The Detrimental Role of Macrophages in PKP2-linked Arrhythmogenic Cardiomyopathy: Lessons from Murine Cytomegalovirus Infection

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic disease covering a wide range of cardiac features, from asymptomatic cases to severe heart failure, frequently linked to mutations in desmosomal cell-cell contact proteins like plakophilin 2 (PKP2). In early stages, some clinically affected patients experience "hot phases", often mistaken for acute myocarditis. The contribution of external triggers, such as common viral infections, to ACM onset and progression remains unclear. To explore this, we investigated the impact of murine cytomegalovirus (MCMV) infection on ACM using mice with a heterozygous cardiomyocyte-specific Pkp2 deficiency (Pkp2^+/-), which showed no differences to wild type (Ctr) mice under basal conditions.

 

Methods and Results: Pkp2^+/- and Ctr mice were infected with MCMV or PBS to investigate heart structure, function and immune response to different time points post infection. Acute infection (5-14 days) caused immune cell plaques in myocardial tissues, comprising cytotoxic CD8⁺ and CD4⁺ T cells, regardless of genotype. While viral DNA load decreased over time, it persisted up to 6 months post infection (6mpi), potentially exposing mice to recurrent virus-related inflammation. Our hypothesis suggesting inflammation amplifying myocardial damage was supported as systolic dysfunction appeared at 3mpi, worsening over time in Pkp2^+/- MCMV but not in Ctr MCMV hearts. Interestingly, fibrosis was absent in cardiac tissues; instead, immune cell infiltration and associated inflammation were predominant. Apart from the expected CD8⁺ T-cell accumulation, we also identified significantly increased monocytes/macrophages, but only in Pkp2^+/- MCMV mice. Single-cell RNA sequencing showed distinct immune cell sub-clusters, including Ly6C^hi monocytes and pro-fibrotic macrophages, typically observed in tissue injury, particularly in Pkp2^+/- MCMV mice. These populations were also present in Pkp2^+/- hearts without infection, indexing a subclinical effect of the Pkp2 mutation and potential involvement of macrophages in the early disease response. Furthermore, results from a similar mouse model with a heterozygous loss of the sarcomeric protein titin underscored the importance of inflammation and immune response in the Pkp2 model, as MCMV infection had no effect on cardiac function or the macrophage population here. Further immune cell analyses in homozygous Pkp2 knockout (KO) mice, displaying pronounced immune cell infiltrations at an early stage, demonstrated a shift from tissue-resident (TIM4⁺ MHCII^-) to pro-inflammatory (TIM4^- MHCII⁺) and tissue-injury-associated macrophages (TIM4^- MHCII^-), suggesting activated states and mediator secretion exacerbating tissue damage, crucial factors in ACM.

 

Conclusion and Perspectives: The heterogeneous clinical manifestations of ACM may result from the interplay between genetic predisposition and immune responses. Our study underscores that disrupted desmosomes, combined with an inflammatory trigger like MCMV infection, can initiate cardiac dysfunction, likely through exaggerated immune activation and progressive tissue damage. In current studies, we want to investigate whether another inflammatory trigger, such as lipopolysaccharide (LPS) has a similar effect in genetically predisposed mice. Further, specific targeting of migrating monocytes (CCR2 antibody) in Pkp2 KO mice might control the pro-inflammatory response in activated disease states necessary to slow down ACM phenotype development.