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bioRxiv · 10.1101/2025.10.08.680826

Epicardial contributions to fibro-inflammatory signaling in a Pkp2-deficient arrhythmogenic cardiomyopathy model

Han, D. D.·Brooks, A.·Baker, C.·Dirkx, R. A.·Mickelsen, D. M.·Fisler, B.·Phadke, K.·Ashton, J.·Delmar, M.·Small, E. M.
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Abstract

BackgroundArrhythmogenic Cardiomyopathy (ACM) is an inherited disease that is characterized by lethal ventricular arrhythmias stemming from myocyte dysfunction. ACM is associated with considerable subepicardial fibrosis and inflammation with right ventricle predominance. Most cases of gene positive ACM are caused by a desmosome protein mutation, with plakophilin-2 (Pkp2) mutations being most common. We hypothesized Pkp2-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis. MethodsWe developed transgenic mice that lack Pkp2 in cardiomyocytes (Pkp2-cKO) or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Non-myocyte populations were isolated 21 days post-tamoxifen injection for single cell RNA-sequencing (scRNA-seq). Immunohistochemistry, flow cytometry, qRT-PCR, and echocardiography were used to interrogate cardiac physiology and cellular composition. ResultsWe identified a population of epicardium-derived fibroblasts characterized by the expression of Ccl2, Ccl7, Thbs1, and Ptx3 that accumulated upon Pkp2 deletion. While pro-inflammatory EPDCs are found in Pkp2-cKO mice, they become significantly enriched in Pkp2-ceKO mice. Pro-inflammatory fibroblasts acquired the senescence-associated secretory phenotype (SASP), correlating with elevated Senescence Associated (SA)-{beta}gal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, that progresses from right to bi-ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared to controls. Antibody-mediated B cell depletion delays the early inflammatory and fibrosis response but did not significantly alter end-stage cardiac physiology. ConclusionPkp2 deletion in EPDC facilitates the emergence of a fibro-inflammatory phenotype that may contribute to ACM pathogenesis.

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Han, D. D., Brooks, A., Baker, C., Dirkx, R. A., Mickelsen, D. M., Fisler, B., Phadke, K., Ashton, J., Delmar, M., Small, E. M.. 2025-10-09. Epicardial contributions to fibro-inflammatory signaling in a Pkp2-deficient arrhythmogenic cardiomyopathy model. https://doi.org/10.1101/2025.10.08.680826

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