**1. Introduction**

386 Myocarditis

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Progressive cardiac dilatation and pump failure of unknown aetiology - termed "idiopathic" dilated cardiomyopathy (DCM) (Richardson et al., 1996; Maron et al., 2006) - represents one of the main causes of severe heart failure in Western populations with an annual incidence of about 100 and a prevalence of 300-400 patients per year (American Heart Association, 2009). The large majority of cases are thought to arise from an initial (mostly viral) infection leading to acute myocardial inflammation. Acute myocarditis may either heal (about one third of the cases) or progress to a chronic inflammatory process with continued fibrotic repair, subsequent dilatation of the left and/or right ventricle and –finally– severe congestive heart failure (about another third of the patients). Progression to DCM appears to occur particularly, when associated (a) with chronic inflammation of the myocardium due to viral persistence (Kühl et al., 2005) and/or (b) with the development of autoantibodies directed against distinct sarcoplasmatic or myocyte membrane proteins that are essential for cardiac function (Freedman & Lefkowitz, 2004; Jahns et al., 2006). The latter findings are further strengthened by the fact that patients with DCM often have alterations in both, their innate and their adaptive immune system (Limas, 1997; Luppi et al., 1998; Jahns et al., 2006; Mahrholdt et al., 2006). Thus, under certain conditions an initial acute inflammatory reaction may proceed into a kind of low-grade inflammation (MacLellan & Lusis, 2003) facilitating the development of abnormal or misled immune responses to the primary (infectious)

<sup>\*</sup> Nikolas Deubner1, Valérie Biovin2, Alida L.P. Caforio3, Stephan B. Felix4, Michael Fu5, Martin J. Lohse2, and Georg Ertl1

*<sup>1</sup> University Hospital of Würzburg, Department of Internal Medicine I,Cardiology, Comprehensive Heart Failure Centre CHFC/IFB, Würzburg, Germany;* 

*<sup>2</sup> University of Würzburg, Institute for Pharmacology and Toxicology, Würzburg, Germany;* 

*<sup>3</sup> University of Padua, Division of Cardiology, Dpt. of Cardiologic, Thoracic and Vascular Sciences, Padua, Italy;* 

*<sup>4</sup> University of Greifswald, Department of Internal Medicine B – Cardiology, Greifswald,Germany;* 

*<sup>5</sup> University of Gothenburg, Department of Molecular and Clinical Medicine, Gothenburg, Sweden.* 

Acute Myocarditis – A Trigger of

autoantibodies (**ETiCS**) study.

**2. Rationale and scope of the ETiCS study** 

their patterns of clearance and/or persistence).

Cardiac Autoimmunity? Expected Insights from the Etiology, Titre-Course… 389

To generate an autoimmune response, myocyte membrane proteins (including receptors) must be degraded to small oligopeptides able to form a complex with a major histocompatibility (MHC) class II or human leukocyte antigen (HLA) molecule of the host (Hoebeke et al., 1996). In previous clinical studies autoimmunity has been found to be associated with certain HLA- and MHC class II-phenotypes (Limas, 1996), and also with the expression and/or activity of the T-lymphocyte antigen 4 (CTLA-4) - known as a potent (indirect) suppressor of the immune system (Golden et al., 2005). Therefore, another important point to consider in the development of (human) post-inflammatory and/or postischemic cardiomyopathy is the patients' genetic pre-disposition, which will determine both, the susceptibility to self-directed immune reactions and the phenotypic expression of

On this background the following book-chapter will review current knowledge and recent experimental and clinical evidence for the potential role of cardiac autoantibodies in the pathogenesis of DCM focussing on the rationale and expected insights from the prospective diagnostic multicentre **E**tiology, **Ti**tre-**C**ourse, and (effect on) **S**urvival of cardiac

Evidence for a pathophysiologic role of autoimmunity in human heart disease has substantially increased during the past two decades, but the true prevalence and clinical impact of cardiac autoantibodies (aabs) in human heart disease are still unclear, as are the events leading to their formation, their frequency of appearance, and their kinetics (that is,

In this regard, the investigator-initiated diagnostic multicentre ETiCS study will prospectively address the hypothesis that a first inflammatory (i.e., acute myocarditis (AMitis)) or ischemic injury of the myocardium (i.e., first acute myocardial infarction (FAMI)) may trigger the development of heart-directed autoimmune reactions (**Fig. 1**).

the myocardial disease (MacLellan & Lusis, 2003; Limas et al., 2004).

