NIH-SPONSORED WORKSHOPCONFERENCE ON DILATED REPORT

National Institutes of Health–Sponsored Workshop on and Immunity in

LESLIE T. COOPER, MD; RENU VIRMANI, MD; NORA M. CHAPMAN, PHD; ANDREA FRUSTACI, MD; RICHARD J. RODEHEFFER, MD; MADELEINE W. CUNNINGHAM, PHD; AND DENNIS M. MCNAMARA, MD

Nonischemic dilated cardiomyopathy (DCM) is an uncommon vances have been made through the use of inbred and cause of but has widespread importance because it is the cause of 45% of heart transplantations. Multiple experimental transgenic rodent models. Advances in clinical treatment and clinical lines of evidence have implicated altered immunity in and diagnosis of and DCM have lagged behind the pathogenesis of DCM. However, advances in the understand­ the advances in our understanding of disease mechanisms. ing of the mechanisms of altered immunity have not affected the diagnosis or treatment of DCM. In recognition of this problem, the The workshop was organized to address the need to National Institutes of Health sponsored an expert workshop with 2 translate advances at the bench into bedside medicine. aims: to review the current understanding of inflammation and Experts from a range of relevant specialties, including immunity as they relate to DCM and to identify the most promising areas for future clinical research efforts in the field. This report virology, biochemistry, immunology, pathology, and clini­ summarizes the scientific opportunities, perceived needs and cal cardiology, met with representatives of the NIH on May barriers, and workshop recommendations on research directions 21 and 22, 2004. The conference was organized into in DCM. The major recommendations from the members of the workshop are organized according to the following themes: cardio­ themes consisting of cardiotropic viruses, innate and ac­ tropic viruses, innate and acquired immune responses, environ­ quired immunity, environmental factors, novel diagnostics, mental factors, novel diagnostics, and novel therapeutics. and novel therapeutics. Within each theme, the participants Mayo Clin Proc. 2006;81(2):199-204 identified perceived challenges and opportunities and made recommendations for support of future research. This re­ DCM = dilated cardiomyopathy; GPx = glutathione peroxidase; MRI = port summarizes the discussions and recommendations magnetic resonance imaging; NIH = National Institutes of Health; qPCR = quantitative polymerase chain reaction; qRT-PCR = quantita­ from that conference. tive reverse transcriptase–PCR; TE = trace element CARDIOTROPIC VIRUSES ilated cardiomyopathy (DCM) is an uncommon cause Viral myocarditis is pathogenically determined by at least 3 Dof heart failure but has widespread importance be­ factors: (1) the virus and its receptor, (2) the host immune cause it is the cause of 45% of heart transplantations.1 The system, and (3) cardiac remodeling.4 In the past 18 years, last National Institutes of Health (NIH)–sponsored expert we have seen important advances in our ability to detect workshop on this subject was held in 1989 and identified cardiotropic viruses from fresh and frozen heart tissue us­ the need for investigation into the immunologic basis of the ing in situ hybridization, quantitative polymerase chain disease.2 Since then, exceptional strides have been made in reaction (qPCR), nested qPCR, and most recently quantita­ our understanding of the immunology and pathophysiology tive reverse transcriptase–PCR (qRT-PCR). The latest tech­ of viral and autoimmune myocarditis.3 Most of these ad- nologies can detect 1 to 10 gene copies present in myocar­ dium. These rapid advances in technology have changed the sensitivity of molecular techniques for viral pathogens and From the Division of Cardiovascular Diseases, Mayo Clinic College of Medi­ cine, Rochester, Minn (L.T.C., R.J.R.); International Registry of Pathology, have provided both challenges and opportunities. Gaithersburg, Md (R.V.); Department of Pathology and Laboratory Medi­ New viral pathogens have emerged in the past decade, cine, University of Nebraska Medical Center, Omaha (N.M.C.); Department of Heart and Great Vessels “Attilio Reale” La Sapienza University and National including adenovirus, Epstein-Barr virus, cytomegalovirus, Institute for Infectious Diseases “Lazzaro Spallanzani,” Rome, Italy (A.F.); , and most recently parvovirus B19.5 Enterovirus University of Oklahoma Health Sciences Center, Oklahoma City (M.W.C.); and Cardiovascular Division, University of Pittsburgh Medical Center, Pittsburgh, serotypes have been identified as different from the 