantibodies in the serum [56]. Another prospective clinical study on patients with idiopathic dilated cardiomyopathy showed significant improvement in NYHA classification and left ventricular function after a 14 weeks of treatment with the HMG-CoA-reductase-inhibitor Simvastatin [57]. Despite the disease-promoting role of TNF-A in EAM, however, TNF-A antagonists have not yet been evaluated in patients with biopsy proven myocarditis, and TNF-A antagonist treatment led to increased morbidity in patients with dilated cardiomyopathy [58, 59]. Despite the fact that the transferability of experimental data to the clinic is often limited by the complexity of the human system, we expect that insights from the EAM model will contribute to the development of novel therapeutic options for DCMi in the future.

Acknowledgements: U.E. holds a Swiss National Foundation professorship and acknowledges research grants from Novartis, Polyphor, Menarini SA, and Astra Zeneca. Astra Zeneca also supports C.B.

41

42 Amelioration Exacerbation Amelioration Amelioration

Rat Rat/Mouse Rat Rat Rat/Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Rat Rat

Anti-CD28

Anti-CD40

ICOSIg*

CCR1* antagonist

IVIG*

Thioredoxin-1

Anti-IL-4

Anti-p19 (IL-23)

Vaccination against IL-17

Anti-IFN-G

Anti-TNF-A

sTNF-AR*

Tyrosinkinase-Inhibitor (Tyrphostin AG-556)

p38 MAPK-Inhibitor (FR167653)

Amelioration

Amelioration

Amelioration

Amelioration

Amelioration

Amelioration

Prevention/amelioration

Amelioration



Amelioration

Mouse

Anti-MIP-1A/anti-MCP-1*

Effect Amelioration

Species Rat

Intervention

Anti-MIF*

References

TNF-A suppression

TNF-A suppression

Blocking activation of heart resident antigen presenting cells?

Blocking activation of heart resident antigen presenting cells?

Inhibition of IFN-G mediated suppression of activated T cells?

Blocking IL-17-mediated effects

Inhibition of Th17 T cell expansion

Suppression of Th2 development

Not clear yet (suppression of leukocyte chemotaxis?)

Induction of T cell anergy?

Inhibition of T cell proliferation, Induction of T cell inactivation

Inhibition of T cell co-stimulation

Blocking of memory T cell survival

Induction of T cell specific anergy

Inhibition of monocyte activation and migration

[64]

[63]

[62]

[61]

[61]

[44]

[44]

[36]

[25]

[34, 60]

[33]

[32]

[14, 31]

[31]

[24]

Inhibition of macrophage migration [23]

Mode of action

Table 1. Effects of specific immunomodulatory interventions in murine experimental autoimmune myocarditis


Rat Rat Rat

IL-13-Ig fusion protein

IL-22-Ig fusion gene

IL10 gene transfer

Amelioration

Amelioration

Amelioration

Amelioration

providing the immunosuppressive cytokine IL-10

Non-specific suppression of various inflammatory mediators

Non-specific suppression of various inflammatory mediators

Inhibition of activation/expansion of autoreactive T cells?

[70]

[69]

[68]

[65–67]

*Abbreviations: MIF macrophage inhibiting factor, MIP macrophage inflammatory protein, MCP monocyte chemotactic protein, ICOS inducible costimulator, CCR Chemokine receptor, sTNF-AR soluble TNF-A receptor, IVIG intravenous immunoglobulines, MAPK mitogen-activated protein kinase, PPARG Peroxisome proliferator-activated receptor-gamma

Rat

PPARG ligand*


43


References 1 2 3

4

5

6

7

8

9

10

11

12

13

44

Drory Y, Turetz Y, Hiss Y, Lev B, Fisman EZ, Pines A and Kramer MR (1991) Sudden unexpected death in persons less than 40 years of age. Am J Cardiol 68: 1388–92 Kawai C (1999) From myocarditis to cardiomyopathy: Mechanisms of inflammation and cell death: Learning from the past for the future. Circulation 99: 1091–100 Wesslen L, Pahlson C, Lindquist O, Hjelm E, Gnarpe J, Larsson E, Baandrup U, Eriksson L, Fohlman J, Engstrand L et al (1996) An increase in sudden unexpected cardiac deaths among young Swedish orienteers during 1979–1992. Eur Heart J 17: 902–10 Caforio AL, Mahon NJ, Tona F and McKenna WJ (2002) Circulating cardiac autoantibodies in dilated cardiomyopathy and myocarditis: Pathogenetic and clinical significance. Eur J Heart Fail 4: 411–7 Caforio AL, Stewart JT, Bonifacio E, Burke M, Davies MJ, McKenna WJ and Bottazzo GF (1990) Inappropriate major histocompatibility complex expression on cardiac tissue in dilated cardiomyopathy. Relevance for autoimmunity? J Autoimmun 3: 187–200 Neumann DA, Burek CL, Baughman KL, Rose NR and Herskowitz A (1990) Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy. J Am Coll Cardiol 16: 839–46 Neu N, Rose NR, Beisel KW, Herskowitz A, Gurri-Glass G and Craig SW (1987) Cardiac myosin induces myocarditis in genetically predisposed mice. J Immunol 139: 3630–6 Kodama M, Matsumoto Y, Fujiwara M, Masani F, Izumi T and Shibata A (1990) A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin fraction. Clin Immunol Immunopathol 57: 250–62 Liao L, Sindhwani R, Leinwand L, Diamond B and Factor S (1993) Cardiac alpha-myosin heavy chains differ in their induction of myocarditis. Identification of pathogenic epitopes. J Clin Invest 92: 2877–82 Pummerer CL, Luze K, Grassl G, Bachmaier K, Offner F, Burrell SK, Lenz DM, Zamborelli TJ, Penninger JM and Neu N (1996) Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice. J Clin Invest 97: 2057– 62 Wegmann KW, Zhao W, Griffin AC and Hickey WF (1994) Identification of myocarditogenic peptides derived from cardiac myosin capable of inducing experimental allergic myocarditis in the Lewis rat. The utility of a class II binding motif in selecting selfreactive peptides. J Immunol 153: 892–900 Eriksson U, Kurrer MO, Schmitz N, Marsch SC, Fontana A, Eugster HP and Kopf M (2003) Interleukin-6-deficient mice resist development of autoimmune myocarditis associated with impaired upregulation of complement C3. Circulation 107: 320–5 Pummerer CL, Grassl G, Sailer M, Bachmaier KW, Penninger JM and Neu N (1996) Cardiac myosin-induced myocarditis: Target recognition by autoreactive T cells requires prior activation of cardiac interstitial cells. Lab Invest 74: 845–52


14

15 16

17 18

19

20 21

22

23

24

25

26 27

Eriksson U, Ricci R, Hunziker L, Kurrer MO, Oudit GY, Watts TH, Sonderegger I, Bachmaier K, Kopf M and Penninger JM (2003) Dendritic cell-induced autoimmune heart failure requires cooperation between adaptive and innate immunity. Nat Med 9: 1484–90 Rose NR, Herskowitz A, Neumann DA and Neu N (1988) Autoimmune myocarditis: A paradigm of post-infection autoimmune disease. Immunol Today 9: 117–20 Afanasyeva M, Georgakopoulos D, Belardi DF, Ramsundar AC, Barin JG, Kass DA and Rose NR (2004) Quantitative analysis of myocardial inflammation by flow cytometry in murine autoimmune myocarditis: Correlation with cardiac function. Am J Pathol 164: 807–15 Fairweather D, Kaya Z, Shellam GR, Lawson CM and Rose NR (2001) From infection to autoimmunity. J Autoimmun 16: 175–86 Kodama M, Zhang S, Hanawa H and Shibata A (1992) Immunohistochemical characterization of infiltrating mononuclear cells in the rat heart with experimental autoimmune giant cell myocarditis. Clin Exp Immunol 90: 330–5 Smith SC and Allen PM (1992) Expression of myosin-class II major histocompatibility complexes in the normal myocardium occurs before induction of autoimmune myocarditis. Proc Natl Acad Sci USA 89: 9131–5 Meldrum DR (1998) Tumor necrosis factor in the heart. Am J Physiol 274: R577–95 Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ and Feldman AM (1997) Dilated cardiomyopathy in transgenic mice with cardiacspecific overexpression of tumor necrosis factor-alpha. Circ Res 81: 627–35 Bachmaier K, Pummerer C, Kozieradzki I, Pfeffer K, Mak TW, Neu N and Penninger JM (1997) Low-molecular-weight tumor necrosis factor receptor p55 controls induction of autoimmune heart disease. Circulation 95: 655–61 Matsui Y, Okamoto H, Jia N, Akino M, Uede T, Kitabatake A and Nishihira J (2004) Blockade of macrophage migration inhibitory factor ameliorates experimental autoimmune myocarditis. J Mol Cell Cardiol 37: 557–66 Goser S, Ottl R, Brodner A, Dengler TJ, Torzewski J, Egashira K, Rose NR, Katus HA and Kaya Z (2005) Critical role for monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha in induction of experimental autoimmune myocarditis and effective anti-monocyte chemoattractant protein-1 gene therapy. Circulation 112: 3400–7 Liu W, Nakamura H, Shioji K, Tanito M, Oka S, Ahsan MK, Son A, Ishii Y, Kishimoto C and Yodoi J (2004) Thioredoxin-1 ameliorates myosin-induced autoimmune myocarditis by suppressing chemokine expressions and leukocyte chemotaxis in mice. Circulation 110: 1276–83 Smith SC and Allen PM (1991) Myosin-induced acute myocarditis is a T cell-mediated disease. J Immunol 147: 2141–7 Malkiel S, Factor S and Diamond B (1999) Autoimmune myocarditis does not require B cells for antigen presentation. J Immunol 163: 5265–8

45


28

29

30

31

32

33

34

35

36

37

38

39

40

46

Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N et al (2001) Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291: 319–22 Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, Ishida M, Hiai H, Matsumori A, Minato N et al (2003) Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med 9: 1477–83 Penninger JM, Neu N, Timms E, Wallace VA, Koh DR, Kishihara K, Pummerer C and Mak TW (1993) The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules. J Exp Med 178: 1837–42 Matsui Y, Inobe M, Okamoto H, Chiba S, Shimizu T, Kitabatake A and Uede T (2002) Blockade of T cell costimulatory signals using adenovirus vectors prevents both the induction and the progression of experimental autoimmune myocarditis. J Mol Cell Cardiol 34: 279–95 Matsui Y, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Tezuka K, Nakayama Y, Morimoto J et al (2003) Adenovirus-mediated gene transfer of ICOSIg fusion protein ameliorates ongoing experimental autoimmune myocarditis. Hum Gene Ther 14: 521–32 Futamatsu H, Suzuki J, Koga N, Adachi S, Kosuge H, Maejima Y, Haga T, Hirao K, Horuk R and Isobe M (2006) A CCR1 antagonist prevents the development of experimental autoimmune myocarditis in association with T cell inactivation. J Mol Cell Cardiol 40: 853–61 Shioji K, Yuan Z, Kita T and Kishimoto C (2004) Immunoglobulin treatment suppressed adoptively transferred autoimmune myocarditis in severe combined immunodeficient mice. Am J Physiol Heart Circ Physiol 287: H2619–25 Mosmann TR and Coffman RL (1989) TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7: 145–73 Afanasyeva M, Wang Y, Kaya Z, Park S, Zilliox MJ, Schofield BH, Hill SL and Rose NR (2001) Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype. Am J Pathol 159: 193–203 Eriksson U, Kurrer MO, Sebald W, Brombacher F and Kopf M (2001) Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: Induction by IL-12 and protection by IFN-gamma. J Immunol 167: 5464–9 Eriksson U, Kurrer MO, Bingisser R, Eugster HP, Saremaslani P, Follath F, Marsch S and Widmer U (2001) Lethal autoimmune myocarditis in interferon-gamma receptordeficient mice: Enhanced disease severity by impaired inducible nitric oxide synthase induction. Circulation 103: 18–21 Rangachari M, Mauermann N, Marty RR, Dirnhofer S, Kurrer MO, Komnenovic V, Penninger JM and Eriksson U (2006) T-bet negatively regulates autoimmune myocarditis by suppressing local production of interleukin 17. J Exp Med 203: 2009–19 Okura Y, Takeda K, Honda S, Hanawa H, Watanabe H, Kodama M, Izumi T, Aizawa


41

42

43 44

45

46 47

48

49

50 51

52

53

54

Y, Seki S and Abo T (1998) Recombinant murine interleukin-12 facilitates induction of cardiac myosin-specific type 1 helper T cells in rats. Circ Res 82: 1035–42 Watford WT, Hissong BD, Bream JH, Kanno Y, Muul L and O’Shea JJ (2004) Signaling by IL-12 and IL-23 and the immunoregulatory roles of STAT4. Immunol Rev 202: 139–56 Afanasyeva M, Wang Y, Kaya Z, Stafford EA, Dohmen KM, Sadighi Akha AA and Rose NR (2001) Interleukin-12 receptor/STAT4 signaling is required for the development of autoimmune myocarditis in mice by an interferon-gamma-independent pathway. Circulation 104: 3145–51 Brombacher F, Kastelein RA and Alber G (2003) Novel IL-12 family members shed light on the orchestration of Th1 responses. Trends Immunol 24: 207–12 Sonderegger I, Rohn TA, Kurrer MO, Iezzi G, Zou Y, Kastelein RA, Bachmann MF and Kopf M (2006) Neutralization of IL-17 by active vaccination inhibits IL-23-dependent autoimmune myocarditis. Eur J Immunol 36: 2849–56 Zhang S, Kodama M, Hanawa H, Izumi T, Shibata A and Masani F (1993) Effects of cyclosporine, prednisolone and aspirin on rat autoimmune giant cell myocarditis. J Am Coll Cardiol 21: 1254–60 Maeda K, Shioi T, Kosugi R, Yoshida Y, Takahashi K, Machida Y and Izumi T (2005) Rapamycin ameliorates experimental autoimmune myocarditis. Int Heart J 46: 513–30 Kitabayashi H, Isobe M, Watanabe N, Suzuki J, Yazaki Y and Sekiguchi M (2000) FTY720 prevents development of experimental autoimmune myocarditis through reduction of circulating lymphocytes. J Cardiovasc Pharmacol 35: 410–6 Yuan Z, Shioji K, Kihara Y, Takenaka H, Onozawa Y and Kishimoto C (2004) Cardioprotective effects of carvedilol on acute autoimmune myocarditis: Anti-inflammatory effects associated with antioxidant property. Am J Physiol Heart Circ Physiol 286: H83–90 Matsui S, Zong ZP, Han JF, Katsuda S, Yamaguchi N and Fu ML (2003) Amiodarone minimizes experimental autoimmune myocarditis in rats. Eur J Pharmacol 469: 165– 73 Godsel LM, Leon JS, Wang K, Fornek JL, Molteni A and Engman DM (2003) Captopril prevents experimental autoimmune myocarditis. J Immunol 171: 346–52 Yuan Z, Kishimoto C, Shioji K, Nakamura H, Yodoi J and Sasayama S (2002) Temocapril treatment ameliorates autoimmune myocarditis associated with enhanced cardiomyocyte thioredoxin expression. Cardiovasc Res 55: 320–8 Nimata M, Kishimoto C, Yuan Z and Shioji K (2004) Beneficial effects of olmesartan, a novel angiotensin II receptor type 1 antagonist, upon acute autoimmune myocarditis. Mol Cell Biochem 259: 217–22 Yuan Z, Nimata M, Okabe TA, Shioji K, Hasegawa K, Kita T and Kishimoto C (2005) Olmesartan, a novel AT1 antagonist, suppresses cytotoxic myocardial injury in autoimmune heart failure. Am J Physiol Heart Circ Physiol 289: H1147–52 Azuma RW, Suzuki J, Ogawa M, Futamatsu H, Koga N, Onai Y, Kosuge H and Isobe M

47


55

56

57

58

59

60

61 62

63

64

65

66

48

(2004) HMG-CoA reductase inhibitor attenuates experimental autoimmune myocarditis through inhibition of T cell activation. Cardiovasc Res 64: 412–20 Li WM, Liu W, Gao C and Zhou BG (2006) Immunoregulatory effects of atorvastatin on experimental autoimmune myocarditis in Lewis rats. Immunol Cell Biol 84: 274– 80 Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G and Maseri A (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107: 857–63 Node K, Fujita M, Kitakaze M, Hori M and Liao JK (2003) Short-term statin therapy improves cardiac function and symptoms in patients with idiopathic dilated cardiomyopathy. Circulation 108: 839–43 Chung ES, Packer M, Lo KH, Fasanmade AA and Willerson JT (2003) Randomized, double-blind, placebo-controlled, pilot trial of infliximab, a chimeric monoclonal antibody to tumor necrosis factor-alpha, in patients with moderate-to-severe heart failure: Results of the Anti-TNF Therapy Against Congestive Heart Failure (ATTACH) trial. Circulation 107: 3133–40 Kwon HJ, Cote TR, Cuffe MS, Kramer JM and Braun MM (2003) Case reports of heart failure after therapy with a tumor necrosis factor antagonist. Ann Intern Med 138: 807–11 George J, Barshack I, Malka E, Goldberg I, Keren P, Laniado S and Keren G (2001) The effect of intravenous immunoglobulins on the progression of experimental autoimmune myocarditis in the rat. Exp Mol Pathol 71: 55–62 Smith SC and Allen PM (1992) Neutralization of endogenous tumor necrosis factor ameliorates the severity of myosin-induced myocarditis. Circ Res 70: 856–63 Kubota T, Bounoutas GS, Miyagishima M, Kadokami T, Sanders VJ, Bruton C, Robbins PD, McTiernan CF and Feldman AM (2000) Soluble tumor necrosis factor receptor abrogates myocardial inflammation but not hypertrophy in cytokine-induced cardiomyopathy. Circulation 101: 2518–25 George J, Barshack I, Goldberg I, Keren P, Gazit A, Levitzki A, Keren G and Roth A (2004) The effect of early and late treatment with the tyrphostin AG-556 on the progression of experimental autoimmune myocarditis. Exp Mol Pathol 76: 234–41 Maruyama S, Kato K, Kodama M, Okura Y, Hirono S, Fuse K, Hanawa H, Nakagawa O, Nakazawa M, Miida T et al (2003) FR167653 suppresses the progression of experimental autoimmune myocarditis. Mol Cell Biochem 246: 39–44 Hasegawa H, Takano H, Zou Y, Qin Y, Hizukuri K, Odaka K, Toyozaki T and Komuro I (2005) Pioglitazone, a peroxisome proliferator-activated receptor gamma activator, ameliorates experimental autoimmune myocarditis by modulating Th1/Th2 balance. J Mol Cell Cardiol 38: 257–65 Maruyama S, Kato K, Kodama M, Hirono S, Fuse K, Nakagawa O, Nakazawa M, Miida T, Yamamoto T, Watanabe K et al (2002) Fenofibrate, a peroxisome proliferatoractivated receptor alpha activator, suppresses experimental autoimmune myocarditis by stimulating the interleukin-10 pathway in rats. J Atheroscler Thromb 9: 87–92


67

68

69

70

Yuan Z, Liu Y, Liu Y, Zhang J, Kishimoto C, Wang Y, Ma A and Liu Z (2003) Peroxisome proliferation-activated receptor-gamma ligands ameliorate experimental autoimmune myocarditis. Cardiovasc Res 59: 685–94 Elnaggar R, Hanawa H, Liu H, Yoshida T, Hayashi M, Watanabe R, Abe S, Toba K, Yoshida K, Chang H et al (2005) The effect of hydrodynamics-based delivery of an IL-13-Ig fusion gene for experimental autoimmune myocarditis in rats and its possible mechanism. Eur J Immunol 35: 1995–2005 Chang H, Hanawa H, Liu H, Yoshida T, Hayashi M, Watanabe R, Abe S, Toba K, Yoshida K, Elnaggar R et al (2006) Hydrodynamic-based delivery of an interleukin-22Ig fusion gene ameliorates experimental autoimmune myocarditis in rats. J Immunol 177: 3635–43 Watanabe K, Nakazawa M, Fuse K, Hanawa H, Kodama M, Aizawa Y, Ohnuki T, Gejyo F, Maruyama H and Miyazaki J (2001) Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation. Circulation 104: 1098–100

49

Coxsackievirus-induced murine myocarditis and immunomodulatory interventions Michel Noutsias1 and Peter Liu 2 1

Department of Internal Medicine – Cardiology, University Hospital of Marburg and Giessen / UKGM GmbH, Faculty of Medicine, Philipps University Marburg, Marburg, Germany 2 Heart & Stroke Medicine and Physiology, Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research, Toronto General Hospital, University of Toronto, Toronto, Canada

Abstract Human acute myocarditis (AMC) and its sequelae, inflammatory cardiomyopathy (DCMi), are mostly caused by cardiotropic viral infections in the Western world. Insights from coxsackievirus B (CVB)-induced experimental myocarditis have substantially contributed to a better understanding of the complex pathogenesis of myocarditis, and have also helped to explain the highly differential courses of human disease. Several inbred murine strains are available for modeling acute myocarditis and chronic ongoing myocarditis after intraperitoneal CVB inoculation. Acute, subacute and chronic phases can be differentiated. The onset and the differential courses of CVB-induced myocarditis are orchestrated by complex virus-host interactions. Several immunomodulatory regimens targeting key players of the innate and the adaptive immune system have unraveled the underlying mechanisms, and identified promising intervention strategies. The latter may, once they have been clinically evaluated, translate to novel treatment strategies for patients.

Introduction Myocarditis is an important cause of heart failure and sudden cardiac death, and is an indication for ventricular assist devices or heart transplantation, especially in young patients. However, it can also masquerade as dilated cardiomyopathy (DCM) in older adults [1–5]. Acute myocarditis and its sequelae, inflammatory cardiomyopathy (DCMi), are mostly induced by “cardiotropic” viral infections in the Western world [6–8]. In addition to standard heart-failure regimens [9], one major goal of research focusing on myocarditis has been to unravel the underlying pathogenic pathways involved in the detrimental courses of the disease to develop tailored treatment strategies for these selected patients with poor prognosis [10]. In this context, detailed knowledge of the virus-induced myocardial injury and the ensuing antiviral as well as anti-cardiac immune response is essential for the development of immunomodulatory modalities.


51

Michel Noutsias and Peter Liu

Animal (murine) models for virus-induced myocarditis have been established for coxsackievirus B3 (CVB). Insights from CVB-induced experimental myocarditis have substantially contributed to a better understanding of the complex pathogenesis of myocarditis, and have also helped to explain the highly differential course of human disease. Moreover, several immunomodulatory regimens have been published for murine CVB-induced myocarditis. Nonetheless, the complexity of the plethora of pathways and the heterogeneous effects of immunomodulatory treatment at the different stages of the disease have also shown that direct translation to human disease might take time and effort for clinical validation. It is broadly acknowledged that the onset and the differential courses of CVB-induced myocarditis are crucially orchestrated not only by the viral infection, but also by the host response. The latter depends among others on multiple factors, including genetics and multiple environmental factors (i.e., diet [11], exposure to toxic agents [12]).

CVB-induced myocarditis in inbred murine strains – A clue to the genetics of disease susceptibility Several mouse strains are susceptible for CVB-induced myocarditis. A defined dosage of CVB is usually administered intraperitoneally. In BALB/c and DBA/2 mice, acute myocarditis with a predictable course of intramyocardial viral infection and inflammation is induced by CVB [13, 14]. The virus in cleared by 14 days (d14) after intraperitoneal inoculation, when the neutralizing antiviral antibody titer reaches the highest levels. B10.A and C57BL/6J mice develop only mild myocarditis, but myocarditis can be aggravated by lipopolysaccharide injection at d0 and d4 after CVB infection [14–16]. In contrast, SWR/J, A.BY/SnJ, A.CA/SnJ, A.SW/SnJ, C3H/HeJ and NMRI strains develop chronic myocarditis with sustained persistence of lower levels of viral RNA and inflammatory cellular response compared to the acute phase up to d90 after the CVB infection, which is paralleled by a dilatation of the left ventricle (LV) and reduced LV function, resembling the aspects of human DCMi [17–23]. Of note, in addition to the mostly used single CVB infection murine experiments, a few publications have used a repetitive CVB infection protocol [24, 25]. This setup might resemble the situation in human myocarditis more closely since many adult myocarditis patients have had a past CVB infection, evidenced by anti-CVB IgG, before their CVB-associated acute myocarditis event, which is not surprising giving the ubiquitous presence of CVB [26]. These inbred strain-dependent differences clearly indicate the dominant influence of the genetic background of the host’s response to the CVB infection. A large body of evidence has unraveled differential courses of antiviral and anti-cardiac immune responses to the initial CVB infection in the respective murine strains. In addition, male mice are more prone to myocardial damage after CVB infection than their female littermates, which is paralleled by observations on a higher incidence

52

Coxsackievirus-induced murine myocarditis and immunomodulatory interventions

of severe courses of myocarditis and DCM in human males compared to females (ratio ~3:1) [27]. Recent epigenetic investigations on A/J (susceptible) and B10.AH2 (resistant) mice with common H2 haplotype elucidated three gene loci: (1) a locus on chromosome 1 on D1Mit200, and (2) a locus on chromosome 4 centered on D4Mit81, both of which were linked to sarcolemmal disruption in males; and (3) a locus on distal chromosome 3 on D3Mit19, which was linked to both myocardial infiltration and sarcolemmal disruption in females [28].

Pathogenesis and distinct phases of CVB-induced myocarditis The following two major pathogenic pathways have been shown to be involved in myocardial injury in CVB-induced myocarditis. These mechanisms are not mutually exclusive: a) Direct cytopathic effects of the virus (see below, acute phase of myocarditis): viral disruption of the cardiomyocyte’s structural integrity and metabolism (e.g., cleavage of dystrophin by the enteroviral protease 2A), and induction of apoptosis [29, 30]. Such direct cytopathic effects are also relevant in low-level infections without virus progeny, comparable to the situation in chronic human DCMi [31]. b) Virus-induced anti-cardiac immune response: Through molecular mimicry, epitope spreading and possibly other mechanisms, the primarily antiviral humoral immune response can become cross-reactive against myocardial proteins and epitope spreading, and can thus impair cardiac contractility [32–34]. Anti-cardiac T cell response is also thought to play a decisive role, but is less well understood. Anti-cardiac autoimmunity is thought to result from an initially exuberant, tissue destructive and (at lower levels) long-lasting, not self-limiting immune response in the chronic phase. After intraperitoneal injection of CVB, three distinct disease phases have been characterized in murine myocarditis: 1) Acute stage: during this stage, high CVB titers are present in the blood, spleen, pancreas and myocardium, consistent with systemic viremia [20]. Viral entry into the myocardium is mediated by specific receptors used by CVB. The internalizing receptor for CVB and adenoviruses 2 and 5 is the human coxsackie adenovirus receptor (CAR) [35]. Additionally, the decay-accelerating factor (DAF) plays a role as co-receptor for CVB serotypes B1, B2, and B5 [36]. Active CVB replication at d4 post infection (p.i.) is associated with early directly virusinduced cardiomyocyte injury by both necrosis and apoptosis in the absence of inflammatory infiltrates [30, 37]. At this stage, the humoral antiviral immune

53


response is mounted [38]. Innate immunity plays a central role during this stage [39]. The myocardial expression of several cytokines [interleukin (IL)-1, IL-6, IL-18, tumor necrosis factor (TNF) and interferons (IFN)] is elevated prior to immune cell infiltration, including chemokines [40–43]. Cytokine expression is involved in inducing remodeling of the extracellular matrix (ECM) by interacting with gene expressions of metalloproteinases (MMP) and their tissue inhibitors (TIMP) [44]. Furthermore, cytokines are key signaling molecules for innate immunity. Innate immunity is evolutionally conserved and is the first line of the defense mechanisms for protecting the host from invading microbial pathogens. It is activated during the acute stage in viral myocarditis [45, 46]. Myeloid differentiation factor-88 (MyD88), a key adaptor of toll-like receptors (TLR) of innate immune system signaling, plays a critical role in cellular inflammatory infiltration and cytokine expression in CVB-induced myocarditis [39]. Induction of TLR4 promotes antigen-presenting cells (APCs), expression of proinflammatory cytokines and decreases regulatory T cells [47, 48]. 2) Subacute phase: this stage of CVB-induced murine myocarditis spans from d5 to d14 p.i., and is characterized by the release of progeny virus, activation of natural killer (NK) cells, induction of cell adhesion molecules, myocardial infiltration by T cells and macrophages, cell-mediated myocytolysis and further innate immunity responses [39, 49–52]. The T cell response involves antigen-specific T cells with restricted usage of T cell receptor VB expression [53], cytotoxic T cells (CTLs), which can cause overwhelming tissue destruction [54], and regulatory T cells [55]. The cellular infiltrates also comprises NK cells [49] and macrophages [56, 57]. For antigen-specific T cell activation to occur, it is necessary for T cells to receive a costimulatory signal expressed on APCs, as well as the main signal provided by binding of T cell receptors to the antigen. Induction of B7-1, B7-2, and CD40 antigens on cardiomyocytes in CVB-induced myocarditis may convey an APC-like function to cardiomyocytes for the CTL attack by CTLs and NK cells [58]. Intramyocardial expression of various cytokines and chemokines is still increased at this stage. Cytokines orchestrate the type of T cell response, induce and maintain ECM remodeling, regulate cardiomyocyte apoptosis, and can contribute directly to depression of LV function [55, 59–66]. Antiviral neutralizing antibodies titers reach the highest levels at this stage. Efficacy of these immune responses in eliminating myocardial CVB infection and, simultaneously, the absence or limited anti-cardiac immunity are associated with the differential disease courses in the respective murine strains. The antiviral response teleologically aims at effective viral elimination, and is thus self limiting. However, the inflammatory response can abate, leading to healing and recovery; or persist, leading to chronic cardiomyopathy. 3) Chronic phase: this stage of CVB-induced murine myocarditis spans d15–d90 p.i. Whereas myocardial CVB infection is eliminated in both BALB/c and DBA/2 mice by d14 p.i., transition to a DCM/DCMi-like phenotype occurs in SWR/J,

54


A.BY/SnJ, A.CA/SnJ, A.SW/SnJ, C3H/HeJ and NMRI strains. Anti-cardiac autoimmunity can evolve through mechanisms such as molecular mimicry, facilitated by myocytolysis and the ensuing liberation of formerly secluded myocardial antigens, which are presented to and processed by the immune system [25, 32, 67, 68]. At this stage, intramyocardial inflammation persists at lower levels, and infiltration is substantially decreased compared to the subacute phase. Likewise, myocardial cytokine expression persists at lower, but still significantly elevated levels, possibly contributing to the ongoing ECM remodeling by maintaining an imbalance MMP and TIMP expression [22, 23, 66]. Microcalcification of the hearts as a result of irreversible tissue destruction can be observed. Continued inflammatory response with mobilization of fibroblast precursors can lead to fibrosis and scarring of the myocardium. Interestingly, this phase can be accompanied by low-level CVB persistence, detectable by PCR, but viral replication and progeny subside [23, 69], indicating ineffective viral elimination, which may be a further inducer of persisting low-level myocarditis. Dominance of potentially tissue-destructive and inadequate counterbalance by regulatory or protective mechanisms may be involved [55]. The delicate balance between the primarily advantageous antiviral immune response and an excessive activation of the immune system, associated with myocardial injury mediated by humoral and cellular autoimmunity irrespective of the viral infection, can finally lead to persistence of myocarditis, chronic depression of LV function and LV dilatation, resembling human DCMi. Heart-failure pathomechanisms become prominent at this chronic stage both in murine myocarditis and human DCMi. The major pathogenic mechanisms of CVB-induced myocarditis are illustrated in Figure 1.

Specific therapeutic strategies in CVB-induced murine myocarditis Even in the modern era in which effective heart-failure treatment is available, acute myocarditis continues to have a highly variable prognosis [70]. Since the early days of myocarditis research, insights from CVB-induced myocarditis have been intended to translate to human investigations to establish beneficial immunomodulatory options, with the major goal of limiting disease progression in selected patient groups presenting with either acute myocarditis (AMC) or DCMi. Insights from immunomodulatory interventions in CVB-induced myocarditis in rodents are in major part derived by administration of immunomodulatory medication, genetic deficiency (knockout) inbred animals, overexpression (knockin) inbred animals, transfer of immunocompetent cells or exogenous administration of immunoregulatory factors (i.e. cytokines). However, the genetic heterogeneity of human patients and further multiple not-well-defined host and environmental factors that impact

55


Figure 1. Pathogenesis of CVB-induced myocarditis

the highly diverse courses of AMC and DCMi cannot be mirrored in murine CVBinduced myocarditis. Limitations of diagnostic techniques for detecting myocarditis in humans hamper acquisition of potential candidates for clinical research [10, 71]. Furthermore, therapeutic interventions in murine models of CVB-induced myocarditis have focused on the very early stage of acute myocarditis, while patients usually present with substantial latency after the supposed viral infection (AMC: days, weeks or few months; DCMi: several months to years). In this context, “double-edged sword” effects are known for many key players of the antiviral immune response. This antithetic potential, ranging from beneficial (mostly associated with viral elimination) to detrimental (mostly induction of anti-cardiac autoimmunity) effects, depends among others on timing before or after CVB infection. Notwithstanding, the ongoing research progress, one should not neglect that the salutary effects of heart-failure medication, which is prescribed to AMC and DCMi patients, are partly due to additional immunomodulatory and further “pleiotropic” effects [72–75]. AMC and DCMi patients are usually under heart-failure medica-

56


tion when initiation of additional immunomodulation is considered [10, 34], which likely blurs the absolute effects of genuine immunomodulatory treatment. These conditions render direct translation of experimental insights from inbred murine CVB-induced myocarditis to human disease intricate. Feasibility and risk-to-benefit experience of most of the experimental approaches mentioned below have not yet been evaluated in humans.

Interference with viral entry mechanisms and antiviral immunomodulation Interference with viral entry mechanisms might be helpful in preventing CVB infection, and in limiting viral spread especially during the replicative phase. Antiviral strategies target both direct virus-mediated myocardial injury, and the disruption of the vicious circle of virus-induced anti-cardiac humoral response. These approaches might be helpful in the very early stage of acute CVB infection, as deduced from insights with WIN 54954, a compound that inhibits early events of picornavirus infection. It reduces viral loads but does not eradicate CVB completely, reduces early, but not late, mortality and apoptosis, without altering systemic or myocardial inflammation [76–80]. CVB entry to host cells is mediated by receptor endocytosis conveyed by CAR and DAF [35, 36]. CAR is substantially down-regulated on cardiomyocytes in the early postnatal period and remains at low levels in adults [81]. However, the “hen or egg” question is still not answered, since CAR induction on adult cardiomyocytes follows CVB myocardial infection [42]. Nonetheless, several lines of evidence have clearly shown the efficacy of inhibiting interactions between CVB and viral entry receptors on myocardial viral infection in preventing myocarditis and LV dysfunction [82–84]. Active vaccination has proven effective in murine CVB experiments [85], but experience is lacking in humans. IFNs are the key cytokines involved in the antiviral immune response. IFN-A and IFN-B are classified as type I IFNs, while IFN-G is a type II IFN. IFN protects against viral replication and promotes viral elimination in vitro and in vivo [63, 86–88]. However, myocardial CVB titers are not significantly decreased by IFN type I, in contrast to findings in the liver [63, 89], suggesting that IFN type I is involved in limiting CVB replication in the whole host, but has little effect on CVB replication in the heart. Disruption of the IFN type II signaling did not affect mortality and resulted in only a mild increase in CVB titers in the myocardium and liver [89]. Although IFN-B treatment was associated with CVB and adenovirus elimination in human DCMi in a single-center phase I trial, viral elimination was not achieved in the multicenter placebo-controlled phase II BICC (Bioferon in patients with chronic viral cardiomyopathy) trial in patients with parvovirus B19 associated DCMi [90, 91]. Therefore, whereas the systemic antiviral effects of IFN may be prominent, the intramyocardial effects of IFN are not well understood, and cannot be directly transferred from CVB-induced myocarditis to various cardiotropic viruses in human

57


DCMi. Ribavirin, a synthetic antiviral agent, reduces myocardial CVB titers and mortality; however, its clinical efficacy in this setting is yet to be elucidated [92].

Immunosuppression Immunosuppression with prednisone inhibited neutralizing antiviral antibodies, and eventually delayed CVB elimination with a detrimental outcome in murine CVB-induced myocarditis [38]. Immunosuppression with the more potent FK-506 diminished T and B cell functions, and reduced of myocarditic infiltrates; however, it also reduced antiviral neutralizing antibodies, resulting in higher CVB titers, and was associated with higher mortality rate [93]. Immunosuppression with cyclosporine was associated with higher mortality in the early and aggravated heart failure without reducing myocarditic lesions in the recovery phase [94]. Likewise, treatment with cyclophosphamide reduced myocarditic lesions but increased mortality [95]. These insights on immunosuppressive treatment of CVB-induced murine myocarditis are consistent with the results of the American Myocarditis Treatment trial, although no detrimental outcome was attributed to immunosuppression in this trial [96]. Immunosuppression can occasionally have a detrimental outcome in DCMi patients with viral persistence [97]. In selected DCMi patients with specific immunohistological proof of chronic inflammation, and after exclusion of viral persistence, immunosuppression has shown beneficial long-term results [98, 99]. These controversies highlight the importance of timing of immunomodulatory treatment in the natural course of the disease, as well as the necessity of detailed analysis of the underlying pathogenesis in humans [10, 100].

Immunomodulation of T cell response Depletion of CD4+ and CD8+ T lymphocytes, but not of either T cell subpopulation alone, reduced infiltration densities and mortality in CVB-induced myocarditis without, however, substantially affecting myocardial CVB titers [13, 101]. Ablation of T cells by rat anti-mouse monoclonal antibodies Lyt 1 and Lyt 2 during the viremic stage resulted in decreased mortality with less myocardial cellular infiltration and necrosis [102]. The pathogenic role of T cells in CVB-induced myocarditis has also been shown in mice lacking p56lck and CD45-knockout mice [103, 104]. Administration of anti-B7-1 antibody alone or combined with anti-CD40L suppressed myocardial injuries, which, in contrast, were exacerbated by anti-B7-2 antibody [105]. Transfer of T cells from AMC patients to severe combined immunodeficiency (SCID) mice that lack cell-mediated immunity induced myocarditis in the recipients [106]. The restricted usage of T cell receptor VB variants in experimental and human myocarditis indicates antigen specificity of T cell infiltrates [107, 108]. These and fur-

58


ther experiments clearly showed that T cells are key players of the adaptive antiviral immune response. However, specific subsets such as Perforin+ CTLs can be overall detrimental, without substantially affecting the course of CVB elimination [54]. In pediatric AMC patients, treatment with OKT3, a monoclonal anti-CD3 antibody, has been shown to hasten recovery of LV function [109]. Ablation strategies targeting pathogenic T cell clones might be more specific and less prone to adverse effects seen with systemic immunosuppression [110]. Notwithstanding the clear evidence of the antiviral assignment of T cells in CVB-induced myocarditis mentioned above, T cells and macrophages derived from the reticuloendothelial system serving as an extracardiac CVB reservoir might be involved in CVB entry mechanisms in terms of a “Trojan horse scenario” [10, 111]. Furthermore, inflammatory infiltrates are involved in ECM remodeling. In this context, MMP-9 deficiency promoted inflammatory infiltration and fibrosis, and, in contrast to the suggested role of T cells in CVB elimination, was also associated with higher myocardial CVB titers [112], which again confirms the antithetic role of several key players of the immune system in CVB-induced myocarditis.

Immunomodulation of humoral response Neutralizing antiviral antibodies are crucially involved in viral elimination in CVBinduced myocarditis and contribute to resistance in developing the chronic stage [38]. Strain-specific diversity of susceptibility to CVB-induced chronic-persistent myocarditis is partly explained by the induction of anti-cardiac autoantibodies [17]. These antiviral antibodies should not be inhibited by immunosuppression, as exemplified by prednisone delaying CVB elimination [38]. In the subacute and especially chronic stage, however, multiple anti-cardiac autoantibodies are induced [113]. Specific treatment of autoantibodies, such as the immunoadsorption established for human DCMi, does not exist for murine CVB myocarditis [114]. Highdose immunoglobulin treatment suppressed CVB-induced myocarditis in the acute viremic stage and induced an anti-inflammatory effect in the subsequent aviremic stage, paralleled by a reduction of the splenic B cell population and of neurohumoral activity and by improvement of ECM remodeling [115, 116]. In human acute DCM, however, no sustained beneficial hemodynamic results could be achieved in the randomized Intervention in Myocarditis and Acute Cardiomyopathy (IMAC) trial compared to controls [117].

Immunomodulation with cytokines Th1-type immune responses, mediated by IFN and other cytokines such as IL-12, are generally pivotal for antiviral responses. IFN-B deficiency was associated with

59


increased mortality, albeit myocardial viral loads were not altered [63]. IFN-G deficiency exacerbated CVB replication [47]. Elevated IFN-G and decreased TNF-A expression were associated with attenuated myocardial damage in CD4(–/–)CD8(–/–) mice [101]. IL-12-receptor deficiency resulted in decreased myocardial viral replication and inflammation [47]. However, transgenic overexpression of IFN-G induced active myocarditis and DCM [118]. Cardiac-specific overexpression of TNF-A induced DCM [65]. Some cytokines decrease the production of other proinflammatory cytokines and are therefore classified “anti-inflammatory”. As such, IL-10 had protective effects in CVB myocarditis [119]. IL-4 suppressed myocardial inflammation and up-regulated several MMP, ameliorating LV-dysfunction in CVB-induced myocarditis [120]. Transforming growth factor-B (TGF-B) among other cytokines is an important mediator of ECM remodeling [121]. A recent review summarizes the available interventions in murine CVB myocarditis targeting key players of ECM remodeling [122]. These experiments clearly show the potential of cytokines as both effectors and targets, and as indicators of promising immunomodulatory approaches. However, the partly antithetic effects of cytokine interventions depending on dosage, timing within the natural course of the disease, and on other factors such as sex [59] also indicate that therapeutic interventions targeting specific cytokines might induce secondary effects within complex signaling networks, which are hard to control. For instance, MMP-9 deficiency led to higher infiltration densities and higher myocardial expression of IFN-B and IFN-G; however, myocardial CVB titers were also increased in these animals [112]. This clearly shows that categorical classification of cytokines and other key components of the immune system into “bad guys” and “good guys” is too simplistic.

Immunomodulation of the innate immune system TLR4 deficiency significantly reduced inflammatory lesions, myocardial CVB replication, and myocardial expression of IL-1B and IL-18 [47]. Deficiency of MyD88, the adaptor for TLR2, TLR4, TLR5, TLR7 and TLR9 signaling, decreased myocardial CVB titers and improved survival. MyD88 deficiency also led to up-regulation of IFN-B and of IFN regulatory factor-3, while IL-1B, TNF-A, IFN-G and IL-18 were down-regulated. Furthermore, it was accompanied by down-regulation of CAR and p56lck, which are involved in CVB entry of the myocardium [39]. These experiments provided evidence of the complex interactions between the innate and the adaptive immune system in CVB-induced myocarditis. TLR2, TLR3, TLR4, TLR7 and TLR9 induce type I IFN, which partly explains these experimental data [123]. In human tissues, however, CVB induces mainly TLR8 [124]. Further research is warranted to unravel whether modulation of pathways involving the innate immune system might be a promising strategy for human AMC and DCMi.

60


References 1

2

3 4

5

6

7

8 9

10

11

12

Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I et al (1996) Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 93: 841–2 Taylor DO, Edwards LB, Boucek MM, Trulock EP, Waltz DA, Keck BM, Hertz MI (2006) Registry of the International Society for Heart and Lung Transplantation: Twenty-third official adult heart transplantation report – 2006. J Heart Lung Transplant 25: 869–79 Karjalainen J, Heikkila J (1999) Incidence of three presentations of acute myocarditis in young men in military service. A 20-year experience. Eur Heart J 20: 1120–5 D’Ambrosio A, Patti G, Manzoli A, Sinagra G, Di Lenarda A, Silvestri F, Di Sciascio G (2001) The fate of acute myocarditis between spontaneous improvement and evolution to dilated cardiomyopathy: A review. Heart 85: 499–504 Noutsias M, Seeberg B, Schultheiss HP, Kühl U (1999) Expression of cell adhesion molecules in dilated cardiomyopathy: Evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99: 2124–31 Kühl U, Pauschinger M, Bock T, Klingel K, Schwimmbeck CP, Seeberg B, Krautwurm L, Noutsias M, Poller W, Schultheiss HP et al (2003) Parvovirus B19 infection mimicking acute myocardial infarction. Circulation 108: 945–50 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111: 887–93 Andreoletti L, Leveque N, Boulagnon C, Brasselet C, Fornes P (2009) Viral causes of human myocarditis. Arch Cardiovasc Dis 102: 559–68 Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson PA, Stromberg A, van Veldhuisen DJ, Atar D, Hoes AW et al (2008) ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur Heart J 29: 2388–442 Noutsias M, Pauschinger M, Poller WC, Schultheiss HP, Kuhl U (2004) Immunomodulatory treatment strategies in inflammatory cardiomyopathy: Current status and future perspectives. Expert Rev Cardiovasc Ther 2: 37–51 Beck MA, Shi Q, Morris VC, Levander OA (1995) Rapid genomic evolution of a nonvirulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates. Nat Med 1: 433–6 Ilback NG, Wesslen L, Fohlman J, Friman G (1996) Effects of methyl mercury on cytok-

61


13 14 15 16

17

18

19

20

21 22

23

24

25

26 27

62

ines, inflammation and virus clearance in a common infection (coxsackie B3 myocarditis). Toxicol Lett 89: 19–28 Woodruff JF, Woodruff JJ (1974) Involvement of T lymphocytes in the pathogenesis of coxsackie virus B3 heart disease. J Immunol 113: 1726–34 Huber SA, Lodge PA (1986) Coxsackievirus B-3 myocarditis. Identification of different pathogenic mechanisms in DBA/2 and Balb/c mice. Am J Pathol 122: 284–91 Lane JR, Neumann DA, Lafond-Walker A, Herskowitz A, Rose NR (1991) LPS promotes CB3-induced myocarditis in resistant B10.A mice. Cell Immunol 136: 219–33 Heymans S, Pauschinger M, De Palma A, Kallwellis-Opara A, Rutschow S, Swinnen M, Vanhoutte D, Gao F, Torpai R, Baker AH et al (2006) Inhibition of urokinase-type plasminogen activator or matrix metalloproteinases prevents cardiac injury and dysfunction during viral myocarditis. Circulation 114: 565–73 Wolfgram LJ, Beisel KW, Herskowitz A, Rose NR (1986) Variations in the susceptibility to Coxsackievirus B3-induced myocarditis among different strains of mice. J Immunol 136: 1846–52 Chow LH, Gauntt CJ, McManus BM (1991) Differential effects of myocarditic variants of Coxsackievirus B3 in inbred mice. A pathologic characterization of heart tissue damage. Lab Invest 64: 55–64 Mall G, Klingel K, Albrecht M, Seemann M, Rieger P, Kandolf R (1991) Natural history of Coxsackievirus B3-induced myocarditis in ACA/Sn mice: Viral persistence demonstrated by quantitative in situ hybridization histochemistry. Eur Heart J 12 Suppl D: 121–3 Klingel K, Kandolf R (1993) The role of enterovirus replication in the development of acute and chronic heart muscle disease in different immunocompetent mouse strains. Scand J Infect Dis Suppl 88: 79–85 Feldman AM, McNamara D (2000) Myocarditis. N Engl J Med 343: 1388–98 Rutschow S, Leschka S, Westermann D, Puhl K, Weitz A, Ladyszenskij L, Jaeger S, Zeichhardt H, Noutsias M, Schultheiss HP et al (2010) Left ventricular enlargement in coxsackievirus-B3 induced chronic myocarditis – Ongoing inflammation and an imbalance of the matrix degrading system. Eur J Pharmacol 630: 145–51 Gluck B, Schmidtke M, Merkle I, Stelzner A, Gemsa D (2001) Persistent expression of cytokines in the chronic stage of CVB3-induced myocarditis in NMRI mice. J Mol Cell Cardiol 33: 1615–26 Nakamura H, Yamamoto T, Yamamura T, Nakao F, Umemoto S, Shintaku T, Yamaguchi K, Liu P, Matsuzaki M (1999) Repetitive coxsackievirus infection induces cardiac dilatation in post-myocarditic mice. Jpn Circ J 63: 794–802 Takata S, Nakamura H, Umemoto S, Yamaguchil K, Sekine T, Kato T, Nishioka K, Matsuzaki M (2004) Identification of autoantibodies with the corresponding antigen for repetitive coxsackievirus infection-induced cardiomyopathy. Circ J 68: 677–82 Woodruff JF (1980) Viral myocarditis. A review. Am J Pathol 101: 425–84 Olbrich HG (2001) [Epidemiology-etiology of dilated cardiomyopathy]. Z Kardiol 90 Suppl 1: 2–9


28

29

30 31

32

33

34 35

36

37

38 39

40

41

42

Aly M, Wiltshire S, Chahrour G, Osti JC, Vidal SM (2007) Complex genetic control of host susceptibility to coxsackievirus B3-induced myocarditis. Genes Immun 8: 193– 204 Badorff C, Lee GH, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, Knowlton KU (1999) Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med 5: 320–6 Huber SA, Budd RC, Rossner K, Newell MK (1999) Apoptosis in coxsackievirus B3-induced myocarditis and dilated cardiomyopathy. Ann N Y Acad Sci 887: 181–90 Wessely R, Henke A, Zell R, Kandolf R, Knowlton KU (1998) Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: An approach to the study of enteroviral persistence in cardiac myocytes. Circulation 98: 450–7 Gauntt CJ, Arizpe HM, Higdon AL, Wood HJ, Bowers DF, Rozek MM, Crawley R (1995) Molecular mimicry, anti-coxsackievirus B3 neutralizing monoclonal antibodies, and myocarditis. J Immunol 154: 2983–95 Caforio AL, Mahon NJ, Tona F, McKenna WJ (2002) Circulating cardiac autoantibodies in dilated cardiomyopathy and myocarditis: Pathogenetic and clinical significance. Eur J Heart Fail 4: 411–7 Cooper LT Jr (2009) Myocarditis. N Engl J Med 360: 1526–38 Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW (1997) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275: 1320–3 Bergelson JM, Chan M, Solomon KR, St John NF, Lin H, Finberg RW (1994) Decayaccelerating factor (CD55), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses. Proc Natl Acad Sci USA 91: 6245–9 Chow LH, Beisel KW, McManus BM (1992) Enteroviral infection of mice with severe combined immunodeficiency. Evidence for direct viral pathogenesis of myocardial injury. Lab Invest 66: 24–31 Tomioka N, Kishimoto C, Matsumori A, Kawai C (1986) Effects of prednisolone on acute viral myocarditis in mice. J Am Coll Cardiol 7: 868–72 Fuse K, Chan G, Liu Y, Gudgeon P, Husain M, Chen M, Yeh WC, Akira S, Liu PP (2005) Myeloid differentiation factor-88 plays a crucial role in the pathogenesis of Coxsackievirus B3-induced myocarditis and influences type I interferon production. Circulation 112: 2276–85 McManus BM, Chow LH, Wilson JE, Anderson DR, Gulizia JM, Gauntt CJ, Klingel KE, Beisel KW, Kandolf R (1993) Direct myocardial injury by enterovirus: A central role in the evolution of murine myocarditis. Clin Immunol Immunopathol 68: 159–69 Seko Y, Takahashi N, Yagita H, Okumura K, Yazaki Y (1997) Expression of cytokine mRNAs in murine hearts with acute myocarditis caused by coxsackievirus b3. J Pathol 183: 105–8 Ruppert V, Meyer T, Pankuweit S, Jonsdottir T, Maisch B (2008) Activation of STAT1

63


43 44

45 46 47

48

49 50

51

52

53

54

55

56

64

transcription factor precedes up-regulation of coxsackievirus-adenovirus receptor during viral myocarditis. Cardiovasc Pathol 17: 81–92 Kishimoto C, Kawamata H, Sakai S, Shinohara H, Ochiai H (2000) Role of MIP-2 in coxsackievirus B3 myocarditis. J Mol Cell Cardiol 32: 631–8 Li J, Schwimmbeck PL, Tschöpe C, Leschka S, Husmann L, Rutschow S, Reichenbach F, Noutsias M, Kobalz U, Poller W et al (2002) Collagen degradation in a murine myocarditis model: Relevance of matrix metalloproteinase in association with inflammatory induction. Cardiovasc Res 56: 235–47 Akira S, Takeda K, Kaisho T (2001) Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat Immunol 2: 675–80 Huang CH, Vallejo JG, Kollias G, Mann DL (2009) Role of the innate immune system in acute viral myocarditis. Basic Res Cardiol 104: 228–37 Fairweather D, Yusung S, Frisancho S, Barrett M, Gatewood S, Steele R, Rose NR (2003) IL-12 receptor beta 1 and Toll-like receptor 4 increase IL-1 beta- and IL-18associated myocarditis and coxsackievirus replication. J Immunol 170: 4731–7 Frisancho-Kiss S, Davis SE, Nyland JF, Frisancho JA, Cihakova D, Barrett MA, Rose NR, Fairweather D (2007) Cutting edge: Cross-regulation by TLR4 and T cell Ig mucin3 determines sex differences in inflammatory heart disease. J Immunol 178: 6710–4 Godeny EK, Gauntt CJ (1987) Murine natural killer cells limit coxsackievirus B3 replication. J Immunol 139: 913–8 Kishimoto C, Kuribayashi K, Masuda T, Tomioka N, Kawai C (1985) Immunologic behavior of lymphocytes in experimental viral myocarditis: Significance of T lymphocytes in the severity of myocarditis and silent myocarditis in BALB/c-nu/nu mice. Circulation 71: 1247–54 Seko Y, Tsuchimochi H, Nakamura T, Okumura K, Naito S, Imataka K, Fujii J, Takaku F, Yazaki Y (1990) Expression of major histocompatibility complex class I antigen in murine ventricular myocytes infected with Coxsackievirus B3. Circ Res 67: 360–7 Seko Y, Matsuda H, Kato K, Hashimoto Y, Yagita H, Okumura K, Yazaki Y (1993) Expression of intercellular adhesion molecule-1 in murine hearts with acute myocarditis caused by coxsackievirus B3. J Clin Invest 91: 1327–36 Seko Y, Yagita H, Okumura K, Yazaki Y (1994) T-cell receptor V beta gene expression in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3. Circulation 89: 2170–5 Gebhard JR, Perry CM, Harkins S, Lane T, Mena I, Asensio VC, Campbell IL, Whitton JL (1998) Coxsackievirus B3-induced myocarditis: Perforin exacerbates disease, but plays no detectable role in virus clearance. Am J Pathol 153: 417–28 Huber SA, Feldman AM, Sartini D (2006) Coxsackievirus B3 induces T regulatory cells, which inhibit cardiomyopathy in tumor necrosis factor-alpha transgenic mice. Circ Res 99: 1109–16 Li K, Xu W, Guo Q, Jiang Z, Wang P, Yue Y, Xiong S (2009) Differential macrophage polarization in male and female BALB/c mice infected with coxsackievirus B3 defines susceptibility to viral myocarditis. Circ Res 105: 353–64


57 58

59 60

61 62

63 64

65

66 67

68 69

70

71

Huber SA, Job LP (1983) Cellular immune mechanisms in Coxsackievirus group B, type 3 induced myocarditis in Balb/C mice. Adv Exp Med Biol 161: 491–508 Seko Y, Takahashi N, Ishiyama S, Nishikawa T, Kasajima T, Hiroe M, Suzuki S, Ishiwata S, Kawai S, Azuma M et al (1998) Expression of costimulatory molecules B7-1, B7-2, and CD40 in the heart of patients with acute myocarditis and dilated cardiomyopathy. Circulation 97: 637–9 Huber SA, Pfaeffle B (1994) Differential Th1 and Th2 cell responses in male and female BALB/c mice infected with coxsackievirus group B type 3. J Virol 68: 5126–32 Seko Y, Takahashi N, Oshima H, Shimozato O, Akiba H, Kobata T, Yagita H, Okumura K, Azuma M, Yazaki Y (1999) Expression of tumour necrosis factor (TNF) receptor/ ligand superfamily co-stimulatory molecules CD40, CD30L, CD27L, and OX40L in murine hearts with chronic ongoing myocarditis caused by coxsackie virus B3. J Pathol 188: 423–30 Rutschow S, Li J, Schultheiss HP, Pauschinger M (2006) Myocardial proteases and matrix remodeling in inflammatory heart disease. Cardiovasc Res 69: 646–56 Huber SA, Mortensen A, Moulton G (1996) Modulation of cytokine expression by CD4+ T cells during coxsackievirus B3 infections of BALB/c mice initiated by cells expressing the gamma delta+ T-cell receptor. J Virol 70: 3039–44 Deonarain R, Cerullo D, Fuse K, Liu PP, Fish EN (2004) Protective role for interferonbeta in coxsackievirus B3 infection. Circulation 110: 3540–3 Kubota T, Miyagishima M, Frye CS, Alber SM, Bounoutas GS, Kadokami T, Watkins SC, McTiernan CF, Feldman AM (2001) Overexpression of tumor necrosis factor-alpha activates both anti- and pro-apoptotic pathways in the myocardium. J Mol Cell Cardiol 33: 1331–44 Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ, Feldman AM (1997) Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha. Circ Res 81: 627–35 Shioi T, Matsumori A, Sasayama S (1996) Persistent expression of cytokine in the chronic stage of viral myocarditis in mice. Circulation 94: 2930–7 Latif N, Zhang H, Archard LC, Yacoub MH, Dunn MJ (1999) Characterization of anti-heart antibodies in mice after infection with coxsackie B3 virus. Clin Immunol 91: 90–8 Fairweather D, Rose NR (2007) Coxsackievirus-induced myocarditis in mice: A model of autoimmune disease for studying immunotoxicity. Methods 41: 118–22 Kyu B, Matsumori A, Sato Y, Okada I, Chapman NM, Tracy S (1992) Cardiac persistence of cardioviral RNA detected by polymerase chain reaction in a murine model of dilated cardiomyopathy. Circulation 86: 522–30 Kindermann I, Kindermann M, Kandolf R, Klingel K, Bultmann B, Muller T, Lindinger A, Bohm M (2008) Predictors of outcome in patients with suspected myocarditis. Circulation 118: 639–48 Baughman KL (2006) Diagnosis of myocarditis: Death of Dallas criteria. Circulation 113: 593–5

65


72

73

74

75

76

77

78

79 80

81

82

83

84

66

Wang WZ, Matsumori A, Yamada T, Shioi T, Okada I, Matsui S, Sato Y, Suzuki H, Shiota K, Sasayama S (1997) Beneficial effects of amlodipine in a murine model of congestive heart failure induced by viral myocarditis. A possible mechanism through inhibition of nitric oxide production. Circulation 95: 245–51 Azuma RW, Suzuki J, Ogawa M, Futamatsu H, Koga N, Onai Y, Kosuge H, Isobe M (2004) HMG-CoA reductase inhibitor attenuates experimental autoimmune myocarditis through inhibition of T cell activation. Cardiovasc Res 64: 412–20 Yuan Z, Kishimoto C, Shioji K, Nakamura H, Yodoi J, Sasayama S (2003) Temocapril treatment ameliorates autoimmune myocarditis associated with enhanced cardiomyocyte thioredoxin expression. Mol Cell Biochem 248: 185–92 Nishio R, Shioi T, Sasayama S, Matsumori A (2003) Carvedilol increases the production of interleukin-12 and interferon-gamma and improves the survival of mice infected with the encephalomyocarditis virus. J Am Coll Cardiol 41: 340–5 Fohlman J, Pauksen K, Hyypia T, Eggertsen G, Ehrnst A, Ilback NG, Friman G (1996) Antiviral treatment with WIN 54 954 reduces mortality in murine coxsackievirus B3 myocarditis. Circulation 94: 2254–9 Pauksen K, Ilback NG, Friman G, Fohlman J (1993) Therapy of coxsackie virus B3-induced myocarditis with WIN 54954 in different formulations. Scand J Infect Dis Suppl 88: 125–30 Kyto V, Saraste A, Fohlman J, Ilback NG, Harvala H, Vuorinen T, Hyypia T (2002) Cardiomyocyte apoptosis after antiviral WIN 54954 treatment in murine coxsackievirus B3 myocarditis. Scand Cardiovasc J 36: 187–92 See DM, Tilles JG (1993) WIN 54954 treatment of mice infected with a diabetogenic strain of group B coxsackievirus. Antimicrob Agents Chemother 37: 1593–8 Heim A, Pfetzing U, Muller G, Grumbach IM (1998) Antiviral activity of WIN 54954 in coxsackievirus B2 carrier state infected human myocardial fibroblasts. Antiviral Res 37: 47–56 Noutsias M, Fechner H, de Jonge H, Wang X, Dekkers D, Houtsmuller AB, Pauschinger M, Bergelson J, Warraich R, Yacoub M et al (2001) Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: Implications for cardiotropic viral infections. Circulation 104: 275–80 Yanagawa B, Spiller OB, Proctor DG, Choy J, Luo H, Zhang HM, Suarez A, Yang D, McManus BM (2004) Soluble recombinant coxsackievirus and adenovirus receptor abrogates coxsackievirus b3-mediated pancreatitis and myocarditis in mice. J Infect Dis 189: 1431–9 Shi Y, Chen C, Lisewski U, Wrackmeyer U, Radke M, Westermann D, Sauter M, Tschope C, Poller W, Klingel K et al (2009) Cardiac deletion of the coxsackievirusadenovirus receptor abolishes coxsackievirus b3 infection and prevents myocarditis in vivo. J Am Coll Cardiol 53: 1219–26 Pinkert S, Westermann D, Wang X, Klingel K, Dorner A, Savvatis K, Grossl T, Krohn S, Tschope C, Zeichhardt H et al (2009) Prevention of cardiac dysfunction in acute


85 86 87 88

89

90

91

92

93

94

95

96

97

coxsackievirus B3 cardiomyopathy by inducible expression of a soluble coxsackievirusadenovirus receptor. Circulation 120: 2358–66 Dan M, Chantler JK (2005) A genetically engineered attenuated coxsackievirus B3 strain protects mice against lethal infection. J Virol 79: 9285–95 Henke A, Zell R, Martin U, Stelzner A (2003) Direct interferon-gamma-mediated protection caused by a recombinant coxsackievirus B3. Virology 315: 335–44 Matsumori A, Tomioka N, Kawai C (1988) Protective effect of recombinant alpha interferon on coxsackievirus B3 myocarditis in mice. Am Heart J 115: 1229–32 Wang YX, da Cunha V, Vincelette J, White K, Velichko S, Xu Y, Gross C, Fitch RM, Halks-Miller M, Larsen BR et al (2007) Antiviral and myocyte protective effects of murine interferon-beta and -{alpha}2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice. Am J Physiol Heart Circ Physiol 293: H69–76 Wessely R, Klingel K, Knowlton KU, Kandolf R (2001) Cardioselective infection with coxsackievirus B3 requires intact type I interferon signaling: Implications for mortality and early viral replication. Circulation 103: 756–61 Kühl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-beta treatment eliminates cardiotropic viruses and improves left ventricular function in patients with myocardial persistence of viral genomes and left ventricular dysfunction. Circulation 107: 2793–8 Coletta AP, Clark AL, Cleland JG (2009) Clinical trials update from the Heart Failure Society of America and the American Heart Association meetings in 2008: SADHARTCHF, COMPARE, MOMENTUM, thyroid hormone analogue study, HF-ACTION, I-PRESERVE, {beta}-interferon study, BACH, and ATHENA. Eur J Heart Fail 11: 214–9 Kishimoto C, Crumpacker CS, Abelmann WH (1988) Ribavirin treatment of murine coxsackievirus B3 myocarditis with analyses of lymphocyte subsets. J Am Coll Cardiol 12: 1334–41 Hiraoka Y, Kishimoto C, Kurokawa M, Ochiai H, Sasayama S (1992) The effects of FK-506, a novel and potent immunosuppressant, upon murine Coxsackievirus B3 myocarditis. J Pharmacol Exp Ther 260: 1386–91 Kishimoto C, Abelmann WH (1989) Absence of effects of cyclosporine on myocardial lymphocyte subsets in Coxsackievirus B3 myocarditis in the aviremic stage. Circ Res 65: 934–45 Kishimoto C, Thorp KA, Abelmann WH (1990) Immunosuppression with high doses of cyclophosphamide reduces the severity of myocarditis but increases the mortality in murine Coxsackievirus B3 myocarditis. Circulation 82: 982–9 Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE (1995) A clinical trial of immunosuppressive therapy for myocarditis. The Myocarditis Treatment Trial Investigators. N Engl J Med 333: 269–75 Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri A (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107: 857–63

67


98

99

100 101

102 103

104

105

106 107

108

109 110

111

112

68

Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewski P, Niklewski T, Zembala M, Polonski L, Rozek MM, Wodniecki J (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: Two-year follow-up results. Circulation 104: 39–45 Frustaci A, Russo MA, Chimenti C (2009) Randomized study on the efficacy of immunosuppressive therapy in patients with virus-negative inflammatory cardiomyopathy: The TIMIC study. Eur Heart J 30: 1995–2002 Maisch B, Camerini F, Schultheiss HP (1995) Immunosuppressive therapy for myocarditis. N Engl J Med 333: 1713; author reply 1714 Opavsky MA, Penninger J, Aitken K, Wen WH, Dawood F, Mak T, Liu P (1999) Susceptibility to myocarditis is dependent on the response of alphabeta T lymphocytes to coxsackieviral infection. Circ Res 85: 551–8 Kishimoto C, Abelmann WH (1989) Monoclonal antibody therapy for prevention of acute coxsackievirus B3 myocarditis in mice. Circulation 79: 1300–8 Liu P, Aitken K, Kong YY, Opavsky MA, Martino T, Dawood F, Wen WH, Kozieradzki I, Bachmaier K, Straus D et al (2000) The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease. Nat Med 6: 429–34 Irie-Sasaki J, Sasaki T, Matsumoto W, Opavsky A, Cheng M, Welstead G, Griffiths E, Krawczyk C, Richardson CD, Aitken K et al (2001) CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling. Nature 409: 349–54 Seko Y, Takahashi N, Azuma M, Yagita H, Okumura K, Yazaki Y (1998) Effects of in vivo administration of anti-B7-1/B7-2 monoclonal antibodies on murine acute myocarditis caused by coxsackievirus B3. Circ Res 82: 613–8 Schwimmbeck PL, Badorff C, Schultheiss HP, Strauer BE (1994) Transfer of human myocarditis into severe combined immunodeficiency mice. Circ Res 75: 156–64 Seko Y, Ishiyama S, Nishikawa T, Kasajima T, Hiroe M, Kagawa N, Osada K, Suzuki S, Yagita H, Okumura K et al (1995) Restricted usage of T cell receptor V alpha-V beta genes in infiltrating cells in the hearts of patients with acute myocarditis and dilated cardiomyopathy. J Clin Invest 96: 1035–41 Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kühl U, Lehmkuhl H, Hetzer R et al (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 9: 3 Perens G, Levi DS, Alejos JC, Wetzel GT (2007) Muronomab-CD3 for pediatric acute myocarditis. Pediatr Cardiol 28: 21–6 Matsumoto Y, Jee Y, Sugisaki M (2000) Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR. J Immunol 164: 2248–54 Klingel K, Stephan S, Sauter M, Zell R, McManus BM, Bultmann B, Kandolf R (1996) Pathogenesis of murine enterovirus myocarditis: Virus dissemination and immune cell targets. J Virol 70: 8888–95 Cheung C, Marchant D, Walker EK, Luo Z, Zhang J, Yanagawa B, Rahmani M, Cox


113

114 115

116

117

118

119 120

121

122 123 124

J, Overall C, Senior RM et al (2008) Ablation of matrix metalloproteinase-9 increases severity of viral myocarditis in mice. Circulation 117: 1574–82 Latif N, Baker CS, Dunn MJ, Rose ML, Brady P, Yacoub MH (1993) Frequency and specificity of antiheart antibodies in patients with dilated cardiomyopathy detected using SDS-PAGE and western blotting. J Am Coll Cardiol 22: 1378–84 Felix SB, Staudt A (2006) Non-specific immunoadsorption in patients with dilated cardiomyopathy: Mechanisms and clinical effects. Int J Cardiol 112: 30–3 Takada H, Kishimoto C, Hiraoka Y (1995) Therapy with immunoglobulin suppresses myocarditis in a murine coxsackievirus B3 model. Antiviral and anti-inflammatory effects. Circulation 92: 1604–11 Kishimoto C, Takamatsu N, Kawamata H, Shinohara H, Ochiai H (2000) Immunoglobulin treatment ameliorates murine myocarditis associated with reduction of neurohumoral activity and improvement of extracellular matrix change. J Am Coll Cardiol 36: 1979–84 McNamara DM, Holubkov R, Starling RC, Dec GW, Loh E, Torre-Amione G, Gass A, Janosko K, Tokarczyk T, Kessler P et al (2001) Controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy. Circulation 103: 2254–9 Reifenberg K, Lehr HA, Torzewski M, Steige G, Wiese E, Kupper I, Becker C, Ott S, Nusser P, Yamamura K et al (2007) Interferon-gamma induces chronic active myocarditis and cardiomyopathy in transgenic mice. Am J Pathol 171: 463–72 Nishio R, Matsumori A, Shioi T, Ishida H, Sasayama S (1999) Treatment of experimental viral myocarditis with interleukin-10. Circulation 100: 1102–8 Li J, Leschka S, Rutschow S, Schwimmbeck PL, Husmann L, Noutsias M, Westermann D, Poller W, Zeichhardt H, Klingel K et al (2007) Immunomodulation by interleukin-4 suppresses matrix metalloproteinases and improves cardiac function in murine myocarditis. Eur J Pharmacol 554: 60–8 Kania G, Blyszczuk P, Stein S, Valaperti A, Germano D, Dirnhofer S, Hunziker L, Matter CM, Eriksson U (2009) Heart-infiltrating prominin-1+/CD133+ progenitor cells represent the cellular source of transforming growth factor beta-mediated cardiac fibrosis in experimental autoimmune myocarditis. Circ Res 105: 462–70 Marchant D, McManus BM (2009) Matrix metalloproteinases in the pathogenesis of viral heart disease. Trends Cardiovasc Med 19: 21–6 Uematsu S, Akira S (2006) Toll-like receptors and innate immunity. J Mol Med 84: 712–25 Triantafilou K, Orthopoulos G, Vakakis E, Ahmed MA, Golenbock DT, Lepper PM, Triantafilou M (2005) Human cardiac inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR) 8-dependent. Cell Microbiol 7: 1117–26

69

Myocardial proteases and matrix remodeling in acute myocarditis and inflammatory cardiomyopathy Susanne Rutschow1, Michel Noutsias1,2 and Matthias Pauschinger1,3 1

Department of Cardiology and Pneumonology, Charité Centrum 11 for Cardiovascular Medicine, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany 2 Department of Internal Medicine – Cardiology, University Hospital of Marburg and Giessen / UKGM GmbH, Faculty of Medicine, Philipps-Universität Marburg, Baldinger Strasse, 35033 Marburg, Germany 3 Department of Cardiology, Medizinische Klinik 8, Klinikum Nürnberg Süd, Breslauer Strasse 201, 90471 Nürnberg, Germany

Abstract Inflammatory cardiomyopathy (DCMi) is commonly induced and maintained by cardiotropic viruses, and is associated with left ventricular (LV) dysfunction accompanied by myocardial inflammatory cell infiltration and increased release of proinflammatory cytokines. Recent insights have revealed the pivotal role played by the disruption of the extracellular matrix (ECM) architecture, caused mainly by cytokine-driven post-translational modification. Fibrillar collagen turnover results from the equilibrium between synthesis and degradation. The imbalance of the matrix degrading system with induced expression of metalloproteinases (MMPs) and plasminogen activators, and with concomitantly reduced expression of the tissue inhibitors of MMPs, contributes to a pathological collagen turnover. This results in a loss of myocardial structural integrity and impairment of LV function. Interference with ECM regulatory mechanisms may be effective in preventing persistent virus-induced and cytokine-driven cardiac injury. Future investigations are warranted to elucidate the possible effects of established immunomodulatory treatment strategies on the expression, biological activity and architecture of ECM components.

