**3. Design of the ETiCS study**

The prospective ETiCS study complies with the standards of Good Clinical Practice (GCP) and has been approved by the Ethics committees of all participating institutions. Within a two years-period ETiCS will include 400 patients with a first cardiac event, 200 of them with acute myocarditis (AMitis), and 200 patients with a first myocardial infarction (FAMI; acute ST-elevation MI only, without any history or signs of previous myocardial infarction). After inclusion and baseline assessment, the patients will undergo three follow-up visits after 3, 6, and 12 months (Fig. 2). Diagnosis of acute myocarditis is based on at least one major and two minor clinical criteria and/or symptoms (see table 1) and must be confirmed by endomyocardial biopsy (EMB) using either the WHO/ISFC (Richardson et al., 1996; Elliott et al., 2008) or the Dallas criteria (Aretz et al., 1987). This proceeding is in full accordance

Immunogenicity is defined as the property of a molecule to induce an immune response (Hoebeke, 1996). To serve as a potential antigen, myocyte constituents (e.g., cardiac membrane receptors) must be degraded by proteolysis into small fragments (oligopeptides), and one or several of the generated fragments must be able to form a complex with one of the major histocompatibility complexes (class II-MHCs) or human leukocyte antigen (HLA) molecules of the host. When presented to T cells (Harding et al., 1990; Mobini et al., 1999) antigenic parts of such myocyte-derived (self) peptides may engender an immunological response. Acute inflammatory processes are supposed to enhance the occurence of selfdirected immune responses (e.g., acute viral or bacterial myocarditis and/or acute ischemic events (Borda et al., 1984; Latif et al., 1993; Kühl et al., 1996; Limas, 1997; Noutsias et al.,

Therefore, we assume that a first substantial inflammatory or ischemic myocyte damage – through liberation of a "critical amount" of cardiac self-antigens previously hidden to the immune system – might induce and perpetuate a disease-causing and/or -modulating autoimmune reaction that deteriorates cardiac function and ultimately results in progressive

As a consequence, the ETiCS study has been designed to provide a maximum of sequential clinical and serological data from patients after a first inflammatory or ischemic cardiac event. The development/prevalence and titre-course (clearance/persistence) of distinct cardiac aabs after 0, 3, 6, and 12 months of either event will be prospectively assessed and correlated with the corresponding cardiac functional parameters, cardiac imaging (echocardiography, cardiac magnetic resonance imaging), and clinical outcome. A limited number of ETiCS sub-studies will focus on components of the patients' immune system potentially involved in the generation of cardiac receptor-aabs – including a search for predisposing genotypes (Limas et al., 2004; Caforio & Iliceto, 2008) – and on the possible impact

Expanding the scope of ETiCS beyond adrenoceptor-directed autoimmunity other known cardiac aabs will be investigated by the respective expert core centres, including aabs against the muscarinic acetylcholine receptor 2 (Fu, L.X.M. et al., 1993), against troponin I (Göser et al., 2006), organ-specific and skeletal muscle cross-reactive anti-heart-aabs (Caforio et al., 2007), and cardio-depressant aabs (Felix et al., 2002). This joint venture will enable a comprehensive characterisation of heart-directed autoimmunity after inflammatory or ischemic disruption of myocardial integrity, and allow for cross-correlations of the titre-

The prospective ETiCS study complies with the standards of Good Clinical Practice (GCP) and has been approved by the Ethics committees of all participating institutions. Within a two years-period ETiCS will include 400 patients with a first cardiac event, 200 of them with acute myocarditis (AMitis), and 200 patients with a first myocardial infarction (FAMI; acute ST-elevation MI only, without any history or signs of previous myocardial infarction). After inclusion and baseline assessment, the patients will undergo three follow-up visits after 3, 6, and 12 months (Fig. 2). Diagnosis of acute myocarditis is based on at least one major and two minor clinical criteria and/or symptoms (see table 1) and must be confirmed by endomyocardial biopsy (EMB) using either the WHO/ISFC (Richardson et al., 1996; Elliott et al., 2008) or the Dallas criteria (Aretz et al., 1987). This proceeding is in full accordance

of conformational adrenoceptor-aabs on renal function (Boivin et al., 2001).

course and prognostic impact of all cardiac aabs prospectively analyzed.

**3. Design of the ETiCS study** 

1999; Liu & Mason, 2001; Rose, 2001; Caforio et al., 2002)).

heart failure.

with the actual AHA/ACC/ESC scientific statement on the role of EMB in the management of cardiovascular disease (Cooper et al., 2007), which strongly recommends EMB in acute myocarditis because of its potential relevance for outcome and therapeutic decisions (Kindermann et al., 2008; Frustaci et al., 2009). In addition, immunohistology and molecular analysis of the EMBs may also significantly contribute to further elucidate the clinical impact of heart-directed autoimmune reactions.


Table 1. Diagnostic criteria for suspicion of acute myocarditis.

Fig. 2. Time schedule of the follow-up examinations in the ETiCS study.

