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

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, their patterns of clearance and/or persistence).

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**).

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

Acute Myocarditis – A Trigger of

impact of heart-directed autoimmune reactions.

(One major and two minor criteria fulfilled)

Suspicion of acute myocarditis

**Major criteria** 

**Minor criteria** 

**AMitis EAMI**

**MRT 24-96h** after hospitalisation

Clinical state (+ PHQ) Biomaterials (blood, **Cardiac aabs**) ECG Holter-ECG Echocardiography

Cardiac Autoimmunity? Expected Insights from the Etiology, Titre-Course… 391

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

**ST/T wave changes in the electrocardiograms (ECG)** 

**Impairment of LVEF on echocardiography with CAD excluded by coronary** 

Clinical state Biomaterials (blood, **Cardiac aabs**) ECG Holter-ECG Echocardiography

> **MRT 12** months after hospitalisation

**Ventricular arrhythmia (ECG)** 

**angiography (no stenosis >50%)** 

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

**0 2 4 6 8 10 12 months**

Table 1. Diagnostic criteria for suspicion of acute myocarditis.

Clinical state Biomaterials (blood, **Cardiac aabs**) ECG Holter-ECG Echocardiography

**Pericardial effusion on echocardiography** 

**Dyspnea, new onset within the past 30 days Chest pain, new onset within the past 30 days Palpitations, new onset within the past 30 days History of infection within the past 30 days Fever >38.0° C within the past 30 days** 

> Clinical state Biomaterials (blood, **Cardiac aabs**) ECG Holter-ECG Echocardiography

**Baseline Follow-up 1 Follow-up 2 Follow-up 3**

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

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 heart failure.

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

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 titrecourse and prognostic impact of all cardiac aabs prospectively analyzed.
