Background: Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disorder with a wide clinical spectrum, ranging from asymptomatic carriers to patients experiencing heart failure and severe arrhythmias. Desmosomal proteins like plakophilin 2 (PKP2) are responsible for maintaining cardiac cell adhesion and mutations in those genes are the major cause of the disease. Interestingly, clinically affected patients often present with acute myocarditis – called “hot phases” of disease.  Whether triggers, such as common viral infections may contribute to disease onset and progression remains unknown. Therefore, we investigated the impact of a murine cytomegalovirus (MCMV) infection on ACM using mice with a heterozygous cardiomyocyte-restricted loss of Pkp2 (Pkp2^+/-), which were indistinguishable from wild type (Ctr) mice under normal conditions.

Methods and Results: Acute MCMV infection led to the formation of immune cell plaques in myocardial tissue sections, consisting of cytotoxic CD8⁺ and CD4⁺ T cells, regardless of the genotype.  Viral DNA load in infected organs decreased over time but persisted until 6 months post infection (6mpi), potentially exposing the mice to recurring virus-related inflammation. In line with our hypothesis that inflammation might amplify myocardial damage, we detected systolic dysfunction starting at 3mpi and worsening over time in Pkp2^+/- MCMV, but not in Ctr MCMV hearts. However, in cardiac tissue sections, fibrosis was not detected. Instead, immune cell infiltration and associated inflammation emerged as a more prominent feature. Apart from MCMV-induced memory inflation of CD8⁺ T cells in infected mice, accumulation of monocytes/macrophages was observed, but only when genetic predisposition and MCMV infection were combined. Single cell RNA sequencing identified three sub-clusters including Ly6C^high monocytes, pericardial macrophages and macrophages with a pro-fibrotic signature that can be typically observed upon tissue injury in Pkp2^+/- MCMV but not in Ctr MCMV mice. Interestingly, these populations were also present in Pkp2^+/- hearts without MCMV infection, suggesting a subclinical effect of the Pkp2 mutation and a potential involvement of macrophages in the early disease response. Additional immune cell analyses on homozygous Pkp2 knockout (KO) mice, exhibiting pronounced fibrosis and systolic dysfunction at 8 weeks of age, revealed increased monocytes/macrophages, B cells, and T cells. More interestingly, in younger KO hearts, we observed a pronounced increase of pro-inflammatory tissue resident macrophages compared to Ctr, whereas the overall macrophage population did not change, suggesting activated states and secretion of mediators that could exacerbate tissue damage as important factor for onset and progression of ACM.

Conclusion: The clinical heterogeneity of ACM patients and “hot phases” of clinical presentation may arise from the interaction between genetic predisposition and immune responses. In our study, we found that disrupted desmosomes combined with an inflammatory trigger could initiate cardiac dysfunction, likely through sustained immune activation and progressive tissue damage.

Future perspectives: Targeting inflammation and immune responses may help to therapeutically control activated disease states via depletion of specific subsets of macrophages in early disease. Such intervention is part of ongoing studies to prevent or slow down the development of the phenotype in Pkp2 KO mice.