Introduction Inflammatory cardiomyopathy (DCMi) is commonly induced and maintained by cardiotropic viruses, and is associated with acute left ventricular (LV) dysfunction accompanied by myocardial inflammatory cell infiltration and increased release of proinflammatory cytokines [1–9]. Both intramyocardial inflammation and viral persistence have been associated with an adverse prognosis in the setting of chronic dilated cardiomyopathy (DCM) and acute myocarditis (AMC), respectively [8,


71

Susanne Rutschow, Michel Noutsias and Matthias Pauschinger

10, 11]. Recent insights have revealed a pivotal role for matrix metalloproteinases (MMPs) in the disruption of the extracellular matrix (ECM) architecture. Several pathways are involved in these processes, including an augmented myocardial collagen turnover, which ultimately leads to the loss of structural integrity of the myocardium, thereby contributing to LV dysfunction [4, 12, 13]. Data from patients under mechanical unloading conditions have clearly shown the plasticity of these processes during myocardial recovery [14, 15]. Acute LV dysfunction in murine AMC is associated with induction of proinflammatory cytokines and an imbalance of the MMPs and the tissue inhibitors of MMPs (TIMPs) system [4]. Proinflammatory cytokines (i.e. TNF-A and IL-15B) contribute to depression of LV function and cardiomyocyte loss by apoptosis and, moreover, play a critical role in maintaining the balance in ECM remodeling [14, 16–18]. Cytokines therefore constitute an important pathogenic link between myocardial inflammation, ECM remodeling and depression of LV contractility. They regulate cardiac fibroblast function with the expression of collagen types I and III and fibronectin, as well as the matrix degradation system by influencing the expression of MMPs, the TIMPs and their activators like urokinase-type plasminogen activator and tissue-type plasminogen activator (uPA, tPA) [19–22]. Maintenance of the physiological myocardial matrix turnover involves highly regulated interactions between cardiac and non-cardiac cells. An increasing body of evidence has revealed the imbalance of components of the ECM system in murine myocarditis and human DCMi, and is highlighted in this review, after an overview on basics of the myocardial ECM and its regulation by cytokines.

Inflammation and cytokines in AMC and DCM In contrast to the mostly indirect evidence on the interactions between cytokines, ECM remodeling and LV dysfunction in chronic heart failure (CHF) patients based on serological analyses, many human investigations in AMC and DCM patients have focused on the intramyocardial expression of these factors using endomyocardial biopsies (EMBs). EMBs can be obtained by highly trained cardiologists with a fairly low rate of major complications [23]. For the contemporary EMB-based diagnosis of DCMi, standardized histological, immunohistological and molecular biological techniques are pertinent for the characterization of intramyocardial inflammation and the detection of viral infections [5–7, 24–29]. Experimental murine myocarditis has provided substantial insights into the immune-mediated pathogenic mechanisms involved and into the natural course of the disease. Basically, the triphasic model of virus-induced murine myocarditis distinguishes an acute, a subacute and chronic phase. The first phase is dominated by viral infection and replication, followed by an autoimmunity-dominated phase, and finally the late stage of DCM. These insights have also been extrapolated to human

72

Myocardial proteases and matrix remodeling in acute myocarditis and inflammatory cardiomyopathy

AMC and DCM [30–32]. During the acute virus-induced vigorous inflammation, a plethora of proinflammatory cytokines are induced mainly by the immunocompetent infiltrates such as IL-1B, TNF-A and interferon-G. The main goal of this initial vigorous inflammatory response is effective viral elimination, and ultimately spontaneous resolution of the disease [33–36]. This temporarily limited abundance of proinflammatory cytokines is linked to histological alterations, i.e., edema, myocyte necrosis and apoptosis, and remodeling of the ECM [37–39]. After the initial abundance of proinflammatory cytokines, regulatory cytokines such as IL-2, IL-4 and IL-10 are involved in tapering down the inflammatory response [40, 41]. The paradigm of human fulminant myocarditis with initially severely depressed LVEF, which is associated with a significantly better outcome compared to AMC patients with moderately depressed LV function, indicates that the natural course of myocarditis depends profoundly on the complex virus-host interactions, which are still mostly not well understood [42]. More detailed analyses on the key immunocompetent determinants, their interactions with ECM remodeling, the kinetics of viral load and the role of genetic susceptibility are pertinent to unravel these intricate pathways. An inadequate immune response results in the emergence of cross-reactive autoantibodies, and a chronification of the cellular infiltrates, which can target cardiomyocytes. The emergence of autoimmunity endorses the second phase, characterized by a chronic “low-level” inflammation and persistence of viral genomes at low viral loads, a progressive loss of cardiomyocytes, and progressive ECM remodeling [36, 43–46]. Viral persistence can result from the ineffective initial antiviral immune response, which has an adverse prognostic impact for DCMi patients, as well as features of anti-cardiac autoimmunity [8, 11, 47, 48]. After these long-term adverse effects of the immune system, the autoimmune phase transits to the third stage, DCM, characterized by irreversible LV dilatation and dysfunction even in the absence of ongoing anti-cardiac or antiviral immunity, and in the absence of viral persistence. The chronic ECM remodeling is a pivotal hallmark of this third stage of the disease, without substantial plasticity for spontaneous improvement due to persistent loss of structural integrity of the myocardium.

Myocardial ECM: Collagen network, regulation of MMPs and TIMPs, and effects of cytokines Myocardial ECM is crucial for the cellular and structural integrity of the heart. It basically constitutes a cross-linked collagen network. After secretion into the ECM, the fibril-forming collagens aggregate spontaneously after following the processing of procollagens into an ordered fibrillar network, stabilized by covalent cross-links. The structural elements of the ECM are dynamic, and the ECM has multiple regulatory pathways. A highly complex network of receptors and enzymes controls the turnover of this well-orchestrated system. The multiple influences of cytokines on

73


this complex network provide a link to the pivotal role of ECM dysregulation in inflammatory pathological conditions. Collagen is mainly synthesized by fibroblasts, and can be regulated by multiple factors, including cytokines. Proinflammatory cytokines like TNF-A, IL-1B and TGF-B decrease the expression of procollagen types I and III, and increase the expression of fibronectin and non-fibrillar procollagen type IV. Furthermore, several other factors like aldosterone, TGF-B or mechanical stretch induce mRNA synthesis of collagen [19]. Angiotensin II induces ECM protein synthesis and accumulation via AT1-receptor stimulation. These effects are mediated by TGF-B and endothelin-1 [49, 50]. In experimental myocardial infarction, inhibition of the angiotensin-converting enzyme (ACE) prevented collagen accumulation and deposition [51]. Fibrillar collagen turnover results from the equilibrium between the synthesis and the degradation of collagen, mainly types I and III. Whereas ECM replacement amounts to about 5–9% in healthy hearts, in pathological conditions such as myocardial infarction ECM replacement rises dramatically to 50% [52]. The daily rate of collagen synthesis is much slower than of non-collagen proteins. The substantially longer half-life period of collagen implies a fairly slow ECM replacement after degradation and a potential vulnerability for adverse remodeling [53]. This additionally reduces the ability to form the cross-linked collagen network, which leads to alterations in the composition and structure of myocardial collagen. ECM remodeling is substantially effected by collagen degradation, which is mainly mediated by MMPs and serine proteinases. MMPs are zinc-dependent enzymes that are expressed under basal conditions by fibroblasts and myocytes. Their expression is induced in response to inflammatory reactions by infiltrating macrophages and lymphocytes [54]. One group of MMPs is secreted into the extracellular space in a latent or proenzyme form, and the other group is membrane bound. Regarding the catalytic domain of MMPs, a large extracellular binding domain at the C terminus is responsible for the substrate specificity and the specific binding to ECM proteins. Three different MMP groups can be subdivided based on these substrate specificities and functions. Collagenases (MMP-1, MMP-8, MMP13) decompose the insoluble collagen fibrils into soluble fragments. The cleaving of these soluble fragments is continued by gelatinases (MMP-2, MMP-9), which are highly expressed in the LV myocardium [12, 55]. Coker et al. [56] showed an isolated expression of these gelatinases in LV myocytes, which is compatible with a direct processing of matrix remodeling by the synthesis and release of MMPs. MMP-2 is thereby constitutively expressed in the myocardium, whereas MMP-9 is inducible and becomes more relevant under inflammatory conditions [57]. The third group, comprising the stromelysin-like MMP-3, degrades a wide range of ECM components, and can initiate the activation of the inactive pro-MMPs [58]. The membrane-bound MMPs (membrane type-MMP, MT-MMP) also play a critical role in ECM remodeling, which not only cleave intact fibrillar collagen and base-

74


ment membrane components, but also directly activate different pro-MMPs [59]. Of note, the resulting fragmented matrix peptides also have biological activities and have been shown to stimulate new collagen synthesis by cardiac fibroblasts [60]. In addition to the ECM components, the target substrates of the MMPs also comprise non-matrix proteins such as cytokines, receptors and adhesions molecules [61, 62]. This explains multifaceted functions of MMPs apart from ECM remodeling in the context of inflammatory conditions, such as regulation of growth, angiogenesis and metastasis of tumor cells [63]. The capability of activated MMPs to degrade the complete ECM is conditioned by a tightly controlled system. MMP transcription can be regulated by several cytokines and growth factors. At a second regulatory level, pro-MMP activation is mediated by serine proteinases (i.e. the plasmin system, MT-MMPs or stromelysins), and, thirdly, activated MMPs can be inhibited by TIMPs. The synthesis of MMPs at the transcriptional level can be regulated by multiple cytokines, neurohormones and growth factors via multiple signaling pathways [64–66]. Proinflammatory cytokines like IL-1B, TNF-A and IL-6 induce MMP synthesis [19, 20]. Murine failing heart with transgenic expression of TNF-A have increased MMP activity [67]. In concert with TNF-A, interferon-G enhances MMP-1 synthesis in human monocytes, and neutralizing antibodies against TNF-A block the induction of MMP-1 by interferon-G [68]. TGF-B has a differential role in the regulation of MMP transcription. On the one hand, TGF-B decreases the proteolytic activity of MMP-1 and MMP-3 by suppressing MMP gene expression through the TGF-B inhibitory element (TIE). On the other hand, TGF-B increases the expression of MMP-2 and MMP-9 [21, 69]. Beside inflammatory cytokines, several other bioactive molecules also influence the synthesis of MMPs. Angiotensin II induces the expression of MMP-2, MMP-9 and MMP-14 in neonatal rat fibroblasts [70]. Its importance in this regulating process is further shown by an improvement in LV function and myocardial geometry through reduced MMP synthesis after ACE inhibition or AT-1 blockade [71, 72]. Endothelin-1 enhances the production of MMP-2 and MMP-9, and endothelin receptor blockade results in reduced levels of MMP-2 and MMP-9 [73]. Furthermore, natriuretic peptides like brain natriuretic peptide (BNP) are able to induce the MMP synthesis in CHF and myocardial remodeling [74]. MT-MMPs and MMP-3 activate different MMP family members by proteolytic cleavage of the MMP-propeptide [22, 59, 61]. Even more important is the plasminogen system, which activates the inactive pro-MMPs by plasminogen activators (PA), tissue-type PA (tPA), urokinase-type PA (uPA) and the PA inhibitor (PAI) [75]. In uPA-knockout mice with acute pressure overload, PAI gene transfer induced improved LV function by reducing myocardial fibrosis and preserving interstitial matrix [76]. Beside MMP activation, the PA are also able to cleave matrix proteins like fibrin and fibronectin. The expression of uPA, which has the same transcription factor binding elements (AP-1, PEA3) in the promotor region as MMP-1 and MMP-

75


3, is also induced by IL-1B and TNF-A [77, 78]. In fibroblasts, IL-1B increases not only the protein and mRNA expression of uPA, but also its receptor uPAR [79]. MMP-3 neutralizes its physiological inhibitors (PAI, A2-antiplasmin) by cleavage, providing a feedback regulation mechanism between the MMPs and the plasmin system [80, 81]. TIMPs are endogenous specific inhibitors of active MMPs [68, 82, 83]. TIMPs bind to the active site of MMP at a 1:1 ratio, blocking their access to the collagen substrate. They can furthermore bind latent MMPs at the N terminus, thereby preventing their activation. TIMP-2 and TIMP-3 are effective inhibitors of membranebound-MMPs, and TIMP-3 is the sole TIMP that can inhibit TNF-A-converting enzyme [84, 85]. TIMP-1-deficient mice develop increased LV mass and increased end diastolic volume, suggesting its important role in preventing activation of the protease cascade and in the maintenance of a dynamic matrix balance. Inflammatory mediators also influence this third step in the control of MMP activity; IL-1 and TNF-A down-regulate the expression of TIMP-1 [86]. In addition to this specific inhibition, A2 macroglubulin and heparin can nonspecifically inhibit the activity of MMPs [87, 88].

Heart failure, cytokines and ECM remodeling Emerging evidence from the last two decades has demonstrated increased levels of proinflammatory cytokines, especially of TNF-A, in CHF patients [89–92]. Transgenic myocardial expression of TNF-A leads to DCM, clearly showing the cardiodepressive biological effects of cytokines [93]. However, a substantial extracardiac source of circulating cytokines has been elucidated in CHF patients, which results from repeated endotoxinemia due to backward congestion [94–97]. Therefore, increased levels of cytokines in CHF patients, although associated to heart failure status and adverse prognosis, do not necessarily reflect the intramyocardial inflammatory process. Nonetheless, investigations in CHF patient have highlighted several important insights into the plasticity of LV dysfunction paralleled by changing levels of circulating cytokines. Improvement of LV function in CHF patients is accompanied by declining circulating TNF-A levels [98–101]. The neurohumoral activation and the elevated oxidative stress during the development of LV dysfunction in congestive heart failure can be triggered by proinflammatory cytokines. Increased free oxidative radicals can lead to an activation of p38-MAPkinase and nuclear factor-kappa B (NF-KB). These directly affect LV dysfunction by inducing cardiomyocytolysis and by the negative inotropic effects of reduced calcium uptake by the sarcoplasmic reticulum [102]. Furthermore, B-adrenergic stimulation leads to intramyocardial induction of TNF-A, IL-1B and IL-6, in parallel with further features of heart failure, such as hypertrophy and myocyte necrosis [103–105].

76


Remodeling of the ECM in AMC and DCMi In experimental virus-induced AMC, dysfunction and dilatation of the left ventricle is linked to a disruption of the collagen turnover, substantial qualitative changes in the collagen network and the resulting alterations of the ECM [4]. Expression of myocardial mRNA and the protein abundance of collagen type I was unchanged, as was total collagen content measured by picrosirius red staining. However, Western blot analysis demonstrated an increased fraction of soluble collagen, suggesting an initial dominance of post-translational collagen variation contingent on an imbalance in the matrix degradation system. This is compatible to findings by Woodiwiss et al. [106] showing that the reduction of collagen crosslinks is responsible for the decreased native insoluble or increased soluble collagen, which is associated with LV dysfunction and dilatation. MMP-9 depolymerizes the cross-linked collagen type I [107]. LV dilatation after myocardial infarction can be prevented by deletion of the MMP-9 gene, highlighting the importance of ECM remodeling for LV function [108]. Experimental virus-induced AMC is characterized by an induction of proinflammatory cytokines such as IL-1B, TNF-A and TGF-B on day 10 post infection, which is temporarily accompanied by a significant induction of MMP-3 and MMP-9, while their endogenous specific inhibitors TIMP-1 and TIMP-4 are reduced concomitantly. Recently, we showed the pivotal effects of the interactions between the plasminogen and the MMP system for LV dysfunction and dilatation in experimental virus-induced AMC [109]. Both uPA and MMP-9 are significantly induced in the acute stage of myocarditis. Targeted deletion of uPA, resulted in reduced MMP activity and cytokine expression. Inhibition of MMPs by adenoviral gene overexpression of TIMP-1 decreased cardiac inflammation and reduced myocardial necrosis in the acute stage, and decreased cardiac fibrosis at 35 days after viral infection. Most importantly, these interventions on uPA and MMP expression prevented coxsackievirus B3-induced cardiac dilatation and dysfunction. These insights, which provided evidence that interference with ECM regulatory mechanisms may be effective in preventing cardiac injury, dilatation and failure in virus-induced myocarditis, offer possible future therapeutic avenues for human AMC. Leipner et al. [110] have recently confirmed that attenuation of fibrosis is a definite aim in the treatment of virus-induced myocarditis by imatinib treatment, an inhibitor of the platelet-derived growth factor, without directly affecting MMP activity. However, extrapolation of experimental insights to human disease has not been successful so far. In the Prevention of Myocardial Infarction Early Remodeling (PREMIER) trial, the selective MMP inhibitor PG-116800 failed to reduce LV remodeling or improve clinical outcomes after acute myocardial infarction [111]. Induction of the matrix degradation system (MMPs), decreased levels of TIMPs and the activated plasmin system in the acute phase of myocarditis may lead to an imbalance in the matrix degradation system in favor of degradation of ECM pro-

77


Figure 1. Scheme of regulatory pathways involved in the regulation of ECM degradation in DCMi, focusing on the role of cytokines.

tein. This ultimately reduces the matrix integrity and disrupts the three-dimensional collagen network by cleaving the cross-links between the collagen molecules, which then leads to LV dysfunction and dilatation. This pathogenic model of the complex regulation of ECM remodeling is illustrated in Figure 1. As mentioned above, the adrenergic system promotes fibrosis and cellular necrosis. The effects of beta-blockers on myocardial inflammation, ECM remodeling and LV function were examined in experimental virus-induced myocarditis. Improvement of LV contractility by carvedilol treatment was accompanied by a reduced expression of MMPs, especially of MMP-8, and of proinflammatory cytokines (IL1B, TNF-A and TGF-B). The effects of metoprolol were not as potent as those of carvedilol. This might be due to the fact that carvedilol is a potent inhibitor of direct epinephrine actions [112], in addition to its known antioxidative and free-radical scavenger properties [113, 114]. Recently, Kindermann et al. [10] demonstrated that absence of beta-blocker treatment is an independent prognostic factor for adverse

78


long-term outcome in patients presenting with AMC, in addition to the immunohistological proof of myocardial inflammation. The major studies on ECM remodeling in human DCMi have been conducted in EMB samples from patients presenting with chronic DCM. Early findings revealed a significantly different ratio of Col III/Col I mRNA expression in immunohistologically detected DCMi (1.16 ± 0.18) versus DCM (2.77 ± 0.65), suggesting increased myocardial stiffness with impaired systolic and diastolic function [13, 115, 116]. Further analyses showed significantly increased myocardial MMP-3 expression and reduced expression of TIMP-4 in EMB samples from patients with immunohistologically confirmed DCMi [117]. Elevated serum circulating MMP-1 levels are inversely associated with the MMP/TIMP ratio and the degree of LV dilatation in patients presenting with DCM [118]. DCM patients with increased serum markers of collagen metabolism have an increased risk for requiring transplantation, a more severely impaired LV function or advanced clinical heart failure status, and higher mortality [119–122]. In addition, polymorphisms of MMP genes might be involved in the pathogenesis of CHF [123]. Four immunomodulatory treatment options have been established so far for DCMi patients: immunosuppression, antiviral interferon treatment, immunoadsorption and intravenous immunoglobulin treatment [124–128]. In light of the intimate link between the immune system, in particular cytokines and regulation of ECM remodeling, future investigations are warranted to elucidate the possible effects of these immunomodulatory treatment strategies on the expression and activity of ECM components.

Conclusions The myocardial ECM is a complex network, and pivotal for the structural integrity of the heart. Alteration in the matrix degradation system induced by cytokines and neurohumoral reaction leads to an impairment of LV function in AMC and DCMi. The imbalance of the matrix degrading system with induced expression of MMPs and plasminogen activators, with concomitantly reduced expression of TIMPs, contributes to a pathological collagen turnover, with loss of myocardial structural integrity and impairment of LV function.

References 1

2

Pauschinger M, Bowles NE, Fuentes-Garcia FJ, Pham V, Kühl U, Schwimmbeck PL, Schultheiss HP, Towbin JA (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99: 1348–54 Pauschinger M, Doerner A, Kühl U, Schwimmbeck PL, Poller W, Kandolf R, Schultheiss

79


3

4

5

6

7

8

9

10

11

12

13

14

80

HP (1999) Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99: 889–95 Pauschinger M, Chandrasekharan K, Noutsias M, Kühl U, Schwimmbeck LP, Schultheiss HP (2004) Viral heart disease: Molecular diagnosis, clinical prognosis, and treatment strategies. Med Microbiol Immunol (Berl) 193: 65–9 Li J, Schwimmbeck PL, Tschöpe C, Leschka S, Husmann L, Rutschow S, Reichenbach F, Noutsias M, Kobalz U, Poller W et al (2002) Collagen degradation in a murine myocarditis model: Relevance of matrix metalloproteinase in association with inflammatory induction. Cardiovasc Res 56: 235–47 Noutsias M, Seeberg B, Schultheiss HP, Kühl U (1999) Expression of cell adhesion molecules in dilated cardiomyopathy: Evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99: 2124–31 Kühl U, Pauschinger M, Bock T, Klingel K, Schwimmbeck CP, Seeberg B, Krautwurm L, Noutsias M, Poller W, Schultheiss HP et al (2003) Parvovirus B19 infection mimicking acute myocardial infarction. Circulation 108: 945–50 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111: 887–93 Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112: 1965–70 Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kühl U, Lehmkuhl H, Hetzer R et al (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 9: 3 Kindermann I, Kindermann M, Kandolf R, Klingel K, Bultmann B, Muller T, Lindinger A, Bohm M (2008) Predictors of outcome in patients with suspected myocarditis. Circulation 118: 639–48 Caforio AL, Calabrese F, Angelini A, Tona F, Vinci A, Bottaro S, Ramondo A, Carturan E, Iliceto S, Thiene G et al (2007) A prospective study of biopsy-proven myocarditis: Prognostic relevance of clinical and aetiopathogenetic features at diagnosis. Eur Heart J 28: 1326–33 Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ 3rd, Spinale FG (1998) Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation 97: 1708–15 Pauschinger M, Knopf D, Petschauer S, Doerner A, Poller W, Schwimmbeck PL, Kühl U, Schultheiss HP (1999) Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio. Circulation 99: 2750–6 Torre-Amione G, Stetson SJ, Youker KA, Durand JB, Radovancevic B, Delgado RM, Frazier OH, Entman ML, Noon GP (1999) Decreased expression of tumor necrosis factor-alpha in failing human myocardium after mechanical circulatory support: A potential mechanism for cardiac recovery. Circulation 100: 1189–93


15

16

17

18 19

20

21 22 23

24

25

26

27

28

Felkin LE, Birks EJ, George R, Wong S, Khaghani A, Yacoub MH, Barton PJ (2006) A quantitative gene expression profile of matrix metalloproteinases (MMPS) and their inhibitors (TIMPS) in the myocardium of patients with deteriorating heart failure requiring left ventricular assist device support. J Heart Lung Transplant 25: 1413–9 Torre-Amione G, Kapadia S, Lee J, Bies RD, Lebovitz R, Mann DL (1995) Expression and functional significance of tumor necrosis factor receptors in human myocardium. Circulation 92: 1487–93 Torre-Amione G, Kapadia S, Benedict C, Oral H, Young JB, Mann DL (1996) Proinflammatory cytokine levels in patients with depressed left ventricular ejection fraction: A report from the Studies of Left Ventricular Dysfunction (SOLVD). J Am Coll Cardiol 27: 1201–6 Torre-Amione G (2005) Immune activation in chronic heart failure. Am J Cardiol 95: 3C-8C; discussion 38C-40C Siwik DA, Chang DL, Colucci WS (2000) Interleukin-1beta and tumor necrosis factoralpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro. Circ Res 86: 1259–65 Deswal A, Petersen NJ, Feldman AM, Young JB, White BG, Mann DL (2001) Cytokines and cytokine receptors in advanced heart failure: An analysis of the cytokine database from the Vesnarinone Trial (VEST). Circulation 103: 2055–2059 Mauviel A (1993) Cytokine regulation of metalloproteinase gene expression. J Cell Biochem 53: 288–95 Nagase H (1997) Activation mechanisms of matrix metalloproteinases. Biol Chem 378: 151–60 Holzmann M, Nicko A, Kühl U, Noutsias M, Poller W, Hoffmann W, Morguet A, Witzenbichler B, Tschope C, Schultheiss HP et al (2008) Complication rate of right ventricular endomyocardial biopsy via the femoral approach: A retrospective and prospective study analyzing 3048 diagnostic procedures over an 11-year period. Circulation 118: 1722–8 Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I et al (1996) Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the definition and classification of cardiomyopathies. Circulation 93: 841–2 Noutsias M, Pauschinger M, Schultheiss HP, Kühl U (2002) Advances in the immunohistological diagnosis of inflammatory cardiomyopathy. Eur Heart J Supplement 4: I54–I62 Noutsias M, Pauschinger M, Ostermann K, Escher F, Blohm JH, Schultheiss H, Kühl U (2002) Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy. Med Sci Monit 8: MT59–71 Kühl U, Lassner D, Pauschinger M, Gross UM, Seeberg B, Noutsias M, Poller W, Schultheiss HP (2008) Prevalence of erythrovirus genotypes in the myocardium of patients with dilated cardiomyopathy. J Med Virol 80: 1243–1251 Pankuweit S, Moll R, Baandrup U, Portig I, Hufnagel G, Maisch B (2003) Prevalence

81


29 30 31 32

33 34 35 36

37 38

39 40

41

42

43

44 45

82

of the parvovirus B19 genome in endomyocardial biopsy specimens. Hum Pathol 34: 497–503 Pankuweit S, Richter A, Ruppert V, Maisch B (2009) [Classification of cardiomyopathies and indication for endomyocardial biopsy revisited]. Herz 34: 55–62 Kawai C (1999) From myocarditis to cardiomyopathy: Mechanisms of inflammation and cell death: Learning from the past for the future. Circulation 99: 1091–100 Feldman AM, McNamara D (2000) Medical Progress: Myocarditis. N Engl J Med 343: 1388–1398 Noutsias M, Pauschinger M, Poller WC, Schultheiss HP, Kühl U (2004) Immunomodulatory treatment strategies in inflammatory cardiomyopathy: current status and future perspectives. Expert Rev Cardiovasc Ther 2: 37–51 Godeny EK, Gauntt CJ (1987) Murine natural killer cells limit coxsackievirus B3 replication. J Immunol 139: 913–8 Liu PP, Mason JW (2001) Advances in the understanding of myocarditis. Circulation 104: 1076–82 Deonarain R, Cerullo D, Fuse K, Liu PP, Fish EN (2004) Protective role for interferonbeta in coxsackievirus B3 infection. Circulation 110: 3540–3 Noutsias M, Pauschinger M, Poller WC, Schultheiss HP, Kuhl U (2004) Immunomodulatory treatment strategies in inflammatory cardiomyopathy: Current status and future perspectives. Expert Rev Cardiovasc Ther 2: 37–51 Keogh AM, Billingham ME, Schroeder JS (1990) Rapid histological changes in endomyocardial biopsy specimens after myocarditis. Br Heart J 64: 406–8 Mendes LA, Picard MH, Dec GW, Hartz VL, Palacios IF, Davidoff R (1999) Ventricular remodeling in active myocarditis. Myocarditis Treatment Trial. Am Heart J 138: 303–8 Kishimoto C, Kitazawa M, Hiraoka Y, Takada H (1997) Extracellular matrix remodeling in coxsackievirus B3 myocarditis. Clin Immunol Immunopathol 85: 47–55 Seko Y, Takahashi N, Yagita H, Okumura K, Yazaki Y (1997) Expression of cytokine mRNAs in murine hearts with acute myocarditis caused by coxsackievirus b3. J Pathol 183: 105–8 Li J, Leschka S, Rutschow S, Schwimmbeck PL, Husmann L, Noutsias M, Westermann D, Poller W, Zeichhardt H, Klingel K et al (2007) Immunomodulation by interleukin-4 suppresses matrix metalloproteinases and improves cardiac function in murine myocarditis. Eur J Pharmacol 554: 60–8 McCarthy RE 3rd, Boehmer JP, Hruban RH, Hutchins GM, Kasper EK, Hare JM, Baughman KL (2000) Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocarditis. N Engl J Med 342: 690–5 Herzum M, Weller R, Jomaa H, Wietrzychowski F, Pankuweit S, Mahr P, Maisch B (1995) Left ventricular hemodynamic parameters in the course of acute experimental coxsackievirus B 3 myocarditis. J Mol Cell Cardiol 27: 1573–80 Feldman AM, McNamara D (2000) Myocarditis. N Engl J Med 343: 1388–98 Kyu B, Matsumori A, Sato Y, Okada I, Chapman NM, Tracy S (1992) Cardiac persis-


46

47

48

49

50

51

52

53

54 55

56

57

58

tence of cardioviral RNA detected by polymerase chain reaction in a murine model of dilated cardiomyopathy. Circulation 86: 522–30 Klingel K, Hohenadl C, Canu A, Albrecht M, Seemann M, Mall G, Kandolf R (1992) Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: Quantitative analysis of virus replication, tissue damage, and inflammation. Proc Natl Acad Sci USA 89: 314–8 Why HJ, Meany BT, Richardson PJ, Olsen EG, Bowles NE, Cunningham L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89: 2582–9 Fujioka S, Kitaura Y, Ukimura A, Deguchi H, Kawamura K, Isomura T, Suma H, Shimizu A (2000) Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 36: 1920–6 Fakhouri F, Placier S, Ardaillou R, Dussaule JC, Chatziantoniou C (2001) Angiotensin II activates collagen type I gene in the renal cortex and aorta of transgenic mice through interaction with endothelin and TGF-beta. J Am Soc Nephrol 12: 2701–10 Tharaux PL, Chatziantoniou C, Fakhouri F, Dussaule JC (2000) Angiotensin II activates collagen I gene through a mechanism involving the MAP/ER kinase pathway. Hypertension 36: 330–6 Smits JF, van Krimpen C, Schoemaker RG, Cleutjens JP, Daemen MJ (1992) Angiotensin II receptor blockade after myocardial infarction in rats: Effects on hemodynamics, myocardial DNA synthesis, and interstitial collagen content. J Cardiovasc Pharmacol 20: 772–8 Li YY, McTiernan CF, Feldman AM (1999) Proinflammatory cytokines regulate tissue inhibitors of metalloproteinases and disintegrin metalloproteinase in cardiac cells. Cardiovasc Res 42: 162–72 Weber KT, Anversa P, Armstrong PW, Brilla CG, Burnett JC Jr, Cruickshank JM, Devereux RB, Giles TD, Korsgaard N, Leier CV et al (1992) Remodeling and reparation of the cardiovascular system. J Am Coll Cardiol 20: 3–16 Woessner JF Jr (1999) Matrix metalloproteinase inhibition. From the Jurassic to the third millennium. Ann N Y Acad Sci 878: 388–403 Coker ML, Zellner JL, Crumbley AJ, Spinale FG (1999) Defects in matrix metalloproteinase inhibitory stoichiometry and selective MMP induction in patients with nonischemic or ischemic dilated cardiomyopathy. Ann N Y Acad Sci 878: 559–62 Coker ML, Doscher MA, Thomas CV, Galis ZS, Spinale FG (1999) Matrix metalloproteinase synthesis and expression in isolated LV myocyte preparations. Am J Physiol 277: H777–87 Tao ZY, Cavasin MA, Yang F, Liu YH, Yang XP (2004) Temporal changes in matrix metalloproteinase expression and inflammatory response associated with cardiac rupture after myocardial infarction in mice. Life Sci 74: 1561–72 Toth M, Chvyrkova I, Bernardo MM, Hernandez-Barrantes S, Fridman R (2003) Pro-

83


59

60

61

62 63 64 65 66 67

68

69

70

71

72

73

84

MMP-9 activation by the MT1–MMP/MMP-2 axis and MMP-3: Role of TIMP-2 and plasma membranes. Biochem Biophys Res Commun 308: 386–95 Miyamori H, Takino T, Seiki M, Sato H (2000) Human membrane type-2 matrix metalloproteinase is defective in cell-associated activation of progelatinase A. Biochem Biophys Res Commun 267: 796–800 Maquart FX, Pickart L, Laurent M, Gillery P, Monboisse JC, Borel JP (1988) Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyll-histidyl-l-lysine-Cu2+. FEBS Lett 238: 343–6 Diekmann O, Tschesche H (1994) Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9. Braz J Med Biol Res 27: 1865–76 Kridel SJ, Chen E, Kotra LP, Howard EW, Mobashery S, Smith JW (2001) Substrate hydrolysis by matrix metalloproteinase-9. J Biol Chem 276: 20572–8 Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2: 161–74 Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T (2003) Regulation of matrix metalloproteinases: An overview. Mol Cell Biochem 253: 269–85 Levick SP, Brower GL (2008) Regulation of matrix metalloproteinases is at the heart of myocardial remodeling. Am J Physiol Heart Circ Physiol 295: H1375–6 Page-McCaw A, Ewald AJ, Werb Z (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8: 221–33 Sivasubramanian N, Coker ML, Kurrelmeyer KM, MacLellan WR, DeMayo FJ, Spinale FG, Mann DL (2001) Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor. Circulation 104: 826–31 Zhou M, Zhang Y, Ardans JA, Wahl LM (2003) Interferon-gamma differentially regulates monocyte matrix metalloproteinase-1 and -9 through tumor necrosis factor-alpha and caspase 8. J Biol Chem 278: 45406–13 Wahl SM, Allen JB, Weeks BS, Wong HL, Klotman PE (1993) Transforming growth factor beta enhances integrin expression and type IV collagenase secretion in human monocytes. Proc Natl Acad Sci USA 90: 4577–81 Coker ML, Jolly JR, Joffs C, Etoh T, Holder JR, Bond BR, Spinale FG (2001) Matrix metalloproteinase expression and activity in isolated myocytes after neurohormonal stimulation. Am J Physiol Heart Circ Physiol 281: H543–51 Reinhardt D, Sigusch HH, Hensse J, Tyagi SC, Korfer R, Figulla HR (2002) Cardiac remodelling in end stage heart failure: Upregulation of matrix metalloproteinase (MMP) irrespective of the underlying disease, and evidence for a direct inhibitory effect of ACE inhibitors on MMP. Heart 88: 525–30 Masutomo K, Makino N, Fushiki MS (2001) Effects of losartan on the collagen degradative enzymes in hypertrophic and congestive types of cardiomyopathic hamsters. Mol Cell Biochem 224: 19–27 Rosano L, Varmi M, Salani D, Di Castro V, Spinella F, Natali PG, Bagnato A (2001)


74

75

76

77 78

79 80

81 82 83

84 85

86 87

88

Endothelin-1 induces tumor proteinase activation and invasiveness of ovarian carcinoma cells. Cancer Res 61: 8340–6 Tsuruda T, Boerrigter G, Huntley BK, Noser JA, Cataliotti A, Costello-Boerrigter LC, Chen HH, Burnett JC Jr (2002) Brain natriuretic Peptide is produced in cardiac fibroblasts and induces matrix metalloproteinases. Circ Res 91: 1127–34 He CS, Wilhelm SM, Pentland AP, Marmer BL, Grant GA, Eisen AZ, Goldberg GI (1989) Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc Natl Acad Sci USA 86: 2632–6 Heymans S, Lupu F, Terclavers S, Vanwetswinkel B, Herbert JM, Baker A, Collen D, Carmeliet P, Moons L (2005) Loss or inhibition of uPA or MMP-9 attenuates LV remodeling and dysfunction after acute pressure overload in mice. Am J Pathol 166: 15–25 Saksela O, Rifkin DB (1988) Cell-associated plasminogen activation: Regulation and physiological functions. Annu Rev Cell Biol 4: 93–126 Nerlov C, Rorth P, Blasi F, Johnsen M (1991) Essential AP-1 and PEA3 binding elements in the human urokinase enhancer display cell type-specific activity. Oncogene 6: 1583–92 Ogura N, Tobe M, Tamaki H, Nagura H, Abiko Y (2001) IL-1beta increases uPA and uPA receptor expression in human gingival fibroblasts. IUBMB Life 51: 381–5 Lijnen HR, Arza B, Van Hoef B, Collen D, Declerck PJ (2000) Inactivation of plasminogen activator inhibitor-1 by specific proteolysis with stromelysin-1 (MMP-3). J Biol Chem 275: 37645–50 Lijnen HR, Van Hoef B, Collen D (2001) Inactivation of the serpin alpha(2)-antiplasmin by stromelysin-1. Biochim Biophys Acta 1547: 206–13 Spinale FG (2002) Matrix metalloproteinases: Regulation and dysregulation in the failing heart. Circ Res 90: 520–30 Li YY, McTiernan CF, Feldman AM (2000) Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 46: 214–24 Brew K, Dinakarpandian D, Nagase H (2000) Tissue inhibitors of metalloproteinases: Evolution, structure and function. Biochim Biophys Acta 1477: 267–83 Amour A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knauper V et al (1998) TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 435: 39–44 Nagase H, Suzuki K, Morodomi T, Enghild JJ, Salvesen G (1992) Activation mechanisms of the precursors of matrix metalloproteinases 1, 2 and 3. Matrix Suppl 1: 237–44 Nagase H, Itoh Y, Binner S (1994) Interaction of alpha 2-macroglobulin with matrix metalloproteinases and its use for identification of their active forms. Ann N Y Acad Sci 732: 294–302 Moutsiakis D, Mancuso P, Krutzsch H, Stetler-Stevenson W, Zucker S (1992) Characterization of metalloproteinases and tissue inhibitors of metalloproteinases in human plasma. Connect Tissue Res 28: 213–30

85


89 90 91

92

93

94

95 96

97 98 99

100

101

102 103 104

86

Levine B, Kalman J, Mayer L, Fillit HM, Packer M (1990) Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 323: 236–41 Ferrari R (1999) The role of TNF in cardiovascular disease. Pharmacol Res 40: 97–105 Feldman AM, Combes A, Wagner D, Kadakomi T, Kubota T, Li YY, McTiernan C (2000) The role of tumor necrosis factor in the pathophysiology of heart failure. J Am Coll Cardiol 35: 537–44 Aukrust P, Ueland T, Lien E, Bendtzen K, Muller F, Andreassen AK, Nordoy I, Aass H, Espevik T, Simonsen S et al (1999) Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 83: 376–82 Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ, Feldman AM (1997) Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha. Circ Res 81: 627–35 Niebauer J, Volk HD, Kemp M, Dominguez M, Schumann RR, Rauchhaus M, PooleWilson PA, Coats AJ, Anker SD (1999) Endotoxin and immune activation in chronic heart failure: A prospective cohort study. Lancet 353: 1838–42 Rauchhaus M, Coats AJ, Anker SD (2000) The endotoxin-lipoprotein hypothesis. Lancet 356: 930–3 Sharma R, Bolger AP, Li W, Davlouros PA, Volk HD, Poole-Wilson PA, Coats AJ, Gatzoulis MA, Anker SD (2003) Elevated circulating levels of inflammatory cytokines and bacterial endotoxin in adults with congenital heart disease. Am J Cardiol 92: 188–93 Anker SD, von Haehling S (2004) Inflammatory mediators in chronic heart failure: An overview. Heart 90: 464–70 Liu L, Zhao SP (1999) The changes of circulating tumor necrosis factor levels in patients with congestive heart failure influenced by therapy. Int J Cardiol 69: 77–82 Torre-Amione G, Stetson SJ, Youker KA, Durand JB, Radovancevic B, Delgado RM, Frazier OH, Entman ML, Noon GP (1999) Decreased expression of tumor necrosis factor-alpha in failing human myocardium after mechanical circulatory support: A potential mechanism for cardiac recovery. Circulation 100: 1189–93 Bruggink AH, van Oosterhout MF, De Jonge N, Gmelig-Meyling FH, De Weger RA (2008) TNFalpha in patients with end-stage heart failure on medical therapy or supported by a left ventricular assist device. Transpl Immunol 19: 64–8 Adamopoulos S, Parissis J, Karatzas D, Kroupis C, Georgiadis M, Karavolias G, Paraskevaidis J, Koniavitou K, Coats AJ, Kremastinos DT (2002) Physical training modulates proinflammatory cytokines and the soluble Fas/soluble Fas ligand system in patients with chronic heart failure. J Am Coll Cardiol 39: 653–63 Barnes PJ, Karin M (1997) Nuclear factor-kappaB: A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–71 Murray DR, Prabhu SD, Chandrasekar B (2000) Chronic beta-adrenergic stimulation induces myocardial proinflammatory cytokine expression. Circulation 101: 2338–41 Chappel CI, Rona G, Balazs T, Gaudry R (1959) Severe myocardial necrosis produced by isoproterenol in the rat. Arch Int Pharmacodyn Ther 122: 123–8


105 Boluyt MO, Long X, Eschenhagen T, Mende U, Schmitz W, Crow MT, Lakatta EG (1995) Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart. Am J Physiol 269: H638–47 106 Woodiwiss AJ, Tsotetsi OJ, Sprott S, Lancaster EJ, Mela T, Chung ES, Meyer TE, Norton GR (2001) Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circulation 103: 155–60 107 Okada Y, Naka K, Kawamura K, Matsumoto T, Nakanishi I, Fujimoto N, Sato H, Seiki M (1995) Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: Implications for bone resorption. Lab Invest 72: 311–22 108 Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P et al (2000) Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 106: 55–62 109 Heymans S, Pauschinger M, De Palma A, Kallwellis-Opara A, Rutschow S, Swinnen M, Vanhoutte D, Gao F, Torpai R, Baker AH et al (2006) Inhibition of urokinase-type plasminogen activator or matrix metalloproteinases prevents cardiac injury and dysfunction during viral myocarditis. Circulation 114: 565–73 110 Leipner C, Grun K, Muller A, Buchdunger E, Borsi L, Kosmehl H, Berndt A, Janik T, Uecker A, Kiehntopf M et al (2008) Imatinib mesylate attenuates fibrosis in coxsackievirus b3-induced chronic myocarditis. Cardiovasc Res 79: 118–26 111 Hudson MP, Armstrong PW, Ruzyllo W, Brum J, Cusmano L, Krzeski P, Lyon R, Quinones M, Theroux P, Sydlowski D et al (2006) Effects of selective matrix metalloproteinase inhibitor (PG-116800) to prevent ventricular remodeling after myocardial infarction: Results of the PREMIER (Prevention of Myocardial Infarction Early Remodeling) trial. J Am Coll Cardiol 48: 15–20 112 Nishio R, Shioi T, Sasayama S, Matsumori A (2003) Carvedilol increases the production of interleukin-12 and interferon-gamma and improves the survival of mice infected with the encephalomyocarditis virus. J Am Coll Cardiol 41: 340–5 113 Lopez BL, Christopher TA, Yue TL, Ruffolo R, Feuerstein GZ, Ma XL (1995) Carvedilol, a new beta-adrenoreceptor blocker antihypertensive drug, protects against freeradical-induced endothelial dysfunction. Pharmacology 51: 165–73 114 Yue TL, McKenna PJ, Gu JL, Cheng HY, Ruffolo RE Jr, Feuerstein GZ (1994) Carvedilol, a new vasodilating beta adrenoceptor blocker antihypertensive drug, protects endothelial cells from damage initiated by xanthine-xanthine oxidase and neutrophils. Cardiovasc Res 28: 400–6 115 Pauschinger M, Doerner A, Remppis A, Tannhauser R, Kühl U, Schultheiss HP (1998) Differential myocardial abundance of collagen type I and type III mRNA in dilated cardiomyopathy: Effects of myocardial inflammation. Cardiovasc Res 37: 123–9 116 Pauschinger M, Chandrasekharan K, Li J, Poller WC, Noutsias M, Tschöpe C, Schultheiss HP (2002) Inflammation and extracellular matrix protein metabolism: Two sides of myocardial remodelling. Eur Heart J 4: I49–I55

87


117 Pauschinger M, Hoppe K, Schwimmbeck PL (2003) Relevance of myocardial inflammation for the activation of matrix metalloproteinase expression in patients with inflammatory cardiomyopathy. Circulation 108: 24 118 Schwartzkopff B, Fassbach M, Pelzer B, Brehm M, Strauer BE (2002) Elevated serum markers of collagen degradation in patients with mild to moderate dilated cardiomyopathy. Eur J Heart Fail 4: 439–4 119 Klappacher G, Franzen P, Haab D, Mehrabi M, Binder M, Plesch K, Pacher R, Grimm M, Pribill I, Eichler HG (1995) Measuring extracellular matrix turnover in the serum of patients with idiopathic or ischemic dilated cardiomyopathy and impact on diagnosis and prognosis. Am J Cardiol 75: 913–8 120 Frantz S, Stork S, Michels K, Eigenthaler M, Ertl G, Bauersachs J, Angermann CE (2008) Tissue inhibitor of metalloproteinases levels in patients with chronic heart failure: An independent predictor of mortality. Eur J Heart Fail 10: 388–95 121 George J, Patal S, Wexler D, Roth A, Sheps D, Keren G (2005) Circulating matrix metalloproteinase-2 but not matrix metalloproteinase-3, matrix metalloproteinase-9, or tissue inhibitor of metalloproteinase-1 predicts outcome in patients with congestive heart failure. Am Heart J 150: 484–7 122 Kelly D, Khan S, Cockerill G, Ng LL, Thompson M, Samani NJ, Squire IB (2008) Circulating stromelysin-1 (MMP-3): A novel predictor of LV dysfunction, remodelling and all-cause mortality after acute myocardial infarction. Eur J Heart Fail 10: 133–9 123 Mizon-Gerard F, de Groote P, Lamblin N, Hermant X, Dallongeville J, Amouyel P, Bauters C, Helbecque N (2004) Prognostic impact of matrix metalloproteinase gene polymorphisms in patients with heart failure according to the aetiology of left ventricular systolic dysfunction. Eur Heart J 25: 688–93 124 Kühl U, Schultheiss HP (1995) Treatment of chronic myocarditis with corticosteroids. Eur Heart J 16: 168–72 125 Kühl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-beta treatment eliminates cardiotropic viruses and improves left ventricular function in patients with myocardial persistence of viral genomes and left ventricular dysfunction. Circulation 107: 2793–8 126 Felix SB, Staudt A (2006) Non-specific immunoadsorption in patients with dilated cardiomyopathy: Mechanisms and clinical effects. Int J Cardiol 112: 30–3 127 Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewski P, Niklewski T, Zembala M, Polonski L, Rozek MM, Wodniecki J (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: Two-year follow-up results. Circulation 104: 39–45 128 Pankuweit S, Funck R, Grimm W, Maisch B (2006) [Diagnosis and treatment of inflammatory heart diseases: Role of endomyocardial biopsy]. Herz 31: 361–5

88

Alterations of the immune system in human viral and inflammatory cardiomyopathy

Molecular genetics of cardiomyopathies and myocarditis Jeffrey A. Towbin1 and Matteo Vatta2 1

Departments of Pediatrics (Cardiology), Molecular and Human Genetics, and Cardiovascular Sciences, Baylor College of Medicine, Texas Children’s Hospital, 6621 Fannin Street, MC 19345-C, Houston, TX 77030, USA 2 Department of Pediatrics (Cardiology), Baylor College of Medicine, Texas Children’s Hospital, 6621 Fannin Street, MC 19345-C, Houston, TX 77030, USA

Glossary AMPK: AMP kinase; cMyBP: cardiac myosin-binding protein; cTn: cardiac troponin; cTnC/ cTnI/cTnT: cTn C, I, T; CVB: coxsackievirus B; DAPC: dystrophin-associated protein complex; DCM: dilated cardiomyopathy; ECM: extracellular matrix; EFE: endocardial fibroelastosis; EMB: endomyocardial biopsy; FDCM: familial DCM; FHC: familial HCM; GSD: glycogen storage disease; HCM: hypertrophic cardiomyopathy; ICD: internal cardioverter-defibrillator; IF: intermediate filament; LGMD: limb girdle muscular dystrophy; LVH: left ventricular hypertrophy; LVNC: left ventricular noncompaction; MLC: myosin light chain; A-TM: A-tropomyosin; WPW: WolffParkinson-White; XDCM: X-linked DCM

Introduction Cardiomyopathies are major causes of morbidity and mortality and over the past 20 years, limited improvements in outcome have been reported [1–3]. However, improvement in the understanding of the major forms of cardiomyopathy has occurred over that time, in large part due to advances in genetics and genomics [4, 5]. In addition, new forms of cardiomyopathy have been described and classified over that time frame, mainly due to our improved genetic-based understanding of heart muscle disease. The improved understanding gained in heart muscle disease has also led to understanding of similarities and differences in heart and skeletal muscle and their often overlapping clinical presentations. A new classification scheme was recently developed for the cardiomyopathies in which five forms of disease were formally classified as distinct forms of cardiomyopathy [6]. These forms include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and left ventricular noncompaction (LVNC). These were further


91

Jeffrey A. Towbin and Matteo Vatta

classified into genetic/inherited forms and acquired/non-inherited forms [6]. The major form of acquired disease is that of viral myocarditis, and this disorder will be discussed along with the genetic-based myocardial diseases. To understand the mechanisms responsible for the development of the clinical phenotypes of cardiomyopathies, an understanding of normal cardiac structure is necessary.

Normal cardiac structure Cardiac muscle fibers are comprised of separate cellular units (myocytes) connected in series [7]. In contrast to skeletal muscle fibers, cardiac fibers do not assemble in parallel arrays but bifurcate and recombine to form a complex three-dimensional network. Cardiac myocytes are joined at each end to adjacent myocytes at the intercalated disc, a specialized area of interdigitating cell membrane (Fig. 1). The intercalated disc contains gap junctions (containing connexins), and mechanical junctions, comprised of adherens junctions (containing N-cadherin, catenins and vinculin) and desmosomes (containing desmin, desmoplakin, desmocollin, desmoglein). Cardiac myocytes are surrounded by a thin membrane (sarcolemma) and the interior of each myocyte contains bundles of longitudinally arranged myofibrils. The myofibrils are formed by repeating sarcomeres, the basic contractile units of cardiac muscle comprised of interdigitating thin (actin) and thick (myosin) filaments (Fig. 1) that give the muscle its characteristic striated appearance [8, 9]. The thick filaments are composed primarily of myosin but additionally contain myosin-binding proteins C (MyBP-C), H and X. The thin filaments are composed of cardiac actin, A-tropomyosin (A-TM), and troponins T, I, and C (cTnT, cTnI, cTnC). In addition, myofibrils contain a third filament formed by the giant filamentous protein, titin, which extends from the Z-disc to the M-line and acts as a molecular template for the layout of the sarcomere. The Z-disc at the borders of the sarcomere is formed by a lattice of interdigitating proteins that maintain myofilament organization by cross-linking antiparallel titin and thin filaments from adjacent sarcomeres (Fig. 2). Other proteins in the Z-disc include A-actinin, nebulette, telethonin/T-cap, capZ, MLP, myopalladin, myotilin, Cypher/ZASP, filamin, and FATZ [8–10]. Finally, the extrasarcomeric cytoskeleton, a complex network of proteins linking the sarcomere with the sarcolemma and the extracellular matrix (ECM), provides structural support for subcellular structures and transmits mechanical and chemical signals within and between cells. The extrasarcomeric cytoskeleton has intermyofibrillar and subsarcolemmal components, with the intermyofibrillar cytoskeleton composed of intermediate filaments (IFs), microfilaments and microtubules [11, 12]. Desmin IFs form a three-dimensional scaffold throughout the extrasarcomeric cytoskeleton with desmin filaments surrounding the Z-disc, allowing for longitu-

92

Molecular genetics of cardiomyopathies and myocarditis

Figure 1. Cardiac myocyte cytoarchitecture. Schematic of the interactions between dystrophin and the dystrophin-associated proteins in the sarcolemma and intracellular cytoplasm (dystroglycans, sarcoglycans, syntrophins, dystrobrevin, sarcospan) at the C-terminal end of the dystrophin. The integral membrane proteins interact with the extracellular matrix via A-dystroglycan-laminin A2 connections. The N terminus of dystrophin binds actin and connects dystrophin with the sarcomere intracellularly, the sarcolemma and extracellular matrix. Additional sarcolemmal proteins include ion channels, adrenergic receptors, integrins and the coxsackie- and adenoviral receptor. Cell-cell junctions, including cadherens, the plakin and other desmosomal family proteins are also notable. Also shown is the interaction between intermediate filament proteins (i.e., desmin) with the nucleus. MLP, muscle LIM protein.

dinal connections to adjacent Z-discs and lateral connections to subsarcolemmal costameres [12]. Microfilaments composed of non-sarcomeric actin (mainly G-actin) also form complex networks linking the sarcomere (via A-actinin) to various components of the costameres. Costameres are subsarcolemmal domains located in a periodic, grid-like pattern, flanking the Z-discs and overlying the I bands, along

93


Figure 2. Z-disc architecture. The Z-disc of the sarcomere is comprised of multiple interacting proteins that anchor the sarcomere. Reported with permission from [9].

the cytoplasmic side of the sarcolemma. These costameres are sites of interconnection between various cytoskeletal networks linking sarcomere and sarcolemma, and are thought to function as anchor sites for stabilization of the sarcolemma and for integration of pathways involved in mechanical force transduction. Costameres contain three principal components: the focal adhesion-type complex, the spectrinbased complex, and the dystrophin/dystrophin-associated protein complex (DAPC) [13, 14]. The focal adhesion-type complex, comprised of cytoplasmic proteins (i.e., vinculin, talin, tensin, paxillin, zyxin), connect with cytoskeletal actin filaments and with the transmembrane proteins A-, B-dystroglycan, A-, B-, G-, D-sarcoglycans, dystrobrevin, and syntrophin. Several actin-associated proteins are located at sites of attachment of cytoskeletal actin filaments with costameric complexes, including A-actinin and the muscle LIM protein, MLP. The C terminus of dystrophin binds B-dystroglycan (Fig. 1), which in turn interacts with A-dystroglycan to link to the

94


ECM (via A-2-laminin). The N terminus of dystrophin interacts with actin. Also notable, voltage-gated sodium channels co-localize with dystrophin, B-spectrin, ankyrin and syntrophins, while potassium channels interact with the sarcomeric Z-disc and intercalated discs [15, 16]. Since arrhythmias and conduction system diseases are common in children and adults with DCM, this could play an important role. Hence, disruption of the links from the sarcolemma to ECM at the dystrophin C terminus and those to the sarcomere and nucleus via N-terminal dystrophin interactions could lead to a “domino effect” disruption of systolic function and development of arrhythmias.

Dilated cardiomyopathy DCM is the most common form of cardiomyopathy. It is characterized primarily by left ventricular (LV) dilation and systolic dysfunction (Fig. 3) with associated right ventricular dysfunction and diastolic abnormalities. In children, the annual incidence of DCM is 0.57 cases per 100,000 per year overall, but is higher in boys than in girls (0.66 vs 0.47 cases per 100,000; p < 0.001), in blacks than in whites (0.98 vs 0.46 cases per 100,000; p < 0.001), and in infants (< 1 year) than in children (4.40 vs 0.34 cases per 100,000; p < 0.001). The majority of children (66%) are thought to have idiopathic disease [17]. The mortality rate in the United States due to cardiomyopathy is greater than 10 000 deaths per annum, with DCM being the major contributor [1]. The total cost of health care in the United States focused on cardiomyopathies is in the billions of dollars and only limited success has been achieved. To achieve improved care and outcomes in children and adults, understanding of the causes of these disorders has been sought. DCM has become a popular target of research over the past 10–15 years, with multiple genes identified during that time period. These genes appear to encode two major subgroups of proteins, cytoskeletal and sarcomeric proteins [4]. The cytoskeletal proteins identified to date include dystrophin, desmin, lamin A/C, D-sarcoglycan, B-sarcoglycan, and metavinculin. In the case of sarcomere-encoding genes, the same genes identified for HCM appear to be culprits, and include B-myosin heavy chain (B-MyHC), MyBP-C, actin, A-TM, and cTnT. A new group of sarcomeric genes, those encoding Z-disk proteins, have also been identified and include cypher/ ZASP [1, 3, 5], muscle LIM protein, and A-actinin-2 [1, 3, 5]. In addition, phospholamban and G4.5/Tafazzin have also been reported. Another form of DCM, the acquired disorder viral myocarditis, has the same clinical features as DCM including heart failure, arrhythmias and conduction block [1, 5]. The most common causes of myocarditis are viral, including the enteroviruses (coxsackieviruses and echovirus), adenoviruses, and parvovirus B19, amongst other cardiotropic viruses [1]. Evidence exists that suggests that viral myocarditis and DCM (genetic) have similar mechanisms of disease based on the proteins targeted.

95


Figure 3. Echocardiography in dilated cardiomyopathy. Apical four chamber view demonstrating a dilated left ventricle. In real-time, the systolic function is depressed.

Clinical genetics of DCM DCM was initially believed to be inherited in a small percentage of cases until Michels et al. [18] showed that approximately 20% of probands had family members with echocardiographic evidence of DCM when family screening was performed. More recently, inherited, familial DCM (FDCM) has been shown to occur in 30–40% of cases [4] with autosomal dominant inheritance being the predominant pattern of transmission; X-linked, autosomal recessive, and mitochondrial inheritance is less common.

96


Molecular genetics of DCM Over the past decade, progress has been made in the understanding of the genetic etiology of FDCM (Tab. 1). Initial progress was made studying families with X-linked forms of DCM, with the autosomal dominant forms of DCM beginning to unravel over the past few years. In the case of X-linked forms of DCM, two disorders have been well characterized, X-linked cardiomyopathy (XLCM), which presents in adolescence and young adults, and Barth syndrome, which is most frequently identified in infancy [19, 20].

Table 1. Dilated cardiomyopathy (DCM) genetics Chr locus

Gene

Protein

Xp21.2

DYS

Dystrophin

Xq28

G4.5

Tafazzin

1q21

LMNA

Lamin A/C

1q32

TNNT2

Cardiac troponin T

1q42-43

ACTN

A-Actinin 2

2q31

TTN

Titin

2q35

DES

Desmin

5q33

SGCD

D-Sarcoglycan

6q22.1

PLN

Phospholamban

10q22.3-23.2

ZASP/Cypher

ZASP

10q22-q23

VCL

Metavinculin

11p11

MYBPC3

Myosin-binding protein C

11p15.1

MLP

Muscle LIM protein

14q12

MYH7

B-Myosin heavy chain

15q14

ACTC

Cardiac actin

15q22

TPM1

A-Tropomyosin

X-linked cardiomyopathies X-linked dilated cardiomyopathy First described in 1987 by Berko and Swift [19] as DCM occurring in males in the teen years and early twenties, with rapid progression from chronic heart failure (CHF) to death due to ventricular tachycardia/fibrillation (VT/VF) or transplan-

97


tation, these patients are distinguished by elevated serum creatine kinase muscle isoforms (CK-MM). Female carriers tend to develop mild to moderate DCM in the fifth decade and the disease is slowly progressive. Towbin and colleagues [21] were the first to identify the disease-causing gene and characterize the functional defect. In this report, the dystrophin gene was shown to be responsible for the clinical abnormalities, and protein analysis by immunoblotting demonstrated severe reduction or absence of dystrophin protein in the heart of these patients. These findings were later confirmed by Muntoni et al. [22] when a mutation in the muscle promoter and exon 1 of dystrophin was identified in another family with XLCM. Subsequently, multiple mutations have been identified in dystrophin in patients with XLCM. Dystrophin is a cytoskeletal protein that provides structural support to the myocyte by creating a lattice-like network to the sarcolemma [23]. In addition, dystrophin plays a major role in linking the sarcomeric contractile apparatus to the sarcolemma and ECM [23–26]. Furthermore, dystrophin is involved in cell signaling, particularly through its interactions with nitric oxide synthase. The dystrophin gene is also responsible for Duchenne and Becker muscular dystrophy (DMD/BMD) when mutated [27]. These skeletal myopathies present early in life (DMD is diagnosed before the age of 12 years, while BMD is seen in teenage males older than 16 years) and the vast majority of patients develop DCM before their 25th birthday. In most patients, CK-MM is elevated similar to that seen in XLCM; in addition, manifesting female carriers develop disease late in life, similar to XLCM. Immunohistochemical analysis demonstrates reduced levels (or absence) of dystrophin, as seen in the hearts of patients with XLCM. Murine models of dystrophin deficiency demonstrate abnormalities of muscle physiology based on membrane structural support abnormalities [28]. In addition to the dysfunction of dystrophin, mutations in dystrophin secondarily affect proteins that interact with dystrophin. At the N terminus, dystrophin binds to the sarcomeric protein actin, a member of the thin filament of the contractile apparatus. At the C terminus, dystrophin interacts with A-dystroglycan, a dystrophin-associated membrane-bound protein involved in the function of the DAPC, which includes B-dystroglycan, the sarcoglycan subcomplex (A-, B-, G-, D-, and E-sarcoglycan), syntrophins, and dystrobrevins [29–31] (Fig. 1). In turn, this complex interacts with A2-laminin and the ECM [32]. Like dystrophin, mutations in these genes lead to muscular dystrophies with or without cardiomyopathy, supporting the contention that this group of proteins is important to the normal function of the myocytes of the heart and skeletal muscles [32, 33]. In both cases, mechanical stress [28] appears to play a significant role in the age-onset dependent dysfunction of these muscles. The information gained from the studies on XLCM, DMD, and BMD, led us to hypothesize that DCM is a disease of the cytoskeleton/sarcolemma and affects the sarcomere [34], a “final common pathway” of DCM [35]. We also have suggested that dystrophin mutations play a role in idiopathic DCM in males. This is supported by our finding that 3 out of 22 boys with DCM had dystrophin mutations

98


and all were later found to have elevated CK-MM [36]. In addition, eight families with DCM and possible X-linked inheritance were also screened, and dystrophin mutations were noted in three of these families. Again, CK-MM was elevated in all subjects carrying mutations [37].

Barth syndrome Initially described as X-linked cardioskeletal myopathy with abnormal mitochondria and neutropenia by Neustein et al. [38] and Barth et al. [39], this disorder typically presents in male infants as CHF associated with neutropenia (cyclic) and 3-methylglutaconic aciduria [40]. Mitochondrial dysfunction is noted on electron microscopy and electron transport chain biochemical analysis. Recently, abnormalities in cardiolipin have been noted [41]. Echocardiographically these infants typically have left ventricular dysfunction with left ventricular dilation, endocardial fibroelastosis (EFE), or a dilated hypertrophic left ventricle. In some cases these infants succumb due to CHF/sudden death VT/VF, or sepsis due to leukocyte dysfunction. The majority of these children survive infancy and do well clinically, although DCM usually persists. In some cases, cardiac transplantation has been performed. Histopathological evaluation typically demonstrates the features of DCM, although EFE may be prominent and the mitochondria are abnormal in shape and abundance. The genetic basis of Barth syndrome was first described by Bione et al. [42] who cloned the disease-causing gene, G4.5. This gene encodes a novel protein called tafazzin, whose gene product is an acyltransferase and results in cardiolipin abnormalities [41]. Mutations in G4.5 result in a wide clinical spectrum, which includes apparent classic DCM, hypertrophic DCM, EFE, or LVNC [4, 43].

Autosomal dominant DCM The most common form of inherited DCM is the autosomal dominant form of disease [1, 4, 5]. These patients present as classic “pure” DCM or DCM associated with conduction system disease (CDDC). In the latter case, patients usually present in the third decade of life with mild conduction system disease, which can progress to complete heart block over decades. DCM usually presents late in the course but is out-of-proportion to the degree of conduction system disease [44]. The echocardiographic and histological findings in both subgroups are classic for DCM, although the conduction system may be fibrotic in patients with CDDC. In both groups of DCM patients, VT, VF, and torsade des pointes (TdP) occur and may result in sudden death. Genetic heterogeneity exists for autosomal dominant DCM with more than 15 loci mapped for pure DCM and five loci for CDDC [3]. In the case of pure DCM, 10 genes have been identified to date, including 3 by our group (D-sarcoglycan,

99


A-actinin-2, ZASP) [4, 5, 45], as well as actin, desmin, TnT, B-MyHC, titin, metavinculin, MyBP-C, A-TM, muscle LIM protein (MLP), B-sarcoglycan and phospholamban [4, 46, 47] (Tab. 1). The majority of genes identified to date encode either cytoskeletal or sarcomeric proteins. In the case of cytoskeletal proteins (desmin, D-sarcoglycan, metavinculin, MLP), defects of force transmission are considered to result in the DCM phenotype, while defects of force generation have been speculated to cause sarcomeric proteininduced DCM [4, 34]. Cardiac actin is a sarcomeric protein that is a member of the sarcomeric thin filament interacting with tropomyosin and the troponin complex. As previously noted, actin plays a significant role in linking the sarcomere to the sarcolemma via its binding to the N terminus of dystrophin, and the mutations in actin that resulted in DCM as described by Olson et al [48] appear to be directly involved in the binding of dystrophin. The DCM-causing mutations are believed to result by causing force transmission abnormalities. Further, actin interacts in the sarcomere with TnT and B-MyHC, two other genes resulting in either DCM or HCM depending on the position of the mutation [49]. In the case of TnT and B-MyHC, force generation abnormalities have been speculated as the responsible mechanism. Desmin is a cytoskeletal protein that forms intermediate filaments specific for muscle [12]. This muscle-specific 53-kDa subunit of class III intermediate filaments forms connections between the nuclear and plasma membranes of cardiac, skeletal and smooth muscle. Desmin is found at the Z lines and intercalated disk of muscle and its role in muscle function appears to involve attachment or stabilization of the sarcomere. Mutations in this gene appear to cause abnormalities of force and signal transmission similar to that believed to occur with actin mutations [50]. Another DCM-causing gene, D-sarcoglycan is a member of the sarcoglycan subcomplex of the DAPC [45, 51]. This gene encodes for a protein involved in stabilization of the myocyte sarcolemma as well as signal transduction. Mutations identified in familial and sporadic cases resulted in reduction of the protein within the myocardium. In the absence of D-sarcoglycan, the remaining sarcoglycans (D, B, G, 3) cannot assemble properly in the endoplasmic reticulum [52]. Mouse models of D-sarcoglycan deficiency demonstrate dilated, HCM, sarcolemmal fragility, and disrupted vasculin smooth muscle, which lead to vascular spasm, including coronary spasm [53, 54]. In addition, mutations in this gene lead to the phenotype of the cardiomyopathic Syrian hamster [55]. Other human mutations in D-sarcoglycan cause a form of autosomal recessive limb girdle muscular dystrophy (LGMD2F), which is rarely associated with heart disease. The final cytoskeletal protein-encoding gene, metavinculin, encodes vinculin and its splice variant metavinculin. Vinculin is ubiquitously expressed and metavinculin is co-expressed with vinculin in heart, skeletal and smooth muscle. This protein complex localized to subsarcolemmal costameres in the heart where they are found in subsarcolemmal costameres in the heart. There, they interact with A-actinin,

100


talin, and G-actin to form a microfilamentous network linking cytoskeleton and sarcolemma. In addition, these proteins are present in adherens junctions in intercalated disks and participate in cell-cell adhesion. Mutations in metavinculin have been shown to disrupt the intercalated disks and alter actin filament cross-linking [56, 57]. Mutations in the sarcomere may produce HCM or DCM. In the latter case, abnormalities in force generation or transmission are thought to contribute to the development of this phenotype [49]. In addition to mutations in the thin filament protein actin, mutations in the thick filament protein-encoding gene B-MyHC has been shown to cause DCM with associated sudden death in at least one infant, as well as DCM in older children and adults [49, 58]. Mutations in this gene are thought to perturb the actin-myosin interaction and force generation or alter crossbridge movement during contraction. Mutations in cTnT, a thin filament protein, have been speculated to disrupt calcium-sensitive TnC binding [49, 58]. Mutations in phospholamban [59, 60] have also been identified, which further support calcium handling as a potentially important mechanism in the development of DCM. Interestingly, Haghihi et al. [60] identified homozygous mutations causing DCM and heart failure, while heterozygotes had cardiac hypertrophy. Recessive mutation in TnI is thought to impair the interaction with TnT, while A-TM mutations have also been identified and were predicted to alter the surface charge of the protein, leading to impaired interaction with actin [61, 62]. A recent area of interest for evaluation at the molecular level is the Z disc [63]. Knoll et al. [64] identified mutations in muscle LIM protein (MLP) and demonstrated that this results in defects in the interaction with telethonin [64]. Using mouse models, they also demonstrated that MLP acts as a stretch sensor and that mutant MLP causes defects in this activity. More recently, mutations in MLP in families and sporadic cases were described, and abnormalities in the T-tubule system [4] and Z disc architecture have been identified by electron microscopy, which correlates with the histopathology seen in MLP-knockout mice [65]. This was further supported by the finding of reduced expression of MLP in human CHF [66, 67]. In addition, mutations in A-actinin-2, which is involved in cross-linking actin filaments and shares a common actin-binding domain with dystrophin, were also identified in familial DCM, disrupting its binding to MLP [5]. Vatta et al. [4] identified mutations in the Z-band alternatively spliced PDZ-motif protein ZASP, the human homolog of the mouse cypher gene that when disrupted leads to DCM [68]. Multiple mutations in this gene were identified in families and sporadic cases of DCM and with LVNC. This protein, which interacts with A-actinin-2, disrupts the actin cytoskeleton when mutated. Another gene, titin, which encodes the giant sarcomeric cytoskeletal protein titin that contributes to the maintenance of the sarcomere organization and myofibrillar elasticity, interacts with these proteins at the Z disc/I band transition zone [69]. Mutations have been identified in familial DCM as well [70].