Fig. 1. Formation of autoantibodies against myocardial self-antigens.

trigger (MacLellan & Lusis, 2003; Freedman & Lefkowitz, 2004; Kühl et al., 2005; Smulski et al., 2006; Maekawa et al., 2007). More recently, a detailed molecular analysis of T cell infiltrates in human endomyocardial biopsies (EMBs) from both, patients with acute myocarditis and patients with (post-)inflammatory DCM revealed an increased expression of CD3d, CD3z, and T cell receptor beta constant region (TRBC) in both disease entities. However, differential expression of functional T cell markers was found in DCM EMBs only (dominance of Th1 markers, regulatory [FoxP3] T cells, and cytotoxic T cells (CTLs)) and not in acute myocarditis. Additionally, in DCM EMBs some Th2 marker genes were increased, indicating that a Th2 response (required for T/B cell interactions) also participates in the T cell infiltrates in DCM (Noutsias et al., 2011). This might explain, why a substantial number of DCM patients have been found to develop cross-reacting antibodies and/or autoantibodies to different cardiac self-antigens, including mitochondrial proteins (e.g., adenine nucleotide translocator, lipoamide and pyruvate dehydrogenase (Schultheiss & Bolte, 1985; Schultheiss et al., 1988; Pohlner et al., 1997; Schulze et al., 1999)), sarcoplasmatic proteins (e.g., actin, laminin, myosin, troponin (Neumann et al., 1990; Caforio et al., 2002; Okazaki et al., 2003; Göser et al., 2006; Li et al., 2006)), and membrane proteins (e.g., cell surface adrenergic or muscarinergic receptors (Fu, L.X.M. et al., 1993; Magnusson et al., 1994; Jahns et al., 1999; Christ et al., 2006)).

Irrespective of whether development of DCM is primarily due to chronic myocardial infection (Kühl et al., 1996) or to abnormalities in the adaptive or innate immune system (Luppi et al., 1998; Eriksson et al., 2003), in both cases cardiac tissue injury is believed to be mediated mainly by cytokines and/or heart-specific autoantibodies (Caforio et al., 1995; Limas, 1997; Eriksson et al., 2003). However, the pathophysiological relevance of each of the aforementioned cardiac autoantibodies (aabs) is far from clear. Low titers of autoantibodies to various house-keeping antigens can also be detected in healthy subjects as a part of the natural immunological repertoire (Rose, 2001).

In addition, under physiological conditions at least the intracellularly localized cardiac antigens are not easily accessible for the immune system. Thus, the mechanisms by which autoimmune-mediated myocardial injury is initiated are mostly based on indirect or circumstantial evidence (Limas, 1997). The potential pathophysiological - and thus clinical – relevance of a heart-specific autoantibody depends on its disease-inducing or -aggravating potential, which in turn is supposed to be associated with both the *accessibility* and the *functional relevance* of its target. Therefore, autoantibodies directed against cell surface key constituents, and in particular aabs that have the potential to affect myocardial contraction and relaxation (e.g. , by interaction with the cardiac beta1-adrenoceptor (beta1-AR)) and/or the M2-muscarinic acetylcholine receptor (M2-AchR)) represent key candidates involved in the initiation and/or progression of DCM (Fu et al. 1993, 2008; Magnusson et al., 1996; Limas, 1997; Engelhardt et al., 2004; Jahns et al., 2004; Freedman & Lefkowitz, 2004). Whereas anti-muscarinic antibodies (exhibiting an agonist-like effect on cardiac M2-AchR) have been associated with negative chronotropic effects at the sinuatrial level (e.g., sinus node dysfunction, atrial fibrillation (Wang et al., 1996; Baba et al., 2004)), functionally activating anti-beta1-AR antibodies have been associated with both the occurrence of severe arrhythmias at the ventricular level, and the development of (maladaptive) left ventricular hypertrophy, finally switching to left ventricular enlargement and progressive heart failure (Jahns et al., 1999, 2006; Iwata et al., 2001; Engelhardt et al., 2004; Störk et al., 2006). In addition, both autoantibodies seem to target the (easily accessible) second extracellular loop of the respective receptors.

