Myeloid cells promoting disease development in a mouse model of arrhythmogenic cardiomyopathy

Background: Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disorder leading to heart failure and arrhythmias with heterogenous disease expression. Mutations in desmosomal proteins like plakophilin 2 (PKP2) are a major cause and affected patients often experience symptoms of acute myocarditis, termed "hot phases" of disease. The role of viral triggers in ACM onset and progression remains unclear. To explore this, we studied the effects of murine cytomegalovirus (MCMV) infection in mice with a heterozygous, cardiomyocyte-specific loss of Pkp2 (Pkp2^+/-), which were indistinguishable from wildtype (Ctr) mice under normal conditions.

Results: Beginning at 3 months post-infection (mpi) and progressively worsening until 6 mpi, we observed systolic dysfunction only in MCMV-challenged Pkp2^+/- mice, suggesting that MCMV infection-induced inflammation triggered the expected effect. Although histological sections showed no visible fibrotic remodeling, computational quantification revealed a genotype-dependent increase in collagenous area relative to heart area in Pkp2^+/- mice, regardless of infection. Elevated numbers of immune cells, including an MCMV-related expansion of CD8⁺ cytotoxic T cells, were found in both Pkp2^+/- MCMV and Ctr MCMV mice. Notably, monocyte/macrophage accumulation occurred only in Pkp2^+/- MCMV mice with genetic and viral hit. In contrast, no functional impairment or significant change in monocyte/macrophage levels was observed in another genetic MCMV-challenged mouse model with a heterozygous loss of the sarcomeric gene titin (Ttn) at 6 mpi. Single-cell RNA sequencing of cardiac leukocytes at 6 mpi revealed compositional changes in immune subpopulations in both mouse models. Notably, CD8 T cell changes linked to memory inflation from MCMV infection were observed in both models, but the Pkp2 model showed a stronger immune response involving dendritic cells (DCs) and CD4 T cells. Genotype-linked differences showed significant changes in monocytes, macrophages, DCs, and B cells in the Pkp2 model, independent of infection, while these changes were absent in the Ttn model. This suggests the Pkp2 mutation alone induces a sub-clinical immune response before detectable heart function impairment. Furthermore, cell-cell communication analysis revealed altered patterns in the Pkp2 model, involving pathways such as neutrophil degranulation, leading to inflammatory mediator release, increased immune cell attraction, tissue damage, and extracellular matrix degradation.

Conclusion: The clinical heterogeneity of ACM patients and the occurrence of "hot phases" may result from the interplay between genetic predisposition and immune responses. In our study, we discovered that the Pkp2 mutation, but not the Ttn mutation, can provoke an exaggerated immune response even before any functional phenotype is apparent in mice. A viral second hit was able to induce a mild ACM-like phenotype, characterized by systolic dysfunction. We observed an accumulation of predominantly myeloid cell types, which likely leads to the activation and dysregulation of various pathways, thereby amplifying myocardial damage and tissue remodeling.

Outlook: The next steps include inferring causal relationships among cell types, performing trajectory calculations, and conducting detailed pathway analysis. Additionally, single nuclei sequencing will be used to uncover signaling from cardiomyocytes and other cardiac cell types involved in the pathology.