101


As seen in pure autosomal dominant DCM, genetic heterogeneity also exists for CDDC. To date, CDDC genes have been mapped to chromosomes 1p1-1q1, 2q1421, 3p25-22, and 6q23. The only gene thus far identified was reported to be lamin A/C on chromosome 1q21, which encodes a nuclear envelope intermediate filament protein [71, 72].

Lamin A/C The lamins are located in the nuclear lamina at the nucleoplasmic side of the inner nuclear membrane, and lamin A and C are expressed in heart and skeletal muscle [73]. Mutations in this gene were initially reported to cause the autosomal dominant form of Emery-Dreifuss muscular dystrophy [74, 75], which has skeletal myopathy associated with DCM and conduction system disease. It has also been found to cause a form of autosomal dominant limb girdle muscular dystrophy (LGMD1B), which is also associated with conduction system disease [76]. Multiple mutations have been identified in patients with DCM and conduction system disease, who, in some cases, had mildly elevated CK. This gene defect appears to be relatively common in patients with CDDC. The mechanism(s) responsible for the development of DCM and conduction system abnormalities, and skeletal myopathy are being determined [77]. Understanding may be aided by understanding how other genes that encode interacting proteins result in disease, such as thymopoietin [78].

Muscle is muscle: Cardiomyopathy and skeletal myopathy genes overlap Interestingly, nearly all of the genes identified for inherited DCM are also known to cause skeletal myopathy in humans and/or mouse models. In the case of dystrophin, mutations cause DMD and BMD, while D-sarcoglycan mutations cause LGMD2F. Lamin A/C has been shown to cause autosomal dominant Emery-Dreifuss muscular dystrophy and LGMD1B, while actin mutations are associated with nemaline myopathy. Desmin, G4.5, A-dystrobrevin, Cypher/ZASP, MLP, A-actinin-2, titin, B-sarcoglycan mutations also have been associated skeletal myopathy, suggesting that cardiac and skeletal muscle function is interrelated and that possibly the skeletal muscle fatigue seen in patients with DCM with and without CHF may be due to primary skeletal muscle disease and not only related to the cardiac dysfunction. It also suggests that the function of these muscles has a “final common pathway” and that both cardiologists and neurologists should consider evaluation of both sets of muscles. Further support for this concept comes from studies of animal models. Mutations in D-sarcoglycan in hamsters results in cardiomyopathy, while mutations in all

102


sarcoglycan subcomplex genes in mice cause skeletal and cardiac muscle disease. Mutations in other DAPC genes as well as dystrophin in murine models also consistently demonstrate abnormalities of skeletal and cardiac muscle function. Arber et al. [65] also produced a mouse deficient in muscle MLP, a structural protein that links the actin cytoskeleton to the contractile apparatus. The resultant mice develop severe DCM, CHF and disruption of cardiac myocyte cytoskeletal architecture. Murine mutations in titin, cypher, A-dystrobrevin, and desmin, all demonstrate cardiac and skeletal muscle disease. Finally, Badorff et al. [79] has shown that the DCM that develops after viral myocarditis has a mechanism similar to the inherited forms. Using coxsackievirus B3 (CVB3) infection of mice, the authors showed that the CVB3 genome encodes for a protease (enteroviral protease 2A) that cleaves dystrophin at the third hinge region of dystrophin, resulting in force transmission abnormalities and DCM. In addition, Xiong et al. [80] showed that abnormal dystrophin increases susceptibility to viral infection and resultant myocarditis. Interestingly, a similar dystrophin mutation, which affects the first hinge region of dystrophin in patients with XLCM, was previously reported by our laboratory, demonstrating a consistent mechanism of DCM development, abnormalities of the cytoskeleton/sarcolemma and sarcomere. In addition, we have shown that N-terminal dystrophin is reduced or absent in hearts of patients with all forms of DCM (ischemic, acquired, genetic, idiopathic) and that reduction of mechanical stress using left ventricular assist devices (LVADS) results in reverse remodeling of dystrophin and of the heart itself [81, 82].

Hypertrophic cardiomyopathy HCM is characterized by asymmetric or concentric wall thickening (Fig. 4), hence the term hypertrophic heart disease [83–85]. However, the major impact of this disorder on human health is its predilection to be inherited, its reputation as the most common cause of sudden death in young, healthy, athletic individuals [86–89], and its potential to develop heart failure [90–92]. In the latter case, heart failure can occur due to diastolic factors or can occur due to the development of systolic dysfunction, so-called “burned out” HCM [90–92]. No matter what the cause of untoward clinical features, HCM plays a significant role in our health care monetary burden as well as the untold price of the tragedies by affected families. In the pediatric age range, the underlying etiologies responsible for the disease and the variable age range of onset differentiates further the childhood form of disease from the adult counterpart [93]. In very young children (i.e., less than 1 year of age), ventricular hypertrophy associated with systolic dysfunction is more the rule than the exception. In addition, overlap disorders in which HCM coexists with other atypical features are also more common during childhood, further confounding the presentations, treatments, and outcomes compared to adult disease.

103


Figure 4. Echocardiography in hypertrophic cardiomyopathy. Left panel: Parasternal short axis echocardiogram. Concentric hypertrophy in a 1-month-old child. Note the small left ventricular chamber. Right Panel: Similar view demonstrates worsening left ventricular hypertrophy and a smaller chamber size at 8 months of age in the same child.

Clinical aspects of HCM HCM is a primary myocardial disorder with an autosomal dominant pattern of inheritance that is characterized by hypertrophy of the left (± right) ventricle with histological features of myocyte hypertrophy, myofibrillar disarray, and interstitial fibrosis [83–85]. HCM is one of the most common inherited cardiac disorders, with a prevalence in young adults of 1 in 500 [85]. Various names have been given to this disorder including hypertrophic obstructive cardiomyopathy and idiopathic subaortic stenosis. These names reflect “textbook” features of asymmetric septal hypertrophy and left ventricular outflow tract obstruction. This description of the disease is based primarily on patients with severe symptoms seen in tertiary hospital referral centers. Epidemiological studies now suggest that a wide spectrum of clinical manifestations of varying severity and prognosis is present in community populations. The first clinical description of HCM was reported in 1869 [94] in France, and was recognized to be a genetic disorder in the late 1950s. Since then, numerous clinical and pathological studies of HCM have been performed. During the last 15 years, molecular genetic studies have given important insights into the pathogenesis of HCM and have provided a new perspective for the diagnosis and management of patients with this disorder [95].

104


Genetics of familial HCM The first gene for familial HCM (FHC) was mapped to chromosome 14q11.2-q12 using genome-wide linkage analysis in a large Canadian family [96]. Soon afterwards, FHC locus heterogeneity was reported [97] and subsequently confirmed by the mapping of the second FHC locus to chromosome 1q3 and of the third locus to chromosome 15q2 [98, 99]. Carrier et al. [100] mapped the fourth FHC locus to chromosome 11p11.2. Multiple other loci were subsequently reported, including loci on chromosomes [101–108] (Fig. 5).

Figure 5. Chromosomal locus positions of the original sarcomere-encoding genes responsible for hypertrophic cardiomyopathy. See Table 2 for the complete list of disease-causing genes and their locus positions.

105


Gene identification in FHC The majority of the disease-causing genes identified to date code for proteins that are part of the sarcomere, which is a complex structure with an exact stoichiometry and multiple sites of protein-protein interactions [95, 109, 110]. These include three myofilament proteins: the B-MyHC, the ventricular myosin essential light chain 1 (MLC-1s/v) and the ventricular myosin regulatory light chain 2 (MLC-2s/v); four thin filament proteins: cardiac actin, cTnT, cTnI, and A-TM; and one MyBP, the cMyBP-C (Tab. 2). Each of these proteins is encoded by multigene families that exhibit tissue-specific, developmental, and physiologically regulated patterns of expression [111]. The giant protein titin [105] and its interactive Z-disc protein,

Table 2. Hypertrophic cardiomyopathy (HCM) genetics Chr locus

Gene

Protein

Xq22

A-Gal

A-Galactosidase

Xq24

LAMP-2

Lysomal-associated membrane protein 2

Xq28

G4.5

Tafazzin

1q32

TNNT2

Cardiac troponin T

1q42-43

ACTN

A-Actinin2

2q31

TTN

Titin

3p21.2

MELC

Myosin essential light chain

3p25

CAV3

Caveolin-3

4q26

MYOZ2

Myozenin

6q13

MYO6

Unconventional myosin VI

7q31

AMPK

AMP kinase

10q22.3

ZASP/Cypher

ZASP

11p11

MYBPC3

Myosin-binding protein C

11p15.1

MLP

Muscle LIM protein

12q23

MRLC

Myosin regulatory light chain

14q12

MYH7

B-Myosin heavy chain

15q14

ACTC

Cardiac actin

15q22

TPM1

A-Tropomyosin

17q12

TCAP

Telethonin

19p13.2

CTNNI

Cardiac troponin I

106


MLP, have also been identified [106]. In addition, the gene located on chromosome 7q3 associated with HCM and Wolff-Parkinson-White (WPW) syndrome was identified as AMP kinase (AMPK), which has been suggested to play a role in energy metabolism and cause infiltration of a glycogen-like substance similar to that seen in Pompe’s disease [112–114].

Thick filament proteins Myosin subunits Myosin is the molecular motor that transduces energy from the hydrolysis of ATP into directed movement and that, by doing so, drives sarcomere shortening and muscle contraction. Cardiac myosin consists of two MyHC and two pairs of light chains (MLC), referred to as essential (or alkali) light chains (MLC-1) and regulatory (or phosphorylatable) light chains (MLC-2), respectively [111]. The myosin molecule is highly asymmetric, consisting of two globular heads joined to a long rod-like tail. The light chains are arranged in tandem in the head-tail junction. Their function is not fully understood. Neither MLC type is required for the adenosine triphosphatase (ATPase) activity of the myosin head, but they probably modulate it in the presence of actin and contribute power stroke. Mutations have been found in the heavy chains and in the two types of ventricular light chains. Concerning the heavy chains, the B isoform (B-MyHC) is the major isoform of the human ventricle and of slow-twitch skeletal fibers. It is encoded by MYH7. This gene appears to be the most commonly mutated HCM gene and hot spots for mutations have been identified [95, 109, 115]. The majority of mutations are missense mutations located either in the head or in the head-rod junction of the molecule. Based on their structural location in the myosin head, the majority of mutations are likely to disrupt both mechanical and catalytic components of actin-myosin interaction, resulting in reduced force generation. Sarcomere assembly is also likely to be disrupted. Mutations in the light meromyosin domain have also been identified, and Blair and colleagues [115] speculated that HCM develops in this case due to abnormalities of myosin filament assembly or interactions with thick filament binding proteins. The MLC isoforms are expressed in the ventricular myocardium and in the slow-twitch muscles and are the so-called ventricular myosin regulatory light chains (MLC-2 s/v) encoded by MYL2, and the ventricular myosin essential light chain (MLC-1 s/v) encoded by MYL3. The MLC are thought to influence the mechanical efficiency of cross-bridge cycling and speed of contraction. It is believed that these proteins regulate power output via a calcium-dependent mechanism, and disruption leads to the HCM phenotype.

107


Myosin-binding protein C MyBP-C is part of the thick filaments of the sarcomere, being located at the level of the transverse stripes, 43 nm apart, seen by electron microscopy in the sarcomere A band. Its function is uncertain, but, for over a decade, evidence has existed to indicate both structural and regulatory roles. Partial extraction of cMyBP-C from rat skinned cardiac myocytes and rabbit skeletal muscle fibers alters Ca2+-sensitive tension [116], and it has been shown that phosphorylation of cMyBP-C alters myosin cross-bridges in native thick filaments, suggesting that cMyBP-C can modify force production in activated cardiac muscles. The cardiac isoform is encoded by the MYBPC3 gene.

Thin filament proteins The thin filament contains actin, the troponin complex and tropomyosin. The troponin complex and tropomyosin constitute the Ca2+-sensitive switch that regulates the contraction of cardiac muscle fibers. Mutations have been found in A-TM and in two of the subunits of the troponin complex: cTnI, the inhibitory subunit, and cTnT, the tropomyosin-binding subunit. A-TM is encoded by TPM1. The cardiac isoform is expressed both in the ventricular myocardium and in fast twitch skeletal muscles [117]. It shares the overall structure of other tropomyosins that are rod-like proteins that possess a simple dimeric A-coiled-coil structure of other tropomyosins that are rod-like proteins that possess a simple dimeric A-coiled-coil structure in parallel orientation along their entire length [117]. It is believed that some mutations in this gene could alter tropomyosin binding to actin. cTnT is encoded by TNNT2. In human cardiac muscle, multiple isoforms of cTnT have been described, which are expressed in the fetal, adult and diseased heart, and which result from alternative splicing of the single gene TNNT2 [118, 119]. The precise physiological relevance of these isoforms is currently poorly understood. Mutations in this gene are predicted to influence the inhibitory regulatory effect of the tropomyosin-troponin complex. cTnI is encoded by TNNl3. The cTnI isoform is expressed only in cardiac muscles [120]. Cooperative binding of cTnI to actin-tropomyosin is a unique property of the cardiac variant, it is thought that mutations disrupt the calcium-sensitive switch mediated by this protein, resulting in increased calcium sensitivity and reduced maximum tension. A-Cardiac actin (ACTC) mutations also cause of FHC. Mogensen et al. [104] identified mutations in a family with heterogeneous phenotypes, ranging from asymptomatic with mild hypertrophy to pronounced septal hypertrophy and left ventricular outflow tract obstruction.

108


Z-disc and other proteins Mutations in titin [105] and MLP [106] have been identified as causes of HCM, suggesting the Z disc to be important in the development of HCM (Figs 1 and 2). More recently, mutations in ZASP, telethonin (TCAP), A-actinin2 [121] and myozenin/calsarcin [122] have also been demonstrated to cause HCM. In addition, disease-causing genes with uncertain mechanisms have been identified including mutations in caveolin-3 and unconventional myosin VI. The functional abnormalities resulting in the clinical phenotype are currently unclear [107, 108].

Genotype-phenotype relations in FHC The pattern and extent of left ventricular hypertrophy in patients with HCM vary greatly, even in first-degree relatives, and a high incidence of sudden death is reported in selected families. An important issue therefore is to determine whether the genotype heterogeneity observed in HCM accounts for the phenotypic diversity of the disease. However, the results must be seen as preliminary, because the available data relate to only a few hundred individuals, and it is obvious that, although a given phenotype may be apparent in a small family, examining large or multiple families with the same mutation is required before drawing unambiguous conclusions. Several concepts have been published for mutations in the MYH7, TNNT2, and MYBPC3 genes. For MYH7, the prognosis for patients with different mutations has been shown to vary. For example, the R403Q mutation was felt to be associated with markedly reduced survival [123], whereas some others, such as V606M, appeared more benign. The disease caused by TNNT2 mutations was reportedly associated with a 20% incidence of non-penetrance, a relatively mild and sometimes subclinical hypertrophy, but a high incidence of sudden death, which occurred even in the absence of significant clinical left ventricular hypertrophy [124, 125]. Mutations in MYBPC3, on the other hand, have been characterized by specific clinical features with a mild phenotype in young subjects, a delayed age at the onset of symptoms and a favorable prognosis before the age of 40 [126–129]. However, despite these assertions, the notion of mutation-specific clinical outcomes was challenged by Van Diest and colleagues [130], who demonstrated that “benign” mutations were uncommon (5/253) and that the mutations studied all had severe clinical disease. Genetic studies have also revealed the presence of clinically healthy individuals carrying the mutant allele, which is associated in first-degree relatives with a typical phenotype of the disease. Several mechanisms could account for the large variability of the phenotypic expression of the mutations: the role of environmental differences and acquired traits (e.g., differences in lifestyle, risk factors, and exercise) and finally the existence of modifier genes and/or polymorphisms that could modulate the phenotypic expression of the disease. Significant results have been obtained thus far

109


regarding the influence of the angiotensin I-converting enzyme (ACE) insertion/deletion (I/D) polymorphism. Association studies showed that, compared to a control population, the D allele is more common in patients with HCM and in patients with a high incidence of sudden cardiac death [131, 132]. An association between the D allele and hypertrophy was seen in the case of MYH7 R403 codon mutations, but not with MYBPC3 mutation carriers [133], raising the concept of multiple genetic modifiers in HCM.

Therapy in HCM The mainstays of therapy in children with HCM have been pharmacological approaches. The two major medication classes used include beta-blockers and calcium-channel blockers [134–136]. In small children, we have used propranolol as our drug of choice due to ease of access, liquid formulation and low side-effect profile. Therapy in these children is monitored by heart rate response, with the goal being approximately 80–100 beats per minute. In older children, we typically treat with atenolol; in children with excessive hypertrophy and severe outflow tract obstruction, we occasionally consider use of combination therapy (beta-blocker plus calcium-channel blocker), although this is not without risk. However, the risk:benefit ratio must be determined for each patient. When standard pharmacologic therapy fails, there are limited options, although the size of the child plays a role. In small children, myomectomy is the only proven option [137, 138]. Again, this is not without risk. In older patients, pacing protocols have been used but are controversial [139–141]. In adults, alcohol septal ablation has been utilized but this has not yet been championed in children due to the uncertainties associated with creating an infarct in a young individual regarding long-term outcome [142–145]. In patients with syncope, ventricular arrhythmias, or other presumed high risk, internal cardioverter-defibrillator (ICD) implantation should be considered [89]. In some patients, pacing is necessary as well. Heart failure in HCM occurs either due to diastolic dysfunction or “burned out” disease with resultant systolic dysfunction. In the latter case, therapy is similar to that used in patients with DCM. This includes use of ACE inhibitor plus B-blocker therapy, with or without diuretic and digoxin [91]. Enalapril and carvedilol are the most common ACE inhibitor/B-blocker combination used. In the case of diastolic dysfunction with heart failure and preserved systolic function, combination therapy with ACE inhibitors, diuretic, with or without angiotensin II-receptor blockers such as candesartan or Losartan are commonly used. In these patients, B-blockers, calcium-channel blockers, and pacing are also considered, along with surgical relief [91]. Finally, in children with metabolic or mitochondrial dysfunction underlying the HCM, metabolic therapies have been successful on occasion. Similar to the therapy

110


in DCM caused by these deficiencies, carnitine, coenzyme Q10, riboflavin and thiamine may be considered [93].

Infiltrative forms of HCM A variety of disorders that have apparent left ventricular hypertrophy and features of HCM occur due to infiltrative disorders. The classic form of infiltrative disease in this category is Pompe’s disease, a disorder typically presenting in the first weeks of life [93]. More recently, other forms of infiltrative disease have been identified with later onset disease, such as Fabry’s disease [146], Danon’s disease [147, 148] and left ventricular hypertrophy due to mutations in AMP-activated protein kinase (AMPK) encoded by the PRKAG2 gene [112–114]. These disorders, along with those caused by mitochondrial abnormalities and genetic dysmorphism syndromes such as Noonan syndrome and LEOPARD syndrome, are caused by abnormalities not primarily affecting the sarcomere [93]. Therapy is similar to the sarcomeric form of the disease, unless systolic dysfunction occurs. In this case, heart failure therapy should be instituted.

Pompe’s disease (type II glycogen storage disease) Genetic deficiency of acid A-1,4 glucosidase, an enzyme involved in the breakdown of glycogen to glucose, results in a wide clinical spectrum ranging from the rapidly fatal infantile onset of type II glycogen storage disease (GSD) to a slowly progressive adult-onset myopathy. The infantile-onset form (Pompe’s disease) typically manifests during the first 5 months of life, and patients usually die before their second year [149]. This rare inborn error of glycogen metabolism occurs in less than 1 per 100 000 births. Massive glycogen accumulation occurs, leading to the clinical findings of enlarged tongue, striking hepatomegaly, hypotonia with decreased deep tendon reflexes, and cardiomyopathy (usually HCM) with congestive heart failure. The glycogen accumulation can be noted histologically in the skeletal muscles, liver, and heart. Children usually succumb in the first 2 years of life. The diagnosis may be predicted from the pathognomonic electrocardiogram (ECG) [93, 150]. The disease has autosomal recessive inheritance; the gene coding for the lysosomal enzyme was originally mapped to chromosome 17 at sub-band 17q23-q25 [151]. Allelic variation at the acid A-glucosidase locus is presumed to be the most important factor in diversity of type II GSD [149, 152]. It has been shown that various combinations of homoallelic and heteroallelic mutant genotypes are the basis for this heteroallelic mutant genotypes are the basis for this clinical heterogeneity. Zhong et al. [153] identified a missense mutation in one allele of a patient with Pompe’s disease. This base pair substitution resulted in a loss of restriction endonuclease sites, which allowed them to demonstrate mRNA expression deficiency

111


from the second allele using polymerase chain reaction (PCR)-amplified RNA. This was the first evidence of single base pair missense mutations in patients with this disease. In addition to molecular analysis, the diagnosis can be made biochemically by analysis of A-glucosidase in blood lymphocytes or skin fibroblasts. Prenatal diagnosis is possible by amniocentesis or chorionic villus sampling by assaying A-glucosidase. Enzyme therapy is now possible and appears to reverse the cardiac phenotype [154].

Fabry’s disease An X-linked recessive disorder with mild expression occasionally seen in carrier females, this entity is caused by deficiency of the enzyme A-galactosidase (A-Gal) and is found in 1 in 40 000 people. Young adults may be prone to renal failure and myocardial infarctions. Fabry’s disease usually has its onset in adolescence and usually manifests with sensations of burning pain in the hands and feet [146, 155]. These sensations tend to be associated with fever, heart, cold, and exercise. With increasing age, multiple angiokeratoma become noticeable, especially around the umbilicus and genitalia. Corneal opacities are often noted. Progressive renal failure develops with age. CNS manifestations include seizures and headaches, as well as hemiplegia associated with an increased risk of stroke. Primary cardiac manifestations in affected males are HCM and mitral insufficiency and the diagnosis depends on echocardiography [156–158]. The left ventricular myocardium and mitral valve tend to be areas of greatest storage of lipid material. On ECG, the PR interval is usually short. Deposition of sphingolipids in the coronary arteries leads to myocardial ischemia and infarction [157, 159]. The disease-causing gene was originally localized to the long arm of the X chromosome in the Xq22 region, and the full-length cDNA was isolated and sequenced [160], demonstrating a 1393-bp cDNA with a 60-nucleotide 5¢ untranslated region, and encoding a precursor peptide of 429 amino acids. The gene was found to contain seven exons; mutations and phenotypic correlation have been described [161, 162]. Nakas and colleagues [163] studied 1603 men by echocardiography and demonstrated significant left ventricular hypertrophy (LVH) in 230 subjects (14%) of which 7 (3%) had proven A-Gal deficiency. These patients had concentric LVH. Linhart et al. [164] studied 30 patients with Fabry’s disease and found 37% had concentric LVH, 10% had asymmetric septal hypertrophy, and 3% had an eccentric pattern of hypertrophy. More recently, Sachdev et al. [165] identified 5 patients with HCM diagnosed after age 40 years and 1 with earlier onset (younger than 40 years) HCM, all with low A-Gal activity. In 5 of the 6 patients, the hypertrophy was concentric, while 1 had asymmetric hypertrophy. In 1 case, LV outflow tract obstruction was noted. Nonsustained ventricular tachycardia occurred in 2 patients and another patient had 2° AV block initially and then atrial fibrillation. Therefore,

112


it appears that these abnormalities in A-Gal activity result in some cases of HCM or “unexplained” LVH.

Danon’s disease Danon’s disease is an X-linked dominant disorder characterized by intracytoplasmic vacuoles containing autophagic material and glycogen in cardiac and skeletal muscle cells, cardiomyopathy and skeletal myopathy, with or without conduction defect, WPW syndrome, or mental retardation [166]. The underlying abnormality affects lysosomal function and is due to mutations in the lysosomal-associated membrane protein 2 (LAMP2) [167]. The clinical phenotypic expression of Danon’s disease is variable. Charron et al. [168] screened 50 cases of HCM for LAMP2 mutations and identified mutations in two patients with HCM and skeletal myopathy. Both of these individuals presented during their teenage years and other younger affected individuals in the family were also identified as young as 7 years of age. WPW syndrome and high voltage QRS complexes on ECG were notable along with high creatine kinase plasma levels. In addition, late LV dilation and dysfunction occurred with symptoms of heart failure. Atrial and ventricular arrhythmias and conduction disease was notable along with death during their twenties. Visual acuity abnormality was also common, due to choriocapillary ocular atrophy. This disease appears to be under-recognized and may play a significant role in pediatric heart failure.

AMP-activated protein kinase AMPK, encoded by the G2 regulatory subunit of the PRKAG2 gene on chromosome 7q31 [101], is an enzyme that modulates glucose uptake and glycolysis [169, 170]. Dominant mutations in this gene were first identified by Gollob et al. [112] and Blair et al. [113] in 2001 in subjects with HCM, WPW pre-excitation, and atrioventricular block. The genetic locus on chromosome 7q3 was first described by MacRae et al. in 1995 [101] in families with HCM and WPW, and was felt to be clinically different than the patients with other forms of adult HCM. Blair et al. [113] made the case that mutations in AMPK resulted in HCM due to compromise of energy production and utilization, but Arad and colleagues [114] provided evidence that this disorder is a form of GSD. Cardiac pathology differed from other forms of HCM, with no myocyte and myofibrillar disarray seen, but instead pronounced formation of vacuoles filled with glycogen-associated granules. The myocytes were enlarged and interstitial fibrosis was minimal. Utilizing a yeast system in which a similar enzyme is functional, Arad et al. [171] introduced the same mutations found in the patients and showed that the enzyme activity is persistent (i.e., does not turn off), leading to glycogen accumulation. The authors confirmed these findings by developing a murine model that mimics the human disorder.

113


Energy-dependent forms of HCM Mitochondrial cardiomyopathies The human mitochondrial genome [172] is a small, circular DNA molecule that is maternally inherited. Mitochondrial DNA (mtDNA) encodes 13 of the 69 proteins required for oxidative metabolism, and 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs) required for their translation. Because mtDNA has much less redundancy than the nuclear genome (in which essentially identical information is received from both parents), and tRNAs and rRNAs are present in multiple copies, the mitochondrial genome is an excellent target for mutations giving rise to human disease [173–175]. Mitochondria enjoy a symbiotic relationship with the cell. These subcellular organelles are dependent on nucleocytoplasmic mechanisms for most structural components, but do contribute vital peptides that are central to cellular respiration. Mitochondria contain a permeable outer membrane and a highly restrictive inner membrane that guards the chemical microenvironment of the matrix compartment. Adaptive mechanisms exist for the passage of large and small molecules across the inner membrane. Translocases shuttle monocarboxylic acids, amino acids, acyl-carnitine conjugates, small ions, and other metabolites in and out of the mitochondrial matrix. Energy is required for importation of proteins into the mitochondria because the nuclear gene-synthesized mitochondrial proteins are precursor molecules that require presequence cleavage. The 13 mtDNA genes are located in the respiratory chain [175–177] and include seven complex I subunits, (ND1, 2, 3, 4L, 4, 5, and 6); one complex III subunit (cytochrome b); three complex IV subunits (COI, II, III); and two complex V subunits (ATPase 6 and 8). Coordination must exist between nuclear and mitochondrial genomes to permit assembly of the complex holoenzymes. Each cell contains numerous mitochondria and each mitochondrion contains multiple copies of mtDNA. This genetic material derives exclusively from the female gamete and any mutation must be passed from female parent to all progeny, male and female. The replicative segregation of mutant mtDNA copies within the cell determines whether this biological disadvantage is expressed. In most mitochondrial disorders, patients carry a mix of mutant and normal mitochondria, a condition known as heteroplasmy, with the proportions varying from tissue-to-tissue and individual-to-individual within a pedigree in a manner correlating with severity of phenotype [174, 175]. Mitochondrial diseases often produce disturbances of brain and muscle function, presumably because these two organs are so metabolically active, and therefore the metabolic demand is high during growth and development [178]. Cardiac disease is most commonly seen with respiratory chain defects [179, 180]. Ragged red fibers are present in muscle biopsy specimens almost invariably when the molecular defect involves mtDNA (except in infants) [173]. These defects represent the genetics of ATP production. The diverse clinical syndromes associated with various respiratory chain complexes are thought to result from involvement of tissue-nonspecific

114


(generalized) subunits in other cases, and the residual enzyme activity in affected tissues [181]. The cardiac diseases associated with mitochondrial defects include both HCM and DCM, and LVNC [182, 183]. No theory has thus far been advanced to explain the cause of these phenotypically different cardiac abnormalities. It is possible, however, that the dilated form occurs after an initial hypertrophic response (i.e., it is a “burned-out” dilated form of HCM).

Kearns-Sayre syndrome This mitochondrial myopathy is characterized by ptosis, chronic progressive external ophthalmoplegia, abnormal retinal pigmentation, and cardiac conduction defects, as well as DCM. Channer et al. [184] reported a case of rapidly developing progressive congestive heart failure and DCM requiring transplantation in a patient with Kearns-Sayre syndrome. Approximately 20% of Kearns-Sayre syndrome patients have cardiac involvement and the majority usually have conduction defects causing progressive heart block. These patients generally have large, heterogeneous deletions in the mitochondrial chromosome. Poulton et al. [185] showed germ line deletions of mtDNA in a family with Kearns-Sayre syndrome using PCR to amplify across the deletion, with primers flanking these deletions. The patient was shown to have a deletion in muscle mtDNA and at low levels in blood that was identical to that found in the mother and sister. The probands, however, had more deleted DNA, correlating with more severe symptomatology. Other mutations have also been described [186, 187].

MERRF syndrome This syndrome is characterized by myoclonic epilepsy with ragged red muscle fibers (MERRF) and is caused by a single nucleotide substitution in tRNA LYS that apparently interferes with mitochondrial translation [188, 189]. Shoffner et al. [190] showed an A to G transition mutation as the cause of the disease associated with defects in complexes I and IV. This abnormality causes decline in ATP-generating capacity, with onset of disease that includes cardiomyopathy. Other reports outline various disease-causing mutations [191–193].

Overlap disorders Left ventricular noncompaction This disorder has previously been considered to be a rare disease and has been identified by a variety of names including spongy myocardium, fetal myocardium, and noncompaction of the left ventricular myocardium [194–198]. The abnormality

115


is believed to represent an arrest in the normal process of myocardial compaction, the final stage of myocardial morphogenesis, resulting in persistence of multiple prominent ventricular trabeculations and deep intertrabecular recesses (Fig. 6). This cardiomyopathy is somewhat difficult to diagnose unless the physician has a high level of suspicion during echocardiographic evaluation. In fact, on careful review of echocardiograms and other clinical data, it appears that LVNC is relatively common in children and is also seen in adults [194, 197]. In the most recent AHA/ACC Cardiomyopathy Classification, LVNC was recognized for the first time as a formal form of classified cardiomyopathy. Two forms of LVNC occur: (1) isolated noncompaction and (2) noncompaction associated with congenital heart disease such as septal defects (ventricular and/or atrial septal defect), pulmonic stenosis, hypoplastic left heart syndrome, amongst others [110, 195–199]. In the isolated form and the form associated with congenital heart disease, metabolic derangements may be notable [183, 196].

Figure 6. Echocardiographic features of left ventricular noncompaction. Top left Panel: A parasternal long axis view, demonstrates a hypertrophic posterior left ventricular wall with a moth-eaten pattern. The left ventricular chamber is small. Top right Panel: A parasternal short axis view with color Doppler demonstrates hypertrophy of the left ventricular apex with deep trabeculations filled with blood (Doppler flow).

116


Clinical features LVNC most commonly presents in infancy with signs and symptoms of heart failure but some patients are identified during later childhood, adolescence or adulthood. Pignatelli et al. [194] recently reported their findings on 36 children identified over a 5-year period, with a median age at presentation of 90 days (range 1 day to 17 years). In this study, 40% of the children presented with low cardiac output or congestive heart failure and only one child (3%) presented with syncope. The most common presenting symptom other than heart failure was asymptomatic ECG or radiographic abnormalities, with 42% being asymptomatic. In addition, 14% of children had associated dysmorphic features, while 19% of affected children had first-degree relatives with cardiomyopathy. Of the children with dysmorphic features, one was diagnosed with DiGeorge syndrome and one with congenital adrenal hyperplasia.

Genetics When LVNC is inherited, it can be transmitted as an X-linked, mitochondrial, autosomal recessive or autosomal dominant trait [110]. In approximately 20–30% of cases, familial inheritance has been identified. The X-linked form usually is associated with isolated noncompaction and a mutation in the G4.5 (tafazzin) gene located on chromosome Xq28 [199]. This gene has also been identified in patients with Barth syndrome. In autosomal dominant inherited cases, mutations in the Z-line protein encoding ZASP, located on chromosome 10q22, have been identified in isolated noncompaction [200], while mutations in the gene encoding a-dystrobrevin, a cytoskeletal protein located on chromosome, have been identified in patients with noncompaction associated with congenital heart disease [199]. No genes have been identified thus far for autosomal recessive-inherited noncompaction, while mutations in mtDNA have been seen in patients with noncompaction [183, 196].

Therapy and outcome The specific therapy depends on the clinical and echocardiographic findings. In patients with systolic dysfunction and heart failure, anti-congestive therapy identical to that used in patients with DCM is appropriate. In particular, ACE inhibitors such as captopril and enalapril, as well as B-adrenergic blocking agents such as metoprolol or carvedilol are useful. Diuretics may also be needed. However, in those patients exhibiting findings more consistent with an HCM or diastolic dysfunction physiological phenotype, B-blocker therapy alone with propranolol or atenolol is more appropriate. In patients with either of these forms of noncompaction with associated mitochondrial or metabolic dysfunction some investigators add a “vitamin cocktail” to the cardiac therapy with coenzyme Q10, carnitine, riboflavin, and thiamine commonly used alone or in combination.

117


In patients having associated congenital heart disease, appropriate therapeutic approaches may include simple pharmacological therapy with diuretics for volume overload associated with left to right shunts, more complex pharmacological therapy for patients with restrictive physiology and pulmonary hypertension, or invasive therapy with catheter intervention or surgical repairs, depending on the lesions. Intimate understanding of the cardiac function abnormalities, evidence of thrombi (which should be treated with anti-coagulation), and the metabolic status of the patient must be attended to by the interventional cardiologist, cardiac anesthesiologist, and surgeon in approaching these patients invasively. In addition, cardiac rhythm disturbances need to be identified and therapies such as pacemakers, ICD, and intracardiac ablations considered. The clinical outcome of patients with noncompaction has been reported to be poor with death occurring due to heart failure or sudden death presumably arrhythmia-related or stroke-related due to embolization of left ventricular thrombi [195, 198]. However, Pignatelli et al. [194] demonstrated a 5-year survival rate of 86%; when transplanted patients were added, the 5-year survival free of death or transplantation was 75%.

Myocarditis Myocarditis is a process characterized by inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of the ischemic damage associated with coronary artery disease. This definition does not take into account the underlying etiology [201].

Etiology Most cases of myocarditis in the United States and Western Europe result from viral infections [202]. In the 1970s and 1980s, coxsackievirus was the most common virus identified, but in the 1990s and early 21st century, the most common viral causes included adenovirus especially serotypes 2 and 5 [203–206] and enterovirus (coxsackieviruses A and B, echovirus, poliovirus), particularly coxsackievirus B (CVB) [207, 208] (Tab. 3). Most recently, parvovirus B19 has become a commonly identified virus in subjects with suspected myocarditis, supplanting adenovirus as the most commonly identified etiological agent [210–213]. However, a wide variety of other viral causes of myocarditis [214, 215] in children have been described, including influenza [216], cytomegalovirus [217], herpes simplex virus [218], hepatitis C [219–221], rubella [222], varicella [223], mumps [224], Epstein-Barr virus [225], human immunodeficiency virus (HIV) [226], and respiratory syncytial virus [227], among others. Other nonviral etiologies include other infectious agents such

118


Table 3. Viral causes of myocarditis Enterovirus Coxsackie A Coxsackie B Echovirus Poliovirus

Varicella

Adenovirus

Rubella

Parvovirus B19

Rubeola

Cytomegalovirus (CMV)

Respiratory syncytial virus (RSV)

Herpesvirus (HSV)

Human immunodeficiency virus

Influenza A

Epstein–Barr virus (EBV)

Mumps Measles Rabies Hepatitis B, C

as rickettsiae, bacteria, protozoa, and other parasites, fungi, and yeasts [228–239]; various drugs, including antimicrobial medications [201]; hypersensitivity, autoimmune, or collagen–vascular diseases [240–242] such as systemic lupus erythematosus, mixed connective tissue disease, rheumatic fever, rheumatoid arthritis, and scleroderma; toxic reactions to infectious agents [243] (e.g., mumps or diphtheria); or other disorders such as Kawasaki disease and sarcoidosis [244, 245]. In most cases, however, idiopathic myocarditis is encountered.

Epidemiology Myocarditis is a disorder that is under-diagnosed [201, 204], but the incidence of the usual lymphocytic form of myocarditis reportedly ranges from 4–5% (as obtained from reports of young men dying of trauma) [246] to as high as 16–21% (as found in autopsy series of children dying suddenly). In adults with unexplained DCM, the incidence ranges from 3% to 63% [201, 247], although the large multicenter Myocarditis Treatment Trial, which was strictly based on the Dallas criteria, reported a 9% incidence [248]. Usually sporadic, viral myocarditis can also occur as an epidemic [249]. Epidemics are usually seen in newborns, most commonly in association with CVB. Intrauterine myocarditis occurs during epidemics as well as sporadically [250]. Postnatal spread of coxsackievirus is via the fecal/oral or airborne route [251, 252]. The World Health Organization (WHO) reports that this ubiquitous family of viruses results in cardiovascular sequelae in less than 1% of infections, although this increases to 4% when CVB is considered [249]. Other important viral causes, such as adenovirus [253, 254] and influenza A, are transmitted through the air.

119


Clinical manifestations Presentation depends on the age of the affected individual [203, 215, 251]. Nonspecific flu-like illness or episodes of gastroenteritis may precede symptoms of congestive heart failure.

Newborns and infants Newborns or infants present with poor appetite, fever, irritability or listlessness, periodic episodes of pallor, and diaphoresis. Sudden death may occur in this subgroup of children [215, 251, 255]. On physical examination, pallor and mild cyanosis in addition to classic symptoms of congestive heart failure are commonly noted. It is important to keep in mind that the younger the child, the more likely that intrauterine myocarditis is now expressed as a chronic disease [215, 243].

Children, adolescents, and adults Older children, adolescents, and adults commonly have a recent history of viral disease 10–14 days prior to presentation [251]. Initial symptoms include lethargy, low-grade fever, and pallor; the child usually has decreased appetite and may complain of abdominal pain. Diaphoresis, palpitations, rashes, exercise intolerance, and general malaise are common signs and symptoms. Later in the course of illness, respiratory symptoms become predominant; syncope or sudden death may occur due to cardiac collapse. Physical examination findings are consistent with congestive heart failure [206, 251], as described above. Unlike newborns, jugular venous distention and pulmonary rales may be observed, and resting tachycardia may be prominent. Arrhythmias, including atrial fibrillation, supraventricular tachycardia, or ventricular tachycardia, as well as atrioventricular block, may occur [251, 256].

Diagnostic tests The diagnosis of myocarditis is often difficult to establish but should be suspected in any patient who presents with unexplained congestive heart failure or ventricular tachycardia. Appropriate diagnostic studies include the following.

Chest radiography Cardiomegaly with pulmonary edema is classically seen.

120


Electrocardiography Sinus tachycardia with low-voltage QRS complexes with or without low-voltage or inverted T waves are classically described. A pattern of myocardial infarction with wide Q waves and ST-segment changes also may be seen [257] (Fig. 7). Ventricular tachycardia, supraventricular tachycardia, atrial fibrillation, or atrioventricular block occurs in some children [256].

Echocardiography A dilated and dysfunctional left ventricle consistent with DCM is seen on twodimensional (Fig. 8) and M-mode echocardiography (Fig. 8). Segmental wall motion abnormalities are relatively common, but global hypokinesis is predominant. Pericardial effusion frequently occurs. Doppler and color Doppler commonly demonstrate mitral regurgitation. Dilation of other chambers also may be seen.

Figure 7. Electrocardiogram in a child with myocarditis. Sinus tachycardia and low-voltage QRS complexes with inverted T waves and a pattern of myocardial infarction with wide Q waves in leads I and aVL, and ST-segment changes consistent with ischemia noted throughout.

121


Figure 8. Echocardiographic features of myocarditis. (A) Two-dimensional parasternal long-axis view demonstrating left ventricular (LV) dilation and a pericardial effusion (PE). Color Doppler interrogation provides evidence of mitral regurgitation. (B) Parasternal long-axis view demonstrating LV dilation and normal papillary muscles (P). (C) M mode demonstrating systolic dysfunction with flattened interventricular septal motion (IVS), fair LV posterior wall excursion (LVPW), LV dilation with increased LV end-diastolic dimension (D), and reduced systolic function (S) and PE.

Endomyocardial biopsy Right ventricular endomyocardial biopsy (EMB) (Fig. 9) is used to evaluate possible inflammatory infiltrate (Fig. 10), which is usually patchy and scattered in the ventricular myocardium. A mononuclear cell infiltrate is diagnostic of myocarditis, although this does not delineate etiology. Myocardial biopsy is diagnostically sensitive in 3–63% of cases [251, 258–261]. Because there is risk associated with biopsy,

122


Figure 9. Endomyocardial biopsy (EMB) technique. The bioptome is advanced via the superior vena cava into the right atrium, across the tricuspid valve into the right ventricle, and finally situated against the interventricular septum, where the biopsy is performed. The bioptome also can be advanced via the inferior vena cava with similar results. (From [150] with permission.)

particularly in young children or those with severe ventricular dilation, centers have abandoned this procedure.

The Dallas criteria The Dallas Criteria define myocarditis as “a process characterized by an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of ischemic damage” due to coronary artery or other disease [262]. At the time of initial biopsy, a specimen may be classified either as active myocarditis, borderline myocarditis, or no myocarditis, depending on whether an

123


Figure 10. EMB histology demonstrates lymphocytic infiltrates, myocardial edema, and necrosis.

inflammatory infiltrate occurs in association with myocyte degeneration or necrosis (active), or too sparse of an infiltrate or no myocyte degeneration occurs (borderline) [248, 262]. Repeat EMB may be appropriate in cases where strong suspicion of myocarditis exists clinically; on repeat EMB, histology may be classified as ongoing myocarditis, resolving myocarditis, or resolved myocarditis.

Viral studies A positive viral culture from myocardium has been considered the diagnostic standard in the past. Viral culture of peripheral specimens, such as blood, stool, or urine, is commonly performed but is unreliable at identifying the causative infection. A fourfold increase in antibody titer correlates with infection [263, 264]. However, these studies are nonspecific because prior infection with the causative virus is commonplace. Molecular analysis using in situ hybridization identified CVB sequences in myocardial samples [265], but this method never gained widespread popularity. PCR [204–206] amplifies viral sequences from cardiac tissue samples, is extremely sensitive, and typically specific.

124


Molecular diagnostics First reported in 1986, in situ hybridization was performed on myocardial tissue using probes for coxsackievirus [265, 266]. This technique is difficult to use in a hospital setting and, therefore, lost favor and never gained widespread use. The PCR amplification process, which identifies specific portions of a viral genome, is sensitive and specific [204–206] (Fig. 11). Using PCR, 20–50% of cases initially identified enterovirus genome; however, no other viral genome was analyzed in these early studies [267–269]. Subsequently, PCR was used to screen other viral genomes within cardiac tissue specimens, and adenovirus (Fig. 12) was identified as commonly as enterovirus (Fig. 13) in heart tissue specimens of patients with myocarditis or DCM (Tab. 4) [204–206, 215]. Additional viral genomes identified using PCR include cytomegalovirus, parvovirus B19, respiratory syncytial virus, Epstein-Barr virus, herpes simplex virus, and influenza A virus [204, 215, 270,

Figure 11. Method for performing polymerase chain reaction (PCR) from myocardial specimens. Using a bioptome to obtain an EMB or scalpel to obtain tissue from an autopsy or explanted heart, the specimen is homogenized, nucleic acid is extracted and, along with dNTPs and viral primers, placed into a PCR thermocycler. Once complete, the sample is placed in an agarose gel and electrophoresed before being visualized as a “band” of a predictable size.

125


Figure 12. PCR for adenovirus genome using nested PCR. The agarose gel demonstrates a 308-bp PCRpositive band in the adenovirus-positive control lane, as well as in lanes 14 and 22, samples obtained from patients with myocarditis. The remaining patients and the negative control lanes are negative for adenoviral genome.

Figure 13. PCR for enterovirus using nested primers. Note the 298-bp band on this agarose gel in the enterovirus-positive (+) control and lanes 26 and 30. The negative control is devoid of any band, excluding contamination. The other patients are negative for enterovirus.

271]. We also were able to show that mumps virus (Fig. 14) was responsible for EFE, a previously important cause of heart failure in children that has disappeared over the past 20 years [243]. Over the past 5 years, parvovirus B19 has become the predominant viral genome identified in the heart but a cause-and-effect has been not been proved, only strongly suggested thus far [209–213, 215]. In Japan,

126


Table 4. Myocarditis etiologies in children by PCR analysis Diagnosis

No. of Samples

No. of PCR+ samples

PCR amplimer

Myocarditis

624

239 (38%)

Adenovirus 142 (23%) Enterovirus 85 (14%) CMV 18 (3%) Parvovirus 6 (< 1%) Influenza A 5 (< 1%) HSV 5 (< 1%) EBV 3 (< 1%) RSV 1 (< 1%)

DCM

149

30 (20%)

Adenovirus 18 (12%) Enterovirus 12 (8%)

Controls

215

3 (1.4%)

Enterovirus 1 (< 1%) CMV 2 (< 1%)

Figure 14. Kaplan-Meier survival curve of adenovirus-positive versus adenovirus-negative PCR from EMB of cardiac transplant recipients (From [297] with permission).

127


hepatitis C virus has been shown to be a common etiological agent, with the other viral genomes typically seen in North America and Europe playing a lesser role [219–221]. PCR analysis does not usually identify viral genome in peripheral blood of patients with myocarditis, but the viral genome can be identified in tracheal aspirates of intubated children with myocarditis, potentially reducing the need for EMB [253].

Differential diagnosis Any cause of acute circulatory failure may mimic myocarditis.

Pathophysiology Viral infection triggers interstitial inflammation or myocardial injury, resulting in cardiac enlargement and an increase in the ventricular end-diastolic volume [201, 246, 251, 254]. Normally, this increase in volume results in an increased force of contraction, improved ejection fraction, and improved cardiac output as described by the Starling mechanism. The infected myocardium is unable to respond to these stimuli, resulting in reduced cardiac output. A domino effect of changes occurs and this results in the pathophysiological response in patients with myocarditis. 1. Interactions with the sympathetic nervous system may preserve systemic blood flow via vasoconstriction and elevated afterload. This sympathetic nervous system input results in tachycardia and diaphoresis. 2. Congestive heart failure ensues with disease progression. Progressive increase in ventricular end-diastolic volume and pressure results in increased left atrial pressure. This is transmitted to the pulmonary venous system, causing increasing hydrostatic forces that overcome the colloid osmotic pressure that normally prevents fluid transudation across capillary membranes. This results in pulmonary edema. 3. Concomitantly, all cardiac chambers dilate, particularly the left ventricle. This dilation, in addition to poor ventricular function, creates worsening pulmonary edema and worsening cardiac function. The ventricular dilation also results in stretching of the mitral annulus and resultant mitral regurgitation, further increasing left atrial volume and pressure. 4. During the healing stages of myocarditis, fibroblasts replace normal myofibers and result in scar formation. Reduced elasticity and ventricular performance can result in persistent heart failure. In addition, ventricular arrhythmias commonly accompany this fibrosis.

128


Pathology Gross and microscopic findings Pathological findings are nonspecific, with similar gross and microscopic changes noted irrespective of the causative agent [201, 213, 251]. The heart weight is increased. and all four chambers are affected. The muscle is flabby and pale, with petechial hemorrhages often seen on the epicardial surfaces, especially in cases of CVB infection. A bloody pericardial effusion also may be seen relating to the often combined finding of pericarditis. The ventricular wall is frequently thin, although hypertrophy may be found as well. The valves and endocardium are not involved. In cases of chronic myocarditis, the valves may be glistening white, suggesting that EFE may be the result of an in utero viral myocarditis [272]. Among children with myocarditis, compared to those with EFE, those with myocarditis typically had symptoms for less than 2 weeks, whereas those with EFE had symptoms for more than 4 months. Mumps and CVB3 have been identified in the myocardium of infants with EFE [243]. Mural thrombi occur in the left ventricle, and small emboli are often found in the coronary and cerebral vessels [273]. Coronary emboli, although rare, may produce areas of ischemia or injury with resultant production of the cardiac arrhythmias that sometimes occur during the acute disease. An interstitial collection of mononuclear cells, including lymphocytes, plasma cells, and eosinophils (Fig. 10) is typical of early myocarditis [207]. Polymorphonuclear cells are rare, as are viral particles. Extensive necrosis of the myocardium, with loss of cross-striation in the muscle fibers and edema, is seen in severe infections, but especially with coxsackievirus. Perivascular accumulation of lymphocytes and plasma cells has been described with CVB myocarditis but is usually a minor finding. In disease due to rickettsiae, varicella, trypanosomes, or other parasites, and in reactions to sulfonamides, this is a much more prominent finding [247]. Diphtheria myocarditis is frequently complicated by arrhythmias and complete atrioventricular block [274]. Diphtheria exotoxin attaches to conductive tissue and interferes with protein synthesis by inhibiting a translocating enzyme in the delivery of amino acids [274]. Triglyceride accumulates, producing fatty changes of the myofibers. Bacterial myocarditis produces microabscesses and patchy focal suppurative changes. A combined perimyocarditis is also frequently encountered. Paracytic myocarditis caused by trichinella has a focal infiltrate with lymphocytes and eosinophils, but larvae are usually not identified [232]. A severe myocarditis caused by Trypanosoma cruzi [229, 230] results in Chagas’ disease. Rare in North America, Chagas’ is endemic in South America, affecting up to 50% of the population. Microscopic examination shows the organism as well as neutrophils, lymphocytes, macrophages, and eosinophils.

129


Sudden death in infancy may result from myocardial inflammation. James [275] described a resorptive, degenerative process in the His bundle and left margin of the atrioventricular node with the absence of inflammatory cells in cases he studied of infants who died in northern Ireland. Giant cell myocarditis [245, 276–279] occurs with tuberculosis, syphilis, rheumatoid arthritis, rheumatic heart disease, sarcoidosis, and fungal or parasitic infections. Giant cells also occur in idiopathic (Fiedler’s) myocarditis. There are two types of giant cells: cells originating from the myocardium and cells derived from interstitial histiocytes.

Long-term sequelae In those cases in which resolution of cardiac dysfunction does not occur, chronic DCM results [280–284]. It has been unclear what the underlying etiology of these long-term sequelae could be, but viral persistence and autoimmunity have been widely speculated. Enteroviral protease 2A directly cleaves the cytoskeletal protein dystrophin, resulting in dysfunction of this protein [285]. Because mutations in dystrophin are known to cause an inherited form of DCM (as well as the DCM associated with the neuromuscular diseases DMD and BMD), it is likely that this is to a large extent responsible for the chronic DCM seen in enteroviral myocarditis [286, 287]. Other viruses, such as adenoviruses, also have enzymes that cleave membrane structural proteins or result in activation or inactivation of transcription factors, cytokines, or adhesion molecules to cause chronic DCM [288, 289]. Therefore, it appears as if a complex interaction between the viral genome and the heart occurs and results in the long-term outcome of affected patients. As in mice, myocarditis in humans may have a genetic basis [290]. Support for this includes the frequent finding of a myocardial lymphocytic infiltrate in patients with familial and sporadic DCM, as well as the few reports of families in which two or more individuals have been diagnosed with myocarditis on EMB. The recent finding of a common receptor for the four most common viral causes of myocarditis (CVB3 and CVB4 and adenoviruses 2 and 5), the human coxsackievirus and adenovirus receptor (HCAR) [291, 292], which if mutated could be responsible for the differences in the host, leading to myocarditis, is intriguing and requires study.

Support for viral cause-and-effect relationship with myocarditis Despite the increasingly common association of viral genome within the myocardium in patients with myocarditis, limited definitive data exist that prove the virus causes ventricular disturbance directly leading to the clinical phenotype. Many

130


physicians and scientists hold to the concept that myocarditis is a primary inflammatory disorder but definitive data are also limited in support of this hypothesis. Further limiting our understanding of myocarditis and the role of viruses is the low percentage of biopsies performed and the even fewer attempts at performing PCR on the myocardium. An excellent human model exists, however, that can be used to study this question. Cardiac transplant recipients undergo routine surveillance biopsies for rejection and, in all cases, histopathology is performed. In our institution, all subjects also undergo myocardial PCR analysis with screening of adenovirus, enterovirus including coxsackievirus, parvovirus B19, cytomegalovirus, and EBV [293, 294]. We have shown that identification of viral genome in these samples correlated with outcome, with a 96% 5-year survival in subjects not having PCR-positive studies anytime during the 5-year follow-up, while those with a single PCR-positive result (or more) have a 5-year survival of 67% (Fig. 14) [295–297]. Interestingly, survival does not closely correlate with the level of inflammatory infiltrate seen on histopathology. This appears, at least in part, to do with the specific viruses identified in the myocardium. For instance, adenovirus tends to cause a lower level of inflammatory infiltrate than enteroviruses or parvovirus B19 [297]. Similar findings have been found in lung transplant recipients [298].

Treatment Care of a patient presenting with a clinical picture and history strongly suggestive of myocarditis depends on the severity of myocardial involvement [299]. Many patients present with relatively mild disease, with minimal or no respiratory compromise and only mild signs of congestive heart failure. These patients require close monitoring to assess whether the disease will progress to worsening heart failure and the need for intensive medical care. Experimental animal studies suggest that bed rest may prevent an increase in intramyocardial viral replication in the acute stage; thus, it appears prudent to place patients under this restriction at the time of diagnosis. Normal arterial blood oxygen levels should be maintained for any patient with compromised hemodynamics resulting in hypoxemia. The current strategy for therapy includes supporting the blood pressure to achieve end-organ perfusion and urine output without “driving” the myocardium with inotropic agents. Phosphodiesterase inhibitors such as intravenous milrinone have been used to provide both inotropy and afterload reduction, and in typical cases is used in association with diuretics. More recently, nesiritide has been used either along with one or both of these agents or in place of this combination. For blood pressure support, calcium and/or vasopressin infusions may be used. The use of intravenous agents such as high-dose dopamine, dobutamine, norepinephrine and epinephrine may improve blood pressure but have the associated cost

131


of increasing heart rate and increasing mechanical stress on the heart, as well as increasing the possibility for arrhythmias. Therefore, these agents should be used cautiously. In our institution, these agents are used typically as a bridge to the placement of mechanical assist device placement. When chronic oral therapy is possible and hypotension is not present, an afterload reducing drug such as captopril or enalapril [300] may be used with beta-blockers such as carvedilol or metoprolol with or without diuretics. Arrhythmias should be vigorously treated [256]. Supraventricular tachyarrhythmias respond to digitalis. Ventricular arrhythmias respond to lidocaine or intravenous amiodarone. Despite aggressive treatment of these arrhythmias, rapid deterioration to ventricular fibrillation, especially in the very young, may occur and should be treated immediately by direct-current cardioversion. In some cases an ICD is necessary. Complete atrioventricular block requires a temporary transvenous pacemaker. Chronic arrhythmias may persist long after the acute disease has passed [256]. Thus, children who recover from myocarditis, regardless of etiology, should be followed indefinitely. The use of immunosuppressive agents in suspected or proven viral myocarditis is controversial [301–303]. Some animal studies have suggested an exacerbation of virus-induced cytotoxicity in the presence of immunosuppressive drugs, possibly due to interference with interferon production. The Myocarditis Treatment Trial analyzed the use of immunosuppressive and steroid therapy [248]. Although the study was performed in adult patients, the results are potentially applicable to children. There was no difference among patients treated with azathioprine and prednisone, cyclosporine and prednisone, and conventional therapy. Immunosuppressive therapy was not beneficial in most patients with histologically confirmed myocarditis. Another important therapeutic option is the use of intravenous D-globulin in children with myocarditis. This option is based on the early results of Drucker et al. [304], who investigated the use of this agent in 21 of 46 children with myocarditis. Patients who received this drug had better left ventricular function at follow-up. In addition, survival tended to be higher at 1 year, although the data did not reach statistical significance because of the small number of patients in the study. Whether this proves to be beneficial or whether these early results mirror the early published experience with corticosteroids remains to be seen [305, 306]. Recently, the use of interferon has been reported. Kuhl and colleagues [306] reported efficacy of interferon-B treatment in myocarditis with viral clearance and prevention of progressive deterioration of left ventricular function. In this Phase II study, 22 consecutive patients with chronic left ventricular dysfunction and PCR-proven enteroviral or adenoviral infection were injected subcutaneously with interferon-B three times weekly for approximately 6 months, with all 22 patients demonstrating viral clearance, reduced left ventricular dilation and improved systol-

132


ic function. Daliento and colleagues [307] reported similar success with interferon-A in patients with enterovirus-induced myocarditis. Finally, left ventricular assist devices and aortic balloon pumps have been used to support the cardiovascular system in some cases, whereas extracorporeal membrane oxygenator therapy has been used in others [308–311]. When necessary, the devices may be life saving and should be considered an option in children large enough to allow placement of the device successfully. In some circumstances, transplantation becomes necessary [295, 312]. The prognosis of acute myocarditis in newborns is poor [313–315]. A 75% mortality rate was observed in 25 infants with suspected CVB myocarditis [313]. Most deaths occurred in the first week of the illness. Older infants and children have a better prognosis, with a mortality rate between 10% and 25% in clinically recognizable cases; however, a subgroup of patients present to the emergency center dead. In other cases, the children present with signs and symptoms of very common childhood disorders such as a viral respiratory illness, gastroenteritis, and/or dehydration and are, therefore, initially treated for these disorders. However, over hours or days, these children may very rapidly deteriorate and succumb, usually after a cardiac or respiratory arrest; on autopsy, myocarditis is diagnosed. These patients are extremely difficult to diagnose and, even if identified, have limited therapeutic options. Hastreiter and Miller reported complete recovery in 50% of their patients [314]; 25% of the patients continued to have an abnormal ECG or chest radiograph even though they were clinically asymptomatic. Abnormalities in the resting ECG may not be seen, but may be brought out with exercise. Adult patients who recover may be asymptomatic at rest or with light exertion but may demonstrate a reduced working capacity with exercise stress testing.

Vaccination Vaccination has been used successfully to prevent diseases. The efficacy of the polio vaccine has led to the suggestion that a broadly specific enteroviral vaccine, or at least a CVB-specific vaccine, could be beneficial for reducing the incidence of myocarditis or DCM. Immunization is protective in mice [316]. The possibility of success in this regard is supported by the study of EFE. This form of DCM was the most common form identified in children until the late 1960s, with an incidence of 1 in 5000 live births in the United States. Since that time, the incidence has declined significantly. Mumps virus genomic RNA sequences were found in 90% of myocardial samples from EFE patients analyzed [243] (Fig. 15). Thus, EFE may result from persistent in utero mumps infection of the myocardium. The mumps virus vaccine has all but eliminated this form of DCM. Although a CVB-specific vaccine may reduce the incidence of myocarditis, other viruses, particularly adenovirus, are potential etiological agents.

133


Figure 15. PCR analysis of fixed heart samples obtained from infants with endocardial fibroelastosis (EFE). Note the PCR-positive bands at 223 bp indicative of mumps virus. Sequence analysis confirmed the viral genome as that consistent with mumps.

Conclusions The cardiomyopathies appear to occur due to disruption of “final common pathways”. These disruptions may be due to purely genetic causes, such as mutations in a single gene that results in a dysfunctional protein, leading to a domino effect of downstream protein interaction abnormalities and ultimately a phenotype. In other situations, multiple mutations in the same gene (compound heterozygosity) or in different genes (digenic heterozygosity) may lead to a phenotype that may be classic, more severe, or even overlapping with other disease forms. In some cases, different intersecting pathways may become disturbed, resulting in complex phenotypes. Further, acquired causes may play a role by causing to disruption of these functional

134


pathways. For instance, DCM results from disruption in the “final common pathway” linking the sarcomere and sarcolemma, and mutations in the affected genes are responsible for cardiac and skeletal muscle dysfunction. The mechanisms of disease, which include disruption of the linkage due to protein-protein interaction abnormalities that occur from dysfunctional proteins, as well as the interplay of other factors such as mechanical stress and stretch, are being elucidated in detail with the development of animal models of the human disease. Genetic mutations and viral infections, in some cases, have overlapping mechanisms. Many of the genes identified, as well as the viral causes, are now clinically available in fee-forservice laboratories (www.bcm.edu/pediatrics/welsh). Novel therapies have resulted from the improved understanding of this clinical phenotype, as noted above. Similarly, the genetic basis and mechanistic understanding of HCM as a disturbance of sarcomeric function has occurred over the past two decades and genetic tests are clinically available. The development of novel targeted therapies has been somewhat slow in coming, but is expected to develop in the near future. In the case of LVNC, the “new kid on the block”, genetic understanding is in the early phases. The future of cardiomyopathy care is poised to shift in the next decade due to these new developments, as well as the growing science of stem cell therapy. Since children have “pure” disease states, unfettered by co-morbidities, the dream of “cures” of muscle disease (cardiac, skeletal muscle) will likely be realized more fully in this population. We must move toward that goal.

