Background: Genetic heart muscle diseases such as arrhythmogenic (ACM) or dilated cardiomyopathy (DCM) can be caused by mutations in desmosomal proteins or titin, respectively. Both diseases manifest in a highly heterogeneous manner with incomplete penetrance and variable clinical outcomes, even for the same gene mutations, indicating that a genetic predisposition is necessary yet not sufficient to induce the cardiac phenotype. Different environmental “second hits” are being discussed to contribute to disease development in addition to genetic factors. Here, we examined the influence of a murine cytomegalovirus (MCMV) infection on cardiomyopathy onset and progression using mouse models of plakophilin 2 (Pkp2^+/-) and titin (Ttn^+/-) haploinsufficiency, which in human patients are associated with ACM and DCM, respectively.

Methods and Results: Pkp2^+/- and Ttn^+/- mice were characterized at baseline and did not develop any cardiac structural or functional phenotype compared to wild-type littermates (Ctr).  Application of MCMV resulted in infiltration of cytotoxic CD8⁺ T-cells into the myocardium and other organs, which persisted until 6 months post infection (6mpi) in Pkp2^+/- (Pkp2^+/- MCMV), Ttn^+/- (Ttn^+/- MCMV) and respective Ctr infected mice (Ctr MCMV). Echocardiography at different time points post infection showed progressive impairment of systolic function starting at 3mpi in Pkp2^+/- MCMV but not in Ttn^+/- MCMV mice, not even at 6mpi. Histological examinations however did not reveal a fibrotic response in seropositive Pkp2^+/- MCMV mice. Flow cytometry targeting T-cell subsets of cardiac leucocytes showed a significant increase of CD8⁺ T-cells in Pkp2^+/- MCMV hearts compared to respective Ctr. MCMV and Ttn^+/- MCMV mice. In addition, the counts of monocytes/macrophages were exclusively elevated in Pkp2^+/- MCMV hearts, pointing towards ongoing immunological processes in the myocardium, which seem to arise from a specific combined effect of Pkp2 mutation and MCMV infection. Preliminary single-cell RNA sequencing data of 6mpi hearts supported the presence of virus-specific KLRG1⁺ CD8⁺ T-cells with a cytotoxic and pro-inflammatory profile in all infected animals. Sub-clustering unraveled a specific accumulation of macrophages with a pro-fibrotic “tissue-injury” signature in a genotype dependent context already in mock-infected Pkp2^+/- animals, which was more pronounced in Pkp2^+/- MCMV mice supporting the hypothesis, that MCMV infections might amplify minor myocardial damages caused by genetic predisposition and thereby lead to a clinical manifestation. However, this effect appeared to be specifically linked to certain genetic alterations such as disturbed cellular adhesion (Pkp2^+/-), as alterations of the sarcomere component Titin (Ttn^+/-) did not evoke cardiac changes.

Conclusion: We implicate inflammation and immune response as major drivers for cardiomyopathy onset and that both, genetic predisposition and common viral infections, may contribute to these processes. We showed that Pkp2 haploinsufficiency specifically changed the presence and composition of myocardial immune cells and that an infection with MCMV resulted in a progressive cardiac dysfunction at organ level in this model. Whether inflammatory immune responses function as a primary cause or occur downstream to the viral hit remains subject of further investigations.