Background:Arrhythmogenic cardiomyopathy (ACM) is a progressive hereditary myocardial disease primarily caused by mutations in desmosomal genes, most frequently plakophilin-2 (Pkp2). These mutations compromise cardiac cell-cell adhesion, resulting in progressive cardiomyocyte loss, extensive fibrotic replacement, and malignant arrhythmias. The cardiac-specific heterozygous Pkp2 knockout (het_KO_Pkp2) mouse model recapitulates key ACM disease features and enables mechanistic studies of disease progression, including age-related progression.
Methods:We performed single-nucleus RNA sequencing (snRNA-seq) on ventricular myocardium from n=3 control and n=3 het_KO_Pkp2 mice. Following nuclei isolation and magnetic bead purification, approx. 20,000 nuclei/sample were sequenced (10x Genomics v3.1). After quality check(removal of doublets and high-mitochondrial content nuclei), 102,880 nuclei remained for analysis. Cell clusters were annotated by differential gene expression (DGE). Cell-type compositional changes were assessed using differential abundance analysis (Milo). Intercellular signaling was mapped by ligand–receptor inference (LIANA), focusing on pathological populations. Biological pathway alterations were defined by gene set enrichment analysis (GSEA).
Results:The het_KO_Pkp2 hearts compared to Ctr showed significant depletion of mature contractile/oxidative cardiomyocytes and increased numbers of stressed cardiomyocytes, fibroblasts, endothelial cells, and immune populations (B-lymphocytes, macrophages), supporting observed systolic dysfunction and ventricular dilation. DA analysis by Milo confirmed significant depletion of the functional cardiomyocyte CM pool (structural, contractile and oxidative metabolism CMs) , the expansion of fibroblast- and immune-rich tissue neighborhoods. DGE revealed downregulation of structural genes (Dmd, Ttn, Ryr2) in cardiomyocytes and upregulation of ECM genes (Postn, Col1a1/3a1) in fibroblasts. LIANA identified key pathological cell–cell signaling: stressed cardiomyocytes release damage-associated molecular patterns (DAMPs) that activated immune cells, driving chronic inflammation; fibroblast-derived extracellular matrix (ECM) components activated immune-cell integrins, perpetuating fibrosis;macrophages secreted ligands sustaining a profibrotic state. GSEA revealed suppression of Wnt/cell cycle and anti-apoptotic pathways in cardiomyocytes, with upregulation of ECM-remodeling, and proinflammatory pathways in stromal and immune compartments. These transcriptional signatures paralleled wet lab findings of increased cardiac fibrosis and immune infiltrates.
Conclusion:These integrated findings demonstrate that Pkp2 deficiency disrupts cardiomyocyte integrity, amplifies immune activation, and drives fibroblast-mediated fibrosis, collectively fueling adverse cardiac remodeling in ACM. The convergence of suppressed regenerative signaling, chronic inflammation, and pathological ECM deposition highlights actionable molecular targets for therapeutic intervention. Modulating these pathways may attenuate disease progression and improve outcomes for carriers of desmosomal mutations, aligning with the goal of setting new standards in cardiovascular medicine.