References 1

2 3

4 5 6

Towbin JA, Bowles NE (2005) Cardiomyopathy. In: McMillian JA, Feigin RA, DeAngelis CD, Jones MD, Jr (eds) Oski’s Pediatrics. Principles and Practice, 4th Edition. Lippincott Williams & Wilkins, Philadelphia. Chapter 283, 1624–1648 Abelman WH, Lorrell BH (1989) The challenge of cardiomyopathy. J Am Coll Cardiol 13: 1219 Towbin JA (2006) Hypertrophic cardiomyopathy. In: Chang AC, Towbin JA (eds) Heart Failure in Children and Young Adults. From molecular mechanisms to medical and surgical strategies. Saunders Elsevier, Philadelphia, Chapter 20, 278–297 Towbin JA, Bowles NE (2002) The failing heart. Nature 415: 227–233 Morita H, Seidman CE (2005) Genetic causes of human heart failure. J Clin Invest 115: 518–526 Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ, Seidman CE, Young JB (2006) Contemporary definitions and classification of the cardiomyopathies: An American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdis-

135


7 8 9 10 11

12 13

14 15

16 17

18

19 20 21

22

136

ciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 113: 1807–1816 Schwartz SM, Duffy JY, Pearl JM, Nelson DP (2001) Cellular and molecular aspects of myocardial dysfunction. Crit Care Med 29: S214–S219 Gregorio CC, Antin PB (2000) To the heart of myofibril assembly. Trends Cell Biol 10: 355–362 Clark KA, McElhinny AS, Beckerle MC, Gregorio CC (2002) Striated muscle cytoarchitecture: An intricate web of form and function. Annu Rev Cell Dev Biol 18: 637–706 Vigoreaux JO (1994) The muscle Z band: Lessons in stress management. J Muscle Res Cell Motil 15: 237–255 Barth AL, Nathke IS, Nelson WJ (1997) Cadherins, catenins and APC protein; interplay between cytoskeletal complexes and signaling pathways. Curr Opin Cell Biol 9: 683–690 Capetanaki Y (2002) Desmin cytoskeleton: A potential regulator of muscle mitochondrial behaviour and function. Trends Cardiovasc Med 12: 339–348 Sharp WW, Simpson DG, Borg TK, Samarel AM, Terracio L (1997) Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes. Am J Physiol 273: H546–556 Straub V, Campbell KP (1997) Muscular dystrophies and the dystrophin-glycoprotein complex. Curr Opin Neurol 10: 168–175 Furukawa T, Ono Y, Tsuchiya H, Katayama Y, Bang ML, Labeit D, Labeit S, Inaagki N, Gregorio CC (2001) Specific interaction of the potassium channel beta-subunit minK with the sarcomeric protein T-cap suggests a T-tubule-myofibril linking system. J Mol Biol 313: 775–784 Kucera JP, Rohr S, Rudy Y (2002) Localization of sodium channels in intercalated disks modulates cardiac conduction. Circ Res 91: 1176–1182 Towbin JA, Lowe AM, Colan SD, Sleeper LA, Orav EJ, Clunie S, Messere J, Cox GF, Lurie PR, Hsu D et al (2006) Incidence, causes, and outcome of dilated cardiomyopathy. JAMA 296: 1867–1876 Michels VV, Moll PP, Miller FA, Tajik AJ, Chu JS, Driscoll DJ, Burnett JC, Rodeheffer RJ, Chesebro JH, Tazelaar HD (1992) The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. N Engl J Med 326: 77–82 Berko BA, Swift M (1987) X-linked dilated cardiomyopathy. N Engl J Med 316: 1186– 1191 Towbin JA, Bowles KR, Bowles NE (1999) Etiologies cardiomyopathy and heart failure. Nat Med 5: 266–267 Towbin JA, Hejtmancik JF, Brink P, Gelb B, Zhu XM, Chamberlain JS, McCabe ER, Swift M (1993) X-linked dilated cardiomyopathy (XLCM): Molecular genetic evidence of linkage to the Duchenne muscular dystrophy gene at the Xp21 locus. Circulation 87: 1854–1865 Muntoni F, Cau M, Ganau A, Congiu R, Arvedi G, Mateddu A, Marrosu MG,


23 24 25

26

27

28

29 30

31 32 33 34 35

36

37

Cicanchetti C, Realdi G, Cao A et al (1993) Brief report: Deletion of the dystrophin muscle-specific promoter region associated with X-linked dilated cardiomyopathy. N Engl J Med 329: 921–925 Cox GF, Kunkel LM (1997) Dystrophies and heart disease. Curr Opin Cardiol 12: 329–343 Hoffman EP, Brown RH, Kunkel LM (1987) Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell 51: 919–928 Meng H, Leddy JJ, Frank J, Holland P, Tuana BS (1996) The association of cardiac dystrophin with myofibrils/Z-discs regions in cardiac muscle suggests a novel role in the contractile apparatus. J Biol Chem 271: 12364–12371 Kaprielian RR, Stevenson S, Rothery SM, Cullen MJ, Severs NJ (2000) Distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium. Circulation 101: 2586–2594 Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50: 509–517 Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeny HL (1993) Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci USA 90: 3710–3714 Campbell KP (1995) Three muscular dystrophies: Loss of cytoskeleton-extracellular matrix linkage. Cell 80: 675–679 Klietsch R, Ervasti JM, Arnold W, Campbell KP, Jorgensen AO (1993) Dystrophinglycoprotein complex and laminin colocalize to the sarcolemma and transverse tubules of cardiac muscle. Circ Res 72: 349–360 Ozawa E, Yoshida M, Suzuki A, Mizuno Y, Hagiwara Y, Noguchi S (1995) Dystrophinassociated proteins in muscular dystrophy. Hum Mol Genet 4: 1711–1716 Emery AE (2002) The muscular dystrophies. Lancet 359: 687–695 Davies KE, Nowak KJ (2006) Molecular mechanisms of muscular dystrophies: Old and new players. Nat Rev Mol Cell Biol 7: 762–773 Towbin JA (1998) The role of cytoskeletal proteins in cardiomyopathies. Curr Opin Cell Biol 10: 131–139 Bowles NE, Bowles KR, Towbin JA (2000) The “Final Common Pathway” hypothesis and inherited cardiovascular disease: The role of cytoskeletal proteins in dilated cardiomyopathy. Herz 25: 168–175 Feng J, Yan J, Buzin CH, Towbin JA, Sommer SS (2002) Mutations in the dystrophin gene are associated with sporadic dilated cardiomyopathy. Mol Genet Metab 77: 119– 126 Feng J, Yan J, Buzin CH, Sommer SS, Towbin JA (2002) Comprehensive mutation scanning of the dystrophin gene in patients with nonsyndromic X-linked dilated cardiomyopathy. J Am Coll Cardiol 40: 1120–1124

137


38 39

40

41 42 43

44

45

46 47 48 49

50

51 52 53

138

Neustein HD, Lurie PR, Dahms B, Takahashi M (1979) An X-linked recessive cardiomyopathy with abnormal mitochondria. Pediatrics 64: 24–29 Barth PG, Scholte HR, Berden JA, van der Klei-van Moorsel JM, Luyt-Houwen IE, van’t Veer-Korthof ET, van der Harten JJ, Sobotka-Plojhar MA (1983) An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes. J Neurol Sci 62: 327–355 Kelley RI, Cheatham JP, Clark BJ, Nigro MA, Powell BR, Sherwood GW, Sladky JT, Swisher WP (1991) X-linked dilated cardiomyopathy with neutropenia, growth retardation, and 3–methylglutaconic aciduria. J Pediatr 119: 738–747 Schlame M, Towbin JA, Heerdt PM, Jehle R, DiMauro S, Blanck TJJ (2002) Deficiency of tetralinoleoyl-cardiolipin in Barth syndrome. Ann Neurol 51: 634–637 Bione S, D’Adamo P, Maestrini E, Gedeon AK, Bolhuis PA, Toniolo D (1996) A novel X-linked gene, G4.5, is responsible for Barth syndrome. Nat Genet 12: 385–389 Bleyl SB, Mumford BR, Thompson V, Carey JC, Pysher TJ, Chin TK, Ward K (1997) Neonatal, lethal noncompaction of the left ventricular myocardium is allelic with Barth syndrome. Am J Hum Genet 61: 868–872 Graber HL, Unverferth DV, Baker PB, Ryan JM, Baba N, Wooley CF (1986) Evolution of hereditary cardiac conduction and muscle disorder: A study involving a family with 6 generations affected. Circulation 74: 21–35 Tsubata S, Bowles KR, Vatta M, Zintz C, Titus J, Muhonen L, Bowles NE, Towbin JA (2000) Mutations in the human delta-sarcoglycan gene in familial and sporadic dilated cardiomyopathy. J Clin Invest 106: 655–662 Karkkainen S, Peuhkurinen K (2007) Genetics of dilated cardiodiomyopathy. Ann Med 32: 91–107 Towbin JA, Bowles NE (2006) Dilated cardiomyopathy: A tale of the cytoskeleton and beyond. J Cardiovasc Electrophysiol 17: 919–926 Olson TM, Michels VV, Thibodeau SN, Tai YS, Keating MT (1998) Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science 280: 750–752 Kamisago M, Sharma SD, DePalma SR, Kamisago M, Sharma SD, De Palwa SR, Solomon S, Sharma P, McDonogouh B, Smoot L, Mullen MP, Woolf PK, Wigle ED et al (2000) Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med 343: 1688–1696 Li D, Tapscott T, Gonzalez O, Burch PE, Quinones MA, Zoghbi WA, Hill R, Bachinski LL, Mann DL, Roberts R (1999) Desmin mutations responsible for idiopathic dilated cardiomyopathy. Circulation 100: 461–464 Fatkin D, Graham RM (2002) Molecular mechanisms of inherited cardiomyopathies. Physiol Ref 82: 945–980 Ozawa E, Mizumo Y, Hagiwara Y, Sasaoka T, Yoshida M (2005) Molecular and cellular biology of the sarcoglycan complex. Muscle Nerve 32: 563–576 Coral-Vazquez R, Cohn RD, Moore SA, Hill JA, Weiss RM, Davisson RL, Straub V, Barresi R, Bansal D, Hrstka RF et al (1999) Disruption of the sarcoglycan-sarcospan


54

55

56

57 58 59

60

61

62

63 64

65

66 67

complex in vascular smooth muscle: A novel mechanism for cardiomyopathy and muscular dystrophy. Cell 98: 465–474 Wheeler MT, Allikian MJ, Heydemann A, Hadhazy M, Zarnegar S, McNally EM (2004) Smooth muscle cell-extrinsic vascular spasms arise from cardiomyocyte degeneration in sarcoglycan-deficient cardiomyopathy. J Clin Invest 113: 668–675 Sakamoto A, Ono K, Abe M, Jasmin G, Eki T, Murakami Y, Masaki T, Toyo-Oka T, Hanaoka F (1997) Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, delta-sarcoglycan, in hamster: An animal model of disrupted dystrophin-associated glycoprotein complex. Proc Natl Acad Sci USA 94: 13873–13878 Olson TM, Illenberger S, Kishimoto NY, Huttelmaier S, Keating MT, Jockusch BM (2002) Metavinculin mutations alter actin interaction in dilated cardiomyopathy. Circulation 105: 431–437 Maeda M, Holder E, Lowes B, Valent S, Bies RD (1997) Dilated cardiomyopathy associated with deficiency of the cytoskeletal protein metavinculin. Circulation 95: 17–20 Chang AN, Potter JD (2005) Sarcomeric protein mutations in dilated cardiomyopathy. Heart Fail Rev 10: 225–235 Schmitt JP, Kamisago M, Asahi M, Li GH, Ahmad F, Mende U, Kranias EG, Maclennan DH, Seidman JG, Seidman CE (2003) Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science 299: 1410–1413 Haghighi K, Kolokathis F, Pater L, Lynch RA, Asahi M, Gramolini AO, Fan GC, Tsiapras D, Hahn HS, Adamopoulos S et al (2003) Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J Clin Invest 111: 869–876 Murphy RT, Mogensen J, Shaw A, Kubo T, Hughes S, McKenna WJ (2004) Novel mutation in cardiac troponin I in recessive idiopathic dilated cardiomyopathy. Lancet 363: 371–372 Olson TM, Kishimoto NY, Whitby FG, Michels VV (2001) Mutations that alter the surface charge of alpha-tropomyosin are associated with dilated cardiomyopathy. J Mol Cell Cardiol 33: 723–732 Pyle WG, Solaro RJ (2004) At the crossroads of myocardial signaling: The role of Z-discs in intracellular signaling and cardiac function. Circ Res 94: 296–305 Knoll R, Hoshijima M, Hoffman HM, Person V, Lorenzen-Schmidt I, Bang ML, Hayashi T, Shiga N, Yasukawa H, Schaper W et al (2002) The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell 11: 943–955 Arber S, Hunter JJ, Ross J Jr, Hongo M, Sansig G, Borg J, Perriard JC, Chien KR, Caroni P (1997) MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell 88: 393–403 Zolk O, Caroni P, Bohm M (2000) Decreased expression of the cardiac LIM domain protein MLP in chronic human heart failure. Circulation 101: 2674–2677 Katz AM (2000) Cytoskeletal abnormalities in the failing heart. Out on a LIM? Circulation 101: 2672–2673

139


68

69 70

71

72

73 74

75

76

77 78

79

80

81

140

Zhou Q, Chu PH, Huang C, Cheng CF, Martone ME, Knoll G, Shelton GD, Evans S, Chen J (2001) Ablation of cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy. J Cell Biol 155: 605–612 Granzier H, Labeit S (2004) The grant protein titin: A major player in myocardial mechanics, signaling, and disease. Circ Res 94: 284–295 Gerul B, Gramlich M, Atherton J, McNabb M, Trombitas K, Sasse-Klaassen S, Seidman JG, Seidman C, Granzier H, Labeit S et al (2002) Mutations of TTN encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat Genet 30: 201–204 Fatkin D, MacRae C, Sasaki T, Wolff MR, Porcu M, Frenneaux M, Atherton J, Vidaillet HJ, Spudich S, De Girolami U (1999) Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease. N Engl J Med 34: 1715–1724 Brodsky GL, Muntoni F, Miocic S, Sinagra G, Sewry C, Mestroni L (2000) Lamin A/C gene mutation associated with dilated cardiomyopathy with variable skeletal muscle involvement. Circulation 101: 473–476 Stuurman N, Heins S, Aebi U (1998) Nuclear Iamins: Their structure, assembly and interactions. J Struct Biol 122: 42–66 Bonne G, DiBarletta MR, Varnous S, Becane HM, Hammouda EH, Merlini L, Muntoni F, Greenberg CR, Gary F, Urtizeberea JA et al (1999) Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat Genet 21: 285–288 Di Barletta R, Ricci E, Galluzzi G, Tonali P, Mora M, Morandi L, Romorini A, Voit T, Orstavik KH, Merlini L et al (2000) Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy. Am J Hum Genet 66: 1407–1412 Muchir A, Bonne G, van der Kooi AJ, van Meegen M, Baas F, Bolhuis PA, de Vissser M, Schwartz K (2000) Identification of mutations in the gene encoding lamin A/C in autosomal dominant limb girdle muscular dystrophy with atrioventricular conduction disturbance (LGMD1B). Hum Mol Genet 9: 1453–1459 Decostre V, Ben Yaou R, Bonne G (2005) Laminopathies affecting skeletal and cardiac muscles: Clinical and pathological aspects. Acta Myol 24: 104–109 Taylor MR, Slavov D, Gajewski A, Vlcek S, Ku L, Fain PR, Vlcek S, Ku L, Fain PR, Carniel E, Di Lenarda A, Sinagra G, Boucek MM et al (2005) Thymopoietin (laminaassociated polypeptide 2) gene mutation associated with dilated cardiomyopathy. Hum Mutat 26: 566–574 Badorff C, Lee G-H, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, Knowlton KU (1999) Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med 5: 320–326 Xiong D, Lee GH, Badorff C, Dorner A, Lee S, Wolf P, Knowlton KU (2002) Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: A genetic predisposition to viral heart disease. Nat Med 8: 872–877 Vatta M, Stetson SJ, Perez-Verdra A, Entman ML, Noon GP, Torre-Amione G, Bowles


82

83 84 85

86 87 88

89

90

91 92 93 94 95 96

97

NE, Towbin JA (2000) Molecular remodeling of dystrophin in patients with end-stage cardiomyopathies and reversal for patients on assist device therapy. Lancet 359: 936– 941 Vatta M, Stetson SJ, Jimenez S, Entman ML, Noon GP, Bowles NE, Towbin JA, TorreAmione G (2004) Molecular normalization of dystrophin in the failing left and right ventricle of patients treated with either pulsatile or continuous flow-type ventricular assist devices. J Am Coll Cardiol 43: 811–817 Maron BJ (2002) Hypertrophic cardiomyopathy: A systemic review. JAMA 287: 1308– 1320 Elliott P, McKenna WJ (2004) Hypertrophic cardiomyopathy. Lancet 363: 188–189 Colan SD, Lipshultz SE, Lowe AM, Sleeper LA, Messere J, Cox GF, Lurie PR, Orav EJ, Towbin JA (2007) Epidemiology and cause-specific outcome of hypertrophic cardiomyopathy in children: Findings from the Pediatric Cardiomyopathy Registry. Circulation 115: 773–781 McKenna WJ, Behr ER (2002) Hypertrophic cardiomyopathy: Management, risk stratification, and prevention of sudden death. Heart 87: 169–176 Frenneaux MP (2004) Assessing the risk of sudden cardiac death in a patient with hypertrophic cardiomyopathy. Heart 90: 570–575 Maron BJ, Carney KP, Lever HM, Lewis JF, Barac I, Casey SA, Sherrid MV (2003) Relationship of race to sudden cardiac death in competitive athletes with hypertrophic cardiomyopathy. J Am Coll Cardiol 41: 974–980 Maron BJ (2000) Hypertrophic cardiomyopathy and sudden death: New perspective on risk stratification and prevention with the implantable cardioverter-defibrillator. Eur Heart J 21: 1979–1983 Spirito P, Maron BJ, Bonow RO, Epstein SE (1987) Occurrence and significance of progressive left ventricular wall thinning and relative cavity dilation in hypertrophic cardiomyopathy. Am J Cardiol 60: 123–139 Aurigemma GP, Gaasch WH (2004) Diastolic heart failure. N Engl J Med 351: 1097– 1105 Gaasch WH, Zile MR (2004) Left ventricular diastolic dysfunction and diastolic heart failure. Annu Rev Med 55: 373–394 Towbin JA, Lipshultz SE (1999) Genetics of neonatal cardiomyopathy. Curr Opin Cardiol 14: 250–262 Vulpian A (1868) Contribution à l’étude des rétrécissements de l’orifice ventriculoaortique. Arch Physiol 3: 220–222 Watkins H (2003) Genetic clues to disease pathways in hypertrophic and dilated cardiomyopathies. Circulation 107: 1344–1346 Jarcho JA, McKenna W, Pare JA, Solomon SD, Holcombe RF, Dickie S, Levi T, DonisKeller H, Seidman JG, Seidman CE (1989) Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1. N Engl J Med 321: 1372–1378 Solomon SD, Jarcho JA, McKenna WJ, Geisterfer-Lowrance A, Germain R, Salerni R,

141


98

99

100

101

102

103

104

105

106

107

108

109 110

142

Seidman JG, Seidman CE (1990) Familial hypertrophic cardiomyopathy is a genetically heterogeneous disease. J Clin Invest 86: 993–999 Watkins H, MacRae C, Thierfelder L, Chou YH, Frenneaux M, McKenna W, Seidman JG, Seidman CE (1993) A disease locus for familial hypertrophic cardiomyopathy maps to chromosome 1q3. Nat Genet 3: 333–337 Thierfelder L, MacRae C, Watkins H, Tomfohrde J, Williams M, McKenna W, Bohm K, Noeske G, Schlepper M, Bowcock A (1993) A familial hypertrophic cardiomyopathy locus maps to chromosome 15q2. Proc Natl Acad Sci USA 90: 6270–6274 Carrier L, Hengstenberg C, Beckmann JS, Guicheney P, Dufour C, Bercovici J, Dausse E, Berebbi-Bertrand I, Wisnewsky C, Pulvenis D (1993) Mapping of a novel gene for familial hypertrophic cardiomyopathy to chromosome 11. Nat Genet 4: 311–313 MacRae CA, Ghaisas N, Kass S, Donnelly S, Basson CT, Watkins HC, Anan R, Thierfelder LH, McGarry K, Rowland E (1995) Familial hypertrophic cardiomyopathy with Wolff-Parkinson-White Syndrome maps to a locus on chromosome 7q3. J Clin Invest 96: 1216–1220 Poetter K, Jiang H, Hassanzadeh S, Master SR, Chang A, Dalakas MC, Rayment I, Sellers JR, Fananapazir L, Epstein ND (1996) Mutation in either the essential regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet 13: 63–69 Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T et al (1997) Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet 16: 379–382 Mogensen J, Klausen IC, Pederson AK, Egeblad H, Bross P, Kruse TA, Gregersen N, Hansen PS, Baandrup U, Borglum AD (1999) A-Cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy. J Clin Invest 103: R39–R43 Satoh M, Takahashi M, Sakamoto T, Hiroe M, Marumo F, Kimura A (1999) Structural analysis of the titin gene in hypertrophic cardiomyopathy: Identification of a novel disease gene. Biochem Biophys Res Commun 2625: 411–417 Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Hoffmann K, Pilz B, Martiniak Y, Gehmlich K, van der Ven PF et al (2003) Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation 107: 1390–1395 Mohiddin SA, Ahmed ZM, Griffith AJ, Tripodi D, Friedman TB, Fananapazir L, Morrell RJ (2004) Novel association of hypertrophic cardiomyopathy, sensorineural deafness and a mutation in unconventional myosin VI (MYO6). J Med Genet 41: 309–314 Hayashi T, Armimura T, Ueda K, Shibata H, Hohda S, Takahashi M, Hori H, Koga Y, Oka N, Imaizumi T et al (2004) Identification and functional and of caveolin-3 mutation associated with familial hypertrophic cardiomyopathy. Biochem Biophys Res Commun 313: 178–184 Seidman JG, Seidman C (2001) The genetic basis for cardiomyopathy from mutation identification to mechanistic paradigms. Cell 108: 557–567 Towbin JA, Bowles NE (2002) The failing heart. Nature 415: 227–233


111 Schiaffino S, Reggiani C (1996) Molecular diversity of myofibrillar proteins: Gene regulation and functional significance. Physiol Rev 76: 371–423 112 Gollob MH, Green MS, Tang AS, Gollob T, Karibe A, Ali Hassan AS, Ahmad F, Lozado R, Shah G, Fananapazir L et al (2001) Identification of a gene responsible for familial Wolff-Parkinson-White syndrome. N Engl J Med 344: 1823–1831 113 Blair E, Redwood C, Ashrafian H, Oliveira M, Broxholme J, Kerr B, Salmon A, Ostman-Smith I, Watkins H (2001) Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: Evidence for a central role of energy compromise in disease pathogenesis. Hum Mol Genet 10: 1215–1220 114 Arad M, Benson DW, Perez-Atayde AR, McKenna WJ, Sparks EA, Kanter RJ, McGarry K, Seidman JG, Seigman CE (2002) Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. J Clin Invest 107: 357–362 115 Blair E, Redwood C, de Jesus Oliveira M, Moolman-Smook JC, Brink P, Corfield VA, Ostman-Smith I, Watkins H (2002) Mutations of the light meromyosin domain of the B-myosin heavy chain rod in hypertrophic cardiomyopathy. Circ Res 90: 263–269 116 Hofmann PA, Hartzell HC, Moss RL (1991) Alterations in Ca2+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers. J Gen Physiol 97: 1141–1163 117 Lees-Miller JP, Helfman DM (1991) The molecular basis for tropomyosin isoform diversity. Bioessays 13: 429–437 118 Mesnard L, Logeart D, Taviaux S, Diriong S, Mercadier JJ, Samson F (1995) Human cardiac troponin T: Cloning and expression of new isoforms in the normal and failing heart. Circ Res 76: 687–692 119 Townsend P, Barton P, Yacoub M, Farza H (1995) Molecular cloning of human cardiac troponin T isoforms: Expression in developing and failing heart. J Mol Cell Cardiol 27: 2223–2236 120 Hunkeler NM, Kullman J, Murphy AM (1991) Troponin I isoform expression in human heart. Circ Res 69: 1409–1414 121 Theis JL, Bos JM, Bartleson VB, Will ML, Binder J, Vatta M, Towbin JA, Gersh BJ, Ommen SR, Ackerman MJ (2006) Echocardiograpy-determined septal morphology in Z-disc hypertrophic cardiomyopathy. Biochem Biophys Res Commun 351: 896–902 122 Osio A, Tan L, Chen SN, Lombardi R, Nagueh SF, Shete S, Roberts R, Willerson JT, Marian AJ (2007) Myozenin-2 is a novel gene for human hypertrophic cardiomyopathy. Circ Res 100: 766–768 123 Watkins H, Rosenzweig T, Hwang DS, Levi T, McKenna W, Seidman CE, Seidman JG (1992) Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy. N Engl J Med 326: 1108–1114 124 Moolman JC, Corfield VA, Posen B, Ngumbela K, Seidman C, Brink PA, Watkins H (1997) Sudden death due to troponin T mutations. J Am Coll Cardiol 29: 549–555 125 Nakajima-Taniguchi C, Matsui H, Fujio Y, Nagata S, Kishimoto T, Yamauchi-Takihara

143


126

127

128

129

130

131

132

133

134

135 136

144

K (1997) Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy. J Mol Cell Cardiol 29: 839–843 Bonne G, Carrier L, Bercovici J, Cruaud C, Richard P, Hainque B, Gautel M, Labeit S, James M, Beckmann J (1995) Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat Genet 11: 438–440 Watkins H, Conner D, Thierfelder L, Jarcho JA, MacRae C, McKenna WJ, Maron BJ, Seidman JG, Seidman CE (1995) Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet 11: 434–437 Niimura H, Bachinski LL, Sangwatanaroj S, Watkins H, Chudley AE, McKenna W, Kristinsson A, Roberts R, Sole M, Maron BJ et al (1998) Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy. N Engl J Med 338: 1248–1257 Charron P, Dubourg O, Desnos M, Bennaceur M, Carrier L, Camproux AC, Isnard R, Hagege A, Langlard JM, Bonne G et al (1998) Clinical features and prognostic implications of familial hypertrophic cardiomyopathy related to cardiac myosin-binding protein C gene. Circulation 97: 2230–2236 Van Driest SL, Ackerman MJ, Ommen SR, Shakur R, Will ML, Nishimura RA, Tajik AJ, Gersh BJ (2002) Prevalence and severity of “benign” mutations in the B-myosin heavy chain, cardiac troponin T, and A-tropomyosin genes in hypertrophic cardiomyopathy. Circulation 106: 3085–3090 Yoneya K, Okamoto H, Machida M, Onozuka H, Noguchi M, Mikami T, Kawaguchi H, Murakami M, Uede T, Kitabatake A (1995) Angiotensin-converting enzyme gene polymorphism in Japanese patients with hypertrophic cardiomyopathy. Am Heart J 130: 1089–1093 Tesson F, Dufour C, Moolman JC, Carrier L, al-Mahdawi S, Chojnowska L, Dubourg O, Soubrier E, Brink P, Komajda M et al (1997) The influence of the angiotensin I converting enzyme genotype in familial hypertrophic cardiomyopathy varies with the disease gene mutation. J Mol Cell Cardiol 29: 831–838 Ortlepp JR, Vosberg HP, Reith S, Ohme F, Mahon NG, Schroder D, Klues HG, Hanrath P, McKenna WJ (2002) Genetic polymorphisms in the rennin-angiotensin-aldosterone system associated with expression of left ventricular hypertrophy in hypertrophic cardiomyopathy: A study of five polymorphic genes in a family with a disease causing mutation in the myosin binding protein C gene. Heart 87: 270–275 Doiuchi J, Hamada M, Ito T, Kokubu T (1987) Comparative effects of calcium-channel blockers and beta-adrenergic blocker on early diastolic time intervals and A-wave ratio in patients with hypertrophic cardiomyopathy. Clin Cardiol 10: 26–30 Lorell BH (1985) Use of calcium channel blockers in hypertrophic cardiomyopathy. Am J Med 78: 43–54 Moran AM, Colan SD (1998) Verapamil therapy in infants with hypertrophic cardiomyopathy. Cardiol Young 8: 310–319


137 Nagueh SF, Ommen SR, Lakkis NM, Killip D, Zoghbi WA, Schaff HV, Danielson GK, Quinones MA, Tajik AJ, Spencer WH (2001) Comparison of ethanol septal reduction therapy with surgical myectomy for the treatment of hypertrophic obstructive cardiomyopathy. J Am Coll Cardiol 38: 1701–1706 138 Maron BJ, Dearani JA, Ommen SR, Maron ML, Schaff HV, Gersh BJ, Nishimura RA (2004) The case for surgery in obstructive hypertrophic cardiomyopathy. J Am Coll Cardiol 44: 2043–2053 139 O’Rourke RA (1999) Cardiac pacing. An alternative treatment for selected patients with hypertrophic cardiomyopathy and adjunctive therapy for certain patients with dilated cardiomyopathy. Circulation 100: 786–788 140 Maron BJ (1996) Appraisal of dual-chamber pacing therapy in hypertrophic cardiomyopathy: Too soon for a rush to judgment? J Am Coll Cardiol 27: 431–432 141 Begley D, Mohiddin S, Fananapazir L (2001) Dual chamber pacemaker therapy for mid-cavity obstructive hypertrophic cardiomyopathy. Pacing Clin Electrophysiol 24: 1639–1644 142 Chang SM, Lakkis NM, Franklin J, Spencer WH 3rd, Nagueh SF (2004) Predictors of outcome after alcohol septal ablation therapy in patients with hypertrophic obstructive cardiomyopathy. Circulation 109: 824–827 143 Nielsen CD, Killip D, Spencer WH 3rd (2003) Nonsurgical septal reduction therapy for hypertrophic obstructive cardiomyopathy: Short-term results in 50 consecutive procedures. Clin Cardiol 26: 275–279 144 Park TH, Lakkis NM, Middleton KJ, Franklin J, Zoghbi WA, Quinones MA, Spencer WH 3rd, Nagueh SF (2002) Acute effect of nonsurgical septal reduction therapy on regional left ventricular asynchrony in patients with hypertrophic obstructive cardiomyopathy. Circulation 106: 412–415 145 Hess OM, Sigwart U (2004) New treatment strategies for hypertrophic obstructive cardiomyopathy. J Am Coll Cardiol 44: 2054–2055 146 Cable WJ, Kolodny EH, Adams RD (1982) Fabry disease: Impaired autonomic function. Neurology 32: 498–502 147 Sugie K, Yamamoto A, Murayama K, Oh SJ, Takahashi M, Mora M, Riggs JE, Colomer J, Iturriaga C, Meloni A et al (2002) Clinicopathological features of genetically confirmed Danon disease. Neurology 58: 1773–1778 148 Yang Z, McMahon CJ, Smith LR, Bersola J, Adesina AM, Breinholt JP, Kearney DL, Dreyer WJ, Denfield SW, Price JF et al (2005) Danon disease as an underrecognized cause of hypertrophic cardiomyopathy in children. Circulation 13: 1612–1617 149 Hers HG, van Hoof F, de Barsy T (1989) Glycogen storage diseases. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York, 425–452 150 Towbin JA (1993) Molecular genetic aspects of cardiomyopathy. Biochem Med Metab Biol 49: 283–320 151 D’Ancona GG, Wurm J, Croce CM (1979) Genetics of type II glycogenosis: Assignment

145


152 153

154

155 156 157

158 159

160

161 162

163

164

165

166

146

of the human gene for acid A-glucosidase to chromosome 17. Proc Natl Acad Sci USA 76: 4526–4529 Beratis NG, LaBadie GU, Hirschhorn K (1983) Genetic heterogeneity in acid A-glucosidase deficiency. Am J Hum Genet 35: 21–33 Zhong N, Martiniuk F, Tzall S, Hirschhorn R (1991) Identification of a missense mutation in one allele of a patient with Pompe disease and use of endonuclease digestion of PCR-amplified RNA to demonstrate lack of mRNA expression from the second allele. Am J Hum Genet 49: 635–645 Raben N, Danon M, Gilbert AL, Dwivedi S, Collins B, Thurberg BL, Mattaliano RJ, Nagaraju K, Plotz PH (2003) Enzyme replacement therapy in the mouse model of Pompe disease. Mol Genet Metab 80: 159–169 Masson C, Cisse I, Simon V, Insalaco P, Audran M (2004) Fabry disease: A review. Joint Bone Spine 71: 381–383 Colucci WS, Lorell BH, Schoen FJ, Warhol MJ, Grossman W (1982) Hypertrophic obstructive cardiomyopathy due to Fabry’s disease. N Engl J Med 307: 926–928 Becker AE, Schoorl R, Balk AG, van der Heide RM (1975) Cardiac manifestations of Fabry’s disease: Report of a case with mitral insufficiency and electrocardiographic evidence of myocardial infarction. Am J Cardiol 36: 829–835 Goldman ME, Cantor R, Schwartz MF, Baker M, Desnick RJ (1986) Echocardiographic abnormalities and disease severity in Fabry’s disease. J Am Coll Cardiol 7: 1157–1161 Broadbent JC, Edwards WD, Gordon H, Hartzler GO, Krawisz JE (1981) Fabry cardiomyopathy in the female confirmed by endomyocardial biopsy. Mayo Clin Proc 56: 623–628 Bishop DF, Calhoun DH, Bernstein HS, Hantzopoulos P, Quinn M, Desnick RJ (1986) Human alpha galactosidase A: Nucleotide sequence of a cDNA clone encoding the mature enzyme. Proc Natl Acad Sci USA 83: 4859–4863 Eng CM, Desnick RJ (1994) Molecular basis of Fabry disease. Mutations and polymorphisms in the human A-galactosidase A gene. Hum Mutat 3: 103–111 Okumiya T, Ishii S, Kase R, Sakuraba H, Suzuki Y (1995) A-Galactosidase gene mutations in Fabry disease: Heterogeneous expressions of mutant enzyme proteins. Hum Genet 95: 557–561 Nakao S, Takenaka T, Maeda M, Kodama C, Tanaka A, Tahara M, Yoshida A, Kuriyama M, Hayashibe H, Sakuraba H (1995) An atypical variant of Fabry’s disease in men with left ventricular hypertrophy. N Engl J Med 333: 288–293 Linhart A, Palecek T, Bultas J, Ferguson JJ, Hrudova J, Karetova D, Zeman J, Ledvinova J, Poupetova H, Elleder M, Aschermann M (2000) New insights in cardiac structural changes in patients with Fabry’s disease. Am Heart J 139: 1101–1108 Sachdev B, Takenaka T, Teraguchih H, Tei C, Lee P, McKenna WJ, Elliott PM (2002) Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy. Circulation 105: 1407–1411 Danon MJ, Oh SJ, DiMauro S, Manaligod JR, Eastwood A, Naidu S, Schliselfeld LH


167

168

169

170

171

172 173

174 175 176 177 178 179

180 181 182

(1981) Lysosomal glycogen storage disease with normal acid maltase. Neurology 31: 51–57 Nishino I, Fu J, Tanji K, Yamada T, Shimojo S, Koori T, Mora M, Riggs JE, Oh SJ, Koga Y (2000) Primary LAMP-2 deficiency causes X-linked vascular cardiomyopathy and myopathy (Danon disease). Nature 406: 906–909 Charron P, Villard E, Sebillon P, Laforet P, Maisonobe T, Dubosca-Bidot L, Romero N, Drouin-Garraud V, Frebourg T, Richard P et al (2004) Danon’s disease as a cause of hypertrophic cardiomyopathy: A systematic survey. Heart 90: 842–846 Cheung PC, Salt IP, Davies SP, Hardie DG, Carling D (2000) Characterization of AMPactivated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J 346: 659–669 Kemp BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen ZP, Witters LA (1999) Dealing with energy demand: The AMP-activated protein kinase. Trends Biochem Sci 24: 22–25 Arad M, Moskowitz IP, Patel VV, Ahmad F, Perez-Atayde AR, Sawyer DB, Walter M, Li GH, Burgon PG, Maguire CT, Stapleton D et al (2003) Transgenic mice overexpressing mutant PRKAG2 define the cause of Wolff Parkinson-White syndrome in glycogen storage cardiomyopathy. Circulation 107: 2850–2856 Attardi G (1996) The elucidation of the human mitochondrial genome: A historical perspective. BioEssays 5: 34–39 Wallace DC, Zheng X, Lott MT, Shoffner JM, Hodge JA, Kelley RI, Epstein CM, Hopkins LC (1988) Familial mitochondrial encephalomyopathy (MERRF): Genetic, pathophysiological, and biochemical characterization of a mitochondrial DNA disease. Cell 55: 601–610 Wallace DC (1989) Mitochondrial DNA mutation and neuromuscular disease. Trends Genet 5: 9–13 Clarke A (1990) Mitochondrial genome: Defects, disease, and evolution. J Med Genet 27: 451–456 Grivell LA (1989) Small, beautiful and essential. Nature 341: 569–571 Anderson S, Banker AT, Barrell BG (1981) Sequence and organization of the human mitochondrial genome. Nature 290: 457–465 Petty RKH, Harding AE, Morgan-Hughes JA (1986) The clinical features of mitochondrial myopathy. Brain 109: 915–938 Mariotti C, Tiranti V, Carrara F, Dallapiccola B, DiDonato S, Zeviani M (1994) Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNALeu(UUR) mutation associated with maternally inherited myopathy and cardiomyopathy. J Clin Invest 93: 1102–1107 Vogel H (2001) Mitochondrial myopathies and the role of the pathologist in the molecular era. J Neuropathol Exp Neurol 60: 217–227 Capaldi RA, Halphen DG, Zhang YZ, Yanamura W (1988) Complexity and tissue specificity of the mitochondrial respiratory chain. J Bioenerg Biomembr 20: 291–311 Ozawa T, Tanaka M, Sugiyama S, Hattori K, Ito T, Oho K, Takahashi A, Sato W,

147


183

184 185 186

187

188

189

190

191

192

193 194

195 196

148

Takada G, Mayumi B (1990) Multiple mitochondrial DNA deletions exist in cardiomyocytes of patients with hypertrophic or dilated cardiomyopathy. Biochem Biophys Res Commun 170: 830–836 Scaglia F, Towbin JA, Craigen WJ, Belmont JW, Smith EO, Neish SR, Ware SM, Hunter JV, Fernbach SD, Vladutiu GD et al (2004) Clinical spectrum, morbidity, and mortality in 113 pediatric patients with mitochondrial disease. Pediatrics 114: 925–931 Channer KD, Channer JL, Campbell MJ, Rees JR (1988) Cardiomyopathy in KearnsSayre syndrome. Br Heart J 59: 486–490 Poulton J, Deadman ME, Ramacharan S, Gardiner RM (1991) Germ-line deletions of mtDNA in mitochondrial myopathy. Am J Hum Genet 48: 649–653 Moraes CT, DiMauro S, Zeviani M, Lombes A, Shanske S, Miranda AF, Nakase H, Bonilla E, Werneck LC, Servidei S (1989) Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome. N Engl J Med 320: 1293–1299 Moraes CT, Schon EA, DiMauro S, Miranda AF (1989) Heteroplasmy of mitochondrial genomes in clonal cultures from patients with Kearns-Sayre syndrome. Biochem Biopsy Res Commun 160: 765–771 Suomalainen A, Kollmann P, Octave J-N, Soderlund H, Syvanen AC (1993) Quantification of mitochondrial DNA carrying tRNA8344Lys point mutation in myoclonus epilepsy and ragged-red fiber disease. Eur J Hum Genet 1: 88–95 Tanno Y, Yoneda M, Nonaka I, Tanaka K, Miyatake T, Tsuji S (1991) Quantitation of mitochondrial DNA carrying tRANLys mutation in MERRF patients. Biochem Biophys Res Commun 179: 880–885 Shoffner JM, Lott MI, Lezza AM, Seibel P, Ballinger SW, Wallace DC (1990) Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation. Cell 61: 931–937 Nakamura M, Nakano S, Goto Y, Ozawa M, Nagahama Y, Fukuyama H, Akiguchi I, Kaji R, Kimura J (1995) A novel point mutation in the mitochondrial tRNASer(UCN) gene detected in a family with MERFF/MELAS overlap syndrome. Biochem Biophys Res Commun 214: 86–93 Silvestri G, Moraes CT, Shanske S, Oh SJ, DiMauro S (1992) A new mtDNA mutation in the tRNA(Lys) gene associated with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet 51: 1213–1217 Fukuhara N (1995) Clinicopathologic features of MERRF. Muscle Nerve 3: 590–594 Pignatelli RH, McMahon CJ, Dreyer WJ, Denfield SW, Price J, Belmont JW, Craigen WJ, Wu J, El Said H, Bezold LI et al (2003) Clinical characterization of left ventricular noncompaction in children. A relatively common form of cardiomyopathy. Circulation 108: 2672–2678 Chin TK, Perloff JK, Williams RG, Jue K, Mohrmann R (1990) Isolated noncompaction of left ventricular myocardium. A study of eight cases. Circulation 82: 507–513 Stollberger C, Finsterer J, Blazek G (2002) Left ventricular hypertrabeculation/noncompaction and association with additional cardiac abnormalities and neuromuscular disorders. Am J Cardiol 90: 899–902


197 Stollberger C, Finsterer J (2004) Left ventricular hypertrabeculation/noncompaction. J Am Soc Echocardiogr 17: 91–100 198 Ichida F, Hamamichi Y, Miyawaki T, Ono Y, Kamiya T, Akagi T, Hamada H, Hirose O, Isobe T, Yamada K et al (1999) Clinical features of isolated noncompaction of the ventricular myocardium: Long-term clinical course, hemodynamic properties and genetic background. J Am Coll Cardiol 34: 233–240 199 Ichida F, Tsubata S, Bowles KR, Haneda N, Uese K, Miyawaki T, Dreyer WJ, Messina J, Li H, Bowles NE, Towbin JA (2001) Novel gene mutations in patients with left ventricular noncompaction or Barth syndrome. Circulation 103: 1256–1263 200 Vatta M, Mohapatra B, Jimenez S, Sanchez X, Faulkner G, Perles Z, Sinagra G, Lin JH, Vu TM, Zhou Q et al (2003) Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction. J Am Coll Cardiol 42: 2014–2027 201 Wynn J, Braunwald E (1997) The cardiomyopathies and myocarditides. In: Braunwald E (ed) Heart disease: A textbook of cardiovascular medicine. WB Saunders, Philadelphia, 1404–1463 202 Berkovich S, Rodriguez-Torres R, Lin JS (1968) Virologic studies in children with acute myocarditis. Am J Dis Child 115: 207–221 203 Dec GW, Palacios IF, Fallon JT, Aretz HT, Mills J, Lee DC, Johnson RA (1985) Active myocarditis in the spectrum of acute dilated cardiomyopathies: Clinical features, histologic correlates, and clinical outcome. N Engl J Med 312: 885–890 204 Bowles NE, Bowles KR, Towbin JA (2005) Viral genome detection and outcome in myocarditis. Heart Fail Clin 1: 407–417 205 Martin AB, Webber S, Fricker FJ, Jaffe R, Demmler G, Kearney D, Zhang YH, Bodurtha J, Gelb B, Ni J et al (1994) Acute myocarditis: Rapid diagnosis by PCR in children. Circulation 90: 330–333 206 Bowles NE, Towbin JA (1998) Molecular aspects of myocarditis. Curr Opin Cardiol 13: 179–184 207 Woodruff JF (1980) Viral myocarditis: A review. Am J Pathol 101: 427–484 208 Hirschman ZS, Hammer SG (1974) Coxsackie virus myopericarditis: A microbiological and clinical review. Am J Cardiol 34: 224–232 209 Schowengerdt KO, Ni J, Denfield SW, Gajarski RJ, Bowles NE, Rosenthal G, Kearney DL, Price JK, Rogers BB, Schauer GM et al (1997) Parvovirus B19 infection as a cause of myocarditis and cardiac allograft rejection: Diagnosis using the polymerase chain reaction (PCR). Circulation 96: 3549–3554 210 Francalanci P, Chance JL, Vatta M, Jimenez S, Li H, Towbin JA, Bowles NE (2004) Cardiotropic viruses in the myocardium of children with end-stage heart disease. J Heart Lung Transplant 23: 1046–1052 211 Maisch B, Ristic AD, Portig I, Pankuweit S (2003) Human viral cardiomyopathy. Front Biosci 8: s39–67 212 Wang X, Zhang G, Liu F, Han M, Xu D, Zang Y (2004) Prevalence of human parvovirus B19 DNA in cardiac tissues of patients with congenital heart diseases indicated by nested PCR and in situ hybridization. J Clin Virol 31: 20–24

149


213 Pankuweit S, Lamparter S, Schoppet M, Maisch B (2004) Parvovirus B19 genome in endomyocardial biopsy specimen. Circulation 109: e179 214 Perez Pulido S (1984) Acute and subacute myocarditis. Cardiovasc Rev Rep 5: 912– 926 215 Bowles NE, Ni J, Kearney DL, Pauschinger M, Schultheiss HP, McCarthy R, Hare J, Bricker JT, Bowles KR, Towbin JA (2003) Detection of viruses in myocardial tissues by polymerase chain reaction, evidence of adenovirus as a common cause of myocarditis in children and adults. J Am Coll Cardiol 42: 466–472 216 Proby CM, Hackett S, Gupta S, Cox TM (1986) Acute myopericarditis in influenza A infection. QJ Med 60: 887–892 217 Schonian U, Crombach M, Maser S, Maisch B (1995) Cytomegalovirus associated heart muscle disease. Eur Heart J 16 (Supp 10): 46–49 218].Lowry PJ, Thompson RA, Littler WA (1983) Humoral immunity in cardiomyopathy. Br Heart J 50: 390–394 219 Matsumori A (2006) Role of hepatitis C virus in cardiomyopathies. Ernst Schering Res Found Workshop 55: 99–120 220 Matsumori A (2005) Hepatitis C virus infection and cardiomyopathies. Circ Res 96: 144–147 221 Okabe M, Fukuda K, Arakawa K, Kikuchi M (1997) Chronic variant myocarditis associated with hepatitis C virus infection. Circulation 96: 22–24 222 Ainger LE, Lawyer NG, Fitch CW (1966) Neonatal rubella myocarditis. Br Heart J 28: 691–697 223 Lorber A, Zonis A, Maisuls E, Dembo L, Palant A, Iancu TC (1988) The scale of myocardial involvement in varicella myocarditis. Int J Cardiol 20: 257–262 224 Chaudary S, Jaski BE (1989) Fulminant mumps myocarditis. Ann Intern Med 110: 569–570 225 Frishman W, Kraus ME, Zabkar J, Brooks V, Alonso D, Dixon LM (1977) Infectious mononucleosis and fatal myocarditis. Chest 72: 535–538 226 Barbaro G, Di Lorenzo G, Grisorio B, Barbarini G for the Gruppo Italiano per Lo Studio Cardiologico dei Pazienti Affeti da AIDS (1998) Incidence of dilated cardiomyopathy and detection of HIV in myocardial cells of HIV-positive patients. N Engl J Med 339: 1093–1099 227 Puchkov GF, Minkovich BM (1972) A case of respiratory syncytial infection in a child by interstitial myocarditis with lethal outcome. Arkh Patol 34: 70–73 228 Vargo TA, Singer DB, Gillette PC, Fernbach DJ (1977) Myocarditis due to visceral larva migrans. J Pediatr 90: 322–323 229 Poltera AA, Cox JN, Owor R (1976) Pancarditis affecting the conducting system and all valves in human African trypanosomiasis. Br Heart J 38: 827–837 230 Marsden PD (1971) South American trypanosomiasis (Chagas’ disease). Int Rev Trop Med 4: 97–121 231 Leak D, Meghji M (1979) Toxoplasmic infection in cardiac disease. Am J Cardiol 43: 841–849

150


232 Bessoudo R, Marrie TJ, Smith ER (1981) Cardiac involvement in trichinosis. Chest 79: 698–699 233 Karjalainen J (1989) Streptococcal tonsillitis and acute nonrheumatic myopericarditis. Chest 95: 359–363 234 Braiser AR, Macklis JD, Vaughan D, Warner L, Kirshenbaum JM (1987) Myopericarditis as an initial presentation of meningococcemia. Am J Med 82: 641–644 235 Gross D, Willens H, Zeldis SM (1981) Myocarditis in Legionnaires’ disease. Chest 79: 232–234 236 Lewes D, Rainford DJ, Lane WF (1974) Symptomless myocarditis and myalgia in viral and Myocoplasma pneumoniae infections. Br Heart J 36: 924–932 237 Marin-Garcia J, Barrett FF (1983) Myocardial function in Rocky Mountain spotted fever: Echocardiographic assessment. Am J Cardiol 51: 341–343 238 McAlister HF, Klemontowicz PT, Andrews C, Fisher JD, Feld M, Furman S (1989) Lyme carditis: An important cause of reversible heart block. Ann Intern Med 110: 339–345 239 Horn H, Saphir O (1935) The involvement of the myocardium in tuberculosis. Annu Rev Tuberc 32: 492–506 240 Ferrans VJ, Rodriguez ER (1985) Cardiovascular lesions in collagen-vascular diseases. Heart Vessels 1: 256–261 241 Lash AD, Wittman AL, Quismorio FP Jr (1986) Myocarditis in mixed connective tissue disease: Clinical and pathologic study of three cases and review of the literature. Semin Arthritis Rheum 15: 288–296 242 Kerr LD, Spiera H (1993) Myocarditis as a complication in scleroderma patients with myositis. Clin Cardiol 16: 895–899 243 Ni J, Bowles NE, Kim Y-H, Demmler G, Kearney D, Bricker JT, Towbin JA (1997) Viral infection of the myocardium in endocardial fibroelastosis: Molecular evidence for the role of mumps virus as an etiological agent. Circulation 95: 133–139 244 Yamamoto LG (2003) Kawasaki disease. Pediatr Emerg Care 19: 422–424 245 Okura Y, Dec GW, Hare JM, Kodama M, Berry GJ, Tazelaar HD, Bailey KR, Cooper LT (2003) A clinical and histopathologic comparison of cardiac sarcoidosis and idiopathic giant cell myocarditis. J Am Coll Cardiol 41: 322–329 246 Sole MJ, Liu P (1993) Viral myocarditis: A paradigm for understanding the pathogenesis and treatment of dilated cardiomyopathy. J Am Coll Cardiol 22: 99A–105A 247 Hudson REB (1965) Myocardial involvement (myocarditis) in infections, infestation and drug therapy. Cardiovasc Pathol 1: 782–854 248 Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE and the Myocarditis Treatment Trial Investigators (1995) A clinical trial of immunosuppressive therapy for myocarditis. N Engl J Med 333: 269–275 249 Grist NR, Reid D (1988) General pathogenicity and epidemiology. In: Bendinelli M, Friedman H (eds) Coxsackieviruses: A general update. Plenum, New York, 241–252 250 Van den Veyver IB, Ni J, Bowles N, Carpenter RJ, Weiner CP, Yankowitz J, Moise KJ, Henderson J, Towbin JA (1998) Detection of intrauterine viral infection using the polymerase chain reaction (PCR). Mol Genet Metab 63: 85–95

151


251 Friedman RA, Schowengerdt KO, Towbin JA (1998) Myocarditis. In: Bricker JT, Garson A Jr, Fisher DJ, Neish SR (eds) The science and practice of pediatric cardiology, 2nd ed. Williams & Wilkins, Baltimore, 1777–1794 252 Baboonian C, Davies MJ, Booth JC, McKenna WJ (1997) Coxsackie B viruses and human heart disease. Curr Top Microbiol Immunol 223: 31–52 253 Akhtar N, Ni J, Langston C, Demmler GJ, Towbin JA (1996) PCR diagnosis of viral pneumonitis from fixed- lung tissue in children. Biochem Mol Med 58: 66–76 254 McManus BM, Kandolf R (1991) Evolving concepts of cause, consequence and control in myocarditis. Curr Opin Cardiol 6: 418–427 255 Shimizu C, Rambaud C, Cheron G, Rouzioux C, Lozinski GM, Rao A, Stanway G, Krous HF, Burns JC (1995) Molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis. Pediatr Infect Dis J 14: 584–588 256 Friedman RA, Kearney DL, Moak JP, Fenrich AL, Perry JC (1994) Persistence of ventricular arrhythmia after resolution of occult myocarditis in children and young adults. J Am Coll Cardiol 24: 780–783 257 Towbin JA, Bricker JT, Garson A Jr (1992) Electrocardiographic criteria for diagnosis of acute myocardial infarction in childhood. Am J Cardiol 69: 1545–1548 258 Grogan M, Redfield MM, Bailey KR, Reeders GS, Gersh BJ, Edwards WD, Rodeheffer RJ (1995) Long-term outcome of patients with biopsy-proven myocarditis: Comparison with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 26: 80–84 259 Mason JW (1991) Distinct forms of myocarditis. Circulation 83: 1110–1111 260 Chow LH, Radio SJ, Sears TD et al (1889) Insensitivity of right ventricular biopsy in the diagnosis of myocarditis. J Am Coll Cardiol 14: 915–920 261 Hauck AJ, Kearney DL, Edwards WD (1989) Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: Implications for role of sampling error. Mayo Clin Proc 64: 1235–1245 262 Aretz HT (1987) Myocarditis: The Dallas criteria. Hum Pathol 18: 619–624 263 Shingu M (1989) Laboratory diagnosis of viral myocarditis: A review. Jpn Circ J 53: 87–93 264 Piedra PA, Poveda GA, Ramsey B, McCoy K, Hiatt PW (1998) Incidence and prevalence of neutralizing antibodies to the common adenoviruses in children with cystic fibrosis: Implication for gene therapy with adenovirus vectors. Pediatrics 101: 1013–1019 265 Bowles NE, Richardson PJ, Olsen EGJ, Archard LC (1986) Detection of coxsackie-B virus specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1: 1120–1123 266 Bowles NE, Rose ML, Taylor P, Banner NR, Morgan-Capner P, Cunningham L, Archard LC, Yacoub MH (1989) End-stage dilated cardiomyopathy. Persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response. Circulation 80: 1128–1136 267 Archard LC, Khan MA, Soteriou BA, Zhang H, Why HJ, Robinson NM, Richardson PJ (1998) Characterization of coxsackie B virus RNA in myocardium from patients

152


268

269

270

271

272 273 274 275 276

277 278

279 280

281

282

with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products. Hum Pathol 29: 578–584 Jin O, Sole MJ, Butany JW, Chia WK, McLaughlin PR, Liu P, Liew CC (1990) Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction. Circulation 82: 8–16 Weiss LM, Liu XF, Chang KL, Billingham ME (1992) Detection of enteroviral RNA in idiopathic dilated cardiomyopathy and other human cardiac tissues. J Clin Invest 90: 156–159 Mantke OD, Meyer R, Prosch S, Nitsche A, Leitmeyer K, Kallies R, Niedrig M (2005) High prevalence of cardiotropic viruses in myocardial tissue from explanted hearts of hearts transplant recipients and heart donors: A 3-year retrospective study from a German patients’ pool. J Heart Lung Transplant 24: 1632–1638 Klingel K, Sauter M, Bock CT, Szalay G, Schnorr JJ, Kandolf R (2004) Molecular pathology of inflammatory cardiomyopathy. Med Microbiol Immunol (Berl) 193: 101–107 Hutchins GM, Vie SA (1972) The progression of interstitial myocarditis to idiopathic endocardial fibroelastosis. Am J Pathol 66: 483–496 Saphir O, Field M (1954) Complications of myocarditis in children. J Pediatr 45: 457–463 Burch GE, Sun S-C, Sohal RS, Chu KC, Colcolough HL (1968) Diphtheritic myocarditis. Am J Cardiol 21: 261–268 James TN (1968) Sudden death in babies: New observations on the heart. Am J Cardiol 22: 479–506 Davidoff R, Palacios I, Southern J, Fallon JT, Newell J, Dec GW (1991) Giant cell versus lymphocytic myocarditis. A comparison of their clinical features and long-term outcomes. Circulation 83: 953–961 Rosenstein ED, Zucker MJ, Kramer N (2000) Giant cell myocarditis: Most fatal of autoimmune diseases. Semin Arthritis Rheum 30: 1–16 Stoica SC, Goddard M, Tsuiu S, Dunning J, McNeil K, Parameshwar J, Large SR (2003) Ventricular assist surprise: Giant cell myocarditis or sarcoidosis? J Thorac Cardiovasc Surg 126: 2072–2074 Cooper LT Jr (2003) Idiopathic giant cell myocarditis. In: Cooper LT Jr (Ed) Myocarditis: From Bench to Bedside. Humana Press, New Jersey, 405–420 McCarthy RE 3rd, Boehmer JP, Hruban RH, Hutchins GM, Kasper EK, Hare JM, Baughman KL (2000) Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocarditis. N Engl J Med 342: 690–695 Felker GM, Jaeger CJ, Klodas E, Thiemann DR, Hare JM, Hruban RH, Kasper EK, Baughman KL (2000) Myocarditis and long-term survival in peripartum cardiomyopathy. Am Heart J 140: 785–791 Grogan M, Redfield MM, Bailey KR, Reeder GS, Gersh BJ, Edwards WD, Rodeheffer

153


283 284 285

286 287 288

289 290

291

292

293

294

295 296

297

298

154

RJ (1995) Long-term outcome of patients with biopsy-proven myocarditis: Comparison with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 26: 80–84 Baboonian C, Treasure T (1997) Meta-analysis of the association of enteroviruses with human heart disease. Heart 178: 539–543 Martino TA, Liu P, Sole MJ (1994) Viral infection and the pathogenesis of dilated cardiomyopathy. Circ Res 74: 182–188 Badorff C, Lee G-H, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, Knowlton KU (1999) Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med 5: 320–326 Towbin JA (1998) The role of cytoskeletal proteins in cardiomyopathies. Curr Opin Cell Biol 10: 131–139 Towbin JA, Bowles NE (2002) The failing heart. Nature 415: 227–233 Grumbach IM, Heim A, Pring-Akerblom P, Vonhof S, Hein WJ, Muller G, Figulla HR (1999) Adenoviruses and enteroviruses as pathogens in myocarditis and dilated cardiomyopathy. Acta Cardiol 54: 83–88 Calabrese F, Thiene G (2003) Myocarditis and inflammatory cardiomyopathy: Microbiological and molecular aspects. Cardiovasc Res 60: 11–25 O’Connell JB, Fowles RE, Robinson JA, Subramanian R, Henkin RE, Gunnar RM (1984) Clinical and pathologic findings of myocarditis in two families with dilated cardiomyopathy. Am Heart J 167: 127–135 Bergelson JM, Cunningham JA, Drouguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW (1997) Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275: 1320–1323 Tomko RP, Xu R, Philipson L (1997) HCAR and MCAR: The human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci USA 94: 3352–3356 Poller W, Fechner H, Noutsias M, Tschoepe C, Schultheiss HP (2002) Highly variable expression of virus receptors in the human cardiovascular system. Implications for cardiotropic viral infections and gene therapy. Z Kardiol 91: 978–991 Bowles NE, Javier Fuentes-Garcia F, Makar KA, Li H, Gibson J, Soto F, Schwimmbeck PL, Schultheiss HP, Pauschinger M (2002) Analysis of the coxsackievirus or dilated cardiomyopathy. Mol Genet Metab 77: 257–259 Towbin JA (2002) Cardiomyopathy and heart transplantation in children. Curr Opin Cardiol 17: 274–279 Schowengerdt KO, Ni J, Denfield SW, Gajarski RJ, Radovancevic B, Frazier HO, Demmler GJ, Kearney D, Bricker JT, Towbin JA (1996) Diagnosis surveillance, and epidemiologic evaluation of viral infections in pediatric cardiac transplant recipients with the use of polymerase chain reaction. J Heart Lung Transplant 15: 111–123 Shirali GS, Ni J, Chinnock RE, Johnston JK, Rosenthal GL, Bowles NE, Towbin JA (2001) Association of viral genome with graft loss in children after cardiac transplantation. N Engl J Med 344: 1498–1503 Bridges ND, Spray TL, Collins MH, Bowles NE, Towbin JA (1998) Adenovirus infec-


299 300 301

302

303

304

305

306

307

308

309

310

311

tion in the lung results in graft failure after lung transplantation. J Thorac Cardiovasc Surg 116: 617–623 Parrillo JE (1998) Myocarditis: How should we treat in 1998? J Heart Lung Transplant 17: 941–944 Rezkalla S, Kloner RA, Khatib G, Khatib R (1990) Beneficial effects of captopril in acute coxsackievirus B3 murine myocarditis. Circulation 81: 1039–1046 Weller AH, Hall M, Huber SA (1992) Polyclonal immunoglobulin therapy protects against cardiac damage in experimental coxsackievirus-induced myocarditis. Eur Heart J 13: 115–119 Chan KY, Iwahara M, Benson LN, Wilson GJ, Freedom RM (1991) Immunosuppressive therapy in the management of acute myocarditis in children: A clinical trial. J Am Coll Cardiol 17: 458–460 Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri A (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107: 857–863 Drucker NA, Colan SD, Lewis AB, Beiser AS, Wessel DL, Takahashi M, Baker AL, Perez-Atayde AR, Newburger JW (1994) A-Globulin treatment of acute myocarditis on the pediatric population. Circulation 89: 252–257 Maisch B, Hufnagel G, Kolsch S, Funck R, Richter A, Rupp H, Herzum M, Pankuweit S (2004) Treatment of inflammatory dilated cardiomyopathy and (peri) myocarditis with immunosuppresion and i.v. immunoglobulins. Herz 29: 624–636 Kuhl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-B treatment eliminates cardiotropic viruses and improves left ventricular function in patients with myocardial persistence of viral genomes and left ventricular dysfunction. Circulation 107: 2793–2798 Daliento L, Calabrese F, Tona F, Caforio AL, Tarsia G, Angelini A, Thiene G (2003) Successful treatment of enterovirus-induced myocarditis with interferon alpha. J Heart Lung Transplant 22: 214–217 Schindler E, Muller M, Kwapisz M, Akinturk H, Valeske K, Thul J, Hempelmann G (2003) Ventricular cardiac-assist devices in infants and children: Anesthetic considerations. J Cardiothorac Vasc Anesth 17: 617–621 Undar A, McKenzie ED, McGarry MC, Owens WR, Surprise DL, Kilpack VD, Mueller MW, Stayer SA, Andropoulos DB, Towbin JA, Fraser CD Jr (2004) Outcomes of congenital heart surgery patients after extracorporeal life support at Texas Children’s Hospital. Artif Organs 28: 963–966 Helman DN, Addonizio LJ, Morales DL, Catanese KA, Flannery MA, Quagebeur JM, Edwards NM, Galantowicz ME, Oz MC (2000) Implantable left ventricular assist devices can successfully bridge adolescent patients to transplant. J Heart Lung Transplant 19: 121–126 Deiwick M, Hoffmeier A, Tjan TD, Krasemann T, Schmid C, Scheld HH (2005) Heart failure in children – Mechanical assistance. Thorac Cardiovasc Surg 53 Suppl 2: S135– 140

155


312 O’Connell JB, Dec GW, Goldenberg IF, Starling RC, Mudge GH, Augustine SM, Costanzo-Nordin MR, Hess ML, Hosenpud JD, Icenogle TB (1990) Results of heart transplantation for active lymphocytic myocarditis. J Heart Transplant 9: 351–356 313 Bengtssen E, Lamberger B (1966) Five year follow-up study of cases suggestive of acute myocarditis. Am Heart J 72: 751–763 314 Hastreiter AR, Miller RA (1964) Diagnosis and treatment of myocarditis and other myocardial diseases. Adv Cardiopulm Dis 22: 250–258 315 English RF, Janosky JE, Ettedgui JA, Webber SA (2004) Outcomes for children with acute myocarditis. Cardiol Young 14: 488–493 316 Zhang HY, Morgan-Capner P, Latif N, Pandolfino YA, Fan W, Dunn MJ, Archard LC (1997) Coxsackievirus B3-induced myocarditis: Characterization of stable attenuated variants that protect against infection with the cardiovirulent wild-type strain. Am J Pathol 150: 2197–2207

156

Pathogenic relevance of autoantibodies in dilated cardiomyopathy Roland Jahns 1,2, Valérie Boivin 2, Georg Ertl 1 and Martin J. Lohse 2 1

Department of Internal Medicine, Medizinische Klinik und Poliklinik I, Cardiology, University Hospital of Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, Germany 2 Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany

Abstract Dilated cardiomyopathy (DCM) is a heart muscle disease of unknown origin characterized by progressive cardiac dilatation and loss of contractile function in the absence of coronary artery disease. Genetic causes and cardiotoxic substances account for about one third of the cases, but the etiology of the two other thirds is still poorly understood. However, within the past two decades evidence has grown continuously that autoimmunity to certain cardiac antigens may play an important role in the development of DCM. Recent experiments in rodents even indicate that autoantibodies targeting the cardiac B1-adrenergic (catecholamine) receptor can actually cause the disease. Dependent on the individual genetic predisposition, such harmful autoimmune reactions most likely occur as a result of heart muscle damage induced by viral triggers, ischemia, and/or exposure to cardiotoxins leading to myocyte apoptosis or necrosis, and subsequent liberation of self antigens previously hidden to the immune system. The following article reviews current evidence and recent experimental and clinical findings focusing on the possible role of autoantibodies against a confined number of cardiac self antigens in the pathogenesis of DCM.

Introduction Progressive cardiac dilatation and pump failure of unknown etiology has been termed “idiopathic” dilated cardiomyopathy (DCM) [1]. DCM represents one of the main causes of severe heart failure with an annual incidence of up to 100 patients and a prevalence of 300–400 patients per million [2]. Mutations in genes encoding myocyte structural proteins [3] and several cardiotoxins, including alcohol, anthracyclines, and, more recently, therapeutically used monoclonal antibodies (e.g., trastuzumab) account for about one third of DCM cases [4, 5]. The etiology of the remaining two thirds is still poorly understood, however. At present the large majority of DCM is thought to arise from an initial (mostly viral) infection leading to acute myocarditis, which upon activation of the immune system may progress to (chronic) autoimmune myocarditis, resulting in cardiac dila-


157

Roland Jahns et al.

tation and severe congestive heart failure. The latter progression occurs particularly when associated (a) with the development of autoantibodies against distinct myocyte structural or membrane proteins that are essential for cardiac function [6, 7], or (b) with chronic inflammation of the myocardium and viral persistence [8]. These recent findings are further strengthened by the fact that patients with DCM often have alterations in both cellular and humoral immunity [7, 9–11]. Under such conditions an initial acute inflammatory reaction may proceed to a kind of low-grade inflammation [12], facilitating the development of abnormal or misled immune responses to the primary infectious trigger [6, 8, 12–14]. In the context of their humoral response a substantial number of DCM patients have been found to develop cross-reacting antibodies and/or autoantibodies to various cardiac antigens, including mitochondrial proteins (e.g., adenine nucleotide translocator, lipoamide and pyruvate dehydrogenase [15–18]), sarcoplasmatic proteins (e.g., actin, laminin, myosin, troponin [19–23]), and membrane proteins (e.g., cell surface adrenergic or muscarinergic receptors [24– 27]). However, from these, only a few selected antibodies appear to be able to cause myocardial tissue injury and to induce severe congestive heart failure by themselves. In addition, the individual genetic predisposition, including the respective human leukocyte antigen (HLA) and the major histocompatibility complex (MHC) phenotype [28], may also significantly contribute to the susceptibility to self-directed immune reactions and the phenotypic expression of the disease [12, 29]. The present article reviews current knowledge and recent experimental and clinical evidence focusing on the possible role of cardiac autoantibodies in the pathogenesis of DCM.

Formation of cardiac autoantibodies Homologies between myocyte surface molecules such as membrane receptors and viral or bacterial proteins have been proposed as a mechanism for the elaboration of endogenous cardiac autoantibodies by antigen mimicry [30, 31]. Chagas’ heart disease, a slowly evolving inflammatory cardiomyopathy, is one of the most prominent examples for this mechanism [13, 32]. The disease originates from an infection with the protozoon Trypanosoma cruzi; molecular mimicry between the ribosomal P2B-protein of T. cruzi and the N-terminal half of the second extracellular loop of the B1-adrenergic receptor (B1-AR) results in generation of cross-reacting antibodies in about 30% of the Chagas’ patients [33]. Because receptor-autoantibodies from patients with DCM preferentially recognize the C-terminal half of the same loop [34], it was speculated that these antibodies might originate from molecular mimicry between the B1-AR and a hitherto unidentified viral pathogen [35]. A further example is Chlamydia-mediated heart disease induced in BALB/c mice, assumingly by antigen mimicry between a microbial antigen and the A-myosin heavy chain molecule, resulting in activation of autoreactive T and B cells [36].

158

Pathogenic relevance of autoantibodies in dilated cardiomyopathy

Figure 1. Formation of autoantibodies against myocardial self antigens (e.g., membrane receptors).

Another – probably more relevant – mechanism leading to the production of endogenous cardiac autoantibodies would be primary cardiac injury followed by (sudden or chronic) liberation of a “critical amount” of antigenic determinants from the myocyte membrane or cytoplasm, previously hidden to the immune system. Such injury most likely occurs upon acute infectious (myocarditis), toxic, or ischemic heart disease (myocardial infarction), resulting in myocyte apoptosis or necrosis [20, 37] (Fig. 1). Presentation of myocardial self antigens to the immune system may then induce an autoimmune response, which in the worst case results in perpetuation of immune-mediated myocyte damage involving either cellular (e.g., T cell) or humoral (e.g., B cell) immune responses, or co-activation of both the innate and the adaptive immune system [37, 38].

Pathogenic relevance of cardiac autoantibodies in DCM Following Witebsky’s postulates [39], ‘indirect’ evidence for the autoimmune etiology of a disease requires identification of the trigger (e.g., the responsible self antigen), and induction of a self antigen-directed immune response in an experimental

159

Roland Jahns et al.

animal, which then must develop a similar disease. ‘Direct’ evidence, however, requires reproduction of the disease by transfer of homologous pathogenic antibodies or autoreactive T cells from one animal to another of the same species [40]. From a pathophysiological point of view, it seems reasonable to link the harmful (e.g., cardiomyopathy-inducing) potential of a heart-specific autoantibody to the ‘accessibility’ and to the ‘functional relevance’ of the corresponding target. In the light of the wide panel of autoantibodies found in the sera from DCM patients directed against (a) heat shock proteins [41], (b) mitochondrial proteins, (c) myocyte structural proteins, and (d) membrane receptors and/or channels, for more than half a century it has been disputed whether autoimmunity could cause DCM [42]. However, recent evidence from animal experiments terminated the controversy by demonstrating a cause-and-effect relationship for at least two distinct antibodies out of the DCM-associated autoantibodies (see sections ‘Myocyte structural proteins’, and ‘Membrane receptor proteins’).

Heat-shock proteins Heat-shock proteins (HSP) generally reside in the cytoplasm where they are separated from the immune system [42]. Transgenic expression of the HSP gp96 on the cell surface, however, resulted in an activation of dendritic cells and spontaneous development of autoimmune diseases affecting the kidney, liver, lung, and sometimes the heart (mostly as endocarditis, but rarely as DCM) [43]. Because HSP are well conserved from bacteria to humans and HSP autoantibodies can be found in a variety of infectious and autoimmune diseases (including rheumatoid arthritis and systemic lupus erythematosus) [41], it seems justified to regard such autoantibodies as remnants (and thus markers) of prior viral or bacterial infection rather than specifically DCM-inducing agents [42].

Mitochondrial proteins Mitochondrial proteins are intracellularly localized molecules widely expressed in the body, making it difficult to explain a heart-specific autoimmune reaction induced by autoantibodies targeting such antigens [42]. However, because cardiomyocytes are rich in gap junctions, facilitating penetration of the plasma membrane, it seems possible that such antibodies might act intracellularly under certain conditions [44], and then may influence myocardial energy metabolism to some extent [17, 18]. For example, immunization of guinea pigs with the ADP–ATPcarrier protein (located in the inner mitochondrial membrane) led to the generation of functional antibodies against the ADP–ATP carrier, and also to a reduction in cardiac function. On isolated mitochondria (but not on intact myocytes) these

160


antibodies blocked the activity of the ADP–ATP carrier, and this effect correlated well with the decrease in cardiac performance [17, 45]. However, the possible in vivo effects achieved by transfer of such anti-ADP–ATP antibodies remain to be demonstrated. Moreover, it has been shown that antibodies against the ADP–ATP carrier can cross-react with the calcium-channel complex in the membrane of cardiomyocytes, thereby increasing calcium influx to the cells, which in the worst case may result in calcium-dependent cell lysis [16]. Chronic calcium overload is supposed to induce mechanical overload and apoptosis of cardiomyocytes, and thus significantly contributes to the transition from compensated to decompensated maladaptive growth in heart failure [42, 46]. Again, the in vivo consequences of the above experimental in vitro finding for the induction of human DCM remain to be demonstrated.

Myocyte structural proteins Myocyte structural proteins (e.g. members of the contractile apparatus as actin, myosin, troponin) are specifically expressed in the heart and may thus account for the organ specificity of an autoimmune reaction [42]. However, sarcolemmal proteins of the contractile apparatus are localized in the cytoplasm, making it difficult for antibodies to access these proteins under physiological conditions. Interestingly, in mice, tropinin I (but not troponin T) has been found to be present also on the surface of cardiomyocytes of normal hearts, [21]. Further, it has been shown that programmed cell death (PD-1)-deficient mice (exhibiting hyperactivation of the immune system and augmented B cell proliferation) develop both DCM and high titer antibodies against a heart-specific 30-kDa protein, subsequently identified as cardiac troponin I (cTnI) [47]. Because, in this mouse model, cTnI was found abundantly on the outer surface of t-tubules (known to be critically involved in excitationcontraction coupling), it was speculated that anti-cTnI antibodies might affect the electrophysiological function of cardiomyocytes. Subsequent in vitro experiments revealed that anti-cTnI antibodies significantly augmented the voltage-dependent calcium current of isolated cardiomyocytes, indicating that this mechanism might in fact contribute to the pathogenesis of DCM [21]. Finally, transfer of anti-cTnI mouse antibodies into naïve BALB/c mice within 3 months also induced a cardiomyopathic phenotype in recipients [21], thus accomplishing Witebsky’s postulates of ‘direct’ evidence for a cause-and-effect relationship between anti-cTnI antibodies and DCM. In this context, two drawbacks should be mentioned. First, troponins are believed to be restricted to the cytoplasm in the intact human heart, making it hard to explain how troponin I-directed autoantibodies access these proteins. Second, a recent report addressing the possible pathogenic role of an autoimmune response to troponins by immunizing A/J mice with recombinant murine cardiac troponin I (mc-TnI) clearly demonstrated that high anti-TnI-antibody titers were associated

161

Roland Jahns et al.

with severe inflammation of the myocardium and increased expression of inflammatory cytokines (followed by fibrosis, cardiomegaly, and impaired contractility) [22], whereas the dilated hearts from anti-TnI antibody-positive PD-1-deficient mice had no apparent signs of inflammation [21]. Taken together, it is still unclear whether the symptoms and phenotypes induced with anti-TnI antibodies reflect myocarditis rather than DCM. Besides troponin, another myocyte structural protein has been the focus of research activities during the past 15 years: cardiac myosin (CM). It has been demonstrated that myocarditis and DCM patients have a high prevalence of antibodies against CM, and that their presence was associated with depressed cardiac function [19, 20, 48, 49]. Immunization of mice with CM results in severe myocarditis with a massive infiltration of the myocardium with macrophages and T cells, indicating activation of the cellular immune system, which is followed by a humoral response with production of anti-myosin antibodies (here, it should be mentioned that antimyosin antibodies also emerged 3 months after immunization of mice with mc-TnI and induction of myocardial inflammation [22]). Isogenic transfer of anti-myosin antibodies into naïve mice failed to reproduce myocarditis and/or cardiac dysfunction, however [50]. This finding argues against an “intrinsic” pathogenic role of CM autoantibodies and rather suggests that, in human heart disease, antibodies against myocyte structural (intracellular) proteins are most likely produced secondary to heart muscle damage and not causally involved in the pathogenesis of DCM. Most recent work, however, points to another interesting link between CM antibodies and development of DCM. Anti-CM antibodies induced by immunization of rats with human CM or a CM-derived peptide (S2-16) appear to cross-react with B-adrenergic receptors (B-AR) on the myocyte cell surface and thereby specifically activate cAMP-dependent protein kinase A (PKA) in cardiomyocytes [23]. Since activation of PKA through the B-AR – Gs protein – cAMP cascade has been shown to induce cardiomyocyte apoptosis and to promote development of heart failure, it seems conceivable that cross-reactive mimicry between CM and B-AR might play a pivotal role in the pathogenesis of DCM [6, 51]. In addition, the same investigators were able to show that passive transfer of IgG from CM-immunized cardiomyopathic rats resulted in myocardial IgG-deposition and development of a cardiomyopathic phenotype in recipient rats [23]. Taken together these results point towards cardiac (membrane) B-AR rather than cardiac (sarcoplasmatic) myosin as key molecules in the pathophysiology of DCM.

