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Pro