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
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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
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Myocarditis and inflammatory cardiomyopathy – clinical management, epidemiology and prognosis Bernhard Maisch, Konstantinos Karatolios, Sabine Pankuweit and Arsen Ristic Pathogenesis, diagnosis and treatment of pericarditis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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G. William Dec Epidemiology and prognosis of myocarditis and dilated cardiomyopathy: Predictive value of clinical parameters and biopsy findings . . . . . . . . . . . . . . . . . . . . . . .
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Animal models of myocarditis – autoimmunity and viral infection; therapeutic interventions Christoph Berger und Urs Eriksson Autoimmune murine myocarditis and immunomodulatory interventions . . . . . . . .
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Michel Noutsias and Peter Liu Coxsackievirus-induced murine myocarditis and immunomodulatory interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Susanne Rutschow, Michel Noutsias and Matthias Pauschinger Myocardial proteases and matrix remodeling in acute myocarditis and inflammatory cardiomyopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Alterations of the immune system in human viral and inflammatory cardiomyopathy Jeffrey A. Towbin and Matteo Vatta Molecular genetics of cardiomyopathies and myocarditis . . . . . . . . . . . . . . . . . . . . . . . .
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Roland Jahns, Valérie Boivin, Georg Ertl and Martin J. Lohse Pathogenic relevance of autoantibodies in dilated cardiomyopathy
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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
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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
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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
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List of contributors
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]
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List of contributors
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
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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
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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
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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
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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])
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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
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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.
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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
Pathogenesis, diagnosis and treatment of pericarditis
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
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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-
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Pathogenesis, diagnosis and treatment of pericarditis
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,
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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).
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Pathogenesis, diagnosis and treatment of pericarditis
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-
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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.
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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-
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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.
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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
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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]).
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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
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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
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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
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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
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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
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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
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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].
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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
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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
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Epidemiology and prognosis of myocarditis and dilated cardiomyopathy
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
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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
Epidemiology and prognosis of myocarditis and dilated cardiomyopathy
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
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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
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Epidemiology and prognosis of myocarditis and dilated cardiomyopathy
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.
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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,
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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).
Inflammatory Cardiomyopathy (DCMi) – Pathogenesis and Therapy, edited by Heinz-Peter Schultheiss and Michel Noutsias © 2010 Springer Basel
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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
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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
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[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
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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
Prevention/amelioration
Prevention/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
Christoph Berger and Urs Eriksson
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*
Autoimmune murine myocarditis and immunomodulatory interventions
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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.
Inflammatory Cardiomyopathy (DCMi) – Pathogenesis and Therapy, edited by Heinz-Peter Schultheiss and Michel Noutsias © 2010 Springer Basel
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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
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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
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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,
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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
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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-
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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
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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-
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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
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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.
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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,
Inflammatory Cardiomyopathy (DCMi) – Pathogenesis and Therapy, edited by Heinz-Peter Schultheiss and Michel Noutsias © 2010 Springer Basel
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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
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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
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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-
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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-
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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].
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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-
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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
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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.
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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
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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-
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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
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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
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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.
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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.
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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-
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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
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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,
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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,
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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].
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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
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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.
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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].
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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.
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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
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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.
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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.
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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
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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
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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
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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,
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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.
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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