64 Pa (D.M.M.). The participating investigators are listed at the end of this serotypes recognized previously.6-8 Given the high rate of article. enterovirus recombination, the current diagnostic method This article was funded by the National Heart, Lung, and Blood Institute and ′ National Institutes of Health Office of Rare Diseases grant R13 HL074877­ of qRT-PCR of conserved regions of the 5 nontranslated 01, the American Heart Association Clinical Cardiology Council, and the region cannot discriminate among the currently recognized American Registry of Pathology. enterovirus serotypes.6 Portions of the enterovirus genome Address reprint requests and correspondence to Leslie T. Cooper, MD, other than the 5′ nontranslated region could be explored for Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905 (e-mail: [email protected]). unique and conserved sequences. Such studies may allow © 2006 Mayo Foundation for Medical Education and Research for a molecular identification of the known serotypes or

Mayo Clin Proc. • February 2006;81(2):199-204 • www.mayoclinicproceedings.com 199 For personal use. Mass reproduce only with permission from Mayo Clinic Proceedings. NIH-SPONSORED WORKSHOP ON DILATED CARDIOMYOPATHY even an ability to classify viruses according to genotype. viral receptors, immune mediators, and the cellular cyto­ Such newly identified genotypes may show stronger corre­ architecture.11-13 lations with myocarditis and DCM than seen among the current serotypes.6 Of note, there are currently no standard INNATE AND ACQUIRED IMMUNITY methods for validating the detection of these new viruses. Furthermore, studies that show a direct pathogenic effect of During the past decade, inbred and transgenic rodent mod­ persistent viral genome in human DCM are lacking. A few els of myocarditis and DCM have yielded profound in­ antiviral agents, including interferon beta,7 interferon alfa,8 sights into the pathogenesis of DCM.14-16 The role of spe­ immunoglobulin,9 and pleconaril,10 have been used to treat cific in inflammation was found to vary between enteroviral DCM, but none have been studied in random­ different models that used different viruses and genetic ized trials. backgrounds.17 The pathogenic role of complement T-cell The workshop participants identified major challenges subtypes and mast cells in postviral myocarditis and DCM to the translation of scientific advances. The greatest chal­ has been suggested from several models.18-20 More recently, lenge is a lack of human tissue, which is required for the the role of the Coxsackie-adenoviral receptor, toll-like re­ diagnosis. It is probable that endomyocardial biopsy sam­ ceptor 4, and decay accelerating factor has been identified pling error plays a major role in reports of different rates of as important in viral pathogenesis.21 The ubiquitin and viral heart disease. Because endomyocardial biopsy may proteosome pathways can be used by Coxsackie B virus to not be performed frequently in the routine diagnosis of cause cell arrest and promote viral replication.22 , extra effort should be made to obtain Murine models of coxsackieviral-induced myocarditis specimens from other sources, such as cardiac tissue ex­ have been developed,23-26 and viral-induced myocarditis plants and autopsy specimens. Indirect methods of detect­ has been associated with immune responses against cardiac ing virus infection, such as serology, virus-specific T-cell myosin.27-29 Subsequently, cardiac myosin was shown to be activation, /chemokine levels, and expression pro­ a dominant autoantigen capable of inducing myocarditis in filing, should also be developed and pursued since cardiac both mice30,31 and rats32,33 with identification of epitopes pathology may not occur until well after immune clearance causing myocarditis.34,35 In most animal models of cardiac of virus. The members recommend a study to correlate the myosin–induced myocarditis, disease can be transferred by rate of viral-positive genomes on sham biopsies with ex­ CD4+ T cells. Although the role of antibodies in myocardi­ planted heart and/or autopsy tissue. tis is controversial, antibodies have been shown to induce A second barrier to standard assessment of viruses in disease in genetically predisposed mice.36 There is evi­ cardiac disease is a lack of standard methods of tissue dence of autoantibody responses against cardiac myosin, collection, tissue handling, and virus detection. Genome sarcolemmal Na,K-ATPase, G protein-linked receptors, target signals can degrade if tissues are fixed in formalin, or and