Membrane receptor proteins Myocyte surface receptors are easily accessible to autoantibodies [42]. The two most promising candidates are the cardiac B1-AR (representing the predominant adrenoceptor subtype in the heart) and the M2-muscarinic acetylcholine receptor;

162


autoantibodies have been detected against both receptors in DCM patients [25, 27, 52]. Whereas anti-muscarinic antibodies (exhibiting an agonist-like action on the cardiac M2 acetylcholine receptor) have been mainly associated with negative chronotropic effects at the sinuatrial level (e.g., sinus node dysfunction, atrial fibrillation [53, 54]), agonistic anti-B1-AR antibodies have been associated with both the occurrence of severe arrhythmia at the ventricular level [55, 56], and the development of (maladaptive) left ventricular (LV) hypertrophy, finally switching to LV enlargement and progressive heart failure [25, 46, 57]. Both autoantibodies appear to be directed against the second extracellular loop of the respective receptors. To generate an autoimmune response, myocyte membrane proteins (e.g., receptors) must be degraded to small oligopeptides able to form a complex with an MHC or HLA class II molecule of the host [30]. In case of the human B1-AR, computer-based analysis for potential immunogenic amino acid stretches has shown that the only portion of the receptor molecule containing B and T cell epitopes and being accessible to antibodies was in fact the predicted second extracellular receptor loop (B1-ECII) [30]. This might explain the successful use of second loop-peptides for the generation of B1-specific receptor antibodies in different animal models [57–59]. Moreover, during the last decade several groups have independently demonstrated that second loop antibodies preferentially recognize intact native B1-AR in various immunological assays (whole-cell ELISA, immunoprecipitation, immunofluorescence), indicating that they are “conformational” [7, 30]. Functional testing revealed that the same antibodies also affected receptor function, such as intracellular cAMP production and/or cAMP-dependent protein kinase (PKA) activity, suggesting that they may act as allosteric regulators of B1-AR activity [59]. To analyze the pathogenetic potential of anti-B1-AR antibodies, in our laboratory we choose an experimental in vivo approach, which met the Witebsky criteria for direct evidence of autoimmune diseases. DCM was induced by immunizing inbred rats against B1-ECII (100% sequence homology between human and rat; ‘indirect evidence’); the disease was then reproduced in healthy animals by isogenic transfer of rat anti-B1-AR ‘autoantibodies’ (‘direct evidence’) [60]. The animals developed progressive LV dilatation and dysfunction, a relative decrease in LV wall thickness, and selective down-regulation of B1-AR, a feature that is also seen in human DCM [51]. These results, together with an agonist-like short-term effect of the antibodies in vivo [60], suggest that both the induced and the transferred cardiomyopathic phenotypes can be attributed mainly to the mild but sustained receptor activation achieved by stimulatory anti-B1-AR antibodies. This hypothesis is supported by the large body of data available on the cardiotoxic effects of excessive and/or longterm B1-AR activation seen after genetic or pharmacological manipulation [61, 62]. Therefore, anti-B1-AR induced dilated immune cardiomyopathy can now be regarded as a pathogenetic disease entity of its own, together with other established receptor-directed autoimmune diseases such as myasthenia gravis or Graves’ disease [6, 7, 60, 63].

163

Roland Jahns et al.

Clinical relevance of cardiac B1-AR antibodies and future perspectives The clinical importance of cardiac autoantibodies is difficult to assess, since low titers of such antibodies can also be detected in the healthy population as a part of the natural immunological repertoire [37]. However, regarding functionally active anti-B1-AR antibodies, our earlier data demonstrate that their prevalence is almost negligible in healthy individuals (< 1%) provided that a screening procedure based on cell systems presenting the target (e.g., the B1-AR) in its natural conformation is used [25]. Employing the latter screening method, the occurrence of anti-B1-AR autoantibodies could also be excluded in patients with chronic valvular or hypertensive heart disease [64]. In contrast, the prevalence of stimulating anti-B1-AR was ~10% in ischemic cardiomyopathy and ~30% in DCM [25], which was significantly higher than in healthy controls, but in the lower range of previous reports on DCM collectives (33–95% prevalence) [24, 34, 65]. It seems conceivable that differences in screening methods aiming to detect functionally active anti-B1-AR autoantibodies most likely account for the wide range of prevalences reported in the past [65]. In fact, only a minor fraction of ELISA-defined human anti-B-AR autoantibodies was able to bind to cell surface-located native B-AR. Only this fraction recognized (as determined by immunofluorescence) and activated (as determined by increases in cellular cAMP and/or PKA activity) human B1-AR expressed in the membrane of intact eukaryotic cells [25, 59]. Therefore, cell systems presenting the target in its natural conformation represent an essential tool in the screening for functionally relevant anti-B-AR autoantibodies [66]. Clinically, the presence of anti-B1-AR autoantibodies in DCM has been shown to be associated with a more severely depressed cardiac function [25], the occurrence of more severe ventricular arrhythmia [67], and a higher incidence of sudden cardiac death [55]. Recent data comparing antibody-positive with antibody-negative DCM patients over a follow-up period of more than 10 years not only confirmed a higher prevalence of ventricular arrhythmia in the presence of activating anti-B1-AR, but also revealed that antibody-positivity predicted an almost threefold increased cardiovascular mortality risk [68]. Taken together, the available clinical data underscore the pathophysiological relevance of functionally active anti-B1-AR antibodies in DCM and encourage further research in the evolving field of antibody-directed strategies as a therapeutic principle [6, 63]. In this regard, one aspect of the beneficial effects of B1-receptor blockade in DCM might be the pharmacological counter-regulation of autoantibody-mediated stimulatory effects [6, 59, 66]. Current experimental therapeutic approaches include elimination of activating anti-B1-AR autoantibodies by non-selective or selective immunoadsorption [63, 69], or direct targeting of the autoantibody-producing B cells themselves [70]. Although a recent smaller pilot study in DCM patients was not able to unravel an association between the hemodynamic benefit of immunoadsorption and anti-B1-AR antibody status [71], the results might be difficult to interpret

164


since native human B-AR were not employed for the definition of functional antiB1-AR antibodies. One prerequisite for both an unequivocal interpretation of future clinical trials and the development of novel antibody-targeted therapies would be a standardized screening procedure for functional anti-B1-AR autoantibodies using cell systems presenting the target receptor in its natural conformation [7, 66, 68]. To summarize, even though stimulating anti-B1-AR autoantibodies can clearly be pathogenic, the pathophysiological sequence of events leading to their generation, their relative contribution to the pathogenesis of human DCM, and their relevance for prognosis and therapy still remain to be determined.

Acknowledgements Our work is currently supported by the Deutsche Forschungsgemeinschaft (Grant DFG/Ja 706/2-4), and the Bundesministerium für Bildung und Forschung (BMBF, GoBio-1/FKZ 0315031 and 0315636; MolDiag/FKZ 01ES0816, 01ES0901, and 01ES0902).

References 1

2 3 4 5 6 7 8

9 10

Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen I, Thiene G, Goodwin J, Gyarfas I et al (1996) Report of the WHO/ISFC task force on the definition and classification of cardiomyopathies. Circulation 93: 841–842 American Heart Association (2007) Dallas; Heart disease and stroke statistics – 2007 update. Circulation 115: e69–e171 Morita H, Seidman J, Seidman CE (2005) Genetic causes of human heart failure. J Clin Invest 115: 518–526 Fabrizio L, Regan TJ (1994) Alcoholic cardiomyopathy. Cardiovasc Drugs Ther 8: 89–94 Chien KR (2000) Myocyte survival pathways and cardiomyopathy: Implications for trastuzumab cardiotoxicity. Semin Oncol 27: 9–17 Freedman NJ, Lefkowitz RJ (2004) Anti-beta1–adrenergic receptor antibodies and heart failure: Causation, not just correlation. J Clin Invest 113: 1379–1382 Jahns R, Boivin V, Lohse MJ (2006) Beta1-adrenergic receptor function, autoimmunity, and pathogenesis of dilated cardiomyopathy. Trends Cardiovasc Med 16: 20–24 Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112: 1965–1970 Limas CJ (1997) Cardiac autoantibodies in dilated cardiomyopathy. Circulation 95: 1979–1980 Luppi P, Rudert WA, Zanone MM, Stassi G, Trucco G, Finegold D, Boyle GJ, Del Nido

165

Roland Jahns et al.

11

12 13

14 15

16

17

18

19

20

21

22

23

24

166

P, McGowan FX, Trucco M (1998) Idiopathic dilated cardiomyopathy. A superantigendriven autoimmune disease. Circulation 98: 777–785 Mahrholdt H, Wagner A, Deluggi CC, Kispert E, Hager S, Meinhardt G, Vogelsberg H, Fritz P, Dippon J, Bock C-T et al (2006) Presentation, patterns of myocardial damage, and clinical course of viral myocarditis. Circulation 114: 1581–1590 MacLellan WR, Lusis AJ (2003) Dilated cardiomyopathy: Learning to live with yourself. Nat Med 9: 1455–1456 Smulski C, Labovsky V, Levy G, Hontebeyrie M, Hoebeke J, Levin MJ (2006) Structural basis of the cross-reaction between an antibody to the Trypanosoma cruzi ribosomal P2beta protein and the human beta1-adrenergic receptor. FASEB J 20: 1396–1406 Maekawa Y, Ouzounian M, Opavsky MA, Liu PP (2007) Connecting the missing link between dilated cardiomyopathy and viral myocarditis. Circulation 115: 5–8 Schultheiss HP, Bolte HD (1985) Immunological analysis of autoantibodies against the adenine nucleotide translocator in dilated cardiomyopathy. J Mol Cell Cardiol 17: 603–617 Schultheiss HP, Kühl U, Janda I, Melzner B, Ulrich G, Morad M (1988) Antibodymediated enhancement of calcium permeability in cardiac myocytes. J Exp Med 168: 2102–2109 Schulze K, Witzenbichler B, Christmann C, Schultheiss HP (1999) Disturbance of myocardial energy metabolism in experimental virus myocarditis by antibodies against the adenine nucleotide translocator. Cardiovasc Res 44: 91–100 Pohlner K, Portig I, Pankuweit S, Lottspeich F, Maisch B (1997) Identification of mitochondrial antigens recognized by the antibody in sera of patients with dilated cardiomyopathy by two-dimensional gel electrophoresis and protein sequencing. Am J Cardiol 80: 1040–1045 Neumann DA, Burek CL, Baughman KL, Rose NR, Herskowitz A (1990) Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy. J Am Coll Cardiol 16: 839–846 Caforio AL, Mahon NJ, Tona F, McKenna WJ (2002) Circulating cardiac autoantibodies in dilated cardiomyopathy and myocarditis: Pathogenetic and clinical significance. Eur J Heart Fail 4: 411–417 Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, Ishida M, Hiai H, Matsumori A, Minato N et al (2003) Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med 9: 1477– 1483 Göser S, Andrassy M, Buss SJ, Leuschner F, Volz CH, Öttl R, Zittrich S, Blaudeck N, Hardt SE, Pfitzer G et al (2006) Cardiac troponin I but not cardiac troponin T induces severe autoimmune inflammation in the myocardium. Circulation 114: 1693–1702 Li Y, Heuser JS, Cunningham LC, Kosanke SD, Cunningham MW (2006) Mimicry and antibody-mediated cell signaling in autoimmune myocarditis. J Immunol 177: 8234–8240 Magnusson Y, Wallukat G, Waagstein F, Hjalmarson Å, Hoebeke J (1994) Autoimmunity


25

26

27

28 29

30 31

32

33

34

35 36 37 38

in idiopathic dilated cardiomyopathy. Characterization of antibodies against the beta1– adrenoceptor with positive chronotropic effect. Circulation 89: 2760–2767 Jahns R, Boivin V, Siegmund C, Inselmann G, Lohse MJ, Boege F (1999) Autoantibodies activating human beta1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure. Circulation 99: 649–654 Christ T, Schindelhauer S, Wettwer E, Wallukat G, Ravens U (2006) Interaction between autoantibodies against the beta1-adrenoceptor and isoprenaline in enhancing L-type Ca2+ current in rat ventricular myocytes. J Mol Cell Cardiol 41: 716–723 Fu LXM, Magnusson Y, Bergh CH, Waagstein F, Hjalmarson Å, Hoebeke J (1993) Localization of a functional autoimmune epitope on the second extracellular loop of the human muscarinic acetylcholine receptor 2 in patients with idiopathic dilated cardiomyopathy. J Clin Invest 91: 1964–1968 Limas CJ (1996) Autoimmunity in dilated cardiomyopathy and the major histocompatibility complex. Int J Cardiol 54: 113–116 Limas CJ, Iakovis P, Anyfantakis A, Kroupis C, Cokkinos DV (2004) Familial clustering of autoimmune diseases in patients with dilated cardiomyopathy. Am J Cardiol 93: 1189–1191 Hoebeke J (1996) Structural basis of autoimmunity against G protein-coupled membrane receptors. Int J Cardiol 54: 103–111 Mobini R, Maschke H, Waagstein F (2004) New insights into the pathogenesis of dilated cardiomyopathy: Possible underlying mechanisms and therapy. Autoimmun Rev 3: 277–284 Elies R, Ferrari I, Wallukat G, Lebesgue D, Chiale P, Elizari M, Rosenbaum M, Hoebeke J, Levin MJ (1996) Structural and functional analysis of the B cell epitopes recognized by anti-receptor autoantibodies in patients with Chagas’ disease. J Immunol 157: 4203–4211 Ferrari I, Levin M, Wallukat G, Elies R, Lebesgue D, Chiale P, Elizari M, Rosenbaum M, Hoebeke J (1995) Molecular mimicry between the immunodominant ribosomal protein PO of Trypanosoma cruzi and a functional epitope on the human beta1-adrenergic receptor. J Exp Med 182: 59–65 Wallukat G, Wollenberger A, Morwinski R, Pitschner HF (1995) Anti-beta1-adrenoceptor autoantibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy: Mapping of epitopes in the first and second extracellular loops. J Mol Cell Cardiol 27: 397–406 Magnusson Y, Hjalmarson Å, Hoebeke J (1996) Beta1-adrenoceptor autoimmunity in cardiomyopathy. Int J Cardiol 54: 137–141 Bachmaier K, Neu N, de la Maza LM, Pal S, Hessel A, Penninger JM (1999) Chlamydia infections and heart disease linked through antigenic mimicry. Science 283: 1335–1339 Rose NR (2001) Infection, mimics, and autoimmune disease. J Clin Invest 107: 943– 944 Eriksson U, Ricci R, Hunziker L, Kurrer MO, Oudit GY, Watts TH, Sonderegger I, Bachmaier K, Kopf M, Penninger JM (2003) Dendritic cell-induced autoimmune heart

167

Roland Jahns et al.

39 40 41 42 43

44

45

46 47

48

49

50 51 52

53

54

168

failure requires cooperation between adaptive and innate immunity. Nat Med 9: 1484– 1490 Witebsky E, Rose NR, Terplan K, Paine JR, Egan RW (1957) Chronic thyreoiditis and autoimmunization. J Am Med Assoc 164: 1439–1447 Rose NR, Bona C (1993) Defining criteria for autoimmune diseases (Witebsky’s postulates revisited). Immunol Today 14: 426–430 Portig I, Pankuweit S, Maisch B (1997) Antibodies against stress proteins in sera of patients with dilated cardiomyopathy. J Mol Cell Cardiol 29: 2245–2251 Okazaki T, Honjo T (2005) Pathogenic roles of cardiac autoantibodies in dilated cardiomyopathy. Trends Mol Med 11: 322–326 Liu B, Dai J, Zheng H, Stoilova D, Sun S, Li Z (2003) Cell surface expression of an endoplasmic reticulum resident heat shock protein gp96 triggers MyD88-dependent systemic autoimmune diseases. Proc Natl Acad Sci USA 100: 15824–15829 Reichlin M (1998) Cellular dysfunction induced by penetration of autoantibodies into living cells: Cellular damage and dysfunction mediated by antibodies to dsDNA and ribosomal P protein. J Autoimmun 11: 557–561 Schulze K, Becker BF, Schauer R, Schultheiss HP (1990) Antibodies to ADP-ATP carrier – an autoantigen in myocarditis and dilated cardiomyopathy – impair cardiac function. Circulation 81: 959–969 Khoynezhad A (2007) Promising aspects and caveats of studies on anti-apoptotic therapies in patients with heart failure. Eur J Heart Fail 9: 120–123 Nishimura H, Okazaki T, Tanaka Y, Natakani K, Hara M, Matsumori A et al (2001) Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291: 319–322 Caforio AL, Grazzini M, Mann JM, Keeling PJ, Botazzo GF, McKenna WJ, Schiaffino S (1992) Identification of A- and B-cardiac myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 85: 1734–1742 Lauer B, Schannwell M, Kühl U, Strauer BE, Schultheiss HP (2001) Antimyosin autoantibodies are associated with deterioration of systolic and diastolic left ventricular function in patients with chronic myocarditis. J Am Coll Cardiol 35: 11–18 Neu N, Ploier B, Öfner C (1990) Cardiac myosin-induced myocarditis: Heart autoantibodies are not involved in the induction of the disease. J Immunol 145: 4094–4100 Lohse MJ, Engelhardt S, Eschenhagen T (2003) What is the role of beta-adrenergic signaling in heart failure? Circ Res 93: 896–906 Matsui S, Fu M, Shimizu M, Fukuoka T, Teraoka K, Takekoshi N, Murakami E, Hjalmarson A (1995) Dilated cardiomyopathy defines serum autoantibodies against G protein-coupled cardiovascular receptors. Autoimmunity 21: 85–88 Wang WZ, Zhao RR, Wu BW, Jin XH, Zuh L, Hjalmarson A, Fu LXM (1996) Effects of anti-peptide antibodies against human M2 muscarinic receptors on cardiac function of rats in vivo. Blood Press Suppl 3: 25–27 Baba A, Yoshikawa T, Fukuda Y, Sugiyama T, Shimada M, Akaishi M, Tsuchimoto K, Ogawa S, Fu M (2004) Autoantibodies against M2-muscarinic acetylcholine receptors:


55

56

57

58

59

60

61

62

63 64

65

66

67

New upstream targets in atrial fibrillation in patients with dilated cardiomyopathy. Eur Heart J 25: 1108–1115 Iwata M, Yoshikawa T, Baba A, Anzai T, Mitamura H, Ogawa S (2001) Autoantibodies against the second extracellular loop of beta1-adrenergic receptors predict ventricular tachycardia and sudden death in patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 37: 418–424 Christ T, Wettwer E, Dobrev D, Adolph E, Knaut M, Wallukat G, Ravens U (2001) Autoantibodies against the beta1-adrenoceptor from patients with dilated cardiomyopathy prolong action potential duration and enhance contractility in isolated cardiomyocytes. J Mol Cell Cardiol 33: 1515–1525 Iwata M, Yoshikawa T, Baba A, Anzai T, Nakamura I, Wainai Y, Takahashi T, Ogawa S (2001) Autoimmunity against the second extracellular loop of beta1-adrenergic receptors induces beta-adrenergic receptor desensitization and myocardial hypertrophy in vivo. Circ Res 88: 578–586 Jahns R, Siegmund C, Jahns V, Reiländer H, Maidhof A, Müller-Esterl W, Lohse MJ, Boege F (1996) Probing human beta1- and beta2-adrenoceptors with domain-specific fusion protein antibodies. Eur J Pharmacol 316: 111–121 Jahns R, Boivin V, Krapf T, Wallukat G, Boege F, Lohse MJ (2000) Modulation of beta1-adrenoceptor activity by domain-specific antibodies and heart failure-associated autoantibodies. J Am Coll Cardiol 36: 1280–1287 Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G, Lohse MJ (2004) Direct evidence for a beta1-adrenergic receptor directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy. J Clin Invest 113: 1419–1429 Engelhardt S, Hein L, Wiesmann F, Lohse MJ (1999) Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA 96: 7059–7064 Woodiwiss AJ, Tsotetsi OJ, Sprott S, Lancaster EJ, Mela T, Chung ES, Meyer TE, Norton GR (2001) Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circulation 103: 155–160 Hershko AY, Naparstek Y (2005) Removal of pathogenic autoantibodies by immunoadsorption. Ann NY Acad Sci 1051: 635–646 Jahns R, Boivin V, Siegmund C, Boege F, Lohse MJ, Inselmann G (1999) Activating beta1-adrenoceptor antibodies are not associated with cardiomyopathies secondary to valvular or hypertensive heart disease. J Am Coll Cardiol 34: 1545–1551 Limas CJ, Goldenberg IF, Limas C (1992) Assessment of immune modulation of betaadrenergic pathways in human dilated cardiomyopathy: Influence of methodologic factors. Am Heart J 123: 967–970 Nikolaev VO, Boivin V, Stoerk S, Angermann CE, Ertl G, Lohse MJ, Jahns R (2007) A novel fluorescent method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure. J Am Coll Cardiol 50: 423–31 Chiale PA, Ferrari I, Mahler E, Vallazza MA, Elizari MV, Rosenbaum MB, Levin MJ (2001) Differential profile and biochemical effects of antiautonomic membrane recep-

169

Roland Jahns et al.

68

69

70 71

170

tor antibodies in ventricular arrhythmias and sinus node dysfunction. Circulation 103: 1765–1771 Störk S, Boivin V, Horf R, Hein L, Lohse MJ, Angermann CE, Jahns R (2006) Stimulating autoantibodies directed against the cardiac beta1-adrenergic receptor predict increased mortality in idiopathic cardiomyopathy. Am Heart J 152: 697–704 Wallukat G, Müller J, Hetzer R (2002) Specific removal of beta1-adrenergic antibodies directed against cardiac proteins from patients with idiopathic dilated cardiomyopathy. N Engl J Med 347: 1806 Anderton SM (2001) Peptide-based immunotherapy of autoimmunity: A path of puzzles, paradoxes, and possibilities. Immunology 104: 367–376 Mobini R, Staudt A, Felix SB, Baumann G, Wallukat G, Deinum J, Hjalmarson Å, Fu M (2003) Hemodynamic improvement and removal of autoantibodies against beta1adrenergic receptor by immunoadsorption therapy in dilated cardiomyopathy. J Autoimmunity 20: 345–350

The role of cytokines in inflammation-induced cardiomyopathy: Pathogenesis and therapeutic implications Jesus G. Vallejo1 and Douglas L. Mann 2 1

Section of Infectious Diseases, Department of Pediatrics and Winters Center for Heart Failure Research, Baylor College of Medicine and Texas Children’s Hospital, 6621 Fannin Street, Houston, TX 77030, USA 2 Section of Cardiology, Texas Heart Institute at St. Luke’s Episcopal Hospital, and Winters Center for Heart Failure Research, Baylor College of Medicine, Houston, TX 77030, USA

Abstract Heart failure of diverse etiology is now recognized to have an important immune component, with proinflammatory cytokines such as TNF influencing the process of cardiac remodeling and prognosis. A complex relationship seems to exist between the adaptive and maladaptive immune response, cytoprotective, growth, and contractile effects of inflammatory mediators. Understanding the mechanisms underlying this innate cytoprotection has the potential to identify new strategies and targets to treat cardiac disease in the future.

Introduction Beginning with the original description of inflammatory cytokines in patients with heart failure in 1990 [1], there has been a growing interest in the role that these molecules play in regulating cardiac structure and function, particularly in pathophysiological conditions that are associated with sustained inflammation, such as viral myocarditis. The wider recognition of the pathophysiological consequences of sustained expression of pro-inflammatory mediators in pre-clinical and clinical heart failure models led to a series of multicenter clinical trials in patients with moderate to advanced heart failure that either utilized “targeted” approaches to neutralize tumor necrosis factor (TNF) or intramuscular injections of autologous blood that had been subjected to oxidative stress ex vivo using a proprietary device (immune modulation therapy; CelacadeTM) [2]. Whereas the targeted approaches to neutralize TNF appear to have resulted in worsening heart failure [3], immune modulation therapy had a neutral effect on the pre-specified primary end-point of the trial. Taken together, these negative clinical trial results had raised a number of important questions about what role, if any, pro-inflammatory cytokines play in the


171

Jesus G. Vallejo and Douglas L. Mann

heart. In this chapter we summarize what is currently known about the role of proinflammatory cytokines in the pathogenesis of viral inflammatory heart disease and virus-induced cardiomyopathy, with a particular focus on the duality of cytokine effects in the heart.

Adaptive and maladaptive effects of pro-inflammatory cytokine signaling in the heart Although the exact role that pro-inflammatory cytokines play in the heart has not been completely defined, it has become abundantly clear that these molecules exert both beneficial and deleterious effects in the heart.

Adaptive effects In terms of the adaptive effects of pro-inflammatory cytokine signaling in the heart, two major themes have emerged thus far. The first is that pro-inflammatory cytokines are not expressed constitutively in healthy myocardial tissue [4, 5]. The second is that these molecules are consistently and rapidly expressed in response to a variety of different forms of myocardial injury. The observation that pro-inflammatory cytokine gene expression is not coupled to a specific form of cardiac injury, but is instead observed in all forms of cardiac injury, suggests that these molecules constitute part of a phylogenetically conserved intrinsic or ‘innate’ stress response system in the heart. Support for a beneficial role for pro-inflammatory cytokines in the heart comes from a series of ‘gain-of-function’ studies that have shown that pro-inflammatory cytokines confer cytoprotective responses in the heart (reviewed in [6]). Although the mechanisms for the cytoprotective effects of cytokines are not known, pro-inflammatory cytokines up-regulate the expression of at least two sets of protective proteins in the heart: the free radical scavenger manganese superoxide dismutase (MnSOD) [7, 8] and the cytoprotective heat shock proteins (HSPs) [9, 10]. Indeed, TNF-induced MnSOD expression is very rapid (< 1 h) and requires very low levels of TNF (0.1 ng/ml) [8]. Given that contracting myocardial cells are continually susceptible to oxygen-derived free radicals, TNF and interleukin-1 (IL1) may play important roles in protecting the heart against oxidative stress. TNF has also been shown recently to up-regulate the expression of heat shock protein 72 (HSP 72) [10], a protein that is thought to protect the heart against ischemia reperfusion injury [11, 12]. In addition, TNF and IL-1B have been shown to activate the transcription factor nuclear factor-kappa B (NF-KB), which has been shown to be cytoprotective under certain circumstances, presumably through up-regulation of one or more cytoprotective genes, including MnSOD, the cellular inhibitors of apoptosis 1 and 2, and members of the Bcl-2 family [13–15].

172

The role of cytokines in inflammation-induced cardiomyopathy: Pathogenesis and therapeutic implications

Similar findings have been obtained in gain-of-function studies for the IL-6 family of cytokines, which includes IL-6, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), IL-11, and oncostatin M (OSM). These cytokines triggers downstream signaling pathways either through the homodimerization of the gp130 receptor or through the heterodimerization of gp130 with a related transmembrane receptor. CT-1 has been shown to blunt serum deprivation-induced apoptosis in isolated neonatal cardiac myocytes through a mitogen-activated protein kinase (MAPK)-dependent pathway. Studies have also shown that LIF confers cytoprotective responses in isolated myocytes, as well as in intact myocardial tissue [16, 17]. The mechanisms for LIF-mediated cytoprotective effects appear to be more complex than those reported for CT-1. That is, whereas studies in isolated neonatal myocytes suggest an important role for the Janus kinase (JAK)- and the signal transducer and activator of transcription (STAT)-mediated signaling pathways [18], studies in adult myocytes suggest that the cytoprotective effects of LIF are mediated through activation of the MAPK pathway. One explanation for these ‘apparent’ differences in the cytoprotective mechanism for LIF is that there may be functionally significant cross-talk between the MAPK and the JAK/STAT pathways. Taken together, the gain-of-function and loss-of-function studies suggest that pro-inflammatory cytokines may play an important role in the orchestration and the timing of the myocardial stress response, both by providing early anti-apoptotic cytoprotective signals that are responsible for delimiting tissue injury, but also by providing delayed signals that facilitate tissue repair and /or tissue remodeling once myocardial tissue damage has supervened. In keeping with this latter point of view, previous studies have shown that CT-1, LIF and TNF are all sufficient to provoke modest hypertrophic growth response in cardiac myocytes [19], and that TNF is sufficient to lead to degradation and remodeling of the extracellular matrix in the heart [19].

Maladaptive effects The interest in understanding the role of inflammatory mediators in a variety of cardiac disease states, including viral myocarditis, arises from the observation that many aspects of cardiac decompensation observed in various cardiac disease states can be explained by the known biological effects of pro-inflammatory cytokines (Tab. 1). Negative inotropic effects of TNF have been observed in studies in which rats were infused with pathophysiologically levels of TNF, as well as in transgenic mice with targeted cardiac overexpression of TNF [20–22]. Experimental studies in rats have shown that circulating concentrations of TNF that overlap those observed in patients with heart failure are sufficient to produce persistent negative inotropic effects that are detectable at the level of the cardiac myocyte. Moreover, the negative inotropic effects of TNF were completely reversible when the TNF infusion was

173


Table 1. Maladaptive effects of inflammatory mediators on left ventricular remodeling Alterations in the biology of the myocyte

-

Myocyte hypertrophy Fetal gene expression Negative inotropic effects Increased oxidative stress

Alterations in the biology of the non-myocytes

- Conversion of fibroblasts to myofibroblasts - Up-regulation of AT1 receptors on fibroblasts - Increased MMP secretion by fibroblasts

Alterations in the extracellular matrix

- Degradation of the matrix - Myocardial fibrosis

Progressive myocyte loss

- Necrosis - Apoptosis

stopped [20]. Franco and colleagues [23] used cine-magnetic resonance imaging to demonstrate that there was a significant increase in left ventricular (LV) volume and a significant decrease in LV ejection fraction over time in transgenic mice with targeted cardiac overexpression of TNF. Importantly, these effects were shown to be dependent upon gene dosage (i.e., the level of myocardial TNF expression). With respect to the potential mechanisms for the deleterious effects of TNF on LV function, it has been suggested that TNF modulates myocardial function through at least two different pathways: an immediate pathway that is manifest within minutes and is mediated by activation of the neutral sphingomyelinase pathway [24], and a delayed pathway that requires hours to days to develop, and is mediated by nitric oxide (NO) [25, 26]. It has also been suggested that TNF and IL-1 may produce negative inotropic effects indirectly through activation and/or release of IL-18 [27]. Specific blockade of IL-18 using IL-18-binding protein leads to an improvement in myocardial contractility in atrial tissue subjected to ischemia reperfusion injury [28]. Myocardial IL-18 has also been shown to be increased by lipopolysaccharide (LPS), and LPS-induced myocardial dysfunction is attenuated by specific neutralization of IL-18 [29]. Interestingly, neutralization of IL-18 had no influence on LPSinduced myocardial TNF production, but TNF-deficient mice had attenuated IL-18 production in response to LPS. These findings suggest that the cardio-depressive role of TNF during endotoxemia may be mediated via induction of IL-18. IL-6 has been shown to decrease cardiac contractility via an NO-dependent pathway that is secondary to IL-6-induced phosphorylation of STAT3 [30]. In this study, IL-6 enhanced de novo synthesis of inducible NO synthase (iNOS) protein, increased NO production, and decreased rat cardiac myocyte contractility after 2 h of incubation. Taken together these observations suggest that IL-6 is sufficient to produce negative inotropic effects through STAT3-mediated activation of iNOS [30].

174


Adaptive and maladaptive effects of inflammatory mediators in viral myocarditis As noted above, activation of pro-inflammatory cytokines in the heart represents a phyologenetically conserved innate stress response. What is less well appreciated is that these innate stress responses evolved in organisms with relatively short lifespans (weeks to months), and was never intended to provide long-term adaptive responses to the host organism. Thus, it is perhaps not surprising that the adaptation of the innate stress response to more complex mammalian systems has, in some settings, resulted in sustained and/or dysregulated pro-inflammatory cytokine signaling, insofar as there was relatively less evolutionary pressure on developing the mechanisms to dampen pro-inflammatory cytokine signaling. The duality of the effects of pro-inflammatory cytokine signaling is perhaps most clearly evident in the setting of viral myocarditis.

Maladaptive effects of pro-inflammatory cytokines in viral myocarditis An important role for pro-inflammatory cytokines in myocyte damage associated with inflammatory heart disease is suggested by the high incidence of cytokine expression, especially TNF, in hearts of patients with myocarditis and dilated cardiomyopathy [31]. Matsumori et al. [32] showed that patients with myocarditis have increased circulating levels of TNF, IL-1, and IL-6. A pathophysiological role for TNF in viral myocarditis has been further suggested as neutralization of TNF attenuates viralinduced damage in a murine model and exogenous TNF exacerbated the disease [33, 34]. Moreover, the cardiomyopathy associated with cardiac restricted overexpression of TNF is partially reversed by TNF antibody treatment. Taken together, these data highlight the potential detrimental effects of TNF in myocarditis.

Adaptive effects of pro-inflammatory cytokines in viral myocarditis In contrast to the tissue destruction that occurs with sustained cytokine signaling, TNF also plays an important role in the host defense against microorganisms. This duality of the effects is adeptly illustrated in an experimental study in TNF–/– mice with targeted deletion of TNF, which is a pro-inflammatory cytokine responsible for activating the immune system [35]. In this study mortality following infection of TNF–/– mice with encephalomyocarditis virus (EMCV) was 100% within 14 days. In contrast, 67% of infected wild-type mice (TNF+/+) were alive at 14 days. Further, exogenous administration of TNF prevented the increase in EMCV-induced mortality. Thus, the absence of TNF signaling likely prevented activation of the immune system, which in turn allowed EMCV to multiply unchecked, thereby increasing

175


the cytopathogenic effects of the virus in the cardiac myocytes, which ultimately led to increased mortality. Viewed together, these observations suggest that TNF plays an important role in the early stages of viral replication and may protect the heart from infection. The development of viral myocarditis is associated with the infiltration of the heart by inflammatory cells that secrete IL-1. Animal model have shown that treatment with recombinant IL-1 enhances coxsackievirus B3 (CVB3)-induced myocarditis in mice known to be partially resistant to viral infection [36]. Furthermore, overexpression of IL-1R antagonist in the mouse heart decreases acute myocardial inflammation induced by CVB3, further supporting the detrimental role of IL-1 in myocarditis [37]. Shioi et al. [38] demonstrated that IL-1 gene expression is elevated in the chronic stages of viral myocarditis relative to other cytokines and that this positively correlated with the extent of fibrosis. Interestingly, the expression of TNF did not correlate with the extent of myocardial fibrosis. Recently, Erikkson et al. [39] have provided the first in vivo evidence that IL-1R triggering is critical for expansion of autoreactive CD4+ T cells and subsequent induction of autoimmune heart disease. Mice lacking the IL-1R were protected from myocarditis and showed impaired priming of myosin-specific CD4+ T cells, which resulted from defective activation of dendritic cells. These findings suggest that IL-1 plays an important role in the autoimmune and remodeling phases of myocarditis. IL-6 has been shown to be anti-inflammatory in several animal models, an effect that is attributed to the inhibition of TNF and IL-1 production [40, 41]. Kanda et al. [42] demonstrated that exogenous IL-6 administered at the time of viral inoculation in mice, improved survival rates, decreased cardiac viral titers, and reduced both myocardial necrosis and lymphocytic infiltration. However, the administration of IL-6 either 4 days before virus inoculation or 4 days after did not influence survival or myocardial injury. Serum TNF levels in animals treated with IL-6 were significantly reduced in the early phase of viral myocarditis compared with control animals. Subsequent studies by this same group revealed that mice with targeted overexpression of myocardial IL-6 exhibited accelerated tissue injury and decreased viral clearance following infection with encephalomyocarditis virus [43]. The accelerated tissue injury was linked to decreased TNF production, as administration of TNF resulted in increased viral clearance and significantly less tissue destruction in the IL-6 transgenic mice. Thus, the short term expression of IL-6 may function as an adaptive response to modulate both inflammatory and immune responses, but sustained IL-6 expression may lead to adverse outcomes if IL-6-induced anti-inflammatory and/or immunosuppressive responses are excessive and prevent activation of the appropriate antiviral response. Studies in mice homozygous for a disrupted NOS2 allele suggest that viral myocarditis is more severe when NOS2 is lacking [44]. Badorff et al. [45] have provided a potential molecular mechanism for a protective role for NO in viral infection. The authors demonstrated that CVB3 possesses a protease (2A) that cleaves the

176


membrane glycoprotein, dystrophin, in human and mouse cardiac membranes. Interestingly, NO inhibits the enteroviral protease 2A via S-nitrosylation of the catalytic cysteine residue. The elaboration of NO by cardiac myocytes may prevent dystrophin cleavage by inactivating viral protease 2A. Thus, the cytokine-induced elaboration of NO may have evolved as an important adaptive mechanism in preventing the development of dilated cardiomyopathy following exposure to CVB3.

Therapeutic implications To date, immunosuppressant therapies used in animal models of viral myocarditis have not been shown to be beneficial in humans. One of the reasons for the lack of efficacy may relate to the duality of the effects of the innate immune system, as described above. Whereas numerous anecdotal and small case series suggested that patients with viral myocarditis might benefit from early steroid or immunosuppressive therapy, the results of the Myocarditis Treatment Trial did not show an improvement in ejection fraction when compared with conventional therapy [46]. While this strategy may effectively suppress the deleterious actions of excessive activation of the adaptive immune system, it may do so at the expense of suppressing the beneficial effects of the innate immune system on viral replication and persistence, which has been implicated in the pathogenesis of dilated cardiomyopathy [47, 48]. The current understanding of the role of inflammation in viral myocarditis suggests that ongoing clinical strategies that employ antiviral strategies (e.g., interferon) and/or immune modifying strategies may be beneficial as long as they leave the effector arm(s) of the innate immune system intact.

Conclusion In the foregoing review, we have summarized experimental and clinical material suggesting that activation of pro-inflammatory cytokines within the heart following viral myocarditis may have beneficial or alternatively detrimental consequences for the host, depending on the duration and degree of cytokine exposure. Although activation of the immune system is initially beneficial by limiting the spread of infection, the immune response may act as a double-edged sword, insofar as excessive immune responses can lead to disease progression independent of the initial viral infection. Thus, the outcome in viral-induced cardiomyopathy represents the outcome of the delicate balance between the beneficial and deleterious effects of pro-inflammatory cytokines in the host (Fig. 1). These recent insights into the pathophysiological role of pro-inflammatory cytokines may have important clinical implications, insofar as they suggest that anti-inflammatory strategies may be deleterious in the setting of viral myocarditis unless properly timed and/or combined with antiviral strategies.

177


Figure 1. Duality of the effects of pro-inflammatory cytokines in viral myocarditis. The outcome in viral-induced cardiomyopathy represents the outcome of the delicate balance between the beneficial effects of pro-inflammatory cytokines, including antiviral effects, cytoprotection and tissue repair, and the deleterious effects of pro-inflammatory cytokines, including myocardial inflammation with myocyte cell death and adverse left ventricular (LV) remodeling.

References 1 2. 3 4

5

6 7

178

Levine B, Kalman J, Mayer L, Fillit HM, Packer M (1990) Elevated circulating levels of tumor-necrosis-factor in severe chronic heart-failure. N Engl J Med 323: 236–41 Tang, WH, Francis GS (2006) The year in heart failure. J Am Coll Cardiol: 48(12): 2575–83 Mann DL (2002) Inflammatory mediators and the failing heart – Past, present, and the foreseeable future. Circ Res 91: 988–98 Kapadia S, Lee J, Torre-Amione G, Birdsall HH, Ma TS, Mann DL (1995) Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest 96: 1042–52 Kapadia SR, Oral H, Lee J, Nakano M, Taffet GE, Mann DL (1997) Hemodynamic regulation of tumor necrosis factor-alpha gene and protein expression in adult feline myocardium. Circ Res 81: 187–95 Mann DL (2003) Stress-activated cytokines and the heart: From adaptation to maladaptation. Annu Rev Physiol 65: 81–101 Eddy LJ, Goeddel DV, Wong GHW (1992) Tumor necrosis factor-alpha pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun 184: 1056–9


8 9 10

11

12

13

14

15

16

17

18

19

20

21

22

Wong GHW, Goeddel DV (1998) Induction of manganous superoxide-dismutase by tumor necrosis factor – possible protective mechanism. Science 242: 941–4 Löw-Friedrich I, Weisensee D, Mitrou P, Schoeppe W (1992) Cytokines induce stress protein formation in cultured cardiac myocytes. Basic Res Cardiol 87: 12–8 Nakano M, Knowlton AA, Yokoyama T, Lesslauer W, Mann DL (1996) Tumor necrosis factor-alpha-induced expression of heat shock protein 72 in adult feline cardiac myocytes. Am J Physiol-Heart Circ Physiol 39: H1231–H1239 Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH (1995) Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95: 1446–56 Plumier JCL, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G et al (1995) Transgenic mice expressing the human heat shock protein-70 have improved postischemic myocardial recovery. J Clin Invest 95: 1854–60 Erl W, Hansson GK, de Martin R, Draude G, Weber KSC, Weber C (1999) Nuclear factor-kappa B regulates induction of apoptosis and inhibitor of apoptosis protein-1 expression in vascular smooth muscle cells. Circ Res 84: 668–77 Lee JP, Palfrey HC, Bindokas VP, Ghadge GD, Ma L, Miller RJ et al (1999) The role of immunophilins in mutant superoxide dismutase-1-linked familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 96: 3251–6 Narula J, Pandey P, Arbustini E, Haider N, Narula N, Kolodgie FD et al (1999) Apoptosis in heart failure: Release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci USA 96: 8144–9 Nelson SK, Wong GHW, Mccord JM (1995) Leukemia inhibitory factor and tumor necrosis factor induce manganese superoxide-dismutase and protect rabbit hearts from reperfusion injury. J Mol Cell Cardiol 27: 223–9 Wang F, Seta Y, Baumgarten G, Engel DJ, Sivasubramanian N, Mann DL (2001) Functional significance of hemodynamic overload-induced expression of leukemia-inhibitory factor in the adult mammalian heart. Circulation 103: 1296–302 Fujio Y, Kunisada K, Hirota H, Yamauchi-Takihara K, Kishimoto T (1997) Signals through gp130 upregulate bcl-x gene expression via STAT1-binding cis-element in cardiac myocytes. J Clin Invest 99: 2898–905 Yokoyama T, Nakano M, Bednarczyk JL, McIntyre BW, Entman M, Mann DL (1997) Tumor necrosis factor-alpha provokes a hypertrophic growth response in adult cardiac myocytes. Circulation 95: 1247–52 Bozkurt B, Kribbs S, Clubb FJ Jr, Michael LH, Didenko VV, Hornsby PJ et al (1998) Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats. Circulation 97: 1382– 91 Bryant D, Becker L, Richardson J, Shelton J, Franco F, Pechock RM et al (1998) Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha. Circulation 97: 1375–81 Kubota T, McTiernan CF, Frye CS, Demetris AJ, Feldman AM (1997) Cardiac-specific

179


23

24

25

26

27 28

29

30

31

32 33

34

35

36

180

overexpression of tumor necrosis factor-alpha causes lethal myocarditis in transgenic mice. J Am Coll Cardiol 29: 29165 Franco F, Thomas GD, Giroir B, Bryant D, Bullock MC, Chwialkowski MC et al (1999) Magnetic resonance imaging and invasive evaluation of development of heart failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha. Circulation 99: 448–54 Oral H, Dorn GW, Mann DL (1997) Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte. J Biol Chem 272: 4836–42 Balligand JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T et al (1993) Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage conditioned medium. J Clin Invest 91: 2314–9 Gulick TS, Chung MK, Pieper SJ, Lange LG, Schreiner GF (1989) Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte beta-adrenergic responsiveness. Proc Natl Acad Sci USA 86: 6753–7 Dinarello CA (1999) Interleukin-18. Methods 19: 121–32 Pomerantz BJ, Reznikov LL, Harken AH, Dinarello CA (2001) Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1 beta. Proc Natl Acad Sci USA 98: 2871–6 Raeburn CD, Dinarello CA, Zimmerman MA, Calkins CM, Pomerantz BJ, McIntyre RC et al (2002) Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction. Am J Physiol Heart Circ Physiol 283: H650–H657 Yu XW, Kennedy RH, Liu SJ (2003) JAK2/STAT3, not ERK1/2, mediates interleukin6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes. J Biol Chem 278: 16304–9 Satoh M, Tamura G, Segawa I, Tashiro A, Hiramori K, Satodate R (1996) Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy. Virchows Arch 427: 503–9 Matsumori A, Yamada T, Suzuki H, Matoba Y, Sasayama S (1994) Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br Heart J 72: 561–6 Kubota T, Bounoutas GS, Miyagishima M, Kadokami T, Sanders VJ, Bruton C et al (2000) Soluble tumor necrosis factor receptor abrogates myocardial inflammation but not hypertrophy in cytokine-induced cardiomyopathy. Circulation 101: 2518–25 Yamada T, Matsumori A, Sasayama S (1994) Therapeutic effect of anti-tumor necrosis factor-alpha antibody on the murine model of viral myocarditis induced by encephalomyocarditis virus. Circulation 89: 846–51 Wada H, Saito K, Kanda T, Kobayashi I, Fujii H, Fujigaki S et al (2001) Tumor necrosis factor-alpha (TNF-alpha) plays a protective role in acute viral myocarditis in mice: A study using mice lacking TNF-alpha. Circulation 103: 743–9 Huber SA, Polgar J, Schultheiss P, Schwimmbeck P (1994) Augmentation of pathogene-


37

38 39

40

41

42 43

44

45

46

47

48

sis of coxsackievirus B3 infections in mice by exogenous administration of interleukin-1 and interleukin-2. J Virol 68: 195–206 Lim BK, Choe SC, Shin JO, Ho SH, Kim JM, Yu SS et al(2002) Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis. Circulation 105: 1278–81 Shioi T, Matsumori A, Sasayama S (1996) Persistent expression of cytokine in the chronic stage of viral myocarditis in mice. Circulation 94: 2930–7 Eriksson U, Kurrer MO, Sonderegger I, Iezzi G, Tafuri A, Hunziker L et al (2003) Activation of dendritic cells through the interleukin 1 receptor 1 is critical for the induction of autoimmune myocarditis. J Exp Med 197: 323–31 DiSanto E, Alonzi T, Poli V, Fattori E, Toniatti C, Sironi M et al (1997) Differential effects of IL-6 on systemic and central production of TNF: A study with IL-6-deficient mice. Cytokine 9: 300–6 Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA (1990) Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells – IL-6 suppresses IL-1 and TNF. Blood 75: 40–7 Kanda T, McManus JE, Nagai R, Imai S, Suzuki T, Yang D et al (1996) Modification of viral myocarditis in mice by interleukin-6. Circ Res 78: 848–56 Tanaka T, Kanda T, McManus BM, Kanai H, Akiyama H, Sekiguchi K et al (2001) Overexpression of interleukin-6 aggravates viral myocarditis: Impaired increase in tumor necrosis factor-alpha. J Mol Cell Cardiol 33: 1627–35 Zaragoza C, Ocampo C, Saura M, Leppo M, Wei XQ, Quick R et al (1998) The role of inducible nitric oxide synthase in the host response to Coxsackievirus myocarditis. Proc Natl Acad Sci USA 95: 2469–74 Badorff C, Fichtlscherer B, Rhoads RE, Zeiher AM, Muelsch A, Dimmeler S et al (2000) Nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2A through S-nitrosylation: A protective mechanism against enteroviral cardiomyopathy. Circulation 102: 2276–81 Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME et al (1995) A clinical trial of immunosuppressive therapy for myocarditis. The Myocarditis Treatment Trial Investigators. N Engl J Med 333: 269–75 Bowles NE, Richardson PJ, Olsen EGJ, Archard LC (1986) Detection of coxsackie-Bvirus specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1: 1120–4 Bowles NE, Bayston TA, Zhang HY, Doyle D, Lane RJ, Cunningham L et al (1993) Persistence of enterovirus RNA in muscle biopsy samples suggests that some cases of chronic fatigue syndrome result from a previous, inflammatory viral myopathy. J Med 24: 145–60

181

Diagnosis of inflammatory and viral cardiomyopathy

Histology and immunohistology of myocarditis Annalisa Angelini, Fiorella Calabrese and Gaetano Thiene Department of Medico-diagnostic Sciences and Special Therapies, Special Pathological Anatomy, University of Padua Medical School, Padua, Italy

Abstract The contemporary definition and classification of cardiomyopathies by the American Heart Association recently listed myocarditis (inflammatory cardiomyopathy) among the primary cardiomyopathies (predominantly involving the heart) as opposed to the secondary forms (mainly involving the heart in the setting of generalized systemic disease). Endomyocardial biopsy (EMB) is still considered the gold standard for the in vivo diagnosis of myocarditis, its sensibility and specificity being increased by the application of immunohistochemistry and morphometric analysis. Obtaining tissue samples from multiple areas of the ventricles greatly increases the sensibility of EMB in detecting myocarditis since the process could be focal, regional or diffuse. Dallas criteria, recognizing inflammatory infiltrates with necrosis and/or degeneration of the adjacent myocytes (not typical of the ischemic damage associated with coronary artery disease) as the key for diagnosis, should be complemented by the introduction of new criteria that should include: clinical presentation, histopathology, immunohistochemistry, morphometry, viral polymerase chain reaction, cardiac antibodies assessment, and imaging findings.

Introduction The contemporary definition and classification of cardiomyopathies by the American Heart Association [1], which updates the previous WHO one from 1996 [2], recently listed myocarditis (inflammatory cardiomyopathy) among the primary cardiomyopathies (predominantly involving the heart) as opposed to the secondary forms (mainly involving the heart in the setting of a more generalized systemic disease). Systemic diseases leading to secondary myocarditis are numerous, and listed among them are Loeffler’s endocarditis, sarcoidosis, endocrine diseases, and autoimmune and collagen diseases. Systemic and cardiac involvement may not behave in parallel: heart involvement can be minimal in a systemic disease extensively involving peripheral organs, or the heart could be severely involved when the other organs are only initially concerned by the process. The heart can be the first or the only organ involved in a systemic disease. Thus, it becomes mandatory to character-


185

Annalisa Angelini, Fiorella Calabrese and Gaetano Thiene

ize the inflammatory cell infiltrates and to exclude systemic involvement of other organs to shed some light on the possible pathogenetic origin of the inflammation. Major etiologies of myocarditis are recognized to be ‘infective’, i.e., viral, bacterial, fungal, parasitic, protozoan, or ‘non infective’, i.e., toxic, hypersensitive or immunological. Based only on histopathological criteria, several distinct types of myocarditis have been identified, which, although not adequate to give an etiological diagnosis, are of importance in a flow chart diagnostic evaluation: lymphocytic, eosinophilic, polymorphous, giant cell, and granulomatous myocarditis. Lymphocytic myocarditis is the most common type of myocarditis in Western countries and most cases are documented or presumed to have viral origin. Over 20 years ago, to develop uniform and reproducible morphological criteria for the pathological diagnosis of myocarditis, a panel of cardiac pathologists developed a classification of the disease based on histological features of endomyocardial biopsy (EMB) specimens, known as Dallas criteria [3]. Unfortunately, since then no up date of these early criteria have been introduced. When myocarditis is suspected from the clinical profile, an EMB may resolve an otherwise ambiguous situation by detecting diagnostic inflammatory infiltrate and necrosis (i.e., the Dallas criteria) but it is limited by sampling error and false-negative histological results. It is recognized that the diagnostic yield of myocardial biopsies can be enhanced substantially by molecular analysis with DNA-RNA extraction and polymerase chain reaction (PCR) to detect viral genome [1]. As recently reported, cardiac magnetic resonance represents a non-invasive imaging of myocarditis capable of evaluating myocardial function, edema, hyperemia, capillary leakage, necrosis, and scar, which, beside inflammatory infiltrates, represent the key features for the diagnosis of myocarditis at histology and immunohistochemistry [4]. Obtaining tissue samples from selected area of the ventricles greatly increases the sensibility of EMB in diagnosing myocarditis since the process can be focal, regional or diffuse, and has been shown to mainly affect the free lateral wall of the left ventricle [4–6]. The frequency of lymphocytic myocarditis detected in EMB of patients with dilated cardiomyopathy is reported to be highly variable, ranging from 3 to 63% [7–11]. This variability could be ascribed to patient selection (children/adults), limited number of samples, timing of the EMB, or unselected area of sampling.

Classifications Historically many types of classifications for myocarditis have been introduced on the basis of causal, morphological, temporal, or clinicopathological criteria. In 1947, Gore and Saphir [12] proposed an etiological classification that recognized an infective, non-infective and isolated form; in 1983, Fenoglio [13] first tried to

186

Histology and immunohistology of myocarditis

make clinicopathological correlations, pointing to an acute, progressive and chronic form on the basis of temporal onset of the disease. More recently, in 1991, another temporal clinicomorphological classification by Lieberman et al. [14] distinguished myocarditis as having fulminant, acute, chronic active and chronic persistent forms.

The Dallas classification: Pros and cons According to the classification, known as the Dallas classification, from the city where the pathologists met, myocarditis is defined as an “inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes, not typical of ischemic damage associated with coronary artery disease” [3]. The Dallas classification suggests a biopsy monitoring in patients with a diagnosis of myocarditis, and adopts two different terminologies for the first and second EMB; the second EMB should be scheduled at least 6 weeks after the first EMB (Tab. 1). The first EMB recognizes an ‘active’ form in the presence of inflammatory cell infiltrates associated with necrosis or degeneration of cardiomyocytes; a ‘borderline’ form when only the inflammatory cells and no myocardial damage can be identified at histology and a negative one in the absence of inflammation. The second or subsequent EMBs compares the morphological findings with the previous one, thus referring to persistent, resolving or healed forms. The adjective persistent or resolving can only be used in those instances in which a diagnosis of myocarditis has been already and unequivocally achieved through a previous EMB. Table 1. Myocarditis: Histopathological criteria from Dallas classification [3] 1st EMB

2nd EMB

Active (inflammatory cells+necrosis/degeneration) Persistent (with or without fibrosis) Borderline (inflammatory cells)

Resolving (with or without fibrosis)

Negative

Healed (with or without fibrosis)

The Dallas criteria present the advantage of using a simple, universally accepted and standardized terminology (Tab. 2). However, some important limitations could be identified: the lack of histotype characterization (lymphocytic, eosinophilic, neutrophilic and giant cell) in view of a possible differentiation between idiopathic forms of myocarditis from a process secondary to a known cause; and the terminology (such as active and borderline and healing with or without fibrosis) currently used in the lymphocytic (more common) form is not applied to the other forms of inflammatory cardiomyopathy. Specifications of the type and extent of myocyte damage as

187


Table 2. Major Pros and cons of Dallas classification criteria. Pros

Cons

1. Simple

1. Lack of histotype characterization

2. Universally accepted

2. Lack of type and quantification of myocyte damage

3. Standardized terminology

3. Lack of type and quantification of fibrosis

4. Application of traditional techniques

4. Lack of type and quantification of inflammatory cell infiltrates at immunohistochemistry 5. Lack of application of new technologies for viral genome detection

well as fibrosis are not included in the classification, even though myocyte changes, including frank necrosis, degeneration, and fibrosis were mentioned in the original report by Aretz [3]. Although it is very difficult to forecast the natural history of the disease and its specific timing, when EMB is performed and a thorough morphological evaluation is carried out, it is relevant to add temporal information of the lesion, and highlight the clinical history to provide indications on the course of the disease for taking adequate therapeutic measures. This approach may avoid the unrestricted use of the term borderline myocarditis, which frequently includes chronic forms, as “inflammatory cardiomyopathy” [15].

Quantification of inflammation and the issue of “normal myocardium” The intensity and distribution of the inflammatory infiltrate are highly variable, ranging from a solitary small focus to multifocal aggregates to diffuse myocardial involvement. On routine hematoxylin and eosin-stained sections it may be difficult to characterize interstitial cells as normal components of the myocardium such as mast cells, fibroblast nuclei in cross section, pericytes, histiocytes, and endothelial cells may resemble lymphocytes [15–17]. Moreover, a small number of inflammatory cells, including lymphocytes, may be found in the normal myocardium. The presence of inflammatory cell infiltrates in the interstitium of “normal” myocardium is still a controversial issue. This is partly due to the difficulties in obtaining myocardium not affected by a disease. The previously reported studies were performed on myocardial tissue from patients with disease other than myocarditis or on donor hearts, and demonstrated that rare lymphocytes can be detected in the interstitium under normal conditions [18–21]. Regarding this point, different

188


range values have been reported in the literature. On examination of 20 high-power (s 400) microscope fields on sections of normal myocardium, Edwards and colleagues [18] found the mean number of interstitial cells to be < 5/high-power fields. Linder et al. [19] and Schnitt et al. [20], using immunohistochemistry for leukocyte common antigen, detected an average of 13 immunoreactive cells/mm2 in “uninflamed” biopsy specimens. Tazelaar and Billingham [21] found foci of inflammatory cells (> 5 inflammatory cells/focus) in 9.3% of 86 cardiac transplant donor hearts. Recently, a cut-off of < 14 leukocytes/mm2 with the presence of < 7 T lymphocytes/ mm2 has been considered the most realistic value [22, 23]. Another important limitation of the Dallas classification is that it lacks any reference to etiology. Many questions remains open and represent a challenge for the scientific community. The clinical diagnosis of myocarditis far exceeds the histopathological confirmation of the presence of myocarditis in EMB. It has been estimated that myocardial biopsies are positive in only 30% of cases of clinically suspected myocarditis [24]. Thus, histological diagnosis represents a fundamental step in the diagnostic process of myocarditis. Although the disease is a diffuse process, it may be microscopically focal, causing a low diagnostic yield by biopsy sampling error [18, 25, 26]. The number of tissue pieces procured as well as the size and the processing of specimens have been demonstrated to highly influence the sensitivity of EMB in the detection of myocarditis. The greater sensitivity of the Stanford-Caves and Cordis bioptomes over other smaller bioptomes has been proven [18, 25]. In particular, the number of biopsy samples also directly increases the likelihood of detecting foci of myocarditis. Chow et al. [27] and Hauck et al. [28] independently reported a sensitivity for detection of myocarditis of approximately 50% using 4–5 biopsy samples. When 17 biopsy specimens per case were taken, the sensitivity reached 79% [27]. It has also been shown that serial sectioning and multiple level examination of EMBs increase the sensitivity of EMB in the evaluation of myocarditis [29, 30], thus facilitating the diagnosis of focal myocarditis with a rate of sensitivity similar to that in diffuse myocarditis [30]. Although some authors have demonstrated that biventricular EMB (obtaining several samples from multiple areas of both the left and right ventricles) may improve the sensitivity in the detection of myocarditis [31], this aggressive approach increases the risk of complications.

Immunohistochemistry: A technical implementation The majority of experts in the field agree that the sensitivity of EMB has now been increased by using immunohistochemistry together with routine histology. As reported, with the application of immunohistochemistry the sensibility of EMB increased from 36% to 80% and specificity decrased from 98% to 85% respectively [4].

189


A large panel of monoclonal and polyclonal antibodies has now been adopted to identify and characterize the inflammatory cell population as well as the activated immunological processes (Tab. 3) [15–17, 32–34]. Table 3. Panel of antibodies for the characterization of inflammatory cells. Antibody

Phenotype

Anti-CD45

Leukocytes

Anti-CD20

B lymphocytes

Anti-CD3

T lymphocytes

Anti-CD4

T helper

Anti-CD8

T cytotoxic

Anti-CD45RO

Activated T lymphocytes

Anti-CD68

Macrophages

Anti-CD31/von Willebrand F

Endothelial

Anti-HLA-ABC

Immunological activation

Anti-HLA-DR

Immunological activation

Since myocarditis is at least in part an immunological disorder, immunological markers of inflammation (which are distributed and are up-regulated throughout the entire myocardium) should always be applied. In a recent paper by Wojnicz et al. [35] on 202 patients with dilated cardiomyopathy, 84 (41%) had HLA-positive EMB; of these 84 patients, on the basis of Dallas criteria, only 7 were diagnosed with active myocarditis, 16 with borderline myocarditis and 61 with no myocarditis. Our previous experience showed that, for patients who did not fulfill the Dallas criteria, immunohistochemistry detected inflammatory infiltrates, characterized by T lymphocytes and macrophages, in the myocardium, thus excluding the possibility of acute ischemia, which is characterized by a neutrophil infiltrate (Fig. 1). Semiquantitation or quantitation with morphometric computerized technique could add more useful clinical and prognostic significance to the histopathological diagnosis, giving information on the active or silent phase of the disease, recurrence, and evolution, and on a decrease in edema, extracellular space and inflammation as well as the possibility of function recovery. The presence of an inflammatory infiltrate on immunohistochemical analysis together with a positive PCR for myocardial virus genome increases the diagnostic accuracy of EMB in those cases in which the application of only the Dallas criteria would otherwise have failed to detect and characterize the inflammatory disease [17]. Correlations between histopathology, immunohistochemistry and molecular

190


a

b

c Figure 1. Borderline myocarditis in a 34-year-old female with congestive heart failure of 6-month duration. (a) Note the scarce inflammatory cell infiltrate. Hematoxylin eosin stain. Original magnification s 20; insert s 40. (b) Note the interstitial fibrosis. Masson tricrom stain. Original magnification s 2. (c) Immunohistochemistry for leukocytes (CD45) and activated lymphocytes (CD45RO). No sign of necrosis.

191


analysis have highlighted that in some viral genome-positive cases the inflammatory cell infiltrate is not extensive or would be categorized as mild. However, as already mentioned, the presence of viral genome is more indicative of a worse outcome, and yields a higher prognostic significance than the inflammatory cell infiltrate. The timing of the biopsy influences its sensitivity, both for the presence of inflammation and the chance to detect viral genome. The early phase of the disease, at the time of acute symptoms onset, is characterized by viral invasion, which can produce local myocardial damage with inflammatory response and recruitment of cytolytic T lymphocytes and cytokines production. Friedrich et al. [36], using contrast media-enhanced magnetic resonance, demonstrated that acute myocarditis evolves from a focal to a disseminated process during the first 2 weeks after the onset of symptoms, thus potentially increasing EMB sensitivity performed in the earlier phase of the disease.

“Borderline myocarditis” Our experience to better characterize the clinical and pathological profile of active and borderline myocarditis showed that the timing of EMB is of crucial importance for a correct management of the disease. The clinical profile of the two forms is different (Tab. 4). Patients (pts) with borderline myocarditis are slightly older and on average undergo EMB later than pts with active myocarditis (15). One out of five pts with borderline myocarditis have a family history of dilated cardiomyopathy and also could represent a peculiar familial form of dilated cardiomyopathy, which has been reported to share inflammatory cell infiltrate in the interstitium, a feature considered a non-specific finding by some authors [37]. Pts with borderline myocarditis more often present with congestive heart failure if compared to active myocarditis pts, which have a more heterogeneous clinical presentation, including cardiogenic shock. Table 4. Active vs borderline myocarditis: Clinical data [15]

No. Pts Age NYHA at onset 2

LVEDV (ml/m )

Active

Borderline

p

26 (62%)

16 (38%)

n.s.

37 ± 15

40 ± 13

n.s.

2.4 (1.3)

2.2 ± 1.2

n.s.

90 ± 42

128 ± 50

0.002

LVEF (%)

34 (12)

34 (15)

n.s.

Follow-up death/CT

4 (15%)

2 (12.5%)

n.s.

Follow-up NYHA

1.3 ± 0.6

1.1 ± 0.4

n.s.

192


One third of the pts with borderline myocarditis (BM) had a left bundle branch block at ECG, versus 8% of pts with active myocarditis. The finding was in keeping with the echocardiographic data which showed that borderline myocarditis had more dilated left ventricles with a decreased mass/volume index. In these pts, the pathological substrate showed a less severe inflammatory cell infiltrate with myocytes often presenting the cytological features of dilated cardiomyopathy (Tab. 5). Borderline myocarditis seems to include forms with a chronic evolution, in which inflammation is still ongoing but the pattern of idiopathic dilated cardiomyopathy is already present (Fig. 1) .The time interval between onset of symptoms and EMB correlated with the histological diagnosis and the likelihood of diagnosing a borderline myocarditis increases at the increase of the time interval. These data are strengthened by the observation that EMB is performed earlier and the diagnosis is most often active myocarditis (81% in cases mimicking acute myocardial infarction and 100% in cardiogenic shock) when the clinical presentation is acute (Fig. 2). In spite of the clinical differences, follow-up of active myocarditis and borderline myocarditis did not differ in terms of NYHA class and outcome (death/heart transplantation). However the inadequacy of the Dallas classification needs to be considered. Dallas criteria retain too many ambiguities and are not of real value in identifying pts with a worse outcome. Table 5. Active vs borderline myocarditis: Pathological data [15].

No. of patients Mean days from onset to EMB

Active

Borderline

p

26 (62%)

16 (38%)

n.s.

40 ± 55

90 ± 93

0.04

Inflammatory infiltrate on HE (score 0–3)

1.65 ± 0.8

0.85 ± 0.3

0.004

CD43

11 (38%)

6 (37.5%)

n.s.

CD43 and CD68

7 (27%)

6 (37.5%)

n.s.

CD68

7 (27%)

3 (18.5%)

n.s.

CD45

1 (4%)

1 (6.3%)

n.s.

Myocarditis is classified as active only on the basis of presence of necrosis and inflammation. Other morphological aspects of myocytes and/or interstitium highly suggestive for an evolution towards a chronic form have not been taken into account. Fibrosis should be considered in more detail and type of fibrosis, whether loose or more compact, Collage type I/III, extension and localization (endocardial, interstitial reactive or replacement type), should be assessed and quantified. At presence histological diagnosis, namely active myocarditis vs borderline myocarditis, does not influence the prognosis, while the presence of viral genome seems to carry a more powerful negative prognostic value. In active forms viral genomes can be detected more frequently in the myocardium, although not significantly.

193


Figure 2. Active myocarditis in a 25-year-old male with recent onset of arrhythmias after flu-like syndromes. (a) Hematoxylin eosin stain. Original magnification s 10. (b) Immunohistochemistry for activated lymphocytes (CD45RO). Note the myocyte necrosis.

Diagnosis of viral myocarditis Morphological analysis still today has great limits in the detection of infective pathogens, particularly viral agents, the commonest cause of inflammatory cardiomyopathy. Viral myocardites usually lack specific cytopathic effects, especially those sustained by RNA viruses. With the exception of some rare forms of cytomegaloviral myocarditis, these effects, when observed, neither necessarily imply the presence of viruses nor are useful in detecting the type of virus, since they may represent degenerative changes or myocyte nuclear polyploidia. The development of molecular biological techniques, particularly amplification methods like Polymerase Chain Reaction (PCR) or Nested-PCR, allows the detection of low copy viral genomes even from an extremely small amount of tissue such as EMBs. Numerous studies of patients with myocarditis have demonstrated the usefulness of PCR analysis for etiologic diagnosis [38–50].

Conclusions Practical experience showed that residual inter observer variability and overall low sensitivity represent serious limitations of Dallas criteria [51]. These limitations

194


could have contributed to the failure of the myocarditis treatment trial, in which the diagnosis of myocarditis was entirely based on histology [52–54]. As clinical, angiographic and ECG criteria do not provide a definitive diagnosis of myocarditis, EMB is crucial for definitive diagnosis and may influence the clinical management in these patients. The diagnosis of myocarditis can avoid the chronic inappropriate use of drugs for the management of peculiar forms of ischemic heart disease like coronary artery vasospasm or coronary artery disease undetected at coronary angiography. The risk of complications for EMB procedure is very low as indicated by the results of our center, where no fatal complications and 0.06% non fatal complications in more than 1000 biopsies performed had occurred. Quantitative criteria for inflammatory infiltrate, immunohistochemical and molecular biology techniques are now deemed compulsory and represent all together the diagnostic gold standard. A new morphological classification should be proposed to overcome the limits of the Dallas criteria providing a histopathological diagnosis useful to the clinician for more appropriate patient’s risk stratification and overall for the application of new therapeutic trials. Time has come to redefine viral and autoimmune myocarditis with the use of methodologies available in the 21st century. Clinician, pathologists, immunologist and molecular cardiologists must contribute to the new criteria which should include: clinical presentation, histopathology, immunohistochemistry, viral search by polymerase chain reaction, cardiac antibodies assessment, and imaging results. [55, 56].

