Multifactorial etiology of arrhythmogenic cardiomyopathy: Interactions between gene mutation, viral infection and age in Pkp2+/- mice

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetically driven disorder characterized by variable clinical presentations. The interplay between genetic factors, such as autosomal dominant loss-of-function mutations in desmosomal proteins like plakophilin 2 (PKP2), and environmental factors including viral infections, modulate disease progression, most likely through activation of inflammatory pathways. Our previous studies highlighted a mild onset of ACM-like symptoms post murine cytomegalovirus (MCMV) infection in a young adult mouse model with a cardiomyocyte-restricted heterozygous loss of Pkp2 (Pkp2^+/-). Here, we extend our investigation to an older Pkp2^+/- cohort to elucidate the impact of age on disease phenotype and immune response.

Methods: 12-month-old Pkp2^+/- and control (Ctr) mice were infected with MCMV or received PBS. We assessed cardiac function, collagen deposition, as well as immune cell dynamics and compared the obtained results to our previously studied younger cohort infected at 3 months of age. Techniques included histological analysis, flow cytometry of cardiac leukocytes, and echocardiography at 1 and 3 months post infection (mpi).

Results: Unlike the younger cohort, 12-month-old Pkp2^+/- mice demonstrated increased collagen deposition in both ventricles even without MCMV infection, suggesting age-related changes in the genetically predisposed myocardium. Flow cytometry indicated significant elevations in Ly6C^high monocytes and macrophages in both, non-infected and MCMV-infected Pkp2^+/- mice, with significant increases in tissue-resident, tissue-injury, and pro-inflammatory macrophage subsets. Notably, CD8⁺ T cells, particularly virus-specific memory T cells, were elevated only in MCMV-infected conditions (Ctr MCMV and Pkp2^+/- MCMV), without any changes in the T cell compartment in non-infected Pkp2^+/- mice. 

Furthermore, echocardiography demonstrated reduced ejection fraction in both, non-infected (36.6 % ± 12.9 %)  and MCMV-infected (38.8 % ± 6.4 %) Pkp2^+/- mice (14.5 % and 12.3 % lower than Ctr non-infected., respectively) as early as 1 mpi, indicating early onset of systolic dysfunction. This dysfunction underscores the significant role of genetic predisposition and age in disease progression, given the minimal differences between the two genetically predisposed groups. Interestingly, the younger cohort infected at 3 months did not show systolic impairment at a similar stage post infection, highlighting age as an additional critical factor in disease severity.

Conclusion: The age at which MCMV infection occurs significantly influences onset and severity of the ACM-like phenotype in Pkp2^+/- mice. Aging predisposes these mice to more severe disease, although the overall immune response is exacerbated by viral infection. This underscores a multifactorial model of ACM progression, where genetic predisposition, age and viral factors interact to determine the severity and dynamics of the disease and immune response.

Outlook: Future studies will aim to elucidate the specific immune pathways activated by aging and genetic factors. We plan to use single-nucleus RNA sequencing to directly capture signals from cardiomyocytes with disturbed desmosomes in our Pkp2^+/- mouse model. Additionally, tailored cytokine analyses of both, heart tissue and serum samples, will be conducted to further elucidate the mechanisms driving disease heterogeneity and progression in ACM.