Does MCMV trigger Pkp2-associated arrhythmogenic cardiomyopathy? Immune phenotyping and heart function at a stage of early latency

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic heart disease presenting with a heterogenous clinical phenotype, ranging from asymptomatic mutation-carriers to sudden cardiac death and heart failure. ACM is commonly caused by mutations in desmosomal proteins, especially plakophilin-2 (Pkp2). However, the cause for the variability in clinical phenotypes among carriers of the same mutation, remains unclear. This suggests the involvement of additional external triggers in disease onset and progression, such as intense physical activity or inflammation.
To explore the role of inflammation in ACM, we investigated the impact of murine cytomegalovirus (MCMV) infection in mice with a heterozygous, cardiomyocyte-specific loss of Pkp2 (Pkp2^+/-) at 8 months of age, as previous experiments focused on infection at 3 months of age.

Methods: 8-month-old Pkp2^+/- and Ctr mice were infected i.p. with 5x10⁵ PFU MCMV or treated with PBS. One month after infection (early latency), animals were submitted to 18F-FDG-PET/CT and MRI imaging to assess inflammation and heart function in vivo. To assess the immune cell response in heart, lymph nodes and spleen, we performed flow cytometry using panels focusing on T-cell and macrophage subsets, histology with Picrosirius red (PSR) and HE staining and cytokine measurements using the Olink Target 48 Mouse Cytokine panel.

Results: The FDG-PET/CT scans displayed increased relative standardized uptake values (rSUV) and visual uptake only in the Pkp2^+/- MCMV group. Likewise, in the MRI, we detected increased end-diastolic mass, end-diastolic and end-systolic dilation, and reduced ejection fraction only for hearts of the Pkp2^+/- MCMV group. This suggests functional impairment in early latency based on a critical interaction between genotype and infection in the development of a cardiac phenotype.
In addition, PSR assessment showed increased collagen deposition in both ventricles for Pkp2^+/- MCMV mice compared to Ctr mice, with a trend for increased collagen in non-infected Pkp2^+/- mice as well. This has not been observed in 3-month-old mice so far, indicating the potential role of aging in combination with genetic predisposition in fibrosis development.
Flow cytometry after MCMV infection was associated with genotype-independent leukocyte infiltration of heart tissue with an accumulation of CD4+ and CD8+ T-cells, as well as increased transient and pro-inflammatory macrophages. Notably, Ly6Chigh monocytes were significantly elevated only in the Pkp2^+/- MCMV group, suggesting a genotype driven myeloid response.
Cytokine analyses from serum samples did not indicate active systemic inflammation in either group, although we noticed elevated levels of IL2, IFNL2 and TNFα only in the Pkp2^+/- MCMV group.
In heart lysates, both MCMV-infected groups showed increased levels of IFNɤ, IL16, CCL5 and CCL4 as well as PD-L1 and PD-L2, suggesting that these are mediated by the sub-acute MCMV infection. Notably, only the Pkp2^+/- MCMV group displayed increased levels of IL1ß, CCL2, CCL12 and FGF21, indicating an intensified inflammatory response and metabolic stress.