Acknowledgements This study was supported by the MURST Target Projects (1999–2000, Myocarditis: therapeutic impact of etiological diagnosis based upon molecular and immunological findings; 2003–2005, Myocarditis: identification of clinical, molecular, and immunological markers for risk stratification) and the Ministry of Health Target project (2004–2007, Inflammatory cardiomyopathy), Rome, Italy. We would like to thank Alessandra Cervellin for her assistance in the preparation of this manuscript, Marco Pizzigolotto for his skillful assistance in the acquisition and preparation of photographic material, and Elisabetta Baliello, Alessandra Dubrovich, Giovanna Mattiazzo and Elisa Carturan for their skillful technical assistance in the preparation of histological, immunohistochemical and molecular material.

References 1

Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ,

195


2

3 4 5

6

7

8 9

10 11

12 13 14 15

196

Seidman CE, Young JB (2006) Contemporary definition and classification of the cardiomyopathies: An American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 113(14): 1807–16 Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I, Martin I, Nordet P (1996) Report of the 1995 WHO/ ISFC task force on the definition of cardiomyopathies. Circulation 93: 841–842 Aretz HT (1987) Myocarditis: The Dallas criteria. Hum Pathol 18: 619–624 Skouri HN, Dec GW, Friedrich MG, Cooper LT (2006) Noninvasive imaging in myocarditis. J Am Coll Cardiol 48: 2085–93 Mahrholdt H, Wagner A, Deluigi CC, Kispert E, Hager S, Meinhardt G, Vogelsberg H, Fritz P, Dippon J, Bock CT et al (2006) Presentation, patterns of myocardial damage, and clinical course of viral myocarditis. Circulation 114: 1581–90 Mahrholdt H, Goedecke C, Wagner A, Meinhardt G, Athanasiadis A, Vogelsberg H, Fritz P, Klingel K, Kandolf R, Sechtem U (2004) Cardiovascular magnetic resonance assessment of human myocarditis: A comparison to histology and molecular pathology. Circulation 109: 1250–8 Muller S, Muller P, Meyer R, Volkmann H, Gottschild D, Thoss K, Kalhofer G, Wessel G (1985) Myocardial bioptic findings in correlation with TL-201 imaging and hemodynamic parameters in patients with latent cardiomyopathies suspected of having myocarditis. Heart Vessels Suppl 1: 111–5 Salvi A, Di Lenarda A, Dreas L, Silvestri F, Camerini F (1989) Immunosuppressive treatment in myocarditis. Int J Cardiol 22: 329–38 Hammond EH, Menlove RL, Anderson JL (1987) Predictive value of immunofluorescence and electron microscopic evaluation of endomyocardial biopsies in the diagnosis and prognosis of myocarditis and idiopathic dilated cardiomyopathy. Am Heart J 114: 1055–65 Bolte HD, Ludwig B, Schultheiss HP (1984) Viral myocarditis: Symptomatology, clinical diagnosis and hemodynamics. Internist (Berl) 25(3): 143–9 Magnani JW, Danik HJ, Dec GW Jr, DiSalvo TG (2006) Survival in biopsy-proven mocarditis: A long term retrospective analysis of the histopathologic, clinical, and hemodynamic predictors. Am Heart J 151: 463–470 Gore I, Saphir O (1947) Myocarditis: A classification of 1402 cases. Am Heart J 34: 827–831 Fenoglio JJ Jr, Ursell PC, Kellogg CF, Drusin RE, Weiss MB (1983) Diagnosis and classification of myocarditis by endomyocardial biopsy. N Engl J Med 301: 12–18 Lieberman EB, Hutchins GM, Herskowitz A, Rose NR, Baughman KL (1991) Clinicopathologic description of myocarditis. J Am Coll Cardiol 18: 1617–26 Angelini A, Crosato M, Boffa GM, Calabrese F, Calzolari V, Chioin R, Daliento L,


16

17

18

19

20

21 22

23 24 25

26 27

28 29

30

Thiene G (2002) Active versus borderline myocarditis: Clinicopathological correlates and prognostic implications. Heart 87: 210–215 Hauck AJ, Kearney DL, Edwards WD (1989) Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: Implications for role of sampling error. Mayo Clin Proc 64: 1235–1245 Angelini A, Calzolari V, Calabrese F, Boffa GM, Maddalena F, Chioin R, Thiene G (2000) Myocarditis mimicking acute myocardial infarction: Role of endomyocardial biopsy in the differential diagnosis. Heart 84: 245–50 Edwards WD, Holmes DR, Reeders GS (1982) Diagnosis of active lymphocytic myocarditis by endomyocardial biopsy: Quantitative criteria for light microscopy. Mayo Clin Proc 57: 419–425 Linder J, Cassling RS, Rogler WC, Wilson JE, Markin RS, Sears TD, McManus BM (1985) Immunohistochemical characterization of lymphocytes in uninflamed ventricular myocardium. Implications for myocarditis. Arch Pathol Lab Med 109: 917–921 Schnitt SJ, Ciano PS, Schoen FJ (1987) Quantitation of lymphocytes in endomyocardial biopsies: Use and limitations of antibodies to leukocyte common antigen. Hum Pathol 18: 796–801 Tazelaar HD, Billingham ME (1986) Myocardial lymphocytes: Fact, fancy or myocarditis? Am J Cardiovasc Pathol 1: 47–49 Kuhl U, Noutsias M, Seeber B, Schultheiss HP (1996) Immunohistological evidence for a chronic intramyocardial inflammatory process in dilated cardiomyopathy. Heart 75: 295–300 Maisch B, Herzum M, Hufnagel G, Schonian U (1996) Immunosuppressive and immunomodulatory treatment for myocarditis. Curr Opin Cardiol 11: 310–324 Billingham ME (1989) Acute myocarditis: Is sampling error a contraindication for diagnostic biopsies? J Am Coll Cardiol 14: 921–922 Edwards WD Holmes DR, Reeders GS (1982) Diagnosis of active lymphocytic myocarditis by endomyocardial biopsy: Quantitative criteria for light microscopy. Mayo Clin Proc 57: 419–425 Edwards WD (1984) Myocarditis and endomyocardial biopsy. Cardiol Clin 2: 647– 656 Chow LH, Radio SJ, Sears TD, McManus BM (1989) Insensitivity of right ventricular endomyocardial biopsy in the diagnosis of myocarditis. J Am Coll Cardiol 14: 915– 920 Hauck AJ, Edwards ED (1992) Histopathologic examination of tissues obtained by endomyocardial biopsy. In: Fowles RE (ed): Cardiac biopsy. Mount Kisco, Futura, 95 Thiene G, Bartoloni G, Poletti A, Boffa GM (1996) Tecniche, indicazioni ed utilità della biopsia endomiocardica. In: Baroldi G, Thiene G (eds): Biopsia endomiocardica, Piccin, Padova, 5–45 Burke AP, Farb A, Robinowitz M, Virmani R (1991) Serial sectioning and multiple level examination of endomyocardial biopsies for the diagnosis of myocarditis. Mod Pathol 4: 690–693

197


31

32

33

34 35

36

37

38

39

40

41

42

43

198

Frustaci A, Bellocci F, Olsen EG (1994) Results of biventricular endomyocardial biopsy in survivors of cardiac arrest with apparently normal hearts. Am J Cardiol 74: 890– 895 Herskowitz A, Ahmed-Ansari A, Neumann DA, Beschorner WE, Rose NR, Soule LM, Burek CL, Sell KW, Baughman KL (1990) Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: A nonhistologic marker of myocarditis. J Am Coll Cardiol 15: 624–632 Kuhl U Seeberg B, Schultheiss HP, Strauer BE (1994) Immunohistological characterization of infiltrating lymphocytes in biopsies of patients with clinically suspected dilated cardiomyopathy. Eur Heart J 15 Suppl. C: 62–67 Kuhl U, Noutsias M, Schultheiss HP (1995) Immunohistochemistry in dilated cardiomyopathy. Eur Heart J 16: SO: 100–106 Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewski P, Niklewski T, Zembala M, Polonski L, Rozek MM, Wodniecki J (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: Two-year follow-up results. Circulation 104: 39–45 Friedrich MG , Strohm O, Schulz-Menger J, Marciniak H, Luft FC, Dietz R (1998) Contrast media-enhanced magnetic resonance imaging visualizes myocardial changes in the course of viral myocarditis. Circulation 97: 1802–9 Mestroni L, Rocco C, Gregori D, Sinagra G, Di Lenarda A, Miocic S, Vatta M, Pinamonti B, Muntoni F, Caforio AL et al (1999) Familial dilated cardiomyopathy: Evidence for genetic and phenotypic heterogeneity. J Am Coll Cardiol 34: 181–90 Jin O, Sole MJ, Butany JW, Chia WK, McLaughlin PR, Liu P, Liew CC (1990) Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction. Circulation 82: 8–16 Chapman NM, Tracy S, Gauntt CJ, Fortmueller U (1990) Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J Clin Microbiol 28: 843–50 Weiss LM, Liu XF, Chang KL, Billingham ME (1992) Detection of enteroviral RNA in idiopathic dilated cardiomyopathy and other human cardiac tissues. J Clin Invest 90: 156–9 Grasso M, Arbustini E, Silini E, Diegoli M, Percivalle E, Ratti G, Bramerio M, Gavazzi A, Vigano M, Milanesi G (1992) Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction. Am J Cardiol 69: 658–64 Petitjean J, Kopecka H, Freymuth F, Langlard JM, Scanu P, Galateau F, Bouhour JB, Ferriere M, Charbonneau P, Komajda M (1993) Detection of enterovirus in endomyocardial biopsy by molecular approach. J Med Virol 41: 260 Hilton DA, Variend S, Pringle JH (1993) Demonstration of Coxsackie virus RNA in formalin-fixed tissue sections from childhood myocarditis cases by in situ hybridization and the polymerase chain reaction. J Pathol 170: 45–51


44

45

46

47

48

49 50

51 52

53 54 55 56

Martin AB, Webber S, Fricker FJ, Jaffe R, Demmler G, Kearney D, Zhang YH, Bodurtha J, Gelb B, Ni J, Bricker T, Towbin JA (1994) Acute myocarditis. Rapid diagnosis by PCR in children. Circulation 90: 330–339 Pauschinger M, Bowles NE, Fuentes-Garcia FJ, Pham V, Kuhl U, Schwimmbeck PL, Schultheiss HP, Towbin JA (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99: 1348–54 Calabrese F, Valente M, Thiene G, Angelini A, Testolin L, Biasiolo MA, Soteriou B, Livi U, Palù G (1999) Enteroviral genome in native hearts may influence outcome of patients who undergo cardiac transplantation. Diagn Mol Pathol 8: 39–46 Chimenti C, Calabrese F, Thiene G, Pieroni M, Maseri A, Frustaci A (2001) Inflammatory left ventricular microaneurysms as a cause of apparently idiopathic ventricular tachyarrhythmias. Circulation 104: 168–173 Calabrese F, Rigo E, Milanesi O, Boffa GM, Angelini A, Valente M, Thiene G (2002) Molecular diagnosis of myocarditis and dilated cardiomyopathy in children. Clinicopathologic features and prognostic implications. Diagn Mol Pathol 11: 212–221 Calabrese F, Thiene G (2003) Myocarditis and inflammatory cardiomyopathy: Microbiological and molecular biological aspects. Cardiovasc Res 60: 11–25 Kuhl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 22; 111: 887–93 McKenna WJ, Davies MJ (1995) Immunosuppression for myocarditis. N Engl J Med 333: 312–313 Mason JW, O’connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE and the myocarditis treatment trial investigators (1995) A clinical trial of immunosuppressive therapy for myocarditis. N Engl J Med 333: 269–275 Tazelaar HD, Billingham ME (1987) Myocardial lymphocytes. Fact, fancy, or myocarditis? Am J Cardiovasc Pathol 147–50 Davies MJ, Ward DE (1992) How can myocarditis be diagnosed and should it be treated? Br Heart J 68: 346–347 Baughman K (2006) Diagnosis of myocarditis: Death of Dallas criteria. Circulation 113: 593–5 Magnani JD, Dec GW (2006) Myocarditis: Current trends in diagnosis and treatment. Circulation 113: 876–890

199

Immunohistological diagnosis of inflammatory cardiomyopathy and diagnosis of cardiotropic viral infections Michel Noutsias, Heinz-Peter Schultheiss and Uwe Kühl Department of Cardiology and Pneumonology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Germany

Abstract Inflammatory cardiomyopathy (DCMi) is a specific cardiomyopathy entity of dilated cardiomyopathy (DCM), being defined by the proof of intramyocardial inflammation and/or viral infection in endomyocardial biopsies (EMBs). The immunohistological evaluation of EMBs, comprising the quantification of infiltrates and of cell adhesion molecule expression, has proven substantially more sensitive and specific compared with the histological EMB investigations according to the Dallas criteria. The molecular biological diagnosis of viral genomes comprises PCR for the qualitative evaluation, quantitative PCR for the determination of viral loads, and sequencing for the analysis of viral genotypes. By these techniques, DCMi has been detected in ca. 60% of the patients clinically presenting with either acute myocarditis (AMC) or DCM. DCMi assessed by immunohistology and viral persistence constitutes a prognostic factor for adverse outcome in AMC and DCM patients. Finally, this contemporary diagnostic repertoire is essential for the selection of DCMi patients who will likely benefit from immunosuppression or antiviral interferon treatment.

Definition of inflammatory cardiomyopathy Inflammatory cardiomyopathy (DCMi) was introduced as a specific cardiomyopathy entity in the 1995 report of the WHO/ISFC Task Force on the Definition and Classification of Cardiomyopathies. It is defined by “myocarditis” in association with cardiac dysfunction. Myocardial inflammation can be by established histological, immunological, and immunohistochemical criteria. Idiopathic, autoimmune, and infectious forms of DCMi are recognized [1]. One major goal of such a classification is the establishment of specific therapeutic approaches targeting particular pathogenic pathways of the disease. Research over the last decade has not only revealed novel insights into the pathogenesis of DCMi, but also stimulated a substantial progress of diagnostic techniques for the etiopathogenetic differentiation of DCMi. The analysis of the hallmarks of DCMi, namely intramyocardial inflammation and viral infection/persistence, has revealed the prognostic impact for the adverse outcome of patients presenting with acute


201

Michel Noutsias, Heinz-Peter Schultheiss and Uwe Kühl

myocarditis (AMC) or dilated cardiomyopathy (DCM). Several immunomodulatory treatment strategies aiming at either attenuation of chronic inflammation in DCMi patients without viral persistence, or targeting viral elimination by antiviral approaches, have demonstrated for the first time beneficial effects in selected DCMi patients based on endomyocardial biopsy (EMB) findings with respect to left ventricular (LV) function parameters and heart failure symptoms. This review focuses on contemporary diagnostic procedures of EMBs for DCMi, as well as on technical details, and highlights major insights derived from these procedures, both regarding the etiopathogenetic differentiation of DCM and the decisive importance of these EMB analyses for the choice of immunomodulatory treatment strategies (immunosuppression or antiviral treatment) by the cumulating evidence from recent trials.

Epidemiology and clinical prognostic parameters of AMC and DCM The incidence of DCM has been reported as 29 per 106 persons/year, with a prevalence of 131 cases per 106 persons/year [2]. At present, detailed epidemiological data specifically on DCMi are lacking; however, epidemiological data can be estimated based on the fact that DCMi (inflammation and/or viral persistence) can be proven in ca. 60% of the DCM patients. Albeit these analyses may be affected by referral bias [3], the reported survival rates for DCM patients (5-year survival rates up to 36%) indicate that a substantial proportion of DCM patients have a grave prognosis even under conventional heart failure medication [4]. DCM is a leading indication for cardiac transplantation [5]. DCM affects males more frequently with a sex ratio of 3:1, and manifests predominantly between the third and fifth decade [6]. In a homogeneous population of young military service men, the incidence of AMC was reported 0.17 per 1000 man-years [7]. However, the real numbers are expected to be substantially higher due to the often subclinical presentation of AMC and misinterpretation of unspecific symptoms. Prospective studies have revealed a 10-year survival rate of ca. 45% in AMC patients, thus being similar to the longterm prognosis of AMC [8–11]. Both the initial clinical presentation of AMC and its prognosis, ranging from spontaneous improvement and mild arrhythmias to rapidly progressive heart failure and sudden cardiac death, are highly variable. Evolution of AMC to DCM has been documented in ca. 21% of the cases within a mean follow-up of 33 months [12]. NYHA class III or IV, and lack of B-blocker medication are decisive clinical parameters for adverse prognosis in AMC patients, while left ventricular ejection fraction (LVEF) or cardiac diameters such as the LV end diastolic diameter (LVEDD), are not prognostically relevant [10]. However, patients presenting with fulminant myocarditis, thus severely depressed LVEF, NYHA IV and indication for maximum heart failure treatment including catecholamines and ultimately assist devices, are

202

Immunohistological diagnosis of inflammatory cardiomyopathy and diagnosis of cardiotropic viral infections

characterized by a significantly better prognosis compared to non-fulminant lymphocytic myocarditis [9]. Cardiac enzyme release, unlike the situation of acute coronary syndrome, has no prognostic impact in AMC patients [13]. All major pathogenic hallmarks of DCMi, namely intramyocardial inflammation, viral persistence and presence of anti-cardiac autoantibodies have been associated with adverse prognosis [10, 11, 14–16]. These observations indicate the remarkable plasticity of AMC and DCM, and indicate that the dynamic course of this disease does not depend only on general of remodeling pathways, which likely respond to heart failure medication, but is affected substantially by superimposed, potentially reversible mechanisms not directly related to heart failure mechanisms, i.e. virus-host interactions, as can be deduced from animal experiments [17].

Pathogenic mechanisms of AMC and DCMi The most detailed knowledge on virus-induced myocardial damage has been unraveled with respect to enteroviruses (EV), especially coxsackievirus B (CVB) (Fig. 1)

Figure 1. Pathogenesis of virus-induced DCMi. Reproduced with permission from [62].

203


[17, 18]. After entering the host primarily via the gastrointestinal or respiratory tract, the virus can enter the heart and induce an antiviral immune response. De novo induction of the coxsackie-adenovirus receptor (CAR) on the cardiomyocyte sarcolemma in DCM hearts might be a key molecular determinant for myocardial coinfections by physiologically unrelated, CAR-dependent viruses (coxsackievirus and adenovirus) [19, 20]. EV can reside in the reticuloendothelial system (especially in B lymphocytes and macrophages). From this extracardiac reservoir, CVB may enter the heart secondarily [21]. Intense, but timely limited myocardial inflammation during the acute stage of myocarditis aims at viral elimination, and thereby at spontaneous resolution of the disease [22]. However, the following major mechanisms, which are not mutually exclusive, can contribute to the transition from AMC to DCMi: a) Direct cytopathic effects of the virus (i.e., cleavage of dystrophin by the enteroviral protease A [23]). Such direct cytopathic effects are also relevant in low-level infections without virus progeny [24]). b) Virus-induced anti-cardiac immune response: Teleologically, the immune response aims at viral elimination and is not primarily detrimental to the heart [17]. Hypothetically, an initial vigorous anti-viral immune response succumbs after complete viral elimination and is therefore beneficial for the course of the disease. Myocarditis/DCM patients with higher circulating cardiac-specific IgG have better survival than those with low levels [25]. The significantly better long-term prognosis of patients presenting with fulminant myocarditis may be attributable to the vigorous immune response leading to effective viral elimination, but also severe depression of LV function in the initial stage [9, 26]. On the other hand, an overwhelming immune response can evolve irrespective of the course of viral infection, and may result in anti-cardiac immunity, i.e., by molecular mimicry, leading to progressive myocytolysis and remodeling [18, 28–30]. In addition to the cellular immunity, antibodies primed against viral antigens can turn cross-reactive against various myocardial proteins, and can thus impair cardiac contractility [31–33]. Cytokines exert cardiodepressive effects, and furthermore promote remodeling by inducing an imbalance of metalloproteinases and their tissue inhibitors [27–29]. Moreover, cytokines induce cell adhesion molecules (CAMs), which mediate the migration of infiltrates [30]. Myocytolysis exerted by cytotoxic T lymphocytes (CTLs) is also mediated by CAMs-ligands interactions [31]. So far, the above-mentioned pathogenic mechanisms have been elucidated with respect to coxsackievirus. Among the plethora of viruses associated with myocarditis and DCMi, recent studies have shown that the most prevalent viruses are parvovirus B19 (B19V), human herpes virus type 6 (HHV6), EV (especially coxsackievirus), adenovirus (ADV), and Epstein-Barr virus (EBV) (Fig. 2) [17, 21]. The detailed viral entry and pathogenic mechanisms for the newly identified cardiotropic viruses are less well known than for EV. B19V capsid expression in cardiomyocytes

204


Figure 2. Spectrum of cardiotropic viruses in patients presenting with DCM. Reproduced with permission from [88].

has been observed in EMBs from DCMi patients, consistent with findings in EVinduced DCMi [32, 33]. However, the association of B19V and HHV6 with coronary endothelial dysfunction is interpreted as vasculotropism of these viruses, giving rise to differential entry mechanisms of the newly identified viruses [34–36]. Both latent and replicative infection modes have been reported for EV [15, 37], and active EV replication has been associated with a substantially worse prognosis compared with EV latency [15]. Recent investigations confirmed the adverse prognostic impact of viral persistence, known previously only for EV (i.e., coxsackievirus) [14, 15], also for the other newly identified cardiotropic viruses [38]. These insights infer that viral persistence is detrimental for the natural course of DCMi, irrespective of the infectious agent, and argue for viral persistence being a common target for antiviral therapeutic approaches in chronic DCMi.

205


Non-invasive diagnostic tools for DCMi Serological assays for the detection of anti-CVB humoral immune response have reportedly a low specificity for the myocardial CVB infection [39]. Detailed analysis of the anti-B19V humoral response patterns targeting epitope-specific antibodies may be helpful in differentiating the B19V infection stages (recent, chronic and persistent/reactivating), but proof of B19V DNA in EMBs is mandatory in such analyses as well [40–42]. Systemic Troponin release can be a marker of myocyte necrosis in AMC and is associated with the immunohistological DCMi detection in EMBs [43]. Nonetheless, sensitivity is low, since only a fraction of AMC patients have elevated Troponin levels [44]. Serological myocardial necrosis markers are helpful in the detection of cardiotropic viruses. This also holds true for contrast agent-enhanced cardiac MRI (CMR). However, recent advances have demonstrated that detection and localization of intramyocardial inflammation and necrosis can be non-invasively assessed by CMR, and these findings are associated with the immunohistological proof of DCMi, but not with the proof of cardiotropic viruses [45, 46]. In AMC patients, the combined application of CMR and EMB yields a considerable diagnostic synergy by overcoming some limitations of CMR and EMB as individually applied techniques [47].

Diagnostic EMB procedures EMBs are usually obtained from the right ventricular septum or from the LV. Periprocedural complications of EMB obtainment are fairly rare in experienced centers, and especially major complications (i.e., pericardial tamponade) were reported to be < 0.4% in large series studies [48–50].

Histological assessment of myocarditis By convention, the Dallas criteria differentiate “active” (interstitial infiltrates with myocytolysis with/without fibrosis) from borderline myocarditis (increased infiltrates with/without fibrosis; Fig. 3) [51]. Myocarditis (including both active and borderline forms) can only rarely be revealed by histological assessment (ca. 5–10%). Noticeably, the diagnostic criterion of histologically detected myocarditis has no prognostic and no therapeutic impact [8, 10]. Histological assessment of myocarditis is hampered by the substantial sampling error and interobserver variability [52–54]. Even Billingham, from the Dallas panel of expert pathologists, stated that “these criteria have been misrepresented as a classification that is used as a sine qua non of the histological diagnosis of acute myocarditis” [55]. These diagnostic obstacles, as well as the negligence of viral

206


Figure 3. Active myocarditis (histology). Focal lymphomononuclear infiltrate with adjacent myocytolysis (original magnification s 200).

persistence, likely contributed to the failure of immunosuppression in the Myocarditis Treatment Trial [25, 56–58]. Further characteristics of cardiomyopathies (i.e., hypertrophy, loss of myofibrils) are also detectable by histological assessment; however, they are not pathognomonic for myocarditis or DCMi. Notwithstanding these pitfalls, histological assessment of EMBs is still mandatory for the diagnosis of “active myocarditis”, and also for the differentiation from further pathologies, (i.e., giant-cell myocarditis, storage diseases) [59–63].

Immunohistological diagnosis of DCMi Immunohistological evaluation, as opposed to the histological diagnostic approaches, enables an exact quantification of infiltrates, a differentiation from non-inflam-

207


matory types of interstitial cells (e.g., fibroblasts, pericytes), a detailed phenotypic characterization of infiltrates, and the assessment of endothelial CAM abundance, which is not accessible to histological evaluation. Kindermann et al. demonstrated the independent prognostic impact of the immunohistological detection of intramyocardial inflammation in AMC patients based on increased infiltrates (> 14 leukocytes/mm2) and enhanced HLA DR expression, as opposed to the known lack of prognostic relevance of the histological Dallas criteria [8, 10]. Immunohistological detection of DCMi is associated with cardiac MRI findings, and is a relevant criterion for the selection of DCMi patients who will likely benefit from immunosuppressive treatment [46, 57, 64–66]. EMBs with immunohistologically confirmed DCMi demonstrate significantly higher gene expressions of a series of immunocompetent molecules by preamplified real-time RT-PCR [67]. These data highlight the evidence of the immunohistological diagnosis of DCMi, and constitute strong arguments for the corresponding EMB analyses. The synopsis in Table 1 summarizes the major differences between the histological and immunohistological evaluation of EMBs for myocarditis/DCMi.

Evaluation of infiltrates in EMBs With the advance of immunohistology and the broadening panel of available diagnostic antibodies, a variety of infiltrate phenotypes has been investigated in EMBs for the diagnosis of DCMi, including markers for pan leukocyte (CD45/LCA, CD18), T lymphocytes (CD3+), activated phenotypes of T lymphocytes (i.e., CD11a/LFA-1+, CD58/LFA-3+, CDw49d/VLA-4+, CD69+), memory T cells (CD45R0+), CTLs (i.e., Perforin+, TIA-1+), macrophages (i.e., CD68, CD11b/Mac-1, 27E10+) and NK cells (CD57+) [16, 30, 31, 68–70]. Exact quantification of infiltrates is pertinent for the diagnosis of DCMi, since semiquantitative assessment is not sensitive enough to reveal the distinct but statistically significant differences compared with controls [30, 70–72]. In control hearts (donor hearts, autopsies from non-cardiac deaths and non-DCM cardiomyopathies), the mean infiltration density of CD3+ T lymphocytes is reportedly < 5.5 T lymphocytes/mm2 [30, 68, 72–76]. Hence, there is a considerable statistical distance in the diagnostic criteria of DCMi cited the 1995 WHO/ISFC report (> 2.0 CD3+/ CD2+ lymphocytes/high power field z 7.0 T lymphocytes/mm2) [70] to differentiate DCMi from normal and from non-DCMi diseased (i.e., alcoholic and ischemic cardiomyopathy) hearts [75, 76]. Of note, the diagnostic thresholds must consider the corresponding phenotypes of infiltrates [30, 31, 72]. For instance, substantially higher thresholds must be considered for broadly expressed phenotypes such as CD18 (pan leukocyte marker) than for more restricted phenotypes such as early activated CD69+ lymphocytes. These considerations equally apply to macrophages, with substantially lower thresholds of specific phenotypes such as early activated

208


Table 1. Comparison of the histological and immunohistological evaluation of myocarditis/ inflammatory cardiomyopathy (DCMi). Histological evaluation of myocarditis (Dallas Criteria)

Immunohistological evaluation of DCMi

Interobserver variability

High [25, 53]

Not precisely known, but expected to be substantially lower, especially using DIA [30, 74, 78]

Sampling error

High [54]

Not precisely known, but expected to be substantially lower [78, 79, 81]

Variability of detection of inflammation

High [25, 95]

Lower [30, 78, 96]

Specific identification and quantification of infiltrates

Impossible *

Feasible [30, 70, 75, 97, 98]

Phenotypic characterization of infiltrates

Impossible *

Broad phenotypic characterization feasible (i.e. T lymphocytes, CTLs, macrophages, specific activation markers) [16, 30, 31, 69, 70, 75, 97, 98].

Evaluation of CAMs expression

Impossible *

Feasible [30, 31, 74, 75, 78, 79, 96, 98, 99]

Prognostic relevance

No prognostic relevance [8, 10].

Patients with immunohistologically proven DCMi have a worse prognosis [10, 16]

Clinical relevance

No discrimination of DCM patients who benefit from immunosuppression [25].

Patients with immunohistologically proven DCMi benefit from immunosuppressive treatment [57, 64, 66, 78]

*Histological assessment cannot specifically stain infiltrates or endothelial CAM abundance, nor does it enable phenotypic characterization of infiltrates. Adapted from [100] with permission of expert Reviews Ltd.

27E10+ macrophages compared with pan-macrophage markers such as CD68+, which can be present in controls in up to 55 cells/mm2 [70, 75]. Multivariate analysis of the diverse phenotypes revealed significant correlations between CD3+ T lymphocytes with specifically activated (CD69+) and adhesion ligands bearing (B2-leukocyte integrins+, LFA-3+, VLA-4+) lymphocytic infiltrates,

209


as well as macrophages [68, 69, 76]. In addition, CTLs (Perforin+, TIA-1+) are associated significantly with activated (LFA-1+, VLA-4+) lymphocytes and NK cells (CD57+), but not with CD3+ naïve T lymphocytes and CD11b macrophages [31]. This observation exemplifies that an arbitrary addition of diverse phenotypes to a non-differentiated binary diagnosis “increased” or “not-increased” infiltrates, irrespective of the respective phenotypes, is prone to blend such potentially valuable information as CTLs, which are of special interest since they exert apoptosis of cardiomyocytes by cytotoxic attack. Typically, infiltrates in DCMi depict a homogeneous infiltration pattern (Fig. 4a); however, focal infiltrates consistent with histologically detected “AMC” can also be observed in ca. 10% of the EMBs of patients presenting with DCM (Fig. 4b) [30, 31]. Detailed data of the possible prognostic impact of such immunohistologically detected focally clustered infiltrates are still lacking. Focally clustered infiltrates should be noted particularly in the immunohistological evaluation of EMBs, since they indicate myocytolysis [31].

Assessment of CAMs expression in EMBs Endothelial cell adhesion molecules (CAMs) are induced by proinflammatory cytokines and mediate transendothelial migration of counterreceptor-bearing leukocytes [77]. CAM (immunoglobulin superfamily, selectins, B1- and B2-integrins) induction was observed in ca. 60% of the EMBs for DCM. The typically homogeneous expression pattern of HLA class I, HLA DR, intercellular cell adhesion molecule-1 (ICAM-1) and CD29 (Fig. 4c) might possibly reduce the sampling error [30, 78]. The close association between the expression level of CAMs and the respective counterreceptor-positive infiltrates infers that the abundance of endothelial CAMs (increased immunoreactivity, at best of several CAMs, compared with baseline expression levels observed in controls) constitutes a reliable diagnostic criterion for DCMi even in the absence of significant T lymphocytic infiltration, which might be due to the sampling error of infiltrates [30, 72, 75, 78–80]. Different evaluation scores result from this differential expression pattern of CAM representatives. In contrast to the CAM representatives with baseline expression levels under normal conditions (i.e., HLA, CD54/ICAM-1, CD29), CD106/ vascular cell adhesion molecule-1 (VCAM-1) and CD62E/E-selectin are absent in healthy hearts. Thus, induction of VCAM-1 and E-selectin indicates endothelial activation when any immunoreactivity can be confirmed (Fig. 4d), whereas enhanced expression indicates endothelial CAM abundance with respect to the former CAMs compared with controls [30, 80]. The abundance of several CAMs was confirmed by several independent investigators [78, 80, 81]. Quantitative evaluation of the expression of CAMs by digital image analysis (DIA) might be likely a suitable tool to overcome the discrepancies originating from the subjective, semiquantitative assessment of CAM

210


a

b

c

d

e Figure 4. Immunohistological aspects of DCMi. (a) Diffuse infiltration pattern of LFA-1+ lymphocytes in DCMi (original magnification s 400). Reproduced with permission from [30]. (b) Focal infiltration pattern of Perforin+ CTLs in DCMi, encircling and entering (white arrows) a cross-sectioned cardiomyocyte, suggesting myocytolysis (original magnification s 630). Reproduced with permission from [31]. (c) Homogeneous ICAM-1 abundance in DCMi (original magnification s 200). Reproduced with permission from [31]. (d) Endothelial VCAM-1 induction in DCMi (original magnification s 1000). Reproduced with permission from [30]. (e) HLA class I induction on the sarcolemma and the intercalated discs (arrows) in an AMC patient with pronounced infiltration (original magnification s 200). Reproduced with permission from [101].

211


immunoreactivity [74]. An elegant approach to quantify CAM abundance is to relate the immunoreactivity of CAMs to the density of endothelia present in the EMBs (both quantified by DIA) [75]. In addition to endothelia and interstitial cells, HLA molecules, ICAM-1 and B1-integrins (CD29) can be also induced on the cardiomyocyte sarcolemma (Fig. 4e) [78, 81], which again deserves special reference in the diagnosis of DCMi. The most convincing argument for CAM evaluation in the diagnosis of DCMi is that HLA sarcolemmal expression has been confirmed as an eligible criterion for the selection of DCM patients who could benefit from immunosuppressive treatment, even neglecting infiltrate quantification [78, 82]. This again is consistent with the essential role of CAMs in mediating the transendothelial migration especially of counterreceptor-positive leukocytes [30].

Technical details of the immunohistological proof of DCMi in EMBs EMB samples are embedded in OCT compound after being obtained and snap frozen in liquid nitrogen. EMB samples can be stored at –80°C until processed. Cryosections are typically performed as 5-Mm-thick serial sections and placed on glass slides, which should be coated (i.e., with poly-l-lysine) to prevent detachment of the sections during the staining procedure. The thickness of the serial sections should be standardized since the resulting cell layers increase with growing section thickness, and can thus affect quantification of both infiltrate density and immunoreactivity levels, i.e., of CAMs. In the strict sense, the obtained infiltration density, as well as the CAMs immunoreactivity, are not only related to the section surface area of the EMB samples, but also on the thickness of the section. The fixed (i.e., in cold acetone) and air-dried slides can be directly subjected to the immunostaining procedure or stored until processed at –20°C. Before incubation with antibodies, unspecific binding sites within the tissue should be saturated by an unspecific serum (e.g., heat-inactivated fetal calf serum). In addition, endogenous activity of the enzyme used for immunohistochemistry should be quenched (in the case of horseradish peroxidase by preincubation with 10% H2O2). When using the avidin-biotin-complex (ABC) method, endogenous unspecific avidin- and biotin-biding sites should be saturated by preincubation with avidin and biotin. Basically, immunostaining is carried out by a primary antibody recognizing the antigen of interest (e.g., mouse anti-human CD3), which after incubation with the tissues is rinsed and is labeled by a secondary antibody raised against the immunoglobulins of the species in which the diagnostic antibody was produced (e.g., rabbit anti-mouse). Alternatively, diagnostic antibodies that are directly conjugated with immunohistochemical enzymes can be used. Specific antibody binding is finally detected by a chemical reaction at the chosen enzyme conjugated to the antibody (e.g., by 3-amino-9-ethylcarbazole as a chromogenic substrate of horseradish peroxidase). Special techniques, such as the ABC method, and the EnVision® secondary

212


antibodies, are suitable for enhancing immunohistochemical stainings substantially by increasing the number of enzymes linked to the diagnostic antibody [83]. After completion of the immunostaining, the slides are finally counterstained (e.g., with hematoxylin) and cover slipped with aqueous or non-aqueous mounting media for permanent preservation until evaluation. Immunohistology should be preferentially performed in frozen sections from EMB samples, in which lymphocytic antigens are better preserved than in paraffinembedded EMB material [84]. On the other hand, this detail infers that infiltration densities derived from immunohistology on frozen sections are higher compared with paraffin-embedded EMBs, and are thus not directly comparable. In addition, not all diagnostic antibodies work equally well in paraformaldehyde/paraffin-fixed (denaturated proteins) and snap-frozen material (native proteins). Nonetheless, it should be noted that the immunohistological proof of DCMi was undertaken in paraffin-embedded samples in some landmark publications, based on CD3+, CD68+ macrophages and HLA-DR expression [10, 47, 57, 66]. The obvious advantage of paraffin-embedded EMB material is the consistently higher quality of tissue morphology, as opposed to snap-frozen samples, which are prone to substantial artifacts due to the freezing and thawing processes in ca. 10–20% of the cases [85]. The procedures of the histological and immunohistological analyses of EMBs are summarized in Figure 5.

Molecular biological detection of viral genomes in EMBs The relevance of an infectious etiology in DCM was speculated as early as 1967 [86]. Due to low sensitivity and specificity, classical diagnostic tools like serology or direct virus isolation from the myocardium have a negligible diagnostic value. PCR amplification of viral genomes has confirmed viral etiology in a major proportion of AMC and DCM patients, and has furthermore linked viral persistence to adverse prognosis in DCM [10, 14, 15, 36, 38, 44, 45, 57, 87–91]. In line with insights arising from EV persistence [14, 15], a recent study revealed that viral persistence contributes to progressive cardiac dysfunction with respect to virtually all prevalent viruses, while viral elimination in the natural course of the disease is associated with improvement of LVEF in chronic DCMi patients [38]. A further interesting issue is the mode of viral infection. Viruses may either reside by latent infection, or actively replicate within the host tissue. Both infection modes have been elucidated with respect to EV infection by strand-specific PCR, with minus-strand RNA indicating active viral replication [15, 37]. Notably, especially DCMi patients with active EV replication have a substantially worse prognosis compared to DCMi patients with EV latency [15]. Up to now, published data on the replication mode of cardiotropic viruses other than EV are still lacking.

213


Figure 5. Histological and immunohistological evaluation of myocarditis/DCMi in EMBs.

The viral loads can be quantified by real-time or quantitative PCR (qPCR). The B19V viral loads published so far in cardiac tissues range substantially between 10 and 115.091 viral copies/Mg nucleic acids [92]. In AMC with B19V viremia, a substantial decrease of B19V viral loads was observed in serial investigations during the natural course of the disease, in parallel to the improvement of LVEF, the decline of immunohistologically, DIA-quantified intramyocardial inflammation, and the emergence of B19V antigen-specific T cells and B19V-NS1 antibodies [41]. Detailed determination of B19V genotypes in EMBs from 151 patients revealed genotype 1 in 28.5%, while genotype 2 was present in 71.5% of these patients. Genotype 1 was associated with significantly reduced LVEF compared to genotype 2 cases (24.4 ± 10.4% versus 31.0 ± 9.5%, p = 0.0001) [93]. These insights infer that, in addition to the mere detection of viral genomes as binary information, the quantification of viral loads and determination of the geno-

214


type may be decisively important for the profound understanding of the dynamics of cardiotropic viral infections in the natural course of the disease, as well as under immunomodulatory treatment. Immunohistologically detected viral protein expression shows significant associations with the PCR results in EV and B19V infections in myocardial tissues [32, 33, 94]. Although sensitivity and specificity of immunohistological viral protein expression cannot meet the diagnostic performance of PCR, this additional technique may be helpful for the characterization of viral protein-expressing cells.

Technical details of detection of viral genomes by nested PCR in EMBs Qualitative analysis of viral genomes is based on amplification of nucleic acids extracted from EMB specimens using PCR technology. Samples should be snap frozen in liquid nitrogen immediately after obtainment to avoid degradation, especially of RNA. The enormous synthesizing power of PCR requires many precautions during the treatment of samples from isolation of nucleic acids to final result by gel electrophoresis. A major pitfall is the carry-over contamination of a newly generated PCR product in a negative sample. The risk of contamination by PCR products in water, plastic ware, pipettes and all surrounding areas of the laboratory is considerable. Therefore, the application of PCR in a diagnostic laboratory, especially of nested PCR (nPCR), also requries a strict spatial separation of all processing steps in particular departments to avoid contamination: 1. Pre-PCR department: storage of EMB specimens, isolation of nucleic acids, preparation of stock solutions for nPCR and RT reaction. 2. PCR department: setup of nPCR reaction and/or real time PCR by ready-to-use stock solutions. Notably, the first PCR and the nPCR should be performed in different departments. 3. Post-PCR department: gel electrophoresis and sequencing of PCR products. To exclude contamination and false-negative results, negative and positive controls must be investigated simultaneously with EMB samples. For the exclusion of falsepositive results, reactions lacking template (water blanks, no-template controls) are used to exclude the presence of contamination. A permanent co-amplification of positive controls is necessary to monitor the efficacy of the amplification process in each assay and finally to confirm efficient nucleic acid extraction and absence of enzyme inhibitors in the template preparation. As positive controls, definitely positive samples, or at best external standards (e.g., cloned viral sequences) can be used. Serial dilutions of positive standards should be co-amplified during any PCR assay. At minimum, one low-copy and one high-copy standard should be simultaneously amplified for a post-hoc check of amplification efficiency. In nPCR protocols, one set of controls are used in the first PCR and one additional set for

215


the nPCR, and controls from the first PCR should be co-amplified over both PCR rounds. The PCR analysis of viral genomes basically comprises four major steps (Fig. 6): 1. Extraction of nucleic acids (RNA/DNA) from the EMB samples 2. Reverse transcription of RNA to cDNA for the detection of RNA viruses (e.g., EV, hepatitis C virus) 3. Amplification of viral genomes by PCR and nPCR 4. Further analyses of the PCR products (electrophoresis for the confirmation of correct amplification, sequencing, real-time PCR to determine the viral load). The sensitivity of the qualitative detection of viral genomes by PCR amplification can be enhanced by nPCR. In the first PCR, a longer gene fragment is amplified. These generated PCR products are the templates for a second round of nPCR amplification. A second primer pair starts the process on the copy numbers of the first PCR and reaches the exponential phase of amplification significantly earlier, which

Figure 6. Flow-chart of major steps for the molecular biological proof of viral genomes in EMBs.

216


generates a shorter PCR product with higher specificity and sensitivity than by the first PCR. Qualitative analysis of the presence of viral genomes or gene expression is predominately done by gel electrophoresis containing a dye (e.g., ethidium bromide). Discrimination of specific products from non-template-specific side products is performed by simultaneous separation of a molecular weight marker and positive controls. In addition to the mere qualitative detection of viral genomes by nPCR, real-time qPCR is a suitable tool for the determination of the viral load [34, 41, 88]. Quantification of viral copies per microgram myocardial nucleic acids is achieved by relating of the threshold cycle (Ct) values obtained from EMBs to a standard curve from defined B19V copy numbers (i.e., B19V plasmid DNA at different dilutions). Direct sequencing of positive PCR results allows the identification of the virus genotype and mutations [93]. Moreover, dealing with such ubiquitous viruses, sequence analysis should be performed from the perspective of quality control to rule out contaminations, which may occur especially during nPCR methods. In summary, the clinical value of EMB diagnostics based on the histological Dallas criteria had undergone decisive drawbacks after the negative results of the immunosuppressive US Myocarditis Treatment Trial. The limited therapeutic and prognostic value of the histological evaluation of myocarditis in EMBs is now widely acknowledged. Cumulative evidence over the last decade has shown that contemporary diagnostic procedures of EMBs do not only enable a detailed etiopathogenetic differentiation of DCMi, but moreover are an indispensable prerequisite for the selection of DCMi patients who might benefit from immunomodulatory treatment in addition to conventional heart failure medication. These comprise the immunohistological assessment of DCMi (quantification and phenotypic characterization of infiltrates and CAMs expression, evaluation of sarcolemmal HLA class I induction) and PCR investigations for viral genomes (qualitative evaluation by nPCR, quantitative assessment by qPCR and sequencing for genotype analysis). This arsenal of diagnostic tools has endorsed a new era of EMB-based diagnosis of DCMi. Standardization and uniformity of these promising new diagnostic approaches is a crucial prerequisite for future randomized multi-center clinical trials.

References 1

2

Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I et al (1996) Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 93: 841–2 Andersson B, Caidahl K, Waagstein F (1995) Idiopathic dilated cardiomyopathy among Swedish patients with congestive heart failure. Eur Heart J 16: 53–60

217


3

4

5

6 7 8

9

10

11

12

13

14

15

16

218

Redfield MM, Gersh BJ, Bailey KR, Ballard DJ, Rodeheffer RJ (1993) Natural history of idiopathic dilated cardiomyopathy: Effect of referral bias and secular trend. J Am Coll Cardiol 22: 1921–6 Sugrue DD, Rodeheffer RJ, Codd MB, Ballard DJ, Fuster V, Gersh BJ (1992) The clinical course of idiopathic dilated cardiomyopathy. A population-based study. Ann Intern Med 117: 117–23 Taylor DO, Stehlik J, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, Kucheryavaya AY, Rahmel AO, Hertz MI (2009) Registry of the international society for heart and lung transplantation: Twenty-sixth official adult heart transplant report-2009. J Heart Lung Transplant 28: 1007–22 Olbrich HG (2001) [Epidemiology-etiology of dilated cardiomyopathy]. Z Kardiol 90 Suppl 1: 2–9 Karjalainen J, Heikkila J (1999) Incidence of three presentations of acute myocarditis in young men in military service. A 20-year experience. Eur Heart J 20: 1120–5 Grogan M, Redfield MM, Bailey KR, Reeder GS, Gersh BJ, Edwards WD, Rodeheffer RJ (1995) Long-term outcome of patients with biopsy-proved myocarditis: Comparison with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 26: 80–4 McCarthy RE 3rd, Boehmer JP, Hruban RH, Hutchins GM, Kasper EK, Hare JM, Baughman KL (2000) Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocarditis. N Engl J Med 342: 690–5 Kindermann I, Kindermann M, Kandolf R, Klingel K, Bultmann B, Muller T, Lindinger A, Bohm M (2008) Predictors of outcome in patients with suspected myocarditis. Circulation 118: 639–48 Caforio AL, Calabrese F, Angelini A, Tona F, Vinci A, Bottaro S, Ramondo A, Carturan E, Iliceto S, Thiene G et al (2007) A prospective study of biopsy-proven myocarditis: Prognostic relevance of clinical and aetiopathogenetic features at diagnosis. Eur Heart J 28: 1326–33 D’Ambrosio A, Patti G, Manzoli A, Sinagra G, Di Lenarda A, Silvestri F, Di Sciascio G (2001) The fate of acute myocarditis between spontaneous improvement and evolution to dilated cardiomyopathy: A review. Heart 85: 499–504 Ammann P, Naegeli B, Schuiki E, Straumann E, Frielingsdorf J, Rickli H, Bertel O (2003) Long-term outcome of acute myocarditis is independent of cardiac enzyme release. Int J Cardiol 89: 217–22 Why HJ, Meany BT, Richardson PJ, Olsen EG, Bowles NE, Cunningham L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89: 2582–9 Fujioka S, Kitaura Y, Ukimura A, Deguchi H, Kawamura K, Isomura T, Suma H, Shimizu A (2000) Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 36: 1920–6 Kanzaki Y, Terasaki F, Okabe M, Hayashi T, Toko H, Shimomura H, Fujioka S, Kitaura Y, Kawamura K, Horii Y et al (2001) Myocardial inflammatory cell infiltrates in cases of


17 18

19

20

21

22 23

24

25

26 27 28 29

30

dilated cardiomyopathy as a determinant of outcome following partial left ventriculectomy. Jpn Circ J 65: 797–802 Liu PP, Mason JW (2001) Advances in the understanding of myocarditis. Circulation 104: 1076–82 Poller W, Fechner H, Noutsias M, Tschoepe C, Schultheiss HP (2002) Highly variable expression of virus receptors in the human cardiovascular system Implications for cardiotropic viral infections and gene therapy. Z Kardiol 91: 978–91 Noutsias M, Fechner H, de Jonge H, Wang X, Dekkers D, Houtsmuller AB, Pauschinger M, Bergelson J, Warraich R, Yacoub M et al (2001) Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: Implications for cardiotropic viral infections. Circulation 104: 275–80 Shi Y, Chen C, Lisewski U, Wrackmeyer U, Radke M, Westermann D, Sauter M, Tschope C, Poller W, Klingel K et al (2009) Cardiac deletion of the coxsackievirusadenovirus receptor abolishes coxsackievirus b3 infection and prevents myocarditis in vivo. J Am Coll Cardiol 53: 1219–26 Klingel K, Stephan S, Sauter M, Zell R, McManus BM, Bultmann B, Kandolf R (1996) Pathogenesis of murine enterovirus myocarditis: Virus dissemination and immune cell targets. J Virol 70: 8888–95 Deonarain R, Cerullo D, Fuse K, Liu PP, Fish EN (2004) Protective role for interferonbeta in coxsackievirus B3 infection. Circulation 110: 3540–3 Badorff C, Lee GH, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, Knowlton KU (1999) Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med 5: 320–6 Wessely R, Henke A, Zell R, Kandolf R, Knowlton KU (1998) Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: An approach to the study of enteroviral persistence in cardiac myocytes. Circulation 98: 450–7 Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE (1995) A clinical trial of immunosuppressive therapy for myocarditis. The Myocarditis Treatment Trial Investigators. N Engl J Med 333: 269–75 Poller W, Fechner H, Noutsias M, Tschöpe C, Pauschinger M, Schultheiss HP (2002) The molecular basis of cardiotropic viral infections. Eur Heart J 4: I18–I30 Matsumori A (1996) Cytokines in myocarditis and cardiomyopathies. Curr Opin Cardiol 11: 302–9 Hunter JJ, Chien KR (1999) Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 341: 1276–83 Li J, Schwimmbeck PL, Tschöpe C, Leschka S, Husmann L, Rutschow S, Reichenbach F, Noutsias M, Kobalz U, Poller W et al (2002) Collagen degradation in a murine myocarditis model: Relevance of matrix metalloproteinase in association with inflammatory induction. Cardiovasc Res 56: 235–47 Noutsias M, Seeberg B, Schultheiss HP, Kühl U (1999) Expression of cell adhesion mol-

219


31

32

33

34

35

36 37

38

39

40

41

42

43

220

ecules in dilated cardiomyopathy: Evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99: 2124–31 Noutsias M, Pauschinger M, Schultheiss HP, Kühl U (2003) Cytotoxic perforin+ and TIA-1+ infiltrates are associated with cell adhesion molecule expression in dilated cardiomyopathy. Eur J Heart Fail 5: 469–79 Escher F, Kühl U, Sabi T, Suckau L, Lassner D, Poller W, Schultheiss HP, Noutsias M (2008) Immunohistological detection of parvovirus B19 capsid proteins in endomyocardial biopsies from dilated cardiomyopathy patients. Med Sci Monit 14: CR333–338 Li Y, Bourlet T, Andreoletti L, Mosnier JF, Peng T, Yang Y, Archard LC, Pozzetto B, Zhang H (2000) Enteroviral capsid protein VP1 is present in myocardial tissues from some patients with myocarditis or dilated cardiomyopathy. Circulation 101: 231–4 Tschöpe C, Bock CT, Kasner M, Noutsias M, Westermann D, Schwimmbeck PL, Pauschinger M, Poller WC, Kühl U, Kandolf R et al (2005) High prevalence of cardiac parvovirus B19 infection in patients with isolated left ventricular diastolic dysfunction. Circulation 111: 879–86 Vallbracht KB, Schwimmbeck PL, Kühl U, Rauch U, Seeberg B, Schultheiss HP (2005) Differential aspects of endothelial function of the coronary microcirculation considering myocardial virus persistence, endothelial activation, and myocardial leukocyte infiltrates. Circulation 111: 1784–91 Bultmann BD, Klingel K, Sotlar K, Bock CT, Kandolf R (2003) Parvovirus B19: A pathogen responsible for more than hematologic disorders. Virchows Arch 442: 8–17 Pauschinger M, Doerner A, Kühl U, Schwimmbeck PL, Poller W, Kandolf R, Schultheiss HP (1999) Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99: 889–95 Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112: 1965–70 Strauer BE, Kandolf R, Mall G, Maisch B, Mertens T, Figulla HR, Schwartzkopff B, Brehm M, Schultheiss HP (2001) [ Update 2001. Myocarditis – cardiomyopathy]. Med Klin (Munich) 96: 608–25 Escher F, Modrow S, Sabi T, Kuhl U, Lassner D, Schultheiss HP, Noutsias M (2008) Parvovirus B19 profiles in patients presenting with acute myocarditis and chronic dilated cardiomyopathy. Med Sci Monit 14: CR589–597 Streitz M, Noutsias M, Volkmer R, Rohde M, Brestrich G, Block A, Klippert K, Kotsch K, Ay B, Hummel M et al (2008) NS1 specific CD8+ T-cells with effector function and TRBV11 dominance in a patient with parvovirus B19 associated inflammatory cardiomyopathy. PLoS ONE 3: e2361 Lindner J, Noutsias M, Lassner D, Wenzel J, Schultheiss HP, Kuehl U, Modrow S (2009) Adaptive immune responses against parvovirus B19 in patients with myocardial disease. J Clin Virol 44: 27–32 Lauer B, Niederau C, Kuhl U, Schannwell M, Pauschinger M, Strauer BE, Schultheiss


44

45

46

47

48 49

50

51 52

53

54

55

HP (1997) Cardiac troponin T in patients with clinically suspected myocarditis. J Am Coll Cardiol 30: 1354–9 Kühl U, Pauschinger M, Bock T, Klingel K, Schwimmbeck CP, Seeberg B, Krautwurm L, Noutsias M, Poller W, Schultheiss HP et al (2003) Parvovirus B19 infection mimicking acute myocardial infarction. Circulation 108: 945–50 Mahrholdt H, Goedecke C, Wagner A, Meinhardt G, Athanasiadis A, Vogelsberg H, Fritz P, Klingel K, Kandolf R, Sechtem U (2004) Cardiovascular magnetic resonance assessment of human myocarditis: A comparison to histology and molecular pathology. Circulation 109: 1250–8 Gutberlet M, Spors B, Thoma T, Bertram H, Denecke T, Felix R, Noutsias M, Schultheiss HP, Kühl U (2008) Suspected chronic myocarditis at cardiac MR: Diagnostic accuracy and association with immunohistologically detected inflammation and viral persistence. Radiology 246: 401–9 Baccouche H, Mahrholdt H, Meinhardt G, Merher R, Voehringer M, Hill S, Klingel K, Kandolf R, Sechtem U, Yilmaz A (2009) Diagnostic synergy of non-invasive cardiovascular magnetic resonance and invasive endomyocardial biopsy in troponin-positive patients without coronary artery disease. Eur Heart J 30: 2869–79 Frustaci A, Pieroni M, Chimenti C (2002) The role of endomyocardial biopsy in the diagnosis of cardiomyopathies. Ital Heart J 3: 348–53 Maisch B, Bültman B, Factor S, Invited-Consultants, McKenna WJ, Richardson PJ, Thiene G, Schultheiss HP, Sekiguchi M, Bayes de Luna A et al (1999) World Heart Federation consensus conference’s definition of inflammatory cardiomyopathy (myocarditis): Report from two expert committees on histology and viral cardiomyopathy. Heartbeat 4: 3–4 Holzmann M, Nicko A, Kühl U, Noutsias M, Poller W, Hoffmann W, Morguet A, Witzenbichler B, Tschope C, Schultheiss HP et al (2008) Complication rate of right ventricular endomyocardial biopsy via the femoral approach: A retrospective and prospective study analyzing 3048 diagnostic procedures over an 11-year period. Circulation 118: 1722–8 Aretz HT (1987) Myocarditis: The Dallas criteria. Hum Pathol 18: 619–24 Strauer BE, Kandolf R, Mall G, Maisch B, Mertens T, Schwartzkopff B, Schultheiss HP (1996) Myocarditis – cardiomyopathy. Consensus Report of the German Association for Internal Medicine, presented at the 100th annual meeting, Wiesbaden, 13 April 1994. Acta Cardiol 51: 347–71 Shanes JG, Ghali J, Billingham ME, Ferrans VJ, Fenoglio JJ, Edwards WD, Tsai CC, Saffitz JE, Isner J, Furner S (1987) Interobserver variability in the pathologic interpretation of endomyocardial biopsy results. Circulation 75: 401–5 Hauck AJ, Kearney DL, Edwards WD (1989) Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: Implications for role of sampling error. Mayo Clin Proc 64: 1235–45 Billingham ME (1989) Myocarditis and endomyocardial biopsy. Ann Intern Med 110: 165–6

221


56 57

58

59

60

61

62

63

64 65

66

67

68

69

222

Maisch B, Camerini F, Schultheiss HP (1995) Immunosuppressive therapy for myocarditis. N Engl J Med 333: 1713; author reply 1714 Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri A (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107: 857–63 Kühl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-beta treatment eliminates cardiotropic viruses and improves left ventricular function in patients with myocardial persistence of viral genomes and left ventricular dysfunction. Circulation 107: 2793–8 Cooper LT, Jr., Berry GJ, Shabetai R (1997) Idiopathic giant-cell myocarditis – natural history and treatment. Multicenter Giant Cell Myocarditis Study Group Investigators. N Engl J Med 336: 1860–6 Cooper LT, Baughman KL, Feldman AM, Frustaci A, Jessup M, Kühl U, Levine GN, Narula J, Starling RC, Towbin J et al (2007) The role of endomyocardial biopsy in the management of cardiovascular disease: A scientific statement from the American Heart Association, the American College of Cardiology, and the European Society of Cardiology. Circulation 116: 2216–33 Frustaci A, Chimenti C, Ricci R, Natale L, Russo MA, Pieroni M, Eng CM, Desnick RJ (2001) Improvement in cardiac function in the cardiac variant of Fabry’s disease with galactose-infusion therapy. N Engl J Med 345: 25–32 Schultheiss HP, Noutsias M, Kühl U, Pauschinger M (2006) Myocarditis and viral cardiomyopathy. In: Camm AJ, Lüscher, TF, Serruys PW (eds): The ESC Textbook of Cardiovascular Medicine, Vol. 1. Oxford: Blackwell Publishing Ltd: 490–501 Noutsias M, Pauschinger M, Gross U, Lassner D, Schultheiss HP, Kuhl U (2008) Giantcell myocarditis in a patient presenting with dilated cardiomyopathy and ventricular tachycardias treated by immunosuppression: A case report. Int J Cardiol 128: e58–9 Kühl U, Schultheiss HP (1995) Treatment of chronic myocarditis with corticosteroids. Eur Heart J 16: 168–72 Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewski P, Niklewski T, Zembala M, Polonski L, Rozek MM, Wodniecki J (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: Two-year follow-up results. Circulation 104: 39–45 Frustaci A, Russo MA, Chimenti C (2009) Randomized study on the efficacy of immunosuppressive therapy in patients with virus-negative inflammatory cardiomyopathy: The TIMIC study. Eur Heart J 30: 1995–2002 Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kühl U, Lehmkuhl H, Hetzer R et al (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 9: 3 Mues B, Brisse B, Zwadlo G, Themann H, Bender F, Sorg C (1990) Phenotyping of macrophages with monoclonal antibodies in endomyocardial biopsies as a new approach to diagnosis of myocarditis. Eur Heart J 11: 619–27 Noutsias M, Pauschinger M, Schultheiss H, Kuhl U (2002) Phenotypic characterization


70

71

72 73

74

75

76

77 78

79

80

81

82 83

of infiltrates in dilated cardiomyopathy – diagnostic significance of T-lymphocytes and macrophages in inflammatory cardiomyopathy. Med Sci Monit 8: CR478–87 Kühl U, Noutsias M, Seeberg B, Schultheiss HP (1996) Immunohistological evidence for a chronic intramyocardial inflammatory process in dilated cardiomyopathy. Heart 75: 295–300 Devaux B, Scholz D, Hirche A, Klovekorn WP, Schaper J (1997) Upregulation of cell adhesion molecules and the presence of low grade inflammation in human chronic heart failure. Eur Heart J 18: 470–9 Noutsias M, Pauschinger M, Schultheiss HP, Kühl U (2002) Advances in the immunohistological diagnosis of inflammatory cardiomyopathy. Eur Heart J Suppl 4: I54–I62 Kolbeck PC, Steenbergen C, Wolfe JA, Sanfilippo F, Jennings RB (1989) The correlation of mononuclear cell phenotype in endomyocardial biopsies with clinical history and cardiac dysfunction. Am J Clin Pathol 91: 37–44 Noutsias M, Pauschinger M, Ostermann K, Escher F, Blohm JH, Schultheiss H, Kuhl U (2002) Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy. Med Sci Monit 8: MT59–71 Mahon NG, Madden BP, Caforio AL, Elliott PM, Haven AJ, Keogh BE, Davies MJ, McKenna WJ (2002) Immunohistologic evidence of myocardial disease in apparently healthy relatives of patients with dilated cardiomyopathy. J Am Coll Cardiol 39: 455– 462 Dettmeyer R, Reith K, Madea B (2002) Alcoholic cardiomyopathy versus chronic myocarditis – immunohistological investigations with LCA, CD3, CD68 and tenascin. Forensic Sci Int 126: 57–62 Springer TA (1990) Adhesion receptors of the immune system. Nature 346: 425–34 Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewski P, Niklewski T, Zembala M, Polonski L et al (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy : Twoyear follow-up results. Circulation 104: 39–45 Herskowitz A, Ahmed-Ansari A, Neumann DA, Beschorner WE, Rose NR, Soule LM, Burek CL, Sell KW, Baughman KL (1990) Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: A nonhistologic marker of myocarditis. J Am Coll Cardiol 15: 624–32 Ino T, Kishiro M, Okubo M, Akimoto K, Nishimoto K, Yabuta K, Okada R (1997) Late persistent expressions of ICAM-1 and VCAM-1 on myocardial tissue in children with lymphocytic myocarditis. Cardiovasc Res 34: 323–8 Noutsias M, Seeberg B, Schultheiss HP, Kuhl U (1999) Expression of cell adhesion molecules in dilated cardiomyopathy: Evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99: 2124–31 Parrillo JE (2001) Inflammatory cardiomyopathy (myocarditis): Which patients should be treated with anti-inflammatory therapy? Circulation 104: 4–6 Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, Pieri F, FraternaliOrcioni G, Pileri SA (1998) The EnVision++ system: A new immunohistochemical

223


84

85

86 87

88

89 90

91

92

93

94

95

96

224

method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol 51: 506–11 Chander S, Talwar KK, Chopra P (1995) Immunohistochemical characterisation and quantitative evaluation of lymphomononuclear cells in dilated cardiomyopathy – an endomyocardial biopsy study. Indian Heart J 47: 360–4 Noutsias M, Pauschinger M, Ostermann K, Escher F, Blohm JH, Schultheiss H, Kühl U (2002) Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy. Med Sci Monit 8: MT59–71 Braimbridge MV, Darracott S, Chayen J, Bitensky L, Poulter LW (1967) Possibility of a new infective aetiological agent in congestive cardiomyopathy. Lancet 1: 171–6 Pankuweit S, Moll R, Baandrup U, Portig I, Hufnagel G, Maisch B (2003) Prevalence of the parvovirus B19 genome in endomyocardial biopsy specimens. Hum Pathol 34: 497–503 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111: 887–93 Baboonian C, Treasure T (1997) Meta-analysis of the association of enteroviruses with human heart disease. Heart 78: 539–43 Pauschinger M, Bowles NE, Fuentes-Garcia FJ, Pham V, Kühl U, Schwimmbeck PL, Schultheiss HP, Towbin JA (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99: 1348–54 Bowles NE, Ni J, Kearney DL, Pauschinger M, Schultheiss HP, McCarthy R, Hare J, Bricker JT, Bowles KR, Towbin JA (2003) Detection of viruses in myocardial tissues by polymerase chain reaction. Evidence of adenovirus as a common cause of myocarditis in children and adults. J Am Coll Cardiol 42: 466–72 Lindner J, Barabas S, Saar K, Altmann D, Pfister A, Fleck M, Deml L, Modrow S (2005) CD4(+) T-cell responses against the VP1-unique region in individuals with recent and persistent parvovirus B19 infection. J Vet Med B Infect Dis Vet Public Health 52: 356–61 Kühl U, Lassner D, Pauschinger M, Gross UM, Seeberg B, Noutsias M, Poller W, Schultheiss HP (2008) Prevalence of erythrovirus genotypes in the myocardium of patients with dilated cardiomyopathy. J Med Virol 80: 1243–1251 Dettmeyer R, Baasner A, Schlamann M, Padosch SA, Haag C, Kandolf R, Madea B (2004) Role of virus-induced myocardial affections in sudden infant death syndrome: A prospective postmortem study. Pediatr Res 55: 947–52 Zee-Cheng CS, Tsai CC, Palmer DC, Codd JE, Pennington DG, Williams GA (1984) High incidence of myocarditis by endomyocardial biopsy in patients with idiopathic congestive cardiomyopathy. J Am Coll Cardiol 3: 63–70 Wojnicz R, Nowalany-Kozielska E, Wodniecki J, Szczurek-Katanski K, Nozynski J, Zembala M, Rozek MM (1998) Immunohistological diagnosis of myocarditis. Potential


role of sarcolemmal induction of the MHC and ICAM-1 in the detection of autoimmune mediated myocyte injury. Eur Heart J 19: 1564–72 97 Terasaki F, Okabe M, Hayashi T, Fujioka S, Suwa M, Hirota Y, Kitaura Y, Kawamura K, Isomura T, Suma H (1999) Myocardial inflammatory cell infiltrates in cases of dilated cardiomyopathy: Light microscopic, immunohistochemical, and virological analyses of myocardium specimens obtained by partial left ventriculectomy. J Card Surg 14: 141–6 98 Staudt A, Schaper F, Stangl V, Plagemann A, Bohm M, Merkel K, Wallukat G, Wernecke KD, Stangl K, Baumann G et al (2001) Immunohistological changes in dilated cardiomyopathy induced by immunoadsorption therapy and subsequent immunoglobulin substitution. Circulation 103: 2681–6 99 Noutsias M, Hohmann C, Pauschinger M, Schwimmbeck PL, Ostermann K, Rode U, Yacoub MH, Kühl U, Schultheiss HP (2003) sICAM-1 correlates with myocardial ICAM-1 expression in dilated cardiomyopathy. Int J Cardiol 91: 153–61 100 Noutsias M, Pauschinger M, Poller WC, Schultheiss HP, Kuhl U (2004) Immunomodulatory treatment strategies in inflammatory cardiomyopathy: current status and future perspectives. Expert Rev Cardiovasc Ther 2: 37–51 101 Noutsias M, Kühl U, Lassner D, Gross U, Pauschinger M, Schultheiss HP, Gutberlet M (2007) Parvovirus B19-associated active myocarditis with biventricular thrombi – results of endomyocardial biopsy investigations and cardiac magnetic resonance imaging. Circulation 115: e378–80

225

Cardiac magnetic resonance imaging: A non-invasive approach for the detection of myocardial inflammation – Potentials and limitations Matthias Gutberlet University Leipzig/ Leipzig Heart Center, Department of Diagnostic and Interventional Radiology, Strümpellstrasse 39, 04289 Leipzig, Germany

Abstract Cardiac magnetic resonance imaging (CMR) has evolved to the imaging modality of choice for the volumetric and functional evaluation of the heart in different cardiac diseases. Furthermore, it is capable of tissue characterization using T1- and T2-weighted images with and without the use of contrast agents. Therefore, it is the ideal imaging modality in cardiomyopathies or ischemic heart disease. Currently, the most commonly used CMR technique is the so-called delayed enhancement technique using T1-weighted inversion recovery sequences 10–15 min after the intravenous administration of a contrast agent, usually Gd-DTPA. With this technique viable myocardium can be distinguished from non-viable myocardium in ischemic heart disease, cardiomyopathies or myocarditis. Therefore, delayed myocardial enhancement is not specific for myocarditis, but a typical subepicardial or diffuse appearance allows for the differentiation between ischemic versus inflammatory heart disease. However, delayed myocardial enhancement represents more the irreversibly injured myocardium than inflammation itself. T2-weighted sequences and early myocardial enhancement using published ratios (edema ratio) or the global relative enhancement are best used for the detection of inflammation in comparison to results from endomyocardial biopsies, especially if used in combination. Simple evaluation of reduced global or regional ventricular function by magnetic resonance imaging is helpful for follow-up examinations but not very sensitive or specific for the diagnosis of myocarditis.

Cardiac magnetic resonance in myocarditis One of the first published studies using magnetic resonance imaging (MRI) for the diagnosis of myocarditis was a study by Gagliardi et al. in 1991 [1] using T2-weighted images in children. Friedrich et al. [2] were the first to use contrastenhanced cardiac MRI (CMR) to diagnose acute myocarditis, but did not validate their results with endomyocardial biopsies (EMB) as Gagliardi did. These first reports did not at that time lead to a widespread use of CMR for this indication. However, more recently, with the use of non-contrast [3, 4] and contrast-enhanced


227

Matthias Gutberlet

[3–14] CMR sequences, CMR has become increasingly popular, indicating that CMR might be superior to other imaging modalities for diagnosing myocarditis [15–17]. Nevertheless, the number of included patients is still very small, the inclusion criteria differ substantially and the most studies have not been validated against the gold standard, EMB.

CMR findings in myocarditis In comparison to other diagnostic tools and imaging modalities, CMR delivers a great variety of diagnostic targets and findings in myocarditis patients.