mitochondrial antigens in humans with myocarditis the sample may be contaminated if the tissue is not pro­ and cardiomyopathy.37,38 Protection against myocarditis by tected from exposure. The members recommend that stan­ immunization with myosin-like or cardiac myosin peptides dard operating procedures for tissue acquisition, handling, has been shown to protect against viral or myosin-induced storage, and transportation be adopted. Quantitative PCR myocarditis,39 and neutralization of α and qRT-PCR techniques should be used to assess the also ameliorates disease in mouse models.40 Therefore, in sensitivity of detecting viruses in tissue samples obtained animal models, immune responses against a variety of by core viral laboratories involved in cardiomyopathy re­ cardiac antigens induce an inflammatory state in the heart, search. Standardization of methodology and determination and case-control studies in humans show an association of sensitivity are critical to assess patient samples in differ­ between anticardiac autoantibodies and DCM. Further ent clinical studies. Although this workshop did not define studies are needed to determine the pathogenic role of the methods for standardization, such methods need to be immune responses against cardiac proteins in human myo­ proposed, discussed, and agreed on for the collaboration of carditis and DCM. cardiologists and virologists. A major challenge is that the relevance of these models The types of viruses that affect different ethnic popula­ in human disease is unknown, and the hypotheses that have tions are also unknown. Epidemiological studies that are been generated in animal models need to be tested in hu­ designed to address the rates of viral cardiomyopathy in man tissue. Available human tissue linked to well-charac­ DCM should be performed before large clinical studies of terized disease phenotypes is lacking. A recommendation antiviral therapy are considered. The role of host genetics of the workshop is to form a multicenter human tissue in the susceptibility to viral infection should receive at­ registry consisting of biopsy, surgical, explanted heart, and tention as a means of stratifying risk, including studies of autopsy tissues with standardized tissue acquisition, han-

200 Mayo Clin Proc. • February 2006;81(2):199-204 • www.mayoclinicproceedings.com For personal use. Mass reproduce only with permission from Mayo Clinic Proceedings. NIH-SPONSORED WORKSHOP ON DILATED CARDIOMYOPATHY dling, and storage. These tissues should be linked to clearly effects. Specifically, diversion of selenium into the seleno­ defined clinical and functional variables. cysteines of virally encoded GPx homopeptides would deplete the host cells of selenium that would otherwise be available for normal selenoprotein synthesis.48 Since ENVIRONMENTAL FACTORS selenophosphate synthetase, one of the cellular proteins Deficiencies of selenium or high levels of cobalt, both trace required for selenoprotein synthesis, is itself a seleno­ elements (TEs), can induce clinically progressive cardiac protein, viral diversion of cellular selenium stores into viral dilation and dysfunction that is histologically indistin­ GPx homopeptides could result in irrecoverable elimina­ guishable from DCM. Myocardial TE accumulation or tion of selenoenzyme metabolic processes within cells that deficiency may result from several factors, including have been virally infected. Infected cells would not be able environmental factors, abnormal dietary intake, intestinal to reinitiate selenoenzyme synthesis, resulting in per­ malabsorption, or an acquired viral myopathy. Selenium manent impairment of free radical detoxification and other deficiency is one of the most clinically relevant TE selenium-dependent cellular processes. To establish disorders.41 Selenium is an unusual TE in that it has its own whether this mechanism contributes to DCM, selenium- messenger RNA codon that specifies its insertion into dependent messenger RNA levels could be measured by in proteins as selenocysteine. Selenocysteine, the 21st physio­ situ hybridization with selenoenzyme functional assays in logically essential amino acid, is present at the active site of DCM and healthy myocardium. all known selenium-dependent enzymes where its unique Cobalt accumulation has been associated with excessive redox potential facilitates their biochemical functions in beer drinking, and mercury intoxication with intake of support of human health.41,42 More than 25 mammalian contaminated fish or abnormal handling of dental amal­ selenium-dependent