Echocardiography and cardiac magnetic resonance imaging (cMRI) will be carried out in all patients within 24 to 96 hours of hospitalisation and one year after the respective cardiac

Acute Myocarditis – A Trigger of

patients.

2008).

susceptibility for heart-directed autoimmune reactions.

**4.2 Adrenoceptor-autoantibodies and their impact on kidney function** 

formation and conversion rates (clearance/persistence) of adrenoceptor-aabs.

**5. Conclusion and expected insights from the ETiCS study** 

These data might furnish a rationale for the development of novel therapeutic strategies to protect the kidney(s) from functional adrenoceptor-aabs in aab-positive heart failure

In the last two decades, much knowledge has accumulated with respect to the possible pathophysiologic and clinical implications of heart-directed aabs (Jahns et al., 2006; Fu, M.,

Homologies between cardiomyocyte surface molecules (in particular membrane receptors) and viral or bacterial proteins have been proposed as a mechanism for the elaboration of endogenous cardiac autoantibodies by antigen mimicry (Elies et al., 1996; Hoebeke et al., 1996). Chagas' heart disease, a slowly evolving inflammatory cardiomyopathy, is one of the best investigated examples to highlight this mechanism. The disease is induced by the protozoon *Trypanosoma cruzi*. About 30% of the Chagas' patients develop antibodies that cross-react between the ribosomal P2beta-protein of *T. cruzi* and some specific amino acids present in the second extracellular loop of the human beta1-adrenoceptor (Elies et al., 1996).

Cardiac Autoimmunity? Expected Insights from the Etiology, Titre-Course… 393

antigenic recall assays as well as FACS and Elispot analyses. In addition, the respectively activated immunologic paths will be derived from the Th1/Th2/Th17 cytokine profiles determined in the sera from cardiac autoantibody-positive patients with acute myocarditis (including an analysis of the corresponding EMBs (Noutsias et al, 2011)) or acute transmural myocardial infarction. Since the cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a potent (indirect) suppressor of the immune system, its mutation or hampered expression might promote hyperreactivity to autoantigens (Golden et al., 2005). Thus, in all ETiCS patients CTLA-4 expression and CTLA-4 alleles will be determined by FACS and by PCR, respectively. In addition, in all patients the HLA-DR/DQ and MHC class II haplotypes (Limas et al., 2004; Caforio & Iliceto, 2008) will be determined and correlated with the formation and titre-course of distinct cardiac aabs in order to unravel the individual genetic

The possible clinical implications of adrenoceptor-directed autoimmunity have gained increasing interest in human heart disease. By contrast, the renal effects of functionally active adrenoceptor-aabs have been almost neglected. Previous immuno-histologic studies on the rat kidney strongly suggest that functionally stimulating adrenoceptor aabs are capable of modulating the secretion of renin from the juxtaglomerular cells *via* renal beta1- AR and, also, to increase the sodium-reabsorption in the distal tubulus (Boivin et al., 2001). Thus, through renal beta1-AR-mediated activation of the renin-angiotensin-aldosteronesystem (RAAS) and/or augmentation of the circulating blood volume such aabs might contribute to further worsening of the prognosis of antibody-positive heart failure patients. To assess the impact of adrenoceptor-aabs on kidney function (and clinical outcome), a number of pre-specified renal functional parameters will be sequentially determined in all ETiCS patients (e.g. serum *versus* urine creatinine/sodium/pH; glomerular filtration rate (GFR), urinary protein excretion). Primary endpoints are the relationship between antibodytitres and renal function at baseline and the "time to first cardiovascular event", adjusted for renal function. Secondary endpoints include the change in renal function dependent on the

event. Follow-up visits at 3, 6, and 12 months comprise a detailed patient history including medication, physical examination, a standardized health questionnaire, echocardiography, ECG, Holter-ECG, and blood sampling (**Fig. 2**). The sequentially obtained blood samples will serve to determine the time course of formation and the titre-course (clearance/persistence) of distinct cardiac aabs at 0, 3, 6, and 12 months after inflammatory or ischemic cardiac injury (AMitis, FAMI). Three-hundred healthy subjects with normal blood pressure, ECG, and exercise-stress test will serve as a control collective (male to female ratio 1:1, n=50 per five-years age range, no history of myocardial infarction, diabetes, or peripheral vascular disease). The primary endpoint of the planned cross-sectional analyses is the association of a specific cardiac aab status at diagnosis (aab-positive/aabnegative) with (**a**) the change in cardiac function as derived from sequential echocardiograms and cMRI (baseline *versus* 12 months), and (b) the severity and clinical course of the index disease. Longitudinal primary endpoints are titre-changes of a given cardiac aab over time, conversion rates (persistence/clearance), and the "time to first cardiovascular event" (see table 2 for definition of composite endpoints). Pre-specified secondary endpoints for each of the different cardiac aabs at diagnosis are time to cardiovascular death and time to all-cause death (table 2).


Table 2. Pre-defined endpoints of the ETiCS study.