Functional assessment CMR is the modality of choice for the assessment of right and left ventricular (LV) function. In myocarditis patients global or regional wall motion abnormalities can be observed. Nevertheless, none of these findings is specific for inflammation and can also be found in ischemic heart disease or cardiomyopathies [17]. Furthermore, myocardial inflammation can also occur without any wall motion abnormalities.

Pericardial effusion Pericardial effusion is a common finding in myocarditis patients with a frequency of occurrence of up to 57% [18]. Pericardial effusion is an unspecific finding but a good indicator for an inflammatory process. The typical circular pericardial effusion can easily be detected (Fig. 1) by standard CINE-MRI sequences like steady state free precession (SSFP) sequences.

Tissue characterization The expected tissue pathologies in acute myocarditis are myocardial edema, capillary leakage, hyperemia and, in severe cases, necrosis and fibrosis [19].

Visualization and quantification of myocardial edema T2-weighted imaging is capable of visualization of myocardial edema [20] due to an excellent contrast between local edema and normal myocardium. However, in contrast to acute myocardial infarction, the edema in myocarditis patients is not always focal and is therefore more difficult to detect by MRI. Therefore, the usually

228

Cardiac magnetic resonance imaging: A non-invasive approach for the detection of myocardial inflammation

Figure 1. Typical circular pericardial effusion in a patient with acute perimyocarditis using (A) T2-weighted and (B) steady state free precession (SSFP) sequences in the four-chamber and (D) two-chamber view. (C) The additionally acquired delayed gadolinium-enhanced images using an inversion recovery gradient echo sequence in the two-chamber view show an intense pericardial contrast enhancement, indicating a perimyocarditis.

employed sequences, with an additional fat suppression pulse, are used in short axis orientation and should be assessed by a quantitative signal intensity analysis of the entire myocardium as described previously [4, 13, 14]. These authors calculated the edema ratio (ER) using a region of interest encompassing the entire LV myocardium and a second region of interest encompassing the entire visible right erector spinae or latissimus dorsi muscle (i.e., skeletal muscle), depending on the homogeneity of the muscle intensity, drawn on the same section (Fig. 2). The mean myocardial

229

Matthias Gutberlet

Figure 2. Calculation of the edema ratio (ER) from T2-weighted STIR sequences in the short axis twochamber view using the mean signal intensity of the myocardium and the latissimus dorsi muscle [2, 4, 14].

signal intensity (SImyo) is correlated with the mean skeletal muscle signal intensity (SIskm) using the equation: ER = SImyo/SIskm With the present techniques, fairly good sensitivity and specificity of up to 70% compared to the results from EMB can be achieved [14]. Nevertheless, the current sequences are not very robust and vary from different MRI systems. Newly developed T2-prepared sequences [21] may help to overcome some of these limitations.

Visualization and quantification of hyperemia and capillary leakage Hyperemia is one feature of myocardial inflammation. Contrast-enhanced T1-weighted MRI sequences have been used to assess myocardial hyperemia [22] in an animal model and also to detect inflammation [23] in skeletal muscle. Due to the fact that the currently available MR gadolinium-based contrast agents distribute into the interstitial space within the first few minutes, images have to be acquired in this early phase (“early enhancement”) to detect myocardial hyperemia. In myocarditis, hyperemia is usually not a localized process. Therefore, several authors

230


have proposed a similar approach as for the quantification of the myocardial edema. They calculated the global relative enhancement (gRE). For this, a region of interest encompassing the entire LV myocardium and a second region of interest encompassing the skeletal muscle within the same section were drawn on the pre-contrast T1-weighted images (Fig. 3) and copied to the post-contrast images.

Figure 3. Manually outlined contours (B, D) of the left ventricle and right erector spinae muscle for the calculation of the global relative enhancement (gRE) from transverse T1-weighted images according to Friedrich et al. [2]. An additional saturation section is positioned across the atria to reduce signal from slow-flowing blood. (A, C) The initial pre- (A) and post-contrast (C) images of a patient with acute myocarditis. In the post-contrast image, a focal contrast enhancement at the lateral wall of the left ventricle is obvious. The 3-month follow-up examination (B, D) shows no focal enhancement and a normalization of the gRE from 7.4 to 2.6 (norm: < 4, according to [2, 4, 14]).

231

Matthias Gutberlet

The mean signal intensities of the myocardium and skeletal muscle before and after contrast enhancement were used [14]: gRE = REmyo/REskm = [(postSImyo– preSImyo)/preSImyo]/[(postSIskm– preSIskm)/preSIskm] where REmyo and REskm are relative enhancement values for the myocardium and skeletal muscle, respectively; preSImyo and postSImyo are the pre- and post-contrast signal intensities in the myocardium, respectively; and preSIskm and postSIskm are the pre- and post-contrast signal intensities in the skeletal muscle, respectively. As cut-off values for active inflammation a gRE of 4 or higher was used [4, 14]. The diagnostic value of this approach has been shown already in several studies [4, 13, 14] starting with the study by Friedrich et al. [2]. Using this technique a sensitivity of 60–80% and a specificity of 68–89% was achieved [4, 14].

Visualization of necrosis and fibrosis (“delayed enhancement”) Several studies have already shown that MRI using so-called “late” or “delayed gadolinium enhancement” is capable of visualizing necrosis in acute myocardial infarction or acute myocarditis [7, 13]. This is possible due to a leak of the cellular membrane allowing Gd-DTPA to diffuse into the necrotic cells with a delayed “wash-in” and “wash-out” of the contrast agent. Using special inversion-recovery gradient echo sequences 10–15 min after the administration of a single or double dose (0.1 or 0.2 mmol/kg body weight) of Gd-DTPA intravenously, it is possible to “null” the signal of normal viable myocardium so that the necrotic region appears bright (Fig. 4). In the chronic stage, due to remodeling processes, the viable tissue is

Figure 4. Inversion recovery sequences in two-chamber long (A) and short axis (B, C) showing the typical subepicardial and intramural delayed enhancement of a myocarditis patient.

232


replaced by fibrous tissue with an increased extracellular space, which also leads to a delayed “wash-in” and “wash-out” of the contrast agent. In contrast to the appearance of delayed gadolinium enhancement in myocardial infarction, it usually occurs subepicardially (Fig. 4), whereas the typical appearance of delayed enhancement in ischemic heart disease is subendocardial. This technique can be up to 100% specific [4], but the sensitivity in some studies was as low as 44%, especially in patients with chronic myocardial inflammation [14], with a sensitivity of only 27%.

Limitations of the current studies The number of patients studied so far is low (Tab. 1), and the inclusion criteria and MRI protocols used differ substantially. The findings in the majority of patients have not been validated against the gold standard, EMB. Further multicenter studies with standardized protocols and validation against EMB are needed.

Table 1. Summary of studies Authors

Journal

Number of patients

Number of biopsy-validated patients

Friedrich et al. Roditi et al.

Ciculation 1998 [2]

19

7

Clin Radiol 2000 [5]

12

4

Rieker et al.

RöFo 2002 [6]

9

3

Laissy et al.

Chest 2002 [3]

20

3

Mahrholdt et al.

Circulation 2004 [7]

32

32

Abdel-Aty et al.

J Am Coll Cardiol 2005 [4]

25

-

Laissy et al.

Radiology 2006 [9]

24

-

Ingkanisorn et al.

JCMR 2006 [10]

21

-

DeCobelli et al.

J Am Coll Cardiol 2006 [11]

23

23

Mahrholdt et al.

Circulation 2006 [8]

87

87

Gutberlet et al.

Radiology 2008 [14]

83

83

References 1

Gagliardi MG, Bevilacqua M, Di Renzi P, Picardo S, Passariello R, Marcelletti C (1991) Usefulness of magnetic resonance imaging for diagnosis of acute myocarditis in infants and children, and comparison with endomyocardial biopsy. Am J Cardiol 68(10): 1089–91

233

Matthias Gutberlet

2

3

4

5

6 7

8

9

10

11

12 13

14

234

Friedrich MG, Strohm O, Schulz-Menger J, Marciniak H, Luft FC, Dietz R (1998) Contrast media-enhanced magnetic resonance imaging visualizes myocardial changes in the course of viral myocarditis. Circulation 97(18): 1802–9 Laissy JP, Messin B, Varenne O, Iung B, Karila-Cohen D, Schouman-Claeys E, Steg PG (2002) MRI of acute myocarditis: A comprehensive approach based on various imaging sequences. Chest 122(5): 1638–48 Abdel-Aty H, Boyé P, Zagrosek A, Wassmuth R, Kumar A, Messroghli D, Bock P, Dietz R, Friedrich MG, Schulz-Menger J (2005) Diagnostic performance of cardiovascular magnetic resonance in patients with suspected acute myocarditis: Comparison of different approaches. J Am Coll Cardiol 45(11): 1815–22 Roditi GH, Hartnell GG, Cohen MC (2000) MRI changes in myocarditis – Evaluation with spin echo, cine MR angiography and contrast enhanced spin echo imaging. Clin Radiol 55(10): 752–8 Rieker O, Mohrs O, Oberholzer K, Kreitner KF, Thelen M (2002) Cardiac MRI in suspected myocarditis. Röfo 174(12): 1530–6 Mahrholdt H, Goedecke C, Wagner A, Meinhardt G, Athanasiadis A, Vogelsberg H, Fritz P, Klingel K, Kandolf R, Sechtem U (2004) Cardiovascular magnetic resonance assessment of human myocarditis: A comparison to histology and molecular pathology. Circulation 109(10): 1250–8 Mahrholdt H, Wagner A, Deluigi CC, Kispert E, Hager S, Meinhardt G, Vogelsberg H, Fritz P, Dippon J, Bock CT, Klingel K, Kandolf R, Sechtem U (2006) Presentation, patterns of myocardial damage, and clinical course of viral myocarditis. Circulation 114(15): 1581–90 Laissy JP, Hyafil F, Feldman LJ, Juliard JM, Schouman-Claeys E, Steg PG, Faraggi M (2005) Differentiating acute myocardial infarction from myocarditis: Diagnostic value of early- and delayed-perfusion cardiac MR imaging. Radiology 237(1): 75–82 Ingkanisorn WP, Paterson DI, Calvo KR, Rosing DR, Schwartzentruber DJ, Fuisz AR, Arai AE (2006) Cardiac magnetic resonance appearance of myocarditis caused by high dose IL-2: Similarities to community-acquired myocarditis. J Cardiovasc Magn Reson 8(2): 353–60 De Cobelli F, Pieroni M, Esposito A, Chimenti C, Belloni E, Mellone R, Canu T, Perseghin G, Gaudio C, Maseri A, Frustaci A, Del Maschio A (2006) Delayed gadolinium-enhanced cardiac magnetic resonance in patients with chronic myocarditis presenting with heart failure or recurrent arrhythmias. J Am Coll Cardiol 47(8): 1649–54 Yelgec NS, Dymarkowski S, Ganame J, Bogaert J (2007) Value of MRI in patients with a clinical suspicion of acute myocarditis. Eur Radiol 17(9): 2211–7 Noutsias M, Kuehl U, Lassner D, Gross U, Pauschinger M, Schultheiss HP, Gutberlet M (2007) Images in cardiovascular medicine. Parvovirus-B19-associated active myocarditis with biventricular thrombi. Results of endomyocardial biopsy investigations and cardiac magnetic resonance imaging. Circulation 115(13): e378–80 Gutberlet M, Spors B, Thoma T, Bertram H, Denecke T, Felix R, Noutsias M, Schultheiss HP, Kühl U (2008) Suspected chronic myocarditis at cardiac MR: Diagnostic accuracy


15

16 17 18

19 20 21

22

23

and association with immunohistologically detected inflammation and viral persistence. Radiology 246(2): 401–9 Liu PP, Yan AT (2005) Cardiovascular magnetic resonance for the diagnosis of acute myocarditis: Prospects for detecting myocardial inflammation. J Am Coll Cardiol 45(11): 1823–5 Skouri HN, Dec GW, Friedrich MG, Cooper LT (2006) Noninvasive imaging in myocarditis. J Am Coll Cardiol 48(10): 2085–93 Magnani JW, Dec GW (2006) Myocarditis: Current trends in diagnosis and treatment. Circulation 113(6): 876–90 Ammann P, Naegeli B, Schuiki E, Mury R, Frielingsdorf J, Bertel O (2003) Long-term outcome of acute myocarditis is independent of initial cardiac enzyme release. Int J Cardiol 89(2–3): 217–22 Kishimoto C, Hiraoka Y (1994) Clinical and experimental studies in myocarditis. Curr Opin Cardiol 9(3): 349–56 Simonetti OP, Finn JP, White RD, Laub G, Henry DA (1996) “Black blood” T2-weighted inversion recovery MR imaging of the heart. Radiology 199(1): 49–57 Kellman P, Aletras AH, Mancini C, McVeigh ER, Arai AE (2007) T2-prepared SSFP improves diagnostic confidence in edema imaging in acute myocardial infarction compared to turbo spin echo. Magn Reson Med 57(5): 891–7 Miller DD, Holmvang G, Gill JB, Dragotakes D, Kantor HL, Okada RD, Brady TJ (1989) MRI detection of myocardial perfusion changes by gadolinium-DTPA infusion during dipyridamole herperemia. Magn Reson Med 10(2): 246–55 Paajanen H, Brasch RC, Schmiedl U, Ogan M (1987) Magentic resonance imaging of local tissue inflammation using gadolinium-DTPA. Acta Radiol 28(1): 79–83

235

Treatment strategies in inflammatory cardiomyopathy patients

Clinical management of acute myocarditis and cardiomyopathy Kenneth L. Baughman† Advanced Heart Disease Section, Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, MA, USA Dr. Baughman died during the final preparation of this book. We thank Dr. Baughman and his coworkers for his book chapter, and his internationally acknowledged, significant and unforgettable work in the field of myocarditis. M. Noutsias and H.P. Schultheiss

Abstract Several forms of acute myocarditis may present as new onset cardiomyopathy and congestive heart failure. These include myocarditis characterized as fulminant, giant cell, chronic acute, eosinophilic (particularly necrotizing eosinophilic myocarditis), peripartum, Lyme, small pox vaccine related, HIV-related, and acute myocardial infarction caused by myocarditis with coronary arteritis. Treatment of each of these disorders is dependent upon considering the diagnosis, performing endomyocardial biopsy for histologic confirmation, and use of immune modulating or immunosuppressive therapy in appropriate candidates. Some forms of acute myocarditis may resolve spontaneously without any treatment including fulminant myocarditis, peripartum, and small pox vaccine related myocarditis. Others require immune modulating therapy including giant cell myocarditis, and eosinophilic myocarditis; while others require specific treatment for the infectious or autoimmune pathogen responsible including eosinophilic myocarditis, Lyme myocarditis, and HIV-related. We currently have the technology which may allow us to determine if the 50% of patients with “idiopathic” cardiomyopathy in fact have a viral or post viral autoimmune related compromise of cardiac function.

Introduction Acute heart failure is defined as the sudden onset of the syndrome of congestive heart failure in a patient who has never had a diagnosis of cardiac disease in the past, or who is experiencing a profound deterioration of a known heart disorder. The potential causes of acute cardiomyopathy are diffuse (Tab. 1). Patients may experience the symptomatic progression of a previously unrecognized heart muscle disorder. Heart muscle dysfunction may be due to an acute coronary obstruction, the combination of multiple sub-clinical ischemic events over time (ischemic cardiomyopathy), or due to infarction of a region of the heart causing mechanical or functional abnormalities (right ventricular infarct, ventricular septal defect, or acute mitral regurgitation due


239

Kenneth L. Baughman

Table 1. Causes of acute cardiomyopathy - Previously unrecognized heart disease - Coronary artery disease - Acute MI - Mechanical complications - Ischemic cardiomyopathy - Hypertensive crisis - Tachycardia - Valvular heart disease - Toxins or medications - Endocrine disorders - Sepsis - Myocarditis

to papillary muscle ischemia or muscle rupture). Patients with profound hypertension (hypertensive crisis) may experience acute heart failure and ventricular compromise due to longstanding increases in left ventricular wall thickness and an acute afterload increase. Tachycardia may induce a cardiomyopathy, or exacerbate a borderline compensated patient. Valvular heart disease, including mitral and aortic regurgitation, and aortic stenosis, may present with established left ventricular dysfunction. Some medications are known to be cardiotoxic or cardio-depressant including alcohol, negative inotropic antiarrhythmics, calcium and beta blockers, and chemotherapy agents (particularly adriamycin). Endocrine disorders including pheochromocytoma and thyroid disease may present with left ventricular compromise. Patients with sepsis may suffer from high output demands due to vasodilatation and simultaneous cytokine depression of myocardial function, resulting in the acute onset of heart failure. Longstanding congenital heart disease, particularly shunts such as patent-ductus arteriosus, can also present with left ventricular compromise, heralding recognition of the defects that have been present since birth. Myocarditis may occur in 10–15% of patients presenting with a new onset of a dilated cardiomyopathy or congestive heart failure. Determination of the etiology of cardiomyopathy is important, as this influences treatment and prognosis (Fig. 1) [1]. In keeping with the focus of this text, we concentrate on myocarditis as a cause for the new onset of congestive heart failure or dilated cardiomyopathy. Specific etiologies (explored below) include fulminant, giant cell, chronic active, eosinophilic, lyme, small pox, HIV, and peripartum myocarditis. Whereas heart biopsy-confirmed myocarditis has been shown to account for 10–15% of patients with new onset heart failure and cardiomyopathy, up to 50% of patients, despite a full and complete evaluation, have no etiology identified [1]. The

240

Clinical management of acute myocarditis and cardiomyopathy

Figure 1. Adjusted Kaplan–Meier estimates of survival according to the underlying cause of cardiomyopathy. Only idiopathic cardiomyopathy and cardiomyopathy due to causes for which survival was significantly different from that in patients with idiopathic cardiomyopathy are shown. Taken from [1], with friendly permission by the Massachusetts Medical Society.

demonstration of cardiotropic viruses by polymerase chain reaction analysis in this ‘idiopathic’ population implies that a much broader group of patients may reach the end stages of heart dysfunction through a viral and/or autoimmune pathway [2, 3]. The diagnosis of myocarditis remains problematic. The Dallas criteria were established in 1986 [4] and are based on the finding of an inflammatory infiltrate with associated myocyte necrosis or damage not characteristic of an ischemic event on endomyocardial biopsy (EMB). Investigators [5] demonstrated by biopsying post-mortem hearts that using the Dallas criteria, for sample numbers greater than five, myocarditis was diagnosed in only two thirds of the subjects who had died of the disorder. Magnetic resonance imaging has demonstrated that myocarditis may begin as an inflammation on the left ventricular lateral wall epicaridially, and that biopsy from the “affected” area is much more likely to result in a histological diagnosis of myocarditis than blind biopsies of the right ventricular septum [6, 7]. Interpretation of histological samples is also difficult utilizing these criteria. In the Myocarditis Treatment Trial [8] only 64% of 111 patients diagnosed by local pathologist had their diagnosis confirmed by the expert panel. Seven expert patholo-

241

Kenneth L. Baughman

gist, however, when asked to interpret heart biopsy samples had marked variability in their assessment of histological findings, including the diagnosis of myocarditis [9]. Several forms of myocarditis have distinct and clinical pathological presentations or histological findings on biopsy. Many investigators have demonstrated that viral pathogens may be present in myocardial tissue without the presence of myocardial inflammation [10], particularly in children. Viral pathogens have been identified in 25–40% of patients with cardiomyopathy [2, 3, 10]. The presence of viral pathogens may adversely affect the outcome of patients with cardiomyopathy [11]. Finally, the Dallas criteria do not identify a group of patients who respond to immunosuppressive therapy [8]. Other investigators, utilizing alternative markers of immune up-regulation, have preliminarily identified dilated cardiomyopathy populations who may respond to immunosuppressive agents [12, 13]. Therefore, while this chapter deals with histological or clinical pathological syndromes associated with easily recognizable myocardial inflammation, there may be a much broader category of patients who have cardiomyopathy related to myocardial inflammation. At this point in time the diagnosis of acute myocarditis or myocardial inflammation-related acute cardiomyopathy requires EMB. Biopsy series performed at centers where the procedure is done frequently have demonstrated a complication rate of 6%, including a 0.5% risk of definite perforation and infrequent mortality related to the procedure [14]. Most American investigators prefer right ventricular endomyocardial sampling; however, many European investigators routinely perform also left ventricular EMB. Left ventricular biopsy does require arterial access and predisposes to microembolic events related to endomyocardial sampling and platelet formation at the site of tissue retrieval. Antiplatelet agents and heparin have been recommended (which may increase the risk of pericardial tamponade if perforation does occur). The risk of biopsy is dependent upon patient factors and the skill of the operator, while interpretation of the material is greatly dependent upon the experience of the pathologist. Both of these factors influence clinician’s willingness to submit their patients to this procedure.

Fulminant myocarditis Lieberman et al. [15] classified myocarditis as either fulminant or acute (non-fulminant) on the basis of clinical pathological criteria. McCarthy et al. [16] reported on the outcome of 15 patient with fulminant myocarditis compared with 132 patients with acute myocarditis. Patients with fulminant myocarditis have a history of an acute viral syndrome occurring within 2 weeks of presentation. The patients present with profound heart failure with severe hemodynamic compromise, often requiring vasopressors, intra-aortic balloon, or left ventricular assist device support. The echocardiograms of these patients [17] demonstrate severe left ventricular

242


hypofunction but near normal left ventricular cavity dimensions. Patients display increased wall thickness (likely due to myocardial edema). EMB demonstrates Dallas criteria myocarditis in virtually every sample with associated interstitial edema and absence of interstitial fibrosis. Patients with fulminant myocarditis either die or recover spontaneously within 2 weeks. Symptoms improve dramatically and echocardiograms normalize usually within 2 weeks. Within 6 months all signs of myocardial inflammation and heart failure have resolved on examination, echocardiography, and repeat EMB [16]. In long-term follow-up, patients with fulminant myocarditis have a dramatically better 5-year survival (93%) compared with those with acute myocarditis (45%) (Fig. 2) [16]. A similar syndrome has been demonstrated in children [18]. Patients with fulminant myocarditis must be supported with whatever means necessary to ensure their survival long enough to allow myocyte recovery. In our experience, patients do not benefit, and may in fact worsen, with the application of immunosuppressive therapy, although a number of single case reports have demonstrated use of immunosuppressive agents with cardiac recovery, and declared an association. We believe that these represent the natural history of the disorder,

Figure 2. Unadjusted transplantation-free survival according to clinicopathological classification. Patients with fulminant myocarditis were significantly less likely to die or require heart transplantation during follow-up than were patients with acute myocarditis (P = 0.05 by the logrank test). Taken from [16], with friendly permission by the Massachusetts Medical Society.

243

Kenneth L. Baughman

which fortunately was not altered by the agents, which could potentially enhance viral replication and worsen prognosis.

Giant cell myocarditis Giant cell myocarditis is an equally distinct clinical pathological entity with characteristic histological findings. Patients with giant cell myocarditis present with left ventricular compromise that has failed to improve, or progresses rapidly despite appropriate medical therapy. Patients are usually middle-aged Caucasian men presenting with heart failure (75%), ventricular arrhythmia (14%), or heart block (5%) [19]. Occasionally, patients present with a syndrome similar to acute myocardial infarction. Approximately on third of the patients have an autoimmune disorder [20, 21]. Echocardiographic findings include severe systolic dysfunction and moderate to severe dilatation. EMB is characterized by geographic myocardial necrosis and giant cells within the inflammatory infiltrate. The infiltrates include lymphocytes, histiocytes, and eosinophils. The histological presentation, and clinical history, is distinct from patients with cardiac sarcoidosis [22]. The natural history of giant cell myocarditis is one of profound and rapid deterioration (Fig. 3). The median survival is 5.5 months from the time of onset, despite treatment for congestive heart failure. There is a suggestion that immunosuppressive agents, particularly directed to the T cell population responsible for the illness [23–25], may improve the outlook. We await the prospective trial of high-dose immunosuppressive therapy for this condition.

Chronic active myocarditis One of the histopathological classifications of Lieberman et al. [15] included chronic active myocarditis. This category, much like chronic active hepatitis, is notable for ongoing inflammation and fibrosis. Patients have an indistinct clinical onset and a moderate established cardiomyopathy with mild dilatation and systolic hypofunction at presentation. EMB demonstrates evidence of scattered myocyte inflammation (active or borderline myocarditis) with ongoing active fibrosis. These patients may develop giant cells over time; however, these may not be evident on early tissue sampling. Serial biopsies demonstrate inflammation and fibrosis with giant cells becoming more prevalent as the disease progresses. Within 2–3 years, these patients often develop a restrictive cardiomyopathy that is refractory to medical management. There are no studies of immunosuppressive therapy in this disorder. Finding inflammation, fibrosis, and giant cells in a population with progressive elevation of right- and left-sided diastolic filling pressures confirms the diagnosis and implies that heart transplant will likely be necessary.

244


Figure 3. Kaplan–Meier survival curves for patients with giant-cell myocarditis. Panel A shows the duration of survival from the onset of symptoms; Panel B shows the duration of survival from the time of presentation at the referring institution; and Panel C shows the duration of survival among 38 patients in whom giant-cell myocarditis was diagnosed by endomyocardial biopsy or by examination of a section of ventricular apex. In each case, survival was significantly longer among patients with lymphocytic myocarditis (P < 0.001 by the log-rank test for each comparison). Taken from [19], with friendly permission by the Massachusetts Medical Society.

245

Kenneth L. Baughman

Eosinophilic myocarditis Eosinophilic myocarditis may be due to hypersensitivity myocarditis, or related to hypereosiniphilic syndromes. Hypersensitivity myocarditis may present with a dilated cardiomyopathy, sudden death, or most frequently as the rapid progression of a previously recognized dilated cardiomyopathy. Patients may have peripheral eosinophilia, rash or fever, although these are infrequent and may be delayed in their presentation. Hypersensitivity may be present in 2–7% of patients [26]. This form of myocarditis should be suspected in individuals in whom clinical deterioration is correlated with the initiation of a new medication, particularly if that medication is known to cause allergic reactions (sulfas) or is a pressor agent, particularly dobutamine [27]. The patient’s echocardiogram may not appear to be significantly different, despite a progression of symptoms and elevation of filling pressures. Biopsy reveals interstitial infiltrates of eosinophils with little (if any) myocyte necrosis. Necrotizing eosinophilic myocarditis or the presence of giant cells in granuloma may be present, but are not necessary to make the diagnosis. Hypereosiniphilic syndromes may also involve the myocardium but usually do so after a longer period of time [28]. This form of eosinophilic myocarditis may present with unheralded heart failure or ventricular arrhythmias. Peripheral eosinophilia is virtually always present in these cases, well before the presentation of heart dysfunction. Therefore, patients with an unexplained deterioration in ventricular function, particularly those who have recently had new agents added to their regimen (including dobutamine) should have this diagnosis considered. The biopsy is definitive and should lead to withdrawal of the offending agents, and treatment with corticosteroid therapy.

Peripartum cardiomyopathy with myocarditis Peripartum cardiomyopathy is a distinct entity characterized by the new onset of left ventricular compromise and heart failure in the last month of pregnancy or within 5 months of delivery, with no other cause of the cardiomyopathy identified, and no prior history of heart disease [29]. Many investigators have demonstrated that EMB of patients with peripartum cardiomyopathy may display myocarditis in up to 78% of subjects [29, 30]. Other investigators, who have not confirmed this high rate of inflammation, have analyzed samples many months after presentation, usually at the time of heart transplant or evaluation for heart transplant [31]. Patients with peripartum cardiomyopathy either improve, or fail to improve within 6 months of presentation [32, 33]. Patients who fail to improve tend to have more profound left ventricular compromise, more depressed left ventricular stroke work

246


index, and elevated cytokines compared with those who improve [34]. The rate of spontaneous improvement is over 85% [33] and those with inflammation show no greater or lesser propensity towards improvement compared with those who do not have inflammation. Because of the high rate of spontaneous improvement, we currently delay EMB in this population for 2 weeks, during which time they receive standard medical therapy for heart failure. If patients have failed to improve significantly, not only symptomatically but also with regard to echocardiographic parameters, we then proceed with EMB. Those with residual inflammation are considered for immunosuppressive therapy, although there are no data to support this approach. Patients who recover resting heart function should be treated with angiotensin converting enzyme inhibitors and beta-blockers for at least 6 months to avoid adverse remodeling. If resting ventricular function is normalized at 6 months, heart failure therapy is removed, and patients undergo echocardiographic exercise testing to assess the hearts function with the stress of increased heart rate and blood volume. Those who fail to improve should return to medical therapy and avoid subsequent pregnancies.

Lyme myocarditis Lyme disease [35] may acutely and chronically involve the myocardium. Most cases present with some form of AV block often resulting in complete heart block [36–38]. Patients may also display electrocardiographic and echocardiographic evidence of diffuse myopericarditis [38] and rarely cardiomyopathy [39]. The cardiac involvement usually occurs between 3 days and 6 weeks of the initial infection. Rarely the offending pathogen (Borrelia burgdorferi) has been demonstrated in the myocardium of a patient with cardiomyopathy [39]. Patients usually respond to antibiotic therapy with no evidence of additional myocardial dysfunction.

Acute myocardial infarction with normal coronaries Many investigators have reported patients presenting with evidence of acute myocardial infarction by electrocardiographic and echocardiographic analysis [40–43]. Patients presenting in this fashion, who have normal coronary arteries have occasionally been submitted to heart biopsy. Approximately one third of this population has demonstrated Dallas criteria myocarditis [41, 42]. Most patients [40–42] have return of normal ventricular function in long-term follow-up [41–43]. Some investigators [44] have demonstrated that patients with myocarditis have impaired endothelial function, which may predispose them to coronary vasospasm and associated myocardial infarction.

247

Kenneth L. Baughman

Small pox vaccine-related myocarditis A small number of patients who have received the small pox vaccine have developed a syndrome compatible with acute myocarditis and rarely dilated cardiomyopathy [45, 46]. In over 230,000 vaccinees, 18 cases of probable myocarditis have been reported [46]. This has not been seen in individuals who have been previously vaccinated. These patients present within 1–3 weeks of vaccination and there are no differentiating features by which the group with myocarditis could be identified as being at risk. In over 37,000 public health workers vaccinated, 2 cases of dilated cardiomyopathy were diagnosed 3 months after vaccination [47]. Despite this, the relationship between vaccination and cardiomyopathy has not been established. Virtually all cases of acute myopericarditis have resolved without any long-term myocardial dysfunction, although some continue to have atypical chest pain [48, 49]. EMB in a single patient demonstrated eosinophilic lymphocytic myocarditis responsive to immunosuppressive therapy [50].

HIV-related myocarditis Patients with HIV may develop left ventricular compromise at a higher rate than seen in the normal population [51]. Left ventricular compromise is more frequent in individuals who are immunosupressed (CD4, less than 400) or who are receiving anti-retroviral therapy [52]. Most of those who have depressed ventricular function have demonstrated myocarditis by EMB [53]. Myocarditis may be due to viral pathogens or to protozoa, bacteria, fungal agents or tuberculosis [54–56]. The HIV virus may also be responsible [57] and is often harbored in the dendritic and myocardial tissue. Prognosis for this group is poor.

Treatment The principles of treatment of acute heart failure related to myocarditis are virtually identical to those without myocardial inflammation, with the exception of the consideration of treatment of the inflammation itself. Evidence for initiation of diuretics, angiotensin converting receptor blockers, angiotensin converting enzyme inhibitors, inotropes, vasodilators, or vasoinotropic agents are not discussed in this chapter.

Immunosuppressive therapy Parillo et al. [58] treated 102 patients with dilated cardiomyopathy with prednisone or placebo. While there was initial improvement in the treated group at 3 months,

248


this did not persist in long-term assessment. Patients who had fibroblastic or lymphocytic infiltrate or immunoglobulin deposition on biopsy appeared to respond with a higher frequency than those who were “non-reactive”. This study demonstrates that treatment of patients with dilated cardiomyopathy with immunosuppressive therapy provides no long-term improvement. A number of small, non-randomized, non-placebo-controlled trials of patients with presumed myocarditis demonstrated improvement in ventricular performance and symptoms in those treated with immunosuppressive therapy. Mason et al. [8] demonstrated in the Myocarditis Treatment Trial that immunosuppressive agents, including prednisone with either cyclosporine or azathioprine, did not improve prognosis or ventricular function compared with the control group receiving standard medical therapy when evaluated at 28 weeks. The Dallas criteria for myocarditis were utilized as the diagnostic criteria for this disorder. Frustaci et al. [12] evaluated 112 of 652 biopsied patients with active lymphocytic myocarditis. Of the 112, 41 had progressive congestive heart failure despite standard medical therapy and were treated with immunosuppressive therapy. Approximately half appeared to improve significantly, as demonstrated by increasing their ejection fraction, and displayed evidence of healed myocarditis by repeat biopsy. A retrospective analysis of these samples demonstrated that viral persistence was present in those who failed to respond, while cardiac autoantibodies were present in those who did respond. This raises the question of an alternative histopathological marker of potential treatment response. Wojnicz et al. [13] showed that 84 of 202 patients with dilated cardiomyopathy had immune up-regulation, as evidence by HLA expression on EMB. This group was randomized to immunosuppressive therapy or placebo and evaluated at 3 months and 2 years. While there was no significant difference in the primary endpoint between the two groups, the ejection fraction, echocardiographic volume parameters, and functional class all improved in the immunosuppressive group. Of the patients in the immunosuppressive group, 72% met criteria for improvement at 3 months compared with 21% of the placebo group. These authors demonstrated a potential alternative marker for immunosuppressive therapy, even in patients with established cardiomyopathy.

Intravenous immunoglobulin Intravenous immunoglobulin is the standard treatment for children with acute cardiomyopathy and presumed myocarditis, based on a small randomized trial. McNamara et al. [59] randomized 62 adult patients with recent onset of dilated cardiomyopathy to intravenous immunoglobulin versus control. Patients displayed no difference in ejection fraction or survival, regardless of immunoglobulin therapy. This was in part due to the significant increase in ejection fraction demonstrated in

249

Kenneth L. Baughman

the placebo group. This confirms that many patients with new onset dilated cardiomyopathy have a condition from which they will spontaneously recover.

Immunoadsorption therapy Some patients with a new onset of dilated cardiomyopathy have been treated with immunoadsorption therapy. This usually includes administration of immunoglobulin. Preliminarily data have suggested [60] that this therapy may be beneficial in the short term, but may require repeated treatment to sustain the improvements demonstrated. While enticing, the mechanism and efficacy of this therapy remains ill defined (see the chapter by S.B. Felix and A. Staudt in this volume).

Summary Acute cardiomyopathy and heart failure may be due to myocardial inflammation. There are distinct clinical pathological entities that account for many of these patients including fulminant, giant cell, chronic active, eosinophilic, lyme, small pox, HIV, and peripartum related myocardial inflammation. While fulminant myocarditis will heal spontaneously and outcome is worsened by immunosuppressive therapy, giant cell myocarditis requires high-dose multi-drug immunosuppressive therapy. There are no data to direct management for patients with chronic active myocarditis, whereas those with eosinophilic myocarditis improve with withdrawal of the offending agent and corticosteroid therapy. Lyme myocarditis is improved by treating the offending pathogen. Cardiomyopathy and myocarditis in patients with HIV/AIDS may be due to a multitude of potential pathogens, which, for successful management, must be identified. Patients with small pox vaccine-related myopericarditis should improve spontaneously with only a small likelihood (if any) of the development of an end-stage cardiomyopathy. Patients with peripartum cardiomyopathy probably have myocarditis which resolves spontaneously. Only if this group does not improve with standard heart failure management within 2 weeks, should biopsy be pursued and consideration given to immunosuppressive therapy. The broader category of patients who present with a new or newly recognized cardiomyopathy, who may have inflammation, is of most interest. This may account for 50% of the patients presenting with cardiomyopathy. There are some data that this population may be responsive to immunosuppressive therapy if there is upregulation of the immune system or the presence of autoimmune antibodies and the absence of cardiotropic viruses persisting in the myocardial tissue. We are on the threshold of being able to identify this group of patients and design appropriate trials for their management.

250


References 1

2

3

4

5

6

7

8

9 10

11

12

1 Felker GM, Thompson RE, Hare JM, Hruban RH, Clemetson DE, Howard DL, Baughman KL, Kasper EK (2000) Underlying causes and long-term survival in patients with Initially unexplained cardiomyopathy. N Engl J Med 342(15): 1077–1084 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111(7): 887–893 Bowles NE, Ni J, Kearney DL, Pauschinger M, Schultheiss HP, McCarthy R, Hare J, Bricker JT, Bowles KR, Towbin JA (2003) Detection of viruses in myocardial tissues by polymerase chain reactions: Evidence of adenovirus as a common cause of myocarditis in children and adults. J Am Coll Cardiol 42: 466–472 Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon JT, Fenoglio JJ, Olsen EG, Schoen FJ (1987) Myocarditis. A histopathologic definition and classification. Am J Cardiovasc Pathol 1(1): 3–14 Hauck AJ, Kearney DL, Edwards WD (1989) Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: Implications for role of sampling error. Mayo Clinic Proc 64(10): 1235–45 Friedrich MG, Strohm O, Schulz-Menger L, Marciniak H, Luft FC, Dietz R (1998) Contrast media-enhanced magnetic resonance imagining visualizes myocardial changes in the course of viral myocarditis. Circulation 97(18): 1802–9 Mahrholdt H, Goedecke C, Wagner A, Meinhardt G, Athanasiadis A, Vogelsberg H, Fritz P, Klingel K, Kandolf R, Sechtem U (2004) Cardiovascular magnetic resonance assessment of human myocarditis: A comparison to histology and molecular pathology. Circulation 109(10): 1250–8 Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE (1995) A clinical trial of immunosuppressive therapy for myocarditis. N Engl J Med 333(5): 269–275 Baughman KL (2006) Diagnosis of myocarditis: Death of Dallas criteria. Circulation 113(4): 593–595 Bowles NE, Ni J, Kearney DL, Pauschinger M, Schultheiss HP, McCarthy R, Hare J, Bricker JT, Bowles KR, Towbin JA (2003) Detection of viruses in myocardial tissues by polymerase chain reaction. Evidence of adenovirus as a common cause of myocarditis in children and adults. J Am Coll Cardiol 42(3): 466–472 Why HJ, Meany BT, Richardson PJ, Olsen EG, Bowles NE, Cunningham L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89(6): 2582–9 Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107(6): 857–63

251

Kenneth L. Baughman

13

14

15 16

17

18 19 20

21

22

23

24

25 26 27 28

252

Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewiski P, Niklewski T, Zembala M, Polonski L, Rozek MM, Wodniecki J (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: Two-year follow-up results. Circulation 104(1): 39–45 Deckers JW, Hare JM, Baughman KL (1992) Complications of transvenous right ventricular endomyocardial biopsy in adult patients with cardiomyopathy: A seven-year survey of 546 consecutive diagnostic procedures in a tertiary referral center. J Am Coll Cardiol 19(1): 43–7 Lieberman EB, Hutchins GM, Herskowitz A, Rose NR, Baughman KL (1991) Clinicopathologic description of myocarditis. J Am Coll Cardiol 18(7): 1617–26 McCarthy RE, Boehmer JP, Hruban RH, Hutchins GM, Kasper EK, Hare JM, Baughman KL (2000) Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocarditis. N Engl J Med 342(10): 690–695 Felker GM, Boehmer RH, Hruban GM, Hutchins GM, Kasper EK, Baughman KL, Hare JM (2000) Echocardiographic findings in fulminant and acute myocarditis. J Am Coll Cardiol 36(1): 227–232 Amabile N, Fraisse A, Bouvenot J, Chetaille P, Ovaert C (2006) Outcome of acute fulminant myocarditis in children. Heart 92(9): 1269–1273 Cooper LT, Berry GJ, Shabetal R (1997) Idiopathic giant-cell myocarditis – Natural history and treatment. N Engl J Med 336(26): 1860–1866 Frustaci A, Cuoco L, Chimenti C, Pieroni M, Fioravanti G, Gentiloni N, Maseri A, Gasbarrini G (2002) Celiac disease associated with autoimmune myocarditis. Circulation 105(22): 2611–2618 Nash CL, Panaccione R, Sutherland LR, Meddings JB (2001) Giant cell myocarditis, in a patient with Crohn’s disease, treated with etanercept – A tumor necrosis factor-alpha antagonist. Can J Gastroenterol 15(9): 607–11 Okura Y, Dec GW, Hare JM, Kodama M, Berry GJ, Tazelaar HD, Bailey KR, Cooper LT (2003) A clinical and histopathologic comparison of cardiac sarcoidosis and idiopathic giant cell myocarditis. J Am Coll Cardiol 41(2): 322–9 Hanawa H, Izumi T, Saito Y, Ochiai Y, Okura Y, Inomata T, Hirono S, Ogawa Y, Saito R, Kodama M et al (1998) Recovery from complete atrioventricular block caused by idiopathic giant cell myocarditis after corticosteroid therapy. Jpn Circ J 62(3): 211–4 Menghini VV, Savcenko V, Olson LJ, Tazelaar HD, Dec GW, Kao A, Cooper LT (1999) Combined immunosuppression for the therapy of idiopathic giant cell myocarditis. Mayo Clinic Proc 74(12): 1221–1226 Cooper LT (2000) Giant cell myocarditis: Diagnosis and treatment. Herz 25(3): 291–8 Taliercio CP, Olney BA, Lie JT (1985) Myocarditis related to drug hypersensitivity. Mayo Clinic Proc 60(7): 463–8 Spear GS (1995) Eosinophilic explant carditis with eosinophilia: ? hypersensitivity to dobutamine infusion. J Heart Lung Transplant 14(4): 755–60 Herzog CA, Snover DC, Staley NA (1984) Acute necrotizing eosinophilic myocarditis. Br Heart J 52(3): 343–8


29 30 31 32

33

34

35 36

37

38

39

40 41

42

43 44

Midei MG, DeMent SH, Feldman AM, Hutchins GM, Baughman KL (1990) Peripartum myocarditis and cardiomyopathy. Circulation 81(3): 922–8 Sanderson JE, Olsen EG, Gatel D (1986) Peripartum hear disease: An endomyocardial biopsy study. Br Heart J 56(3): 285–91 Rizeq MN, Rickenbacher PR, Fowler MB, Billingham ME (1994) Incidence of myocarditis in peripartum cardiomyopathy. Am J Cardiol 74(5): 474–7 O’Connell JB, Costanzo-Nordin MR, Subramanian R, Robinson JA, Wallis DE, Scanlon PJ, Gunnar RM (1986) Peripartum cardiomyopathy: Clinical, hemodynamic, histologic and prognostic characteristics. J Am Coll Cardiol 8(1): 52–6 Felker GM, Jaeger CJ, Klodas E, Thiemann DR, Hare JM, Hruban RH, Kasper EK, Baughman KL (2000) Myocarditis and long-term survival in peripartum cardiomyopathy. Am Heart J 140(5): 785–91 Silwa K, Skudicky D, Bergemann A, Candy G, Puren A, Sareli P (2000) Peripartum cardiomyopathy: Analysis of clinical outcome, left ventricular function, plasma levels of cytokines and fas/APO-1. J Am Coll Cardiol 35(3): 701–5 Steere AC (2001) Lyme disease. N Engl J Med 345(2): 115–25 Steere AC, Batsford WP, Weinberg M, Alexander J, Berger HJ, Wolfson S, Malawista SE (1980) Lyme carditis: Cardiac abnormalities of lyme disease. Ann Intern Med 93(1): 8–16 McAlister HF, Klementowicz PT, Andrews C, Fisher JD, Feld M, Furman S (1989) Lyme carditis: An important cause of reversible heart block. Ann Intern Med 110(5): 339–45 van der Linde MR, Crijns HJ, de Koning J, Hoogkamp-Korstanje JA, de Graaf JJ, Piers DA, van der Galien A, Lie KI (1990) Range of atrioventricular conduction disturbances in lyme borreliosis: A report of four cases and review of other published reports. Br Heart J 63(3): 162–8 Stanek G, Klein J, Bittner R, Glogar D (1990) Isolation of Borrelia burgdorferi from the myocardium of a patient with longstanding cardiomyopathy. N Engl J Med 322(4): 249–52 Dec GW, Waldman H, Southern J, Fallon JT, Hutter AM, Palacios I (1992) Viral myocarditis mimicking acute myocardial infarction. J Am Coll Cardiol 20(1): 85–9 Sarda L, Colin P, Boccara F, Daou D, Lebtahi R, Faraggi M, Nguyen C, Cohen A, Slama MS, Steg PG, Le Guludec D (2001) Myocarditis in patients with clinical presentation of myocardial infarction and normal coronary angiograms. J Am Coll Cardiol 37(3): 786–92 Angelini A, Calzolari V, Calabrese F, Boffa GM, Maddalena F, Chioin R, Thiene G (2000) Myocarditis mimicking acute myocardial infarction: Role of endomyocardial biopsy in the differential diagnosis. Heart 84(3): 245–50 Wang K, Asinger RW, Marriott HJ (2003) ST-segment elevation in conditions other than acute myocardial infarction. N Engl J Med 349(22): 128–35 Klein RM, Schwartzkopff R, Strauer BE (1998) Evidence of endothelial dysfunction of

253

Kenneth L. Baughman

45

46

47

48

49

50

51

52

53

54

55

56 57

254

epicardial coronary arteries in patients with immunohistochemically proven myocarditis. Am Heart J 136(3): 389–97 Centers for Disease Control and Prevention: Morbidity and Mortality Weekly Report (2003) Update: Cardiac related events during the civilian smallpox vaccination program – United States 2003. J Am Med Assoc 290(1): 31–34 Halsell JS, Riddle JR, Atwood JE, Gardner P, Shope R, Poland GA, Gray GC, Ostroff S, Eckart RE, Hospenthal DR, Gibson RL et al (2003) Myopericarditis following smallpox vaccination among vaccinia-naïve U.S. military personnel. J Am Med Assoc 289(24): 3283–3289 Centers for Disease Control and Prevention (2003) Update: Cardiac related events during the civilian smallpox vaccination program – United States. MMWR Morb Mortal Wkly Rep 52(27): 639–42 Cassimatis DC, Atwood JE, Engler RM, Linz PE, Grabenstein JD, Vernalis MN (2004) Smallpox vaccination and myopericarditis: A clinical review. J Am Coll Cardiol 43(9): 1503–10 Eckart RE, Love SS, Atwood JE, Arness MK, Cassimatis DC, Campbell CL, Boyd SY, Murphy JG, Swerdlow DL, Collins LC et al (2004) Incidence and follow-up of inflammatory cardiac complications after smallpox vaccination. J Am Coll Cardiol 44(1): 201–5 Murphy JG, Wright RS, Bruce GK, Baddour LM, Farrell MA, Edwards WD, Kita H, Cooper LT (2003) Eosinophilic-lympocytic myocarditis after smallpox vaccination. Lancet 362(9393): 1378–80 Herskowitz A, Vlahov D, Willoughby S, Chaisson RE, Schulman SP, Neumann DA, Baughman KL (1993) Prevalence and incidence of left ventricular dysfunction in patients with human immunodeficiency virus infection. Am J Cardiol 71(11): 955–8 Barbaro G, Di Lorenzo G, Grisorio B, Barbarini G (1998) Incidence of dilated cardiomyopathy and detection of HIV in myocardial cells of HIV-positive patients. N Engl J Med 339(16): 1093–9 Herskowitz A, Wu TC, Willoughby SB, Vlahov D, Ansari AA, Beschorner WE, Baughman KL (1994) Myocarditis and cardiotropic viral infection associated with severe left ventricular dysfunction in late-stage infection with human immunodeficiency virus. J Am Coll Cardiol 24(4): 1025–32 Anderson DW, Virmani R, Reilly JM, O’Leary T, Cunnion RE, Robinowitz M, Macher AM, Punja U, Villaflor ST, Parrillo JE et al (1988) Prevalent myocarditis at necropsy in the acquired immunodeficiency syndrome. J Am Coll Cardiol 11(4): 792–9 Hofman R, Drici MD, Gibelin P, Michiels JF, Thyss A (1993) Prevalence of toxoplasma myocarditis in patients with the acquired immunodeficiency syndrome. Br Heart J 70(4): 376–81 Niedt GW, Schinelle RA (1985) Acquired immunodeficiency syndrome. Clinicopathologic study of 56 autopsies. Arch Pathol Lab Med 109(8): 727–34 Rodriguez ER, Nasim S, Hsia J, Sandin RL, Ferreira A, Hilliard BA, Ross AM, Garrett CT (1991) Cardiac myocytes and dendritic cells harbor human immunodeficiency


58

59

60

virus in infected patients with and without cardiac dysfunction: Detection by multiplex, nested, polymerase chain reaction in individually microdissected called from right ventricular endomyocardial biopsy tissue. Am J Cardiol 68(15): 1511–20 Parrillo JE, Cunnion RE, Epstein SE, Parker MM, Suffredini AF, Brenner M, Schaer GL, Palmeri ST, Cannon RO, Alling D (1989) A prospective, randomized, controlled trial of prednisone for dilated cardiomyopathy. N Engl J Med 321(16): 1119–21 McNamara DM, Holubkov R, Starling RC, Dec GW, Loh E, Torre-Amione G, Gass A, Janosko K, Tokarczyk T, Kessler P et al (2001) Controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy. Circulation 103(18): 2254–9 Staudt A, Schäper F, Stangl V, Plagemann A, Bohm M, Merkel K, Wallukat G, Wernecke KD, Stangl K, Baumann G, Felix SB (2001) Immunohistological changes in dilated cardiomyopathy induces by immunoadsorption therapy and subsequent immunoglobulin substitution. Circulation 103(22): 2681–6

255

Immunosuppressive treatment of inflammatory cardiomyopathy patients Andrea Frustaci and Cristina Chimenti Department of Cardiovascular and Respiratory Sciences, La Sapienza University, Rome, Italy

Abstract Inflammatory cardiomyopathy, defined as myocarditis associated with cardiac dysfunction is a main cause of heart failure. The acknowledgment of the underlying histological and molecular substrate may significantly contribute to improve the treatment of the disease that can be so devastating to require cardiac transplantation. Immunosuppression has been recognized as an effective therapeutic option for eosinophilic, giant cell, granulomatous myocarditis and for lymphocytic myocarditis associated with connective tissue disorders and with rejection of the transplanted heart. Treatment of idiopathic lymphocytic myocarditis particularly in its chronic form is still debated. Recent studies suggest that a negative PCR analysis of endomyocardial tissue for viral genomes and the detection of autoimmune markers, as HLA up-regulation and anti-heart autoantibodies, identify responders to immunosuppression improving the patients’ outcome even in an advanced stage of the disease. Recovery of cardiac function in responders to immunosuppression is associated with inhibition of cell death and with newly synthesized contractile material.

General view Inflammatory cardiomyopathy according to the WHO/ISFC definition is characterized by myocarditis associated with cardiac dysfunction [1]. Clinical presentation includes acute heart failure and cardiogenic shock [2], conduction disturbances or ventricular tachyarrhythmias [3]. In addition, myocardial inflammation may mimic an acute myocardial infarction [4, 5] and may even be responsible of global biventricular dysfunction in patients with severe coronary artery disease [6]. Recently myocarditis, often viral in origin, has been recognized as a cause of electrical and mechanical deterioration in patients with hypertrophic cardiomyopathy [7]. The mortality rate for patients with myocarditis and heart failure is 20% at 1 year and 56% at 4 years [8]. A definite diagnosis of myocarditis is possible only with endomyocardial biopsy [9] as clinical history and findings as well as standard non-invasive techniques usually do not allow identifying the inflammatory origin of myocardial damage. Over the last few years, the histological diagnosis of myocarditis


257

Andrea Frustaci and Cristina Chimenti

has been significantly improved by the introduction of immunohistochemical and molecular biology techniques [3, 10]. Immunohistochemistry on myocardial biopsies is actually considered mandatory for the quantification and characterization of inflammatory infiltrates that represent a main step in the diagnosis of myocarditis. In addition, in myocardial tissue specimens, the fundamental pathogenic role played by cardiotropic viruses and the complex multiple interactions with immune system can be recognized through the systematic application of molecular biology techniques (PCR or in situ hybridization) [11, 12]. Specific surface receptors that allow viruses to enter the myocardial cell [13] and the production of viral proteolytic enzymes capable of cleaving structural myocardial proteins such as dystrophin have been described [14]. With regard to the pathogenic role played by viruses in myocardial damage, important contributions may derive from the combination of molecular biology with recently developed techniques such as laser-capture microdissection. Using this technique, different cellular populations recognized by immunohistochemical staining can be dissected from a same paraffin section; the isolated cluster of cells can, therefore, be used separately for cell-specific molecular biology and proteomic studies [15]. This new technique has been applied in patients with myocarditis and EBV genome detectable by conventional PCR in the endomyocardial biopsies [16]. Nine patients with EBV-related inflammatory cardiomyopathy have been studied: lymphocytes and myocytes were microdissected from paraffin sections and DNA extracted from the collected cells was analyzed separately by PCR. Blood and myocardial samples from patients with positive and negative serology for EBV were used as controls. The EBV genome was detected in myocytes but not in infiltrating lymphocytes of patients, or in myocardial samples of controls. At a mean follow-up of 31 ± 14 months, despite full conventional heart failure therapy, a progressive cardiac dilation and dysfunction was observed in all patients. Intramyocyte detection of EBV genome supports a direct cytopathic role for this virus and suggests the need for an antiviral/immunomodulatory therapy. According to these studies, it is clear that detection of viral genome in myocardial tissue of patients with myocarditis represent the first step in identifying the best treatment for each subgroup of patients.

Immunosuppressive treatment in inflammatory cardiomyopathy Despite the improvement of diagnostic techniques in defining the characteristics and the etiology of the inflammatory process and the more specific comprehension of the mechanisms leading to myocardial damage, a specific standardized treatment of myocarditis is not yet available. In particular, immunosuppressive treatment of myocarditis has drawn much attention in the past, but less enthusiasm in the recent time because of controversial

258

Immunosuppressive treatment of inflammatory cardiomyopathy patients

results obtained both in children [17–20] and in adults [21, 22]. In the absence of specific markers of eligibility for immunosuppressive therapy, large trials produced misleading results [8], showing the absence of an evident improvement of survival in myocarditis patients treated with immunosuppressive drugs versus placebo. Immunosuppression is recommended essentially for the treatment of eosinophilic [23], granulomatous [24], and giant-cell myocarditis [25, 26] as well as lymphocytic myocarditis associated with connective tissue diseases [27] or with the rejection of a transplanted heart [28] (Tab. 1). Table 1. Responsiveness of myocarditis to immunosuppressive therapy Type of myocarditis

Response

Myocarditis associated with hypereosinophilic syndrome

+++

Myocarditis associated with connective tissue disorders

++

Rejection of transplanted heart

++

Giant-cell myocarditis

+/–

Viral/idiopathic myocarditis

+/–

With regard to idiopathic lymphocytic myocarditis, beside the acute phase for which a spontaneous resolution has been reported in up to 40% of the cases [29], there is growing evidence that many patients with idiopathic myocarditis and chronic heart failure are likely to benefit from immunosuppression. Wojnicz et al. [30] in a randomized placebo-controlled study suggested that an up-regulation of HLA antigens in the myocardial tissue of patients with lymphocytic myocarditis may identify a homogeneous subgroup of inflammatory dilated cardiomyopathy sustained by an autoimmune mechanism of damage, and may represent a marker of susceptibility to beneficial effects of immunosuppression. The evidence that only 70% of patients with myocarditis and HLA up-regulation really had an improvement of cardiac function from immunosuppression compared to 30% of the group on placebo could be in part due to the limit of the immunohistochemical semiquantitative method used to evaluate HLA up-regulation. In a retrospective study, the virological and immunological profiles of patients with active lymphocytic myocarditis and chronic heart failure, responders and nonresponders to immunosuppressive therapy, were analyzed [31]. Forty-one patients with a histological diagnosis of active lymphocytic myocarditis and characterized by a progressive heart failure with an ejection fraction (EF) of < 40%, lasting over 6 months in spite of conventional supportive therapy were studied. All patients were similar in terms of duration and severity of cardiac disease, histological findings and poor responsiveness to full conventional therapy. They received immunosuppressive therapy including 1 mg kg–1 day–1 prednisone for 4 weeks followed by 0.33 mg kg–1

259


day–1 for 5 months and 2 mg kg–1 day–1 azathioprine for 6 months. The patients were classified as responders if they had a decrease of at least one New York Heart Association (NYHA) class and an improvement in EF of r 10% compared with baseline measures. The patients were classified as non-responders if NYHA class and EF failed to improve or deteriorated, and if they faced major events like cardiogenic shock, heart transplantation or cardiac death. Of the 41 patients, 21 responded with prompt improvement in left ventricular EF (LVEF) and showed evidence of healed myocarditis at control biopsy. Conversely, 20 patients failed to respond of whom 12 remained stationary, 3 underwent cardiac transplantation and 5 died, showing a histological evolution toward dilated cardiomyopathy. Retrospective PCR on frozen endomyocardial tissue and evaluation of circulating cardiac autoantibodies on patients sera showed that non-responders had a high prevalence (85%) of viral genomes in the myocardium and no detectable cardiac autoantibodies in the serum, whereas 90% of responders were positive for autoantibodies, with only 3 (15%) presenting viral particles on PCR analysis of frozen endomyocardial tissue. Remarkably, responders to immunosuppression showed a recovery of cell myofibrillar content, associated with a decrease of cell death by both apoptosis and necrosis, and an increased in A-myosin heavy chain expression and A/B-myosin heavy chain ratio [32]. Among non-responders the myocardial persistence of enterovirus and adenovirus or their combination was associated with the worst clinical outcome. Pilot studies have demonstrated that these patients may benefit from the administration of B interferon with complete myocardial clearance of viral genome and long-term improvement of clinical symptoms and cardiac function [33]. Interestingly, serology for cardiotropic viruses failed to predict the presence of viral genome in the myocardium, and was positive in both patients with positive and negative PCR results, suggesting as in other studies [34] that this tool cannot be used as an alternative to endomyocardial tissue PCR for the diagnosis of viral myocarditis. To confirm these encouraging results in a prospective manner, we decided to perform a further study enrolling patients with myocarditis and chronic heart failure and submitting all patients showing no evidence of viral genome in myocardial tissue to immunosuppressive treatment. At least two frozen myocardial samples from each patient were used for PCR for the most common cardiotropic viruses (adenovirus, enterovirus, parvovirus B19, Epstein Barr virus, cytomegalovirus, herpes simplex virus, hepatitis C virus, influenza virus). Among 89 consecutive patients, 52 were virus negative on PCR. Among these, 2 patients were excluded because of contraindications to steroidal treatment; 33 were treated with 1 mg kg–1 day–1 prednisone for 4 weeks, followed by 0.33 mg kg–1 day–1 for 5 months and 2 mg kg–1 day–1 azathioprine for 6 months. The patients were classified as responders and non-responders according to the clinical and instrumental criteria adopted in the previously mentioned retrospective study [31]. After 6 months of immunosuppressive treatment 45 patients (86%) showed a significant improvement of cardiac function and dimensions [LVEF from 26.5 ± 4.2

260


to 48.1 ± 6.0%, left ventricular end diastolic diameter (LVEDD) from 66.7 ± 6.0 to 46.5 ± 6.3 mm, fractional shortening from 23.6 ± 4.5 to 46.0 ± 6.3%], while 6 patients maintained stable clinical picture and cardiac function parameters. Remarkably, even patients with severe advanced disease (LVEDD up to 90 mm and LVEF < 20%) could significantly improve (LVEDD decreased by 20–30% and LVEF enhanced up to 40%, while NYHA class changed from IV to II) allowing patients to resume their previous work. Comparing baseline features of responders and non-responders we were unable to identify any clinical, echocardiographic or histological marker predicting inefficacy of treatment. These data obtained prospectively, with the decision to use immunosuppressive treatment based on myocardial evidence of viral genome, confirm our previous findings. The lack of response in a minority (13%) of virus-negative patients raises questions on the possible presence of additional viruses not included in the screen or the persistence of mechanisms inducing myocyte damage and dysfunction not susceptible to immunosuppression.

Conclusions In conclusion, the results of several studies have shown that immunosuppressive therapy is an effective option in a majority of patients with chronic non-viral myocarditis. Molecular studies for the detection of viral genome in myocardial tissue represent the first step to characterize myocarditis patients and to direct therapeutic strategies. Further studies improving our knowledge on viral and immuno-mediated mechanisms of cardiac damage will allow to further personalizing of patients’ treatment.

References 1

2 3

4

5

Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I et al (1996) Report of the 1995 WHO/ISFC task force on the definition and classification of cardiomyopathies. Circulation 93: 841–842 Feldman AM, McNamara D (2000) Myocarditis. N Engl J Med 343: 1388–98 Chimenti C, Calabrese F, Thiene G, Pieroni M, Maseri A, Frustaci A (2001) Inflammatory left ventricular microaneurysms as a cause of apparently idiopathic ventricular tachyarrhythmias. Circulation 104: 168–73 Narula J, Khaw BA, Dec GW Jr, Palacios IF, Southern JF, Fallon JT, Strauss HW, Haber E, Yasuda T (1993) Brief report: Recognition of acute myocarditis masquerading as acute myocardial infarction. N Engl J Med 328;100–104 Angelini A, Calzolari V, Calabrese F, Boffa GM, Maddalena F, Chioin R, Thiene G

261


6

7

8

9

10

11

12

13

14

15

16

17

262

(2000) Myocarditis mimicking acute myocardial infarction: Role of endomyocardial biopsy in the differential diagnosis. Heart 84: 245–50 Frustaci A, Chimenti C, Maseri A (1999) Global bi-ventricular dysfunction in patients with symptomatic coronary artery disease may be caused by myocarditis. Circulation 99: 1295–1299 Frustaci A, Verardo R, Caldarulo M, Acconcia MC, Russo MA, Chimenti C (2007) Myocarditis in hypertrophic cardiomyopathy patients presenting acute clinical deterioration. Eur Heart J 28: 733–740 Mason JW, O’ Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE (1995) A clinical trial of immunosuppressive therapy for myocarditis. N Engl J Med 333: 269–75 Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon JT, Fenoglio JJ, Olsen EG, Schoen FJ (1987) Myocarditis. A histopatologic definition and classification. Am J Cardiovasc Pathol 1: 3–14 Kuhl U, Seeberg B, Schultheiss HP, Strauer BE (1994) Immunohistological characterization of infiltrating lymphocytes in biopsies of patients with clinically suspected dilated cardiomyopathy. Eur Heart J 15: 62–7 Pauschinger M, Bowles NE, Fuentes-Garcia F, Pham V, Kuhl U, Schwimmbeck PL, Schultheiss HP, Towbin JA et al (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99: 1348–54 Pauschinger M, Doerner A, Kuehl U, Schwimmbeck PL, Poller W, Kandolf R, Schultheiss HP (1999) Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99: 889–95 Noutsias M, Fechner H, de Jonge H, Wang X, Dekkers D, Houtsmuller AB, Pauschinger M, Bergelson J, Warraich R, Yacoub M et al (2001) Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: Implications for cardiotropic viral infections. Circulation 104: 275–80 Badorff C, Lee GH, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, Knowlton KU (1999) Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med 5: 320–6 Gjerdrum LM, Lielpetere I, Rasmussen LM, Bendix K, Hamilton-Dutoit S (2001) Laserassisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis. Identification of cell populations using immunohistochemistry and in situ hybridization. J Mol Diagn 3: 105–110 Chimenti C, Russo A, Pieroni M, Calabrese F, Verardo R, Thiene G, Russo MA, Maseri A, Frustaci A (2004) Intramyocyte detection of Epstein-Barr virus genome by laser capture microdissection in patients with inflammatory cardiomyopathy. Circulation 110: 3534–9 Chan KY, Iwahara M, Benson LN, Wilson GJ, Freedom RM (1991) Immunosuppressive


18

19

20

21

22 23

24

25

26 27 28 29

30

31

therapy in the management of acute myocarditis in children; a clinical trial. J Am Coll Cardiol 17: 458–460 Matitiau A, Perez-Atayde A, Sanders SP, Sluysmans T, Parness IA, Spevak PJ, Colan SD (1994) Infantile dilated cardiomyopathy. Relation of outcome to left ventricular mechanics, hemodynamics, and histology at the time of presentation. Circulation 90: 1310–8 Lee KJ, McCrindle BW, Bohn DJ, Wilson GJ, Taylor GP, Freedom RM, Smallhorn JF, Benson LN (1999) Clinical outcomes of acute myocarditis in childhood. Heart 82: 226–33 Kleinert S, Weintraub RG, Wilkinson JL, Chow CW (1997) Myocarditis in children with dilated cardiomyopathy: Incidence and outcome after dual therapy immunosuppression. J Heart Lung Transplant 16: 1248–54 Parrillo JE, Cunnion RE, Epstein SE, Parker MM, Suffredini AF, Brenner M, Schaer GL, Palmeri ST, Cannon RO, Alling D et al (1989) A prospective, randomized, controlled trial of prednisone for dilated cardiomyopathy. N Engl J Med 321: 1061–8 Kuhl U, Schultheiss HP (1995) Treatment of chronic myocarditis with corticosteroids. Eur Heart J 16 Suppl O: 168–72 Frustaci A, Gentiloni N, Chimenti C, Natale L, Gasbarrini G, Maseri A et al (1998) Necrotizing myocardial vasculitis in Churg-Strauss syndrome: Clinicohistologic evaluation of steroids and immunosuppressive therapy. Chest 114: 1484–9 Badorff C, Schwimmbeck PL, Kuhl U, Gerhold M, Stein H, Schultheiss HP (1997) Cardiac sarcoidosis: Diagnostic validation by endomyocardial biopsy and therapy with corticosteroids. Z Kardiol 86: 9–14 Cooper LT Jr, Berry GJ, Shabetai R (1997) Idiopathic giant-cell myocarditis-natural history and treatment. Multicenter Giant Cell Myocarditis Study Group Investigators. N Engl J Med 336: 1860–6 Frustaci A, Chimenti C, Pieroni M, Gentiloni N (2000) Giant cell myocarditis responding to immunosuppressive therapy. Chest 117: 905–907 Frustaci A, Gentiloni N, Caldarulo M (1996) Acute myocarditis and left ventricular aneurysm as presentations of systemic lupus erythematosus. Chest 109: 282–4 Billingham ME (1997) Can histopathology guide immunosuppression for cardiac allograft rejection in the light of new techniques? Transplant Proc 29: 35S-36S Dec GW Jr, Palacios IF, Fallon JT, Aretz HT, Mills J, Lee DC, Johnson RA (1985) Active myocarditis in the spectrum of acute dilated cardiomyopathies. Clinical features, histologic correlates, and clinical outcome. N Engl J Med 312: 885–90 Wojnicz R, Nowalany-Kozielska E, Wojciechowska C, Glanowska G, Wilczewski P, Niklewski T, Zembala M, Polonski L, Rozek MM, Wodniecki J (2001) Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: Two-year follow-up results. Circulation 104: 39–45 Frustaci A Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri A (2003) Active lymphocytic myocarditis: Virologic and immunologic profile of responders vs nonresponders to immunosuppressive therapy. Circulation 107: 857–63

263


32

33

34

264

Frustaci A, Chimenti C, Pieroni M, Salvatori L, Morgante E, Sale P, Ferretti E, Petrangeli E, Gulino A, Russo MA (2006) Cell death, proliferation and repair in human myocarditis responding to immunosuppressive therapy. Mod Pathol 19: 755–65 Kuhl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-beta treatment of patients with enteroviral and adenoviral cardiomyopathy causes effective virus clearance and long-term clinical improvement. Circulation 107: 2793–8 Martin AB, Webber S, Fricker FJ, Jaffe R, Demmler G, Kearney D, Zhang YH, Bodurtha J, Gelb B, Ni J et al (1994) Acute myocarditis. Rapid diagnosis by PCR in children. Circulation 90: 330–9

Antiviral interferon-B treatment in patients with chronic viral cardiomyopathy Heinz-Peter Schultheiss, Michel Noutsias and Uwe Kühl Department of Cardiology and Pneumonology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany

Abstract The etiology of “idiopathic” dilated cardiomyopathy (DCM) is due to persistence of viruses in ca. 60% of the patients presenting with DCM. This can be concluded from the high rate of detectable viral genomes in endomyocardial biopsy (EMB) samples in such patients. The pivotal role of interferons (IFN) as a natural defense system against viruses is well documented by experimental data. In an open-label pilot trial, 22 patients with EMB-proven persistence of viral genomes [enterovirus (EV), 15 patients; adenovirus (ADV), 7 patients] were treated with recombinant IFN-B1a. In parallel to the viral elimination proven after 6-month antiviral treatment, left ventricular ejection fraction (LVEF) improved, end-diastolic diameters declined significantly, and an amelioration of heart failure symptoms was evident. In patients with immunohistologically proven intramyocardial inflammation a substantial decrease of infiltrates and cell adhesion molecule expression was noted after IFN treatment. There were no severe side effects. Based on the favorable results of this pilot study, the randomized European-wide multicenter BICC trial (Betaferon®: Interferon-B in Patients With Chronic Viral Cardiomyopathy) for IFN-B1b treatment of patients with EMB-proven persistence of EV, ADV and parvovirus B19 (B19V) was conducted.