proteins are currently recognized, gams.49 Cobalt and mercury accumulation in myocytes is each with distinct patterns of tissue and subcellular dis­ accompanied by ultrastructural changes of mitochondria tribution and occurrence.41,42 Normal nutritional selenium and other intracellular organelles; however, the mechanism status is necessary to maintain an adequate immune sys­ of cobalt toxicity is unknown. Pronounced accumulation of tem, and there is increasing evidence that augmented heavy metals (mercury and antimony) has been detected selenium status is associated with diminished cancer risk.43 by neutron activation analysis in endomyocardial biopsy Selenium deficiency may derive from its deprivation in specimens from patients with DCM, independent of sys­ the soil of some geographic areas (Keshan disease)41,42,44 or temic disorders or environmental exposure.50 At this time, from intestinal malabsorption or TE loss caused by an it is not known whether administration of a deficient ele­ intestinal bypass aimed to treat severe obesity. Selenium ment or elimination of a toxic source may lead to functional deficiency exerts its pathogenic effect by reducing the anti­ and clinical improvement in DCM. oxidant properties of the heart, causing an impairment of A major obstacle to research is the lack of systemat­ the immune system and increasing viral genome virulence, ically collected tissue linked to well-characterized clinical thus making the heart more susceptible to viral infec­ data. The interaction among viral heart disease, excessive tions.45,46 Recent work has shown that an amyocarditic alcohol intake, and heavy metals has been suggested but strain of Coxsackie B3 virus (CVB3/0) was converted to not investigated substantially. In addition, recent popula­ virulent in selenium-deficient mice. This conversion was tion-based studies suggest an increased risk of DCM in accompanied by changes in the genetic structure of the black vs white Americans51 and in indigenous vs non- virus to resemble the genome of other virulent Coxsackie indigenous Australian children.52 Both the genetic and the B3 virus strains. Similar increases in virulence accom­ environmental basis for these differences need to be pur­ panied by multiple changes in the viral genome by passage sued in future studies. Specific recommendations include through selenium-deficient hosts have been shown in (1) studies in family members of affected individuals to influenza virus A/Bangkok/1/79.47 define host factors responsible for the increased risk of Another possible link between selenium and viral in­ DCM and (2) a clinical study designed to confirm the role fections could be represented by the evidence that several of TE and heavy metals in DCM and to better characterize viral pathogens possess a highly conserved gene sequence the interaction among environmental factors, cardiotropic capable of encoding for a truncated glutathione peroxidase virus infection, and host genetics. (GPx). Such viruses include coxsackievirus, human im­ munodeficiency virus 1, human immunodeficiency virus 2, NOVEL DIAGNOSTICS hepatitis C, hepatitis B, , , and Ebola. Al­ though the target tissues for these viruses vary widely, it is Hematoxylin-eosin stained cardiac tissue from endomyo­ possible that the viruses have some common functional cardial biopsy specimens interpreted by the Dallas criteria

Mayo Clin Proc. • February 2006;81(2):199-204 • www.mayoclinicproceedings.com 201 For personal use. Mass reproduce only with permission from Mayo Clinic Proceedings. NIH-SPONSORED WORKSHOP ON DILATED CARDIOMYOPATHY has been the gold standard for the antemortem diagnosis of state-of-the-art qPCR methods for the detection of myocarditis for more than 20 years.53 Major problems with cardiotropic viruses. The workshop members agreed that this standard include sampling error and lack of expert such trials should have sufficiently large sample sizes and consensus on myocyte necrosis and inflammation criteria. lengthy follow-up times to detect significant treatment ef­ Furthermore, diagnosis of myocarditis based on the Dallas fects on quality of life, functional capacity, or composite criteria is not prognostically useful in acute DCM.54 There­ end points that may include death, cardiovascular death, fore, heart biopsy is currently performed infrequently be­ and heart transplantation. cause it rarely changes prognosis or treatment in DCM.55 Immunoabsorption to remove anticardiac autoantibod­ Uncommon but important exceptions to this general rule ies in patients with evidence of autoimmune