Background

The prevalence and involvement of cardiac autoantibodies (aAB) in dilated cardiomyopathy (DCM) still remains unclear. Immunization of mice using immunogenic peptide sequences from cardiac proteins induces experimental autoimmune myocarditis (EAM) with consecutive DCM phenotype. This study aimed to investigate the prevalence of immunogenic epitopes from cardiac aABs in DCM patients.

Methods

In this retrospective single center study, we enrolled 83 DCM patients endomyocardial biopsy confirmed who presented between May 2010 and July 2013 at university hospital of Heidelberg. A subgroup of healthy volunteer’s served as control. Clinical characteristics from DCM patients were accessed retrospectively. The study was performed in confirmation with the principles of Declaration of Helsinki (2008) and approved by local ethics committee. Peptide-array analysis was performed for IgG antibodies against preselected peptides from sera of DCM patients. Proteins and peptide sequences were chosen after comprehensive review of the literature for proteins inducing EAM, cardiomyopathies, myocardial inflammation or myocardial fibrosis in animal models or human. Proteins were fragmented into peptides to identify immunogenic epitopes within the respective protein. Array analysis was performed using PEPperPRINT peptide-array by PEPperPRINT Heidelberg GmbH. For analysis, the 95^th percentile of the array intensity from healthy individuals was calculated and subtracted from the intensity of each peptide from DCM patients. Positive epitopes exhibiting a positive aAB intensity in at least >12% were selected for analysis. Continuous variables are presented as medians with interquartile range. Statistical analysis was performed using MedCalc 11.1 (MedCalc Software bvba, Ostend Belgium). For generation of figures GraphPad 7 (GraphPad Software La Jolla, California) was used.  

Results

In a total of 130 screened peptide sequences, we identified 10 immunogenic epitopes. Here, aABs against peptide sequences of beta-2-adrenergic receptor (EAINCYANETCCDFFTNQAY) could be detected in 12%, beta-1-adrenergic receptor (β1AR: LMHWWRAESDEARRCYNDP) in 17%, and a total of 8 immunogenic epitopes from cardiac myosin (α-CM) were identified. Immunogenic epitopes of α-CM include aABs against peptide sequence of KRKLEDECSELKRDID in 25%, KLELQSALEEAEASLEH in 19%, KIQLEAKVKEMNERLE in 13%, IMDLENDKQQLDERLK, DQMNEHRSKAEETQRSVND and LQSALEEAEASLEHEEGKI in 13% and aABs against peptide sequences of RVLSKANSEVAQWRTKYE and RQAEEAEEQANTNLSKFR in 12% of DCM patients. For aAB positive patients in β1-AR and α-CM (KRKLEDECSELKRDID) a significant difference in hsTnT serum level and left ventricular ejection fraction (LVEF) after a follow up of 12 [8-22] months could be detected (β1-AR: hsTnT: 11 [9-31] vs. 8 [5-14], p=0.0384, LVEF: 28 [20-40] vs. 40 [30-47], p=0.0304). (α-CM: hsTnT: 10 [9-24] vs. 8 [5-14], p=0.0272, LVEF: 28 [20-41] vs. 40 [30-48], p=0.0095).

Conclusions

In this study we were able to characterize immunogenic epitopes of cardiac aABs in DCM patients. This study offers insights for potential screening mechanisms in order to identify DCM patients at risk.

Sources of Funding

This study was founded by the German Research Foundation (DFG) und the Else-Fresenius Stiftung.