trigger (MacLellan & Lusis, 2003; Freedman & Lefkowitz, 2004; Kühl et al., 2005; Smulski et al., 2006; Maekawa et al., 2007). More recently, a detailed molecular analysis of T cell infiltrates in human endomyocardial biopsies (EMBs) from both, patients with acute myocarditis and patients with (post-)inflammatory DCM revealed an increased expression of CD3d, CD3z, and T cell receptor beta constant region (TRBC) in both disease entities. However, differential expression of functional T cell markers was found in DCM EMBs only (dominance of Th1 markers, regulatory [FoxP3] T cells, and cytotoxic T cells (CTLs)) and not in acute myocarditis. Additionally, in DCM EMBs some Th2 marker genes were increased, indicating that a Th2 response (required for T/B cell interactions) also participates in the T cell infiltrates in DCM (Noutsias et al., 2011). This might explain, why a substantial number of DCM patients have been found to develop cross-reacting antibodies and/or autoantibodies to different cardiac self-antigens, including mitochondrial proteins (e.g., adenine nucleotide translocator, lipoamide and pyruvate dehydrogenase (Schultheiss & Bolte, 1985; Schultheiss et al., 1988; Pohlner et al., 1997; Schulze et al., 1999)), sarcoplasmatic proteins (e.g., actin, laminin, myosin, troponin (Neumann et al., 1990; Caforio et al., 2002; Okazaki et al., 2003; Göser et al., 2006; Li et al., 2006)), and membrane proteins (e.g., cell surface adrenergic or muscarinergic receptors (Fu, L.X.M. et al., 1993; Magnusson et al.,

Irrespective of whether development of DCM is primarily due to chronic myocardial infection (Kühl et al., 1996) or to abnormalities in the adaptive or innate immune system (Luppi et al., 1998; Eriksson et al., 2003), in both cases cardiac tissue injury is believed to be mediated mainly by cytokines and/or heart-specific autoantibodies (Caforio et al., 1995; Limas, 1997; Eriksson et al., 2003). However, the pathophysiological relevance of each of the aforementioned cardiac autoantibodies (aabs) is far from clear. Low titers of autoantibodies to various house-keeping antigens can also be detected in healthy subjects as a part of the

In addition, under physiological conditions at least the intracellularly localized cardiac antigens are not easily accessible for the immune system. Thus, the mechanisms by which autoimmune-mediated myocardial injury is initiated are mostly based on indirect or circumstantial evidence (Limas, 1997). The potential pathophysiological - and thus clinical – relevance of a heart-specific autoantibody depends on its disease-inducing or -aggravating potential, which in turn is supposed to be associated with both the *accessibility* and the *functional relevance* of its target. Therefore, autoantibodies directed against cell surface key constituents, and in particular aabs that have the potential to affect myocardial contraction and relaxation (e.g. , by interaction with the cardiac beta1-adrenoceptor (beta1-AR)) and/or the M2-muscarinic acetylcholine receptor (M2-AchR)) represent key candidates involved in the initiation and/or progression of DCM (Fu et al. 1993, 2008; Magnusson et al., 1996; Limas, 1997; Engelhardt et al., 2004; Jahns et al., 2004; Freedman & Lefkowitz, 2004). Whereas anti-muscarinic antibodies (exhibiting an agonist-like effect on cardiac M2-AchR) have been associated with negative chronotropic effects at the sinuatrial level (e.g., sinus node dysfunction, atrial fibrillation (Wang et al., 1996; Baba et al., 2004)), functionally activating anti-beta1-AR antibodies have been associated with both the occurrence of severe arrhythmias at the ventricular level, and the development of (maladaptive) left ventricular hypertrophy, finally switching to left ventricular enlargement and progressive heart failure (Jahns et al., 1999, 2006; Iwata et al., 2001; Engelhardt et al., 2004; Störk et al., 2006). In addition, both autoantibodies seem to target the (easily accessible) second extracellular loop

1994; Jahns et al., 1999; Christ et al., 2006)).

natural immunological repertoire (Rose, 2001).

of the respective receptors.

To generate an autoimmune response, myocyte membrane proteins (including receptors) must be degraded to small oligopeptides able to form a complex with a major histocompatibility (MHC) class II or human leukocyte antigen (HLA) molecule of the host (Hoebeke et al., 1996). In previous clinical studies autoimmunity has been found to be associated with certain HLA- and MHC class II-phenotypes (Limas, 1996), and also with the expression and/or activity of the T-lymphocyte antigen 4 (CTLA-4) - known as a potent (indirect) suppressor of the immune system (Golden et al., 2005). Therefore, another important point to consider in the development of (human) post-inflammatory and/or postischemic cardiomyopathy is the patients' genetic pre-disposition, which will determine both, the susceptibility to self-directed immune reactions and the phenotypic expression of the myocardial disease (MacLellan & Lusis, 2003; Limas et al., 2004).

On this background the following book-chapter will review current knowledge and recent experimental and clinical evidence for the potential role of cardiac autoantibodies in the pathogenesis of DCM focussing on the rationale and expected insights from the prospective diagnostic multicentre **E**tiology, **Ti**tre-**C**ourse, and (effect on) **S**urvival of cardiac autoantibodies (**ETiCS**) study.