cardiomyopa­ include myocarditis,56 cardiac sarcoidosis, eosi­ thy is a promising new technology.67,68 Current limitations nophilic myocarditis (including hypersensitivity myocardi­ include the lack of an adequate animal model and a clear tis),57 and severe cases of myocarditis after small pox vac­ mechanistic rationale for persistent treatment benefit. Spe­ cine (vaccinia) myocarditis.58 The workshop members rec­ cifically, it is unclear why cardiac autoantibodies do not ommend investigation of the prognostic usefulness of rebound after several weeks or months to pretreatment novel immunohistologic markers and viral genomes levels, and the mechanism of long-term benefit is unclear. coupled with standardized tissue acquisition. Limitations of current data include the lack of adequately Cardiac magnetic resonance imaging (MRI) has emerged controlled studies and the use of different immunoabsorp­ as a promising, noninvasive imaging technique for the tion columns and different treatment protocols in various diagnosis of DCM and myocarditis. Recent studies have reports. The workshop participants agreed that immuno­ correlated the presence of myocarditis with gadolinium absorption is an appealing new technology that deserves uptake on cardiac MRI.59 In these studies, the pattern of further investigation. inflammation is usually distinct from ischemic damage. Therefore, MRI may be useful for identifying regional SUMMARY OF RECOMMENDATIONS abnormalities that occur early in the course of clinical myocarditis. Future studies need to address the prognostic On the basis of the presentations and discussions of the value of MRI in addition to established clinical variables. workshop sections, the participants recommended 6 areas Novel blood and serologic markers of prognosis were that would benefit from increased support from the NIH discussed by the workshop members, including soluble Fas during the next 5 to 10 years: (CD95/Apo1, a tumor necrosis factor receptor),60 soluble 1. Studies to determine the sensitivity and specificity Fas ligand,61 interleukin 10, and genomic studies from of qRT-PCR to detect cardiotropic viruses in a broad popu­ peripheral blood mononuclear cells. All these hold promise lation of heart failure patients with DCM for diagnosis and prognosis in DCM. The workshop 2. Determination of the prognostic value of viral ge­ strongly supported further investigation into these areas. nome detection in acute and chronic DCM—the study should be designed to detect added prognostic value be­ yond that available from clinical and noninvasive variables NOVEL THERAPEUTICS 3. Studies to determine the role of innate and adaptive The workshop members addressed the potential for future immunity in human DCM—these studies should take ad­ randomized treatment trials for acute or chronic DCM. vantage of registries of human tissue Because of the rarity of acute DCM and the large improve­ 4. Familial case-control studies to determine the ge­ ments in the placebo groups of the Myocarditis Treatment netic basis of DCM and the interaction of genetic predispo­ Trial62 and the Intervention in Myocarditis and Acute Myo­ sition with environmental variables cardiopathy trial,63 an adequately powered treatment trial 5. Studies designed to determine the prognostic useful­ of patients with acute DCM of less than 6 months’ dura­ ness of MRI and novel blood and serologic markers for tion was considered infeasible. In contrast, the workshop DCM and myocarditis members noted several positive treatment trials of im­ 6. Studies of immunoabsorption for the treatment of munomodulatory and antiviral therapy for chronic DCM. chronic DCM These included interventions with azathioprine and pred­ 64,65 66 nisone, intravenous immunoglobulin, and interferon Participating Investigators and Institutions. Leslie T. Cooper, 7 beta. Obstacles to an adequately powered antiviral or MD (Chair) and Richard J. Rodeheffer, MD, Mayo Clinic College immunomodulatory treatment trial of chronic DCM in­ of Medicine, Rochester, Minn; Renu Virmani, MD (Cochair), clude the need for a multicenter design, consensus on the International Registry of Pathology, Gaithersburg, Md; Judith optimal diagnostic criteria, and incorporation of current Massicot-Fisher, PhD, Alice Mascette, MD, and Jamie Varghese,

202 Mayo Clin Proc. • February 2006;81(2):199-204 • www.mayoclinicproceedings.com For personal use. Mass reproduce only with permission from Mayo Clinic Proceedings. NIH-SPONSORED WORKSHOP ON DILATED CARDIOMYOPATHY

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