Introduction Advances in heart failure treatment have resulted in improved survival in these patients [1]. However, current heart failure therapy is symptomatic and does not influence specific underlying pathogenic mechanisms. Many patients presenting with dilated cardiomyopathy (DCM) progress to terminal heart failure, and DCM represents the most common heart failure entity requiring heart transplantation [2]. A genetic origin of DCM has been reported in up to 25% of cases, but the majority of these cases are sporadic. A viral or immune pathogenesis is suspected in ca. 60% of patients presenting with “idiopathic” DCM [3, 4]. This is in line with the transition from acute myocarditis (AMC) to DCM [5]. For the proof of a myocardial viral infection, endomyocardial biopsies (EMBs) need to be investigated with the


265

Heinz-Peter Schultheiss, Michel Noutsias and Uwe Kühl

sensitivity of the nested polymerase chain reaction (nPCR) to detect viral genomes even at low copy numbers [6]. In addition to the “classic” myocarditis-inducing coxsackievirus B (CVB), belonging to the group of enteroviruses (EV), further, and partly substantially more prevalent, cardiotropic viruses have been detected in acute myocarditis (AMC) and DCM patients: parvovirus B19 (B19V), human herpes-virus type 6 (HHV6), adenovirus (ADV) and Epstein-Barr virus (EBV) [7–11]. Efficacy of a specific antiviral approach to eliminate these viruses, and thus a major pathogenic key player for progressive deterioration of left ventricular ejection fraction (LVEF) and heart failure symptoms, would constitute be a desirable and feasible approach to synergistically improve the actual hemodynamic situation, and possibly also the long-term prognosis of these patients [12].

Course of viral infection in human AMC and DCM During the stage of AMC, complete or partial recovery of myocardial function may occur even in severe cases of cardiac dysfunction [5]. The retrospective study by McCarthy et al. [13] for the first time demonstrated that a fulminant inflammatory process is actually associated with a better long-term outcome compared with the non-fulminant presentation of myocarditis (survival rates for fulminant and nonfulminant disease were 93% vs 45%, respectively, after 5.6 years of follow-up). Based upon this crucial retrospective study, it may be deduced that intramyocardial inflammation is not detrimental per se; moreover, it is evident that inflammation may merely reflect the attempt of the immune system to eliminate cardiotropic viruses. The reversibility of ventricular dysfunction suggests that initial impairment of myocardial contractility is not caused by an irreversible loss of cardiomyocytes or irreversible destruction of the cell-matrix integrity. The initial deterioration of ventricular function, to some extent, might be caused by the production of negative inotropic mediators of the immune response to viral infection, such as cytokines [e.g., tumor necrosis factor (TNF)-A, interleukin (IL)-1B, IL-6, interferon (IFN)-G], or the induction of inducible NO synthase (iNOS) causing increased amounts of NO, which reversibly interferes with myocardial cell function and matrix integrity [14–17]. Therefore, myocardial function may recover if these cardiodepressive substances decrease during spontaneous virus clearance and resolution of the inflammatory process [18–21]. In a number of patients, however, immunological processes fail to clear viruses completely. Thus, although classically regarded as agents of self-limiting infections, a considerable body of evidence implicates persistent viruses in the etiology of chronic myocarditis and DCM, and with the progressive course of the disease [22–26]. Restricted viral replication, even a relatively small number of infected myocardial cells, has been shown to be sufficient for the maintenance of chronic inflammation, morphological alterations of the structural integrity of the myocardium, and interfer-

266

Antiviral interferon-B treatment in patients with chronic viral cardiomyopathy

ence with myocyte function in animal models of CVB-infected mice and thus may be responsible for progressive myocardial impairment [27, 28]. Active EV replication in the myocardium of DCM patients implies that EVs may alter active cellular metabolism and, consequently, may produce an ongoing myocytopathic effect even in late persistent infection [27, 29, 30]. One can assume that both the direct myocytopathic effect of cardiotropic viruses and the activation of the immune system against viral antigens and/or cryptic myocardial antigens released by viral infection perpetuates the cycle of cardiac dysfunction observed in DCM patients. In addition, virus and immune-mediated disturbance of myocardial cell metabolism, energy production or energy transfer may also contribute to myocardial dysfunction [31–33]. Interference of virus and immune-mediated processes with cell matrix integrity might be of considerable importance during the acute and chronic stage of the disease [34].

Antiviral immunomodulation with IFNs A retrospective analysis by Frustaci et al. [25] confirmed that patients with viral persistence (except for hepatitis C) do not improve, and may even deteriorate during immunosuppressive treatment. These data confirm that DCM patients with chronic viral persistence should not be subjected to immunosuppressive strategies [35], and are in line with insights from animal experiments [36]. The important role of IFNs as a natural defense system against viruses is well documented by three lines of experimental [37] and clinical [38] data: 1. A strong correlation has been established between IFN production and natural recovery in many viral infections. 2. Inhibition of IFN production or its action enhances the severity of infection. 3. Treatment with IFN protects against viral infections. The antiviral effects of IFN are largely independent from the virus type, and result in an intracellular inhibition of the viral replication cycle. IFNs increase the resistance towards viral replication even in cells that neighbor infected cells but have not themselves been infected yet. Immunomodulatory effects include activation of macrophages and natural killer cells as well as enhancement of major histocompatibility complex (MHC) antigen expression [39]. Three types of IFNs have been identified, which differ both in their structure and in their antigenic properties: IFN-A, derived from leukocytes; IFN-B from fibroblasts; and IFN-G, which is derived from lymphocytes. IFNs react with specific receptors on the cell surfaces to activate cytoplasmic signal proteins. These proteins enter the nucleus to stimulate cellular genes that encode a number of further key proteins responsible for the defensive activity, including antiviral, antiproliferative, antitumor and immunomodulatory effects.

267


Anti-viral IFN treatment in human chronic viral cardiomyopathy Early reports on relatively small patient cohorts indicated beneficial hemodynamic effects of IFN-A treatment in DCM patients, and partial elimination of viral infections [40, 41]. These studies also indicated that IFN treatment is well tolerated in DCM patients. However, only EV genomes were investigated in some of the patients in these investigations. Since the spectrum of cardiotropic viruses including the even substantially more prevalent B19V, HHV6, ADV and EBV were not known at the time these studies were conducted, the efficacy of IFN treatment stratified by these newly recognized cardiotropic viruses was not investigated. The antiviral potential of IFN-B against CVB has been demonstrated in vitro and in animal models [42, 43]. In an open-label pilot trial, 22 patients with EMBproven persistence of viral genomes (EV: 15 patients; ADV: 7 patients) were treated with recombinant IFN-B1a [44]. These patients were carefully selected to have a chronic course of the disease with an average duration of cardiac symptoms of 44 ± 27 months (Tab. 1). Acute deterioration of LVEF or patients suggestive of AMC were excluded from the study due to known potential of LVEF recovery in the spontaneous course of the disease [5]. During the screening period preceding the active treatment, left ventricular end-diastolic (LVEDD) and end-systolic diameters (LVESD) increased significantly and left ejection fraction (LVEF) did not improve during the 22 months before treatment, despite extensive conventional heart failure medication. At baseline, most patients were in New York Heart Association functional classes II and III. According to the histological analysis (Dallas Criteria), neither active nor borderline myocarditis were detected in the EMBs. An inflammatory process was detected in 7 patients presenting with increased numbers of CD3+ lymphocytes (> 7.0/mm2) in the immunohistological analysis [45]. The IFN-B therapy (Beneferon®; Renschler, Laupheim, Germany) followed a stepped regimen to reduce the flu-like side effects typical of the initial phase of an IFN therapy. The subcutaneous administration was initiated at a dose of 2 s 106 U IFN-B three times a week on alternate days, and was increased to 12 s 106 U during the second and 18 s 106 U during the third week. By the end of week 24, the IFN-B treatment was discontinued. The protocol was approved by the Human Research Committee of the Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin. All patients gave written informed consent before treatment. Treatment of these 22 patients did not result in any severe adverse effects of the IFN-B therapy necessitating cessation of treatment. The major complaints reported during the initial 2–4 weeks were injection site reactions, fatigue, arthralgia, headache, influenza-like symptoms and reversible progression of pre-existing diseaseassociated symptoms such as dyspnea, angina or arrhythmias (Tab. 2). Most symptoms were mild or moderate and disappeared during the first weeks of treatment.

268


Table 1. Demographic and clinical characteristics of 22 virus-positive patients with persistent LV dysfunction included in the IFN-B treatment Demographic Number of patients Mean (SD) age (years) Sex (male/female)

22 51.8±13.6* 13/9

Clinical characteristics NYHA functional class I II III IV Fatigue Angina Dyspnea Arrhythmias History of cardiac symptoms (months)

1 (5%)§ 10 (45%) 10 (45%) 1 (5%) 18 (82%) 7 (32%) 18 (82%) 11 (50%) 44 ± 27

Endomyocardial biopsy Enterovirus/adenovirus Active/borderline myocarditis Immunohistology (CD3+ T-lymphocytes/HPF)

15/7 0/0 5.7 ± 5.5

Medication ACE inhibitors B-blockers Glycosides Diuretics Warfarin

73% 64% 64% 59% 50%

*mean ± standard deviation § n (percent) Reproduced with permission from [48].

The clinical investigations and the results of EMB diagnostics obtained within a period of 6 weeks after completion of the IFN-B treatment are listed in Table 3 in comparison to the pretreatment data. At follow-up, there was no evidence of either EV or ADV in the EMBs, consistent with viral elimination under antiviral IFN-B treatment. A reduction in LV dimensions and improvement in LVEF occurred in parallel with improvement of clinical symptoms. Patients with regional as well as global contractile dysfunction improved, but improvement was more pronounced in patients with global LV dysfunction at study entry (Fig. 1). At a mean follow-up of 12

269


Table 2. Main side effects reported during the first 4 weeks of IFN-B treatment. Injection site reaction

95.4%

Fatigue

68.2%

Arthralgia

59.1%

Dyspnea

50.0%

Headache

40.1%

Angina pectoris

36.3%

Influenza-like symptoms

31.8%

Dizziness

31.8%

Arrhythmias

27.3%

Myalgia

22.7%

Fever

13.6%

Nausea

13.6%

Table 3. Clinical, hemodynamic, virological, and immunohistological data of patients before and after IFN-B therapy. Before IFN-B

After IFN-B

p value

LV angiography: LVEF (%)

44.7 ± 15.5

53.1 ± 16.8

< 0.001

Echocardiography: LVEDD (mm) LVESD (mm) NYHA

59.7 ± 11.1 43.4 ± 13.6 2.5 ± 0.6

56.5 ± 11.1 39.4 ± 12.1 1.7 ± 0.7

< 0.001 < 0.001 < 0.05

Endomyocardial biopsy: PCR / molecular biology: EV ADV

15 7

0 0

Histology – Myocarditis

0

0

Immunohistology: Inflammation (n = 7) No inflammation (n = 15)

19.2 ± 4.8 2.6 ± 1.8

4.0 ± 4.6 2.9 ± 3.1

< 0.05 n.s.

ADV: adenovirus; EV: enterovirus; LV: left ventricular; LVEF: left ventricular ejection fraction; LVEDD: left ventricular end-diastolic diameter; LVESD: left ventricular end-systolic diameter; NYHA: New York Heart Association functional class; PCR: polymerase chain reaction. Reproduced with permission from [48].

270


Figure 1. Course of LVEF in patients before and after IFN-B treatment in patients with regional wall motion abnormalities (n = 12; LVEF > 50%) and global impairment of LV function (n = 10; LVEF < 50%). Reproduced with permission from [48].

months, recovery of LVEF had been preserved in all patients, without any evidence for a new decline of LVEF. Importantly, 5 patients demonstrated further improvement of LVEF in these additional 6 months of follow-up time, even after cessation of the IFN-B treatment. Furthermore, 67% of patients showed an improvement of at least one NYHA functional class (Fig. 2). None of the patients showed deterioration in NYHA functional class. In parallel with the virus clearance, myocardial inflammation as assessed by immunohistology resolved significantly (Fig. 3). A small series of two patients with EV persistence confirmed the efficacy of IFN treatment in both viral elimination and augmenting LVEF [46]. These findings during IFN-B treatment, together with the lack of improvement before specific antiviral treatment suggest that a substantial proportion of the LV dysfunction and wall motion abnormalities were caused by the persistent viral infection in these carefully selected patients, which eventually resolved after elimination of the responsible injurious agents, and was thus reversible. The data furthermore suggest that the beneficial clinical effect of IFN-B based upon the elimination of cardiotropic viruses may occur even in DCM patients presenting with a long history of heart failure symptoms. Ongoing viral persistence has been associated with an adverse prognosis including early death or requirement for transplantation [23, 24, 26]. Experimental and

271


Figure 2. Clinical improvement after 6 months of IFN-B therapy. NYHA: New York Heart Association functional class. Reproduced with permission from [48].

Figure 3. Lymphocyte infiltration density in EMBs from patients before and after IFN-B treatment. Reproduced with permission from [48].

272


clinical data highlight that an imbalance of the cytokine network and a defect in the cytokine-induced immune response are major causes leading to the development of viral persistence and progressive myocardial dysfunction.

The BICC Trial Based on the favorable results of this pilot study, a randomized, double-blind, placebo-controlled, European-wide multicenter study was initiated. In this BICC trial (Betaferon® / Interferon-B in Patients With Chronic Viral Cardiomyopathy), the efficacy of IFN-B1b (Schering, Berlin) was investigated in patients with EMBproven EV-, ADV- and B19V-persistence. After clinical screening and EMB diagnostics, 143 patients with chronic viral cardiomyopathy were included from 31 medical centers in 7 European countries, and were randomized to three study arms: placebo, 4 s 106 IU IFN-B1b per injection, and 8 s 106 IU IFN-B1b per injection, respectively (phase II trial). The treatment period duration was again 24 weeks, and, after EMBs, patients were followed-up for another 24 weeks, including quality of life and echocardiographic assessment. The trial has not yet been published. However, the study results presented at the American Heart Association meeting in 2008 demonstrated either viral elimination or substantial decrease of the viral load, accompanied by an improvement of the LVEF and heart failure symptoms [47]. The final analysis of the different patient strata and the published trial are still awaited. These promising data argue for a phase III trial with this safe antiviral approach in patients with chronic viral cardiomyopathy. However, the BICC trial also showed that, although at lower viral loads, especially B19V can persist after IFN-B1b treatment in some patients. This finding, which was not observed for EV and ADV in the pilot trial [48], poses a series of questions that need to be addressed in future investigations and therapeutic trials: 1. PCR proof of B19V – is it pathogenically relevant in every case [49]? 2. What is the therapeutic significance of the highly variable B19V copy numbers in EMBs, and of the adaptive immune response [50, 51]? 3. What is the therapeutic relevance of the fairly new finding of diverse B19V genotypes in diverse patient cohorts [52]? 4. Does B19V have diverse viral replication patterns in myocardial tissues, and do these replication patterns have prognostic and therapeutic implications similar to the situation reported for EV [24, 53]?

References 1

Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson

273


2

3

4

5

6

7

8

9

10

11

274

PA, Stromberg A, van Veldhuisen DJ, Atar D, Hoes AW et al (2008) ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: The Task Force for the diagnosis and treatment of acute and chronic heart failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur J Heart Fail 10: 933–89 Taylor DO, Stehlik J, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, Kucheryavaya AY, Rahmel AO, Hertz MI (2009) Registry of the international society for heart and lung transplantation: Twenty-sixth official adult heart transplant report-2009. J Heart Lung Transplant 28: 1007–22 Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi F, Charron P, Dubourg O, Kühl U, Maisch B, McKenna WJ et al (2008) Classification of the cardiomyopathies: A position statement from the European Society Of Cardiology Working Group on Myocardial and Pericardial Diseases. Eur Heart J 29: 270–6 Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I et al (1996) Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 93: 841–2 D’Ambrosio A, Patti G, Manzoli A, Sinagra G, Di Lenarda A, Silvestri F, Di Sciascio G (2001) The fate of acute myocarditis between spontaneous improvement and evolution to dilated cardiomyopathy: A review. Heart 85: 499–504 Bowles NE, Richardson PJ, Olsen EG, Archard LC (1986) Detection of Coxsackie-Bvirus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1: 1120–3 Pauschinger M, Bowles NE, Fuentes-Garcia FJ, Pham V, Kühl U, Schwimmbeck PL, Schultheiss HP, Towbin JA (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99: 1348–54 Kühl U, Pauschinger M, Bock T, Klingel K, Schwimmbeck CP, Seeberg B, Krautwurm L, Noutsias M, Poller W, Schultheiss HP et al (2003) Parvovirus B19 infection mimicking acute myocardial infarction. Circulation 108: 945–50 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111: 887–93 Angelini A, Calzolari V, Calabrese F, Boffa GM, Maddalena F, Chioin R, Thiene G (2000) Myocarditis mimicking acute myocardial infarction: Role of endomyocardial biopsy in the differential diagnosis. Heart 84: 245–50 Caforio AL, Calabrese F, Angelini A, Tona F, Vinci A, Bottaro S, Ramondo A, Carturan E, Iliceto S, Thiene G et al (2007) A prospective study of biopsy-proven myocarditis: Prognostic relevance of clinical and aetiopathogenetic features at diagnosis. Eur Heart J 28: 1326–33


12 13

14

15

16

17

18

19

20

21

22 23

24

Liu PP, Mason JW (2001) Advances in the understanding of myocarditis. Circulation 104: 1076–82 McCarthy RE, Boehmer JP, Hruban RH, Hutchins GM, Kasper EK, Hare JM, Baughman KL (2000) Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocarditis. N Engl J Med 342: 690–5 Freeman GL, Colston JT, Zabalgoitia M, Chandrasekar B (1998) Contractile depression and expression of proinflammatory cytokines and iNOS in viral myocarditis. Am J Physiol 274: H249–H258 Lowenstein CJ, Hill SL, Lafond Walker A, Wu J, Allen G, Landavere M, Rose NR, Herskowitz A (1996) Nitric oxide inhibits viral replication in murine myocarditis. J Clin Invest 97: 1837–43 Bachmaier K, Neu N, Pummerer C, Duncan GS, Mak TW, Matsuyama T, Penninger JM (1997) iNOS expression and nitrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor 1. Circulation 96: 585–91 Li YY, Feng Y, McTiernan CF, Pei W, Moravec CS, Wang P, Rosenblum W, Kormos RL, Feldman AM (2001) Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices. Circulation 104: 1147–52 Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL (1992) Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 257: 387–9 Torre Amione G, Kapadia S, Lee J, Durand JB, Bies RD, Young JB, Mann DL (1996) Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart. Circulation 93: 704–11 Testa M, Yeh M, Lee P, Fanelli R, Loperfido F, Berman JW, LeJemtel TH (1996) Circulating levels of cytokines and their endogenous modulators in patients with mild to severe congestive heart failure due to coronary artery disease or hypertension. J Am Coll Cardiol 28: 964–971 Bozkurt B, Kribbs SB, Clubb FJ Jr, Michael LH, Didenko VV, Hornsby PJ, Seta Y, Oral H, Spinale FG, Mann DL (1998) Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats. Circulation 97: 1382–91 Baboonian C, Treasure T (1997) Meta-analysis of the association of enteroviruses with human heart disease. Heart 78: 539–43 Why HJ, Meany BT, Richardson PJ, Olsen EG, Bowles NE, Cunningham L, Freeke CA, Archard LC (1994) Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89: 2582–9 Fujioka S, Kitaura Y, Ukimura A, Deguchi H, Kawamura K, Isomura T, Suma H, Shimizu A (2000) Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 36: 1920–6

275


25

26

27

28

29

30

31

32

33

34

35

36 37 38 39

276

Frustaci A, Chimenti C, Calabrese F, Pieroni M, Thiene G, Maseri A (2003) Immunosuppressive therapy for active lymphocytic myocarditis: Virological and immunologic profile of responders versus nonresponders. Circulation 107: 857–63 Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112: 1965–70 Wessely R, Henke A, Zell R, Kandolf R, Knowlton KU (1998) Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: An approach to the study of enteroviral persistence in cardiac myocytes. Circulation 98: 450–7 Klingel K, Kandolf R (1993) The role of enterovirus replication in the development of acute and chronic heart muscle disease in different immunocompetent mouse strains. Scand J Infect Dis Suppl 88: 79–85 Liu P, Aitken K, Kong Y, Opavsky MA, Martino T, Dawood F, Wen W, Kozieradzki I, bachmaier K, Straus D et al (2000) The tyrosine kinase p56Ick is essential in coxsackievirus B3-mediated heart disease. Nat Med 6: 429–434 Pauschinger M, Doerner A, Kuehl U, Schwimmbeck PL, Poller W, Kandolf R, Schultheiss HP (1999) Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99: 889–95 Badorff C, Noutsias M, Kühl U, Schultheiss HP (1997) Cell-mediated cytotoxicity in hearts with dilated cardiomyopathy: Correlation with interstitial fibrosis and foci of activated T lymphocytes. J Am Coll Cardiol 29: 429–34 Schulze K, Witzenbichler B, Christmann C, Schultheiss HP (1999) Disturbance of myocardial energy metabolism in experimental virus myocarditis by antibodies against the adenine nucleotide translocator. Cardiovasc Res 44: 91–100 Doerner A, Pauschinger M, Schwimmbeck PL, Kuhl U, Schultheiss HP (2000) The shift in the myocardial adenine nucleotide translocator isoform expression pattern is associated with an enteroviral infection in the absence of an active T-cell dependent immune response in human inflammatory heart disease. J Am Coll Cardiol 35: 1778–84 Heymans S, Pauschinger M, De Palma A, Kallwellis-Opara A, Rutschow S, Swinnen M, Vanhoutte D, Gao F, Torpai R, Baker AH et al (2006) Inhibition of urokinase-type plasminogen activator or matrix metalloproteinases prevents cardiac injury and dysfunction during viral myocarditis. Circulation 114: 565–73 Heim A, Stille-Siegener M, Kandolf R, Kreuzer H, Figulla HR (1994) Enterovirusinduced myocarditis: Hemodynamic deterioration with immunosuppressive therapy and successful application of interferon-alpha. Clin Cardiol 17: 563–5 Matsumori A, Tomioka N, Kawai C (1989) Viral myocarditis: Immunopathogenesis and the effect of immunosuppressive treatment in a murine model. Jpn Circ J 53: 58–60 Alsharifi M, Mullbacher A, Regner M (2008) Interferon type I responses in primary and secondary infections. Immunol Cell Biol 86: 239–45 Manns MP, Wedemeyer H, Cornberg M (2006) Treating viral hepatitis C: Efficacy, side effects, and complications. Gut 55: 1350–9 Baron S (1992) Introduction to the Interferon System. In: Baron S (ed): Interferon:


40 41

42

43 44

45

46

47

48

49

50

51

52

Principles and Medical Applications. Galveston, TX: The University of Texas Medical Branch at Galveston, Dept. of Microbiology: 1–15 Stille-Siegener M, Heim A, Figulla HR (1995) Subclassification of dilated cardiomyopathy and interferon treatment. Eur Heart J 16 Suppl O: 147–9 Miric M, Vasiljevic J, Bojic M, Popovic Z, Keserovic N, Pesic M (1996) Long-term follow up of patients with dilated heart muscle disease treated with human leucocytic interferon alpha or thymic hormones initial results. Heart 75: 596–601 Heim A, Canu A, Kirschner P, Simon T, Mall G, Hofschneider PH, Kandolf R (1992) Synergistic interaction of interferon-beta and interferon-gamma in coxsackievirus B3-infected carrier cultures of human myocardial fibroblasts. J Infect Dis 166: 958–65 Deonarain R, Cerullo D, Fuse K, Liu PP, Fish EN (2004) Protective role for interferonbeta in coxsackievirus B3 infection. Circulation 110: 3540–3 Kuhl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-beta treatment eliminates cardiotropic viruses and improves left ventricular function in patients with myocardial persistence of viral genomes and left ventricular dysfunction. Circulation 107: 2793–8 Kühl U, Noutsias M, Seeberg B, Schultheiss HP (1996) Immunohistological evidence for a chronic intramyocardial inflammatory process in dilated cardiomyopathy. Heart 75: 295–300 Daliento L, Calabrese F, Tona F, Caforio AL, Tarsia G, Angelini A, Thiene G (2003) Successful treatment of enterovirus-induced myocarditis with interferon-alpha. J Heart Lung Transplant 22: 214–7 Coletta AP, Clark AL, Cleland JG (2009) Clinical trials update from the Heart Failure Society of America and the American Heart Association meetings in 2008: SADHARTCHF, COMPARE, MOMENTUM, thyroid hormone analogue study, HF-ACTION, I-PRESERVE, {beta}-interferon study, BACH, and ATHENA. Eur J Heart Fail 11: 214–9 Kühl U, Pauschinger M, Schwimmbeck PL, Seeberg B, Lober C, Noutsias M, Poller W, Schultheiss HP (2003) Interferon-beta treatment eliminates cardiotropic viruses and improves left ventricular function in patients with myocardial persistence of viral genomes and left ventricular dysfunction. Circulation 107: 2793–8 Modrow S (2006) Parvovirus B19: The causative agent of dilated cardiomyopathy or a harmless passenger of the human myocard? Ernst Schering Res Found Workshop 63–82 Streitz M, Noutsias M, Volkmer R, Rohde M, Brestrich G, Block A, Klippert K, Kotsch K, Ay B, Hummel M et al (2008) NS1 specific CD8+ T-cells with effector function and TRBV11 dominance in a patient with parvovirus B19 associated inflammatory cardiomyopathy. PLoS ONE 3: e2361 Lindner J, Noutsias M, Lassner D, Wenzel J, Schultheiss HP, Kuehl U, Modrow S (2009) Adaptive immune responses against parvovirus B19 in patients with myocardial disease. J Clin Virol 44: 27–32 Kühl U, Lassner D, Pauschinger M, Gross UM, Seeberg B, Noutsias M, Poller W,

277


53

278

Schultheiss HP (2008) Prevalence of erythrovirus genotypes in the myocardium of patients with dilated cardiomyopathy. J Med Virol 80: 1243–1251 Pauschinger M, Doerner A, Kühl U, Schwimmbeck PL, Poller W, Kandolf R, Schultheiss HP (1999) Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99: 889–95

Immunoadsorption in dilated cardiomyopathy patients Stephan B. Felix and Alexander Staudt Department of Internal Medicine B, Ernst-Moritz-Arndt-University of Greifswald, Germany

Abstract Abnormalities of the cellular and humoral immune system have been described in patients with dilated cardiomyopathy (DCM). Various circulating cardiac autoantibodies have been detected in DCM patients. Circulating antibodies are extractable by immunoadsorption (IA). Recent open controlled pilot studies have shown that removal of circulating antibodies by IA induces improvement of cardiac function in DCM. IA, furthermore, decreases myocardial inflammation. In vitro data indicate that removal of negative inotropic antibodies may represent the essential mechanism of IA in DCM. These antibodies belong to immunoglobulin G subclass 3 and may play an important role in cardiac dysfunction of DCM patients. Recent data indicate that the Fc fragments of the immunoglobulins that bind to newly detected sarcolemma-specific FcG receptors IIa are involved in the functional effects of cardiac autoantibodies. Removal of various cardiac antibodies through unspecific IA could therefore offer a hopeful treatment approach in DCM for intervention into this autoimmune process.

Abnormalities of the cellular and humoral immune system in dilated cardiomyopathy Dilated cardiomyopathy (DCM) is characterized by progressive dilatation and impairment of contractile functions of the left ventricle or both ventricles [1]. Medical treatment of heart failure such as administration of ACE inhibitors, B-blockers, and spironolactone is the generally accepted therapeutic principle. Despite advances in medical treatment of heart failure, the general prognosis for DCM is poor. In many cases heart transplantation remains the only therapeutic option when disease progression cannot be inhibited by pharmacotherapy. Alternative therapeutic strategies for treatment of DCM are consequently of essential interest. An association between myocarditis and DCM has been hypothesized for a subset of patients with DCM [2, 3]. Viral genomes are frequently detected in endomyocardial biopsies of patients with DCM [4]. Both experimental and clinical data indicate that viral infection and inflammatory processes may be involved in


279

Stephan B. Felix and Alexander Staudt

the pathogenesis of DCM [5–9]. Viral persistence is associated with progression of cardiac dysfunction [10]. Abnormalities of the cellular immune system are a common feature of myocarditis and DCM. For patients with DCM, immunohistological methods have been introduced for diagnosis of myocardial inflammation. Infiltration with lymphocytes and mononuclear cells, as well as increased expression of cell adhesion molecules are frequent phenomena in DCM [11–13]. These findings support the hypothesis that the immune process is still active. In many patients with DCM, the term “inflammatory cardiomyopathy” may therefore be applicable in describing the pathogenesis of the disease process [1]. Patients with myocarditis and DCM also demonstrate abnormalities of the humoral immune system. Furthermore, various autoantibodies have been detected in DCM patients: these include antibodies against mitochondrial proteins [14], cardiac myosin [15], cardiac B1-adrenergic receptors [16], muscarinergic receptors [17], and the sarcolemmal Na-K-ATPase [18]. The functional role of cardiac autoantibodies in DCM is under debate. Antibodies may reflect an inflammatory response to myocyte necrosis, thereby representing an epiphenomenon or, alternatively, may play an active role in the disease process. In patients with chronic myocarditis and cardiomyopathy, the prevalence of antibodies against cardiac myosin is associated with the deterioration of cardiac function [19]. Experimental data indicate that certain cardiac autoantibodies may play a functional role in DCM: antibodies against the ADP/ATP carrier interact with the calcium channel and possess cardiotoxic properties [14]. In the plasma of patients with DCM, negative inotropic antibodies are detectable that decrease the calcium transients of isolated cardiomyocytes [20]. A recent study by Li et al. [21] demonstrated that anti-cardiac myosin antibodies induced by immunization of rats with cardiac myosin target the B-adrenergic receptor on the heart cell surface and induce cAMP-dependent protein kinase A activity in heart cells. Passive transfer of purified antibodies from cardiac myosinimmunized rats results in IgG deposition and apoptosis in the heart, leading to a cardiomyopathic heart disease phenotype in recipients. Immunization of rodents against peptides derived from cardiovascular G protein receptors induces morphological changes of myocardial tissue resembling DCM [22, 23]. A recent study by Jahns et al. [24] investigated the pathogenic significance of autoantibodies that target cardiac B1-adrenoceptors in DCM: rats immunized against the second extracellular loop of cardiac B1-receptors developed progressive left ventricular dilatation and dysfunction. Interestingly, sera transferred from these immunized animals to unsensitized rats induced a similar cardiomyopathic phenotype, thus demonstrating the pathogenic potential of a particular antibody for development of DCM. Further confirmation of the principle that autoantibodies contribute to induction of the disease process and to progression to DCM has been provided in a study by Nishimura et al. [25]. The authors of this study showed that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory core-

280

Immunoadsorption in dilated cardiomyopathy patients

ceptor develop autoimmune DCM with production of high-titer circulating IgG autoantibodies reactive to a 33-kDa protein expressed specifically on the surface of cardiomyocytes. Okazaki et al. [26] were recently able to identify this antigen as cardiac troponin I.

Immunoadsorption as a new therapeutic principle for treatment of DCM Circulating antibodies are extractable by immunoadsorption (IA). Removal of circulating antibodies by IA has been successfully used for treatment of a number of autoimmune diseases such as Goodpasture’s syndrome or lupus erythematodes [27, 28]. If cardiac antibodies do in fact contribute to cardiac dysfunction in DCM, their removal by IA would be expected to improve the hemodynamic of patients with DCM. We conducted an initial uncontrolled pilot study with the purpose of ascertaining the short-term hemodynamic effects of IA in patients with severe heart failure due to DCM [29]. Immunoglobulin (Ig) extraction from the plasma of these patients induced a significant increase in cardiac index (CI), accompanied by simultaneous fall in systemic vascular resistance (SVR). These data suggest that removal of antibodies may improve the hemodynamics in DCM. We then conducted an open randomized study to investigate the hemodynamic influence of IA in patients with DCM-symptomatic heart failure [New York Heart Association (NYHA) III–IV, left ventricular ejection fraction (LVEF) < 30%, CI < 2.5 l/min/m2] who had previously been on a stable regimen of oral medication for treatment of heart failure [30]. The patients were randomly assigned either to the treatment group with IA, or to the control group without IA. In the IA group, IA was performed in four courses at 1-month intervals. The patients were initially treated in one IA session daily, on 3 consecutive days. IA was then repeated for three further courses on 2 consecutive days, until the third month. Following each course, we substituted IgG to reduce infection risk and to block rebound of antibody production in B cells following IgG depletion. Hemodynamics and LVEF did not change throughout the 3 months in the control group. In contrast, CI and stroke volume index (SVI) rose significantly (30%) in the treatment group, accompanied by concomitant parallel reduction in SVR. The improvement in CI and SVI persisted after 3 months. LVEF likewise increased significantly in the treatment group (Fig. 1), which indicates that IA followed by IgG substitution improves cardiovascular function in DCM patients. In a case-controlled study performed by others, IA was conducted in one course of 5 consecutive days without IA substitution and after depletion [31]. IA was not repeated during follow-up. In this study, LVEF had increased from 22% to 40% 1 year after IA, which was significantly different from the control group without IA therapy. Interestingly, no relapse of cardiac autoantibodies occurred. It is unclear, however, why IA – which took place in one course without repetition – apparently

281


Figure 1. Changes in left ventricular ejection fraction, as assessed by echocardiography in the immunoadsorption/immunoglobulin group (IA/IgG group, filled bars, n = 9) and in the control group (controls, open bars, n = 9). ** p < 0.01 significantly different from baseline, ++ p < 0.01 significantly different from controls. Reproduced from [30].

induced prolonged improvement in left ventricular function, although in other autoimmune diseases IA or plasmapheresis is generally repeated in periodic intervals. It is feasible that IA not merely enhances hemodynamics, but likewise influences myocardial inflammation in patients with DCM. We accordingly conducted a randomized study to investigate immunohistological changes induced by IA therapy and subsequent IgG substitution. IA was performed in four courses, at 1-month intervals, in patients with DCM, and in comparison with controls without immunomodulatory therapy [32]. For immunohistological analysis, right ventricular biopsies were obtained from all patients at baseline and after 3 months. Among control patients, the number of lymphocytes (CD3, CD4, and CD8) and the number of leukocyte common antigen (LCA)-positive cells in the myocardium remained stable over 3 months. Furthermore, no changes in expression of HLA class II antigens were observed. In contrast, IA therapy and subsequent IgG substitution induced significant decrease in lymphocytes and LCA-positive cells in the myocardium during follow-up, which was paralleled by significant decline in HLA class-II antigen expression [32].

282


Further confirmation of the therapeutic concept of IA therapy for treatment of DCM was obtained from a recent experimental study by Matsui et al. [33] that investigated the effects of specific IA of anti-B1-adrenoceptor autoantibodies in rabbits with autoimmune cardiomyopathy induced by active immunization with a B1-adrenoceptor peptide. Specific removal of anti-B1-adrenoceptor autoantibodies improved cardiac structure and function of these animals.

The beneficial effects of IA are related to removal of cardiac antibodies The contribution of a particular antibody to myocardial damage in DCM remains to be elucidated. Experimental studies have demonstrated that antibodies against the B1-adrenergic receptor [22–24] and antibodies against troponin I [25, 26] in fact induce myocardial dysfunction and left ventricular dilatation resembling DCM. On the other hand, a recent study demonstrated that the beneficial hemodynamic effects of nonspecific IA are not directly associated with the removal of B1-adrenoreceptor autoantibodies [34]. Recently, several novel putative DCM-specific antigens were identified, by applying profiling of the autoantibody repertoire of DCM patients with the aid of protein array and microarray technology [35]. Speculation has arisen that different cardiac antibodies contribute to cardiac dysfunction in DCM. The role of a particular antibody to the disease process may be evaluated by specific IA or, alternatively, by specific blockade or inactivation of a particular antibody. A further study investigated potential mechanisms of beneficial acute hemodynamic effects induced by IA, by testing the effects of human antibodies in isolated rat cardiomyocytes [20]. Confocal laser scanning microscopy was applied to analyze the effects of antibodies on cell contraction and on calcium-dependent fluorescence in isolated field-stimulated rat cardiomyocytes. Purified antibodies obtained from the blood of healthy blood donors did not influence calcium transients and cell shortening of the cardiomyocytes. In contrast, purified antibodies obtained from DCM patients brought about an immediate and concentration-dependent decrease in calcium transients and cell shortening. Acute hemodynamic improvement among the patients correlated with the cardiodepressant effect of their antibodies on the isolated cardiomyocytes. These data indicate that removal of circulating negative inotropic antibodies from plasma may contribute to the early beneficial hemodynamic effects of IA in patients with DCM [20]. A recent study performed by others demonstrated that autoantibodies removed by IA from DCM patients induce positive chronotropic effects and exhibit complement-dependent cytotoxicity in neonatal rat cardiomyocytes [36]. Detection of cardiodepressant antibodies may be of essential therapeutic relevance, since the contribution of humoral activity, with production of cardiodepressant antibodies, may differ among DCM patients. For treatment of patients with severe heart failure due to DCM, it is important to identify those patients

283


who will receive hemodynamic benefit from IA. This means that predictors for hemodynamic improvement play a key role. A recent study accordingly systematically investigated whether detection of the cardiodepressant effects of antibodies obtained from patients’ plasma before IA may be predictive for short- and longterm hemodynamic improvement during IA [37]. Since IA effectively removes cardiac autoantibodies from plasma, such a procedure furthermore enables evaluation of the role played by the humoral immune system in cardiac dysfunction among DCM patients. Before IA, antibodies were purified from the plasma of DCM patients with heart failure and left ventricular dysfunction (LVEF < 30%, n = 45). The functional effects of antibodies (300 mg/l) on calcium transients and on systolic cell shortening were analyzed in rat cardiomyocytes. After this in vitro analysis, IA was performed in four courses at 1-month intervals until month 3. Antibodies of the majority of these patients (n = 29) induced cell shortening of cardiomyocytes as well as significant reduction in calcium transients (cardiodepressant group). Antibodies from 16 patients did not influence calcium transients and cell shortening (non-cardiodepressant group). Only the cardiodepressant group demonstrated significant hemodynamic improvement after IA. In contrast, IA did not show any beneficial effects in the non-cardiodepressant group. These data indicate that evidence of cardiodepressant antibodies predicts hemodynamic benefits during IA [37].

Role of IgG subclass 3 antibodies IgG subclasses differ from one another immunologically and functionally. The antibodies that trigger effector functions and that are most likely to be involved in immunoregulatory activity are IgG-3 and IgG-1. IgG-3 is the most active complement-fixing IgG subclass [38]. Furthermore, IgG-3 antibodies are more efficient than IgG-1 as mediators of antibody-dependent cellular cytotoxicity. It was recently shown that DCM patients have elevated levels of IgG-3 antibodies against A- and B-myosin heavy chains. The level of these antibodies correlates with the degree of left ventricular dysfunction [39]. A recent study investigated the role of antibodies belonging to various IgG subclasses with respect to cardiac dysfunction in DCM. According to the data of this study, the negative inotropic effect of antibodies obtained from DCM patients is primarily attributable to antibodies belonging to IgG-3 subclass [40]. In DCM patients, the acute and prolonged beneficial hemodynamic effects of IA therapy depend on effective removal of IgG-3 antibodies [40, 41]. Removal of IgG-3 antibodies may, therefore, represent the essential mechanism of IA in DCM. However, the underlying mechanism of the negative inotropic effect of the antibodies remains to be elucidated. Further studies are necessary to ascertain the mechanism involved in the functional effects of cardiodepressant antibodies.

284


FcG receptors IIa on cardiomyocytes and their potential functional relevance in DCM A recent study revealed a new potential mechanism for antibody-induced impairment of cardiac function in DCM patients [42]. FcG receptor IIa (CD32) was demonstrated by immunofluorescence in rat and human ventricular cardiomyocytes. Furthermore, cardiodepressant antibodies obtained from DCM patients bind to their cardiac antigen(s) via the F(ab’)2 fragment but the Fc part of the antibodies triggers the negative inotropic effects via the newly detected FcG receptor on cardiomyocytes (Fig. 2). FcG receptor IIa can induce an activating signal via its cytoplasmic domains, thereby possibly triggering the negative inotropic effect. The proposed model of FcG receptor-dependent activation of cardiomyocytes by DCM autoantibodies provides an explanation of why antibodies directed against different antigens on cardiomyocytes can induce the same functional effects [42]. Therefore, unspecific IA may be a novel effective therapeutic intervention that removes numerous cardiac antibodies that bind to different cardiac epitopes but may share a common effect via binding of their Fc fragment to a specific FcG receptor IIa located on the sarcolemma of cardiomyocytes.

Figure 2. IgG of dilated cardiomyopathy (DCM) patients induce negative inotropic effects by binding via their Fab part to the antigenic epitope on cardiomyocytes, and then via their Fc part to the Fc receptor IIa (FcR). Reproduced with modifications from [42].

285


In conclusion, assumption is justified that activation of the humoral immune system, with production of cardiac autoantibodies, may play a functional role in cardiac dysfunction of patients with DCM. Nonspecific IA that removes various circulating antibodies against different cardiac epitopes may represent a therapeutic option for a subset of patients with DCM. However, the above-cited studies, which included a small number of patients with DCM and heart failure, require confirmation by a larger, randomized, prospective, multi-centre study – one which would reveal the long-term effect of IA. Since studies published on the cardiovascular effects of IA in patients with DCM were performed according to an open-controlled design, a multi-center study is necessary to confirm these data on the basis of a placebo-controlled design. It will likewise be necessary to evaluate the effects of IA on prognostic clinical endpoints such as mortality and morbidity of patients with DCM.

References 1

2

3 4

5

6

7

286

Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ, Seidman CE, Young JB (2006) Contemporary definitions and classification of the cardiomyopathies: An American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 113: 1807–1816 Dec GW, Palacios IF, Fallon JT, Aretz HT, Mills J, Lee DC, Johnson RA (1985) Active myocarditis in the spectrum of acute dilated cardiomyopathies. Clinical features, histologic correlates, and clinical outcome. N Engl J Med 312: 885–890 Feldman AM, McNamara DM (2000) Myocarditis. N Engl J Med 343: 1388–1398 Kühl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111: 887–893 Bowles NE, Richardson PJ, Olsen EG, Archard LC (1986) Detection of coxsackie-Bvirus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1: 1120–1123 Kandolf R, Ameis D, Kirschner P, Canu A, Hofschneider PH (1987) In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: An approach to the diagnosis of viral heart disease. Proc Natl Acad Sci USA 84: 6272–6276 Pauschinger M, Bowles NE, Fuentes-Garcia FJ, Pham V, Kühl U, Schwimmbeck PL, Schultheiss HP, Towbin JA (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99: 1348– 1354


8 9 10

11 12

13

14

15

16

17

18

19

20

21

Liu PP, Mason JW (2001) Advances in the understanding of myocarditis. Circulation 104: 1076–1082 Magnani JW, Dec GW (2006) Myocarditis: Current trends in diagnosis and treatment. Circulation 113: 876–890 Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112: 1965–1970 Kühl U, Noutsias M, Schultheiss HP (1995) Immunohistochemistry in dilated cardiomyopathy. Eur Heart J 16 Suppl O: 100–106 Wojnicz R, Nowalany-Kozielska E, Wodniecki J, Szczurek-Katanski K, Nozynski J, Zembala M, Rozek MM (1998) Immunohistological diagnosis of myocarditis. Potential role of sarcolemmal induction of the MHC and ICAM-1 in the detection of autoimmune mediated myocyte injury. Eur Heart J 19: 1564–1572 Noutsias M, Seeberg B, Schultheiss HP, Kühl U (1999) Expression of cell adhesion molecules in dilated cardiomyopathy: Evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99: 2124–2131 Schultheiss HP, Kühl U, Janda I, Melzner B, Ulrich G, Morad M (1988) Antibodymediated enhancement of calcium permeability in cardiac myocytes. J Exp Med 168: 2102–2119 Caforio AL, Grazzini M, Mann JM, Keeling PJ, Bottazzo GF, McKenna WJ, Schiaffino S (1992) Identification of alpha- and beta-cardiac myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 85: 1734–1742 Wallukat G, Wollenberger A, Morwinski R, Pitschner HF (1995) Anti-beta1–adrenoceptor autoantibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy: Mapping of epitopes in the first and second extracellular loops. J Mol Cell Cardiol 27: 397–406 Fu LX, Magnusson Y, Bergh CH, Liljeqvist JA, Waagstein F, Hjalmarson A, Hoebeke J (1993) Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy. J Clin Invest 91: 1964–1968 Baba A, Yoshikawa T, Ogawa S (2002) Autoantibodies produced against sarcolemmal Na-K-ATPase: Possible upstream targets of arrhythmias and sudden death in patients with dilated cardiomyopathy. J Am Coll Cardiol 40: 1153–1159 Lauer B, Schannwell M, Kühl U, Strauer BE, Schultheiss HP (2000) Antimyosin autoantibodies are associated with deterioration of systolic and diastolic left ventricular function in patients with chronic myocarditis. J Am Coll Cardiol 35: 1106–1110 Felix SB, Staudt A., Landsberger M, Grosse Y, Stangl V, Spielhagen T, Wallukat, G, Wernecke K-D, Baumann G, Stangl K (2002) Removal of cardiodepressant antibodies in dilated cardiomyopathy by immunoadsorption. J Am Coll Cardiol 39: 646–652 Li Y, Heuser JS, Cunningham LC, Kosanke SD, Cunningham MW (2006) Mimicry and antibody-mediated cell signaling in autoimmune myocarditis. J Immunol 177: 8234–8240

287


22

23

24

25

26

27

28 29

30

31

32

33

288

Fu ML, Schulze W, Wallukat G, Hjalmarson A, Hoebeke J (1996) A synthetic peptide corresponding to the second extracellular loop of the human M2 acetylcholine receptor induces pharmacological and morphological changes in cardiomyocytes by active immunization after 6 months in rabbits. Clin Immunol Immunopathol 78: 203–207 Matsui S, Fu ML, Katsuda S, Hayase M, Yamaguchi N, Teraoka K, Kurihara T, Takekoshi N, Murakami E, Hoebeke J, Hjalmarson A (1997) Peptides derived from cardiovascular G-protein-coupled receptors induce morphological cardiomyopathic changes in immunized rabbits. J Mol Cell Cardiol 29: 641–655 Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G, Lohse MJ (2004) Direct evidence for a beta1–adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy. J Clin Invest 113: 1419–1429 Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N, Honjo T (2001) Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291: 319–322 Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, Ishida M, Hiai H, Matsumori A, Minato N, Honjo T (2003) Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med 9: 1477– 1483 Bygren P, Freiburghaus C, Lindholm T, Simonsen O, Thysell H, Wieslander J (1985) Goodpasture’s syndrome treated with staphylococcal protein A immunoadsorption. Lancet 2: 1295–1296 Palmer A, Gjorstrup P, Severn A, Welsh K, Taube D (1988) Treatment of systemic lupus erythematodes by extracorporeal immunoadsorption. Lancet 2: 272 Dörffel WV, Felix SB, Wallukat G, Brehme S, Bestvater K, Hofmann T, Kleber FX, Baumann G (1997) Short-term hemodynamic effects of immunoadsorption in dilated cardiomyopathy. Circulation 95: 1994–1997 Felix SB, Staudt A, Dörffel WV, Stangl V, Merkel K, Pohl M, Döcke WD, Morgera S, Neumayer HH, Wernecke KD, Wallukat G, Stangl K, Baumann G (2000) Hemodynamic effects of immunoadsorption and subsequent immunoglobulin substitution in dilated cardiomyopathy: Three-month results from a randomized study. J Am Coll Cardiol 35: 1590–1598 Müller J, Wallukat G, Dandel M, Bieda H, Brandes K, Spiegelsberger S, Nissen E, Kunze R, Hetzer R (2000) Immunoglobulin adsorption in patients with idiopathic dilated cardiomyopathy. Circulation 101: 385–391 Staudt A, Schaper F, Stangl V, Plagemann A, Böhm M, Merkel K, Wallukat G, Wernecke K-D, Stangl K, Baumann G, Felix SB (2001) Immunohistological changes in dilated cardiomyopathy induced by immunoadsorption therapy and subsequent immunoglobulin substitution. Circulation 103: 2681–2686 Matsui S, Larsson L, Hayase M, Katsuda S, Teraoka K, Kurihara T, Murano H, Nishikawa K, Fu M (2006) Specific removal of beta1-adrenoceptor autoantibodies by immunoabsorption in rabbits with autoimmune cardiomyopathy improved cardiac structure and function. J Mol Cell Cardiol 41: 78–85


34

35

36

37

38

39

40

41

42

Mobini R, Staudt A, Felix SB, Baumann G, Wallukat G, Deinum J, Svensson H, Hjalmarson A, Fu M (2003) Hemodynamic improvement and removal of autoantibodies against beta1-adrenergic receptor by immunoadsorption therapy in dilated cardiomyopathy. J Autoimmun 20: 345–350 Horn S, Lueking A, Murphy D, Staudt A, Gutjahr C, Schulte K, König A, Landsberger M, Lehrach H, Felix SB, Cahill DJ (2006) Profiling humoral auto-immune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip. Proteomics 6: 605–613 Chen J, Larsson L, Haugen E, Fedorkova O, Angwald E, Waagstein F, Fu M (2006) Effects of autoantibodies removed by immunoadsorption from patients with dilated cardiomyopathy on neonatal rat cardiomyocytes. Eur J Heart Fail 8: 460–467 Staudt A, Staudt Y, Dörr M, Böhm M, Knebel F, Hummel A, Wunderle L, Tiburcy M, Wernecke K-D, Baumann G, Felix SB (2004) Potential role of humoral immunity in cardiac dysfunction of patients suffering from dilated cardiomyopathy. J Am Coll Cardiol 44: 829–836 Bruggemann M, Williams GT, Bindon CI, Clark MR, Walker MR, Jefferis R, Waldmann H, Neuberger MS (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med 166: 1351–1361 Warraich RS, Noutsias M, Kazak I, Seeberg B, Dunn MJ, Schultheiss HP, Yacoub MH, Kühl U, Kasac I (2002) Immunoglobulin G3 cardiac myosin autoantibodies correlate with left ventricular dysfunction in patients with dilated cardiomyopathy: Immunoglobulin G3 and clinical correlates. Am Heart J 143: 1076–1084 Staudt A, Böhm M, Knebel F, Grosse Y, Bischoff C, Hummel A, Dahm JB, Borges A, Jochmann N, Wernecke K-D, Wallukat G, Baumann G, Felix SB (2002) Potential role of autoantibodies belonging to the immunoglobulin G-3 subclass in cardiac dysfunction among patients with dilated cardiomyopathy. Circulation 106: 2448–2453 Staudt A, Dörr M, Staudt Y, Böhm M, Probst M, Empen K, Plötz S, Maschke HE, Hummel A, Baumann G, Felix SB (2005) Role of immunoglobulin G3 subclass in dilated cardiomyopathy – Results from protein-A immunoadsorption. Am Heart J 150: 729–36 Staudt A, Eichler P, Trimpert C, Felix SB, Greinacher A (2007) Fc(gamma) receptors IIa on cardiomyocytes and their potential functional relevance in dilated cardiomyopathy. J Am Coll Cardiol 49: 1684–92

289

Index

active myocarditis definition

187, 188, 193, 206, 207

anti-muscarinic M2-receptor (auto)antibodies

188

antiviral strategy

(anti-M2-abs)

187

histopathology

acute dilated cardiomyopathy

27, 28

apoptosis

160–163 177

54

acute heart failure, definition 240

autoantibodies/ receptor-autoantibodies

acute left ventricular dysfunction 71, 72

autoantibody-targeted therapy

acute myocarditis (AMC) 71–73, 77–79,

autoimmune heart disease 176 autoimmune myocarditis

202–205, 242, 266, 267 clinical presentation epidemiology

Barth syndrome

202, 203

adenovirus (ADV)

72, 73

Bcl-2 family BICC Trial

77–79

pathogenic mechanism viral infection

157

202

inflammation and cytokines matrix remodeling

203–205

99

172 273

biopsy, see endomyocardial biopsy borderline myocarditis

266, 267

definition

118, 204

B1-adrenergic receptor (B1-AR)

187, 192, 193, 206

187

157

AMP-activated protein kinase 113

capillary leakage, visualization

anti-ADP/ATP carrier (auto)antibodies

cardiac antibodies

(anti-ADP/ATP-abs)

160–163

anti-B1-adrenergic receptor (auto)antibodies (anti-B1-AR abs) 160–163 anti-cardiac autoimmunity

53

anti-cardiac immune response 53 anti-cardiac myosin-(auto)antibodies (anti-CMabs)

anti-cardiac troponin I-(auto)antibodies 160–163

antigen-presenting cell (APC) 54 anti-heat shock protein (auto)antibodies (anti-HSP-abs)

160–163

231

159

cardiac autoantibodies

159, 280, 281, 283

cardiac magnetic resonance (CMR) 206, 227–233 cardiac tamponade

13

cardiomyopathy arrhythmogenic

91

classification scheme

160–163

(anti-cTnI-abs)

158

164, 165

etiology of hypertrophic peripartum restrictive

91

240 91, 103–106, 109–115 246 91

cardiotrophin-1 (CT-1)

173

291

Index

cardiotropic viral infection 201–217

enteroviral protease 2A 177

cardiovascular mortality

enterovirus (EV)

CD4+ T cell

164, 165

118, 203

eosinophilic myocarditis

176

cell adhesion molecule (CAM) 54, 204, 210,

246

Epstein-Barr virus (EBV) 204 experimental autoimmune myocarditis

212 cellular inhibitor of apoptosis 1 and 2 172

(EAM)

37–41

chronic active myocarditis 244

extracellular matrix (ECM) 54, 73–79

ciliary neurotrophic factor (CNTF) 173

ECM remodeling

54, 74, 76–79

clinical prognostic parameter 202 congenital heart disease 116

Fabry’s disease

coxsackie adenovirus receptor (CAR) 53, 204

familial HCM (FHC)

coxsackievirus B (CVB) 51–60, 118, 176, 203

FcG receptor IIa 285

cytokines

fibrosis, visualization

59, 60, 72–76, 171

cytokines, immunomodulation cytokines, proinflammatory cytopathic effect cytoskeleton

59, 60

112 105, 106, 109 232

fulminant myocarditis

204, 242, 243

genetic predisposition

158

giant cell myocarditis

29, 130, 244

171

53, 204

92

cytotoxic T lymphocyte (CTL) 54, 204, 208 heart failure Dallas classification Dallas criteria

123, 206, 209, 241

Danon’s disease

76

heat shock protein (HSP) 172

187–189

histological evaluation of myocarditis 209 histopathology

113

25, 188

decay-accelerating factor (DAF) 53

HIV-related myocarditis

digital image analysis (DIA) 210

human herpes virus type 6 (HHV6) 204

dilated cardiomyopathy (DCM) 71–73, 77–79,

humoral immune system 279, 280

91, 95–103, 157, 175, 202, 268–272,

humoral immunity

279–286

humoral response

chronic

71–73, 77–79, 268–272

epidemiology incidence

231 246

103–106, 109–115

202 72, 73

HCM, familial (FHC)

105, 106, 109

96

survival rate viral infection dystrophin

hyperemia, visualization

hypertrophic cardiomyopathy (HCM) 91,

98

inflammation and cytokines inherited

158 59

hypersensitivity myocarditis

202, 203

final common pathway

248

IL-6

202

174

IL-18

266–272

174

immune response, anti-cardiac 53

98

immune response, humoral antiviral 54 encephalomyocarditis virus (EMCV) 175 endomyocardial biopsy (EMB)

31, 32, 122,

immunoadsorption

164, 165, 250, 281–284,

286

187–195, 202, 206–217, 227, 228

immunoadsorption therapy

Dallas classification

immunoglobulin, intravenous

sensitivity of

292

187

189, 190, 192

immunohistochemistry

250 249

189–192

Index

immunohistological diagnosis

207–213

murine strain

immunomodulation, antiviral

57

myocarditis 26–32, 51–60, 95, 118–135,

immunosuppression

173–177, 187–193, 228–233, 257, 258

58

immunosuppressive treatment infiltrate

248, 258–261

active

187, 188, 193

borderline

204, 208–210

inflammatory cardiomyopathy (DCMi) 37,

30, 187, 192, 193

clinical manifestation

27

77–79, 201–217

cardiac magnetic resonance 228–233

definition

CVB-induced

201

matrix remodeling

77–79

pathogenic mechanism

203–206

immunohistological evaluation inflammatory cell antibodies

209

diagnosis

257, 258

giant cell

29

lymphocytic

188

magnetic resonance imaging 32

188–190

190

quantification on tissue

51–60, 176

188

mortality rate

257

natural history

28

innate immune system 60

outcome predictor

29, 30

innate immunity

pathologic finding

26

54, 60

innate stress response 175

sampling error

intercalated disc

viral

92

interferon-B treatment

myocarditis treatment trial 28, 177

265–273

internal cardioverter-defibrillator (ICD) 110

myocardium myosin

Kearns-Sayre syndrome

26

95, 173–177, 194 188

107, 108

myosin-binding protein C

115

108

left ventricular ejection fraction 174

natural killer (NK) cell 54

left ventricular noncompaction 91, 115–118

necrosis, visualization

leukemia inhibitory factor (LIF) 173

neutral sphingomyelinase pathway

LPS-induced myocardial dysfunction 174

nitric oxide (NO)

Lyme myocarditis

nuclear factor-kappa B (NF-KB) 172

macrophage

247

208

232 174

174

oxidative stress

172

parvovirus B19

118, 204

magnetic resonance imaging (MRI) 32, 227 manganese superoxide dismutase (MnSOD)

pericardial effusion

172

MERRF syndrome metalloproteinase

pericarditis

115

acute

54

mitochondrial cardiomyopathy

114

mitogen-activated protein kinase (MAPK)dependent pathway

173

10–13

3–21

6–9

autoreactive bacterial chronic

20

19 7, 9–16

molecular mimicry

53

constrictive

murine myocarditis

37–41, 51–60, 72

infectious

4

15, 16

autoimmune

37–41

neoplastic

21

experimental

72

recurrent

9, 10

293

Index

renal failure traumatic tuberculous viral

T cell

19, 20

39, 40, 53, 54

T cell receptor 54

5 19

17

T cell response

53

T lymphocyte

208

polymerase chain reaction (PCR) 125–128

therapy, (auto)antibody-directed

Pompe’s disease

thick filament protein

111

post-cardiac injury syndrome 20

thin filament protein

prognostic impact

toll-like receptor

208

164, 165

107, 108 108–115

54

traumatic pericardial effusion 21 receptor autoantibodies regulatory T cell

tumor necrosis factor (TNF) 171

158

54 viral genome

renal failure, pericarditis in 19, 20

viral load sampling error, myocarditis sarcolemma sarcomere

26

92

viral myocarditis

95, 173–177, 194

viral pericarditis

17

viral protease 2A

92

signal transducer and activator of transcription (STAT)-mediated signaling pathway

31, 213

214

173

viral replication

177 213

virus-host interaction

small pox vaccine-related myocarditis 248

virus-induced anti-cardiac immune response 204

STAT3

vitamin cocktail, in cardiac therapy 117

294

174

The PIR-Series Progress in Inflammation Research Homepage: www.birkhauser.ch Up-to-date information on the latest developments in the pathology, mechanisms and therapy of inflammatory disease are provided in this monograph series. Areas covered include vascular responses, skin inflammation, pain, neuroinflammation, arthritis cartilage and bone, airways inflammation and asthma, allergy, cytokines and inflammatory mediators, cell signalling, and recent advances in drug therapy. Each volume is edited by acknowledged experts providing succinct overviews on specific topics intended to inform and explain. The series is of interest to academic and industrial biomedical researchers, drug development personnel and rheumatologists, allergists, pathologists, dermatologists and other clinicians requiring regular scientific updates.

Available volumes: T Cells in Arthritis, P. Miossec, W. van den Berg, G. Firestein (Editors), 1998 Medicinal Fatty Acids, J. Kremer (Editor), 1998 Cytokines in Severe Sepsis and Septic Shock, H. Redl, G. Schlag (Editors), 1999 Cytokines and Pain, L. Watkins, S. Maier (Editors), 1999 Pain and Neurogenic Inflammation, S.D. Brain, P. Moore (Editors), 1999 Apoptosis and Inflammation, J.D. Winkler (Editor), 1999 Novel Inhibitors of Leukotrienes, G. Folco, B. Samuelsson, R.C. Murphy (Editors), 1999 Metalloproteinases as Targets for Anti-Inflammatory Drugs, K.M.K. Bottomley, D. Bradshaw, J.S. Nixon (Editors), 1999 Gene Therapy in Inflammatory Diseases, C.H. Evans, P. Robbins (Editors), 2000 Cellular Mechanisms in Airways Inflammation, C. Page, K. Banner, D. Spina (Editors), 2000 Inflammatory and Infectious Basis of Atherosclerosis, J.L. Mehta (Editor), 2001 Neuroinflammatory Mechanisms in Alzheimer’s Disease. Basic and Clinical Research, J. Rogers (Editor), 2001 Inflammation and Stroke, G.Z. Feuerstein (Editor), 2001 NMDA Antagonists as Potential Analgesic Drugs, D.J.S. Sirinathsinghji, R.G. Hill (Editors), 2002 Mechanisms and Mediators of Neuropathic pain, A.B. Malmberg, S.R. Chaplan (Editors), 2002 Bone Morphogenetic Proteins. From Laboratory to Clinical Practice, S. Vukicevic, K.T. Sampath (Editors), 2002 The Hereditary Basis of Allergic Diseases, J. Holloway, S. Holgate (Editors), 2002 Inflammation and Cardiac Diseases, G.Z. Feuerstein, P. Libby, D.L. Mann (Editors), 2003 Mind over Matter – Regulation of Peripheral Inflammation by the CNS, M. Schäfer, C. Stein (Editors), 2003 Heat Shock Proteins and Inflammation, W. van Eden (Editor), 2003 Pharmacotherapy of Gastrointestinal Inflammation, A. Guglietta (Editor), 2004 Arachidonate Remodeling and Inflammation, A.N. Fonteh, R.L. Wykle (Editors), 2004 Recent Advances in Pathophysiology of COPD, P.J. Barnes, T.T. Hansel (Editors), 2004 Cytokines and Joint Injury, W.B. van den Berg, P. Miossec (Editors), 2004

Cancer and Inflammation, D.W. Morgan, U. Forssmann, M.T. Nakada (Editors), 2004 Bone Morphogenetic Proteins: Bone Regeneration and Beyond, S. Vukicevic, K.T. Sampath (Editors), 2004 Antibiotics as Anti-Inflammatory and Immunomodulatory Agents, B.K. Rubin, J. Tamaoki (Editors), 2005 Antirheumatic Therapy: Actions and Outcomes, R.O. Day, D.E. Furst, P.L.C.M. van Riel, B. Bresnihan (Editors), 2005 Regulatory T-Cells in Inflammation, L. Taams, A.N. Akbar, M.H.M Wauben (Editors), 2005 Sodium Channels, Pain, and Analgesia, K. Coward, M. Baker (Editors), 2005 Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation, A.B Malmberg, K.R. Bley (Editors), 2005 The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis and Cancer, Z. Zukowska, G.Z. Feuerstein (Editors), 2005 Toll-like Receptors in Inflammation, L.A.J. O’Neill, E. Brint (Editors), 2005 Complement and Kidney Disease, P.F. Zipfel (Editor), 2006 Chemokine Biology – Basic Research and Clinical Application, Volume 1: Immunobiology of Chemokines, B. Moser, G.L. Letts, K. Neote (Editors), 2006 The Hereditary Basis of Rheumatic Diseases, R. Holmdahl (Editor), 2006 Lymphocyte Trafficking in Health and Disease, R. Badolato, S. Sozzani (Editors), 2006 In Vivo Models of Inflammation, 2nd Edition, Volume I, C.S. Stevenson, L.A. Marshall, D.W. Morgan (Editors), 2006 In Vivo Models of Inflammation, 2nd Edition, Volume II, C.S. Stevenson, L.A. Marshall, D.W. Morgan (Editors), 2006 Chemokine Biology – Basic Research and Clinical Application. Volume II: Pathophysiology of Chemokines, K. Neote, G.L. Letts, B. Moser (Editors), 2007 Adhesion Molecules: Function and Inhibition, K. Ley (Editor), 2007 The Immune Synapse as a Novel Target for Therapy, L. Graca (Editor), 2008 The Resolution of Inflammation, A.G. Rossi, D.A. Sawatzky (Editors), 2008 Bone Morphogenetic Proteins: From Local to Systemic Therapeutics, S. Vukicevic, K.T. Sampath (Editors), 2008 Angiogenesis in Inflammation: Mechanisms and Clinical Correlates, M.P. Seed, D.A. Walsh (Editors), 2008 Matrix Metalloproteinases in Tissue Remodelling and Inflammation, V. Lagente, E. Boichot (Editors), 2008 Microarrays in Inflammation, A. Bosio, B. Gerstmayer (Editors), 2009 Th 17 Cells: Role in Inflammation and Autoimmune Disease, B. Ryffel, F. Di Padova (Editors), 2009 New Therapeutic Targets in Rheumatoid Arthritis, P.P. Tak (Editor), 2009 The Hygiene Hypothesis and Darwinian Medicine, G.A.W. Rook (Editor), 2009 Occupational Asthma, T. Sigsgaard, D. Heederik (Editors), 2010

Inflammatory Cardiomyopathy (DCMi) - Pathogenesis and Therapy (Progress in Inflammation Research)

Microarrays in Inflammation (Progress in Inflammation Research)

Antirheumatic Therapy: Actions and Outcomes (Progress in Inflammation Research)

The Resolution of Inflammation (Progress in Inflammation Research) (Progress in Inflammation Research)

Proteases and Their Receptors in Inflammation (Progress in Inflammation Research)

Endothelial Dysfunction and Inflammation (Progress in Inflammation Research)

Regulatory T Cells in Inflammation (Progress in Inflammation Research)

The Inflammasomes (Progress in Inflammation Research)

Lymphocyte Trafficking in Health and Disease (Progress in Inflammation Research)

Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation (Progress in Inflammation Research)

Matrix Metalloproteinases in Tissue Remodelling and Inflammation (Progress in Inflammation Research)

Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation (Progress in Inflammation Research)

Th 17 Cells: Role in Inflammation and Autoimmune Disease (Progress in Inflammation Research)

The Hygiene Hypothesis and Darwinian Medicine (Progress in Inflammation Research)

The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis, and Cancer (Progress in Inflammation Research)

Signaling Pathways in Cancer Pathogenesis and Therapy

Caveolins in Cancer Pathogenesis, Prevention and Therapy

The Hereditary Basis of Rheumatic Diseases (Progress in Inflammation Research)

The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis, and Cancer (Progress in Inflammation Research)

Toll-like Receptors in Inflammation (Progress in Inflammation Research, Volume 57)

The Immune Synapse as a Novel Target for Therapy (Progress in Inflammation Research)

Autologous And Cancer Stem Cell Gene Therapy (Progress in Gene Therapy) (Progress in Gene Therapy)

Chemokine Biology - Basic Research and Clinical Application: Vol. 1: Immunobiology of Chemokines (Progress in Inflammation Research)

Chemokine Biology: Basic Research and Clinical Application: Vol. 2: Pathophysiology of Chemokines (Progress in Inflammation Research)

Gene Therapy for Autoimmune and Inflammatory Diseases

Behcet's Disease (Inflammatory Disease and Therapy)

Advances in Targeted Cancer Therapy (Progress in Drug Research)

Chronic Viral and Inflammatory Cardiomyopathy (Ernst Schering Research Foundation Workshop 55)

Molecular and Cellular Basis of Inflammation (Current Inflammation Research)

Progress in nonlinear analysis research

Inflammatory Cardiomyopathy (DCMi) - Pathogenesis and Therapy (Progress in Inflammation Research)

Microarrays in Inflammation (Progress in Inflammation Research)

Antirheumatic Therapy: Actions and Outcomes (Progress in Inflammation Research)

The Resolution of Inflammation (Progress in Inflammation Research) (Progress in Inflammation Research)

Proteases and Their Receptors in Inflammation (Progress in Inflammation Research)

Endothelial Dysfunction and Inflammation (Progress in Inflammation Research)

Regulatory T Cells in Inflammation (Progress in Inflammation Research)

The Inflammasomes (Progress in Inflammation Research)

Lymphocyte Trafficking in Health and Disease (Progress in Inflammation Research)

Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation (Progress in Inflammation Research)

Matrix Metalloproteinases in Tissue Remodelling and Inflammation (Progress in Inflammation Research)

Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation (Progress in Inflammation Research)

Th 17 Cells: Role in Inflammation and Autoimmune Disease (Progress in Inflammation Research)

The Hygiene Hypothesis and Darwinian Medicine (Progress in Inflammation Research)

The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis, and Cancer (Progress in Inflammation Research)

Signaling Pathways in Cancer Pathogenesis and Therapy

Caveolins in Cancer Pathogenesis, Prevention and Therapy

The Hereditary Basis of Rheumatic Diseases (Progress in Inflammation Research)

The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis, and Cancer (Progress in Inflammation Research)

Toll-like Receptors in Inflammation (Progress in Inflammation Research, Volume 57)

The Immune Synapse as a Novel Target for Therapy (Progress in Inflammation Research)

Autologous And Cancer Stem Cell Gene Therapy (Progress in Gene Therapy) (Progress in Gene Therapy)

Chemokine Biology - Basic Research and Clinical Application: Vol. 1: Immunobiology of Chemokines (Progress in Inflammation Research)

Chemokine Biology: Basic Research and Clinical Application: Vol. 2: Pathophysiology of Chemokines (Progress in Inflammation Research)

Gene Therapy for Autoimmune and Inflammatory Diseases

Behcet's Disease (Inflammatory Disease and Therapy)

Advances in Targeted Cancer Therapy (Progress in Drug Research)

Chronic Viral and Inflammatory Cardiomyopathy (Ernst Schering Research Foundation Workshop 55)

Molecular and Cellular Basis of Inflammation (Current Inflammation Research)

Progress in nonlinear analysis research

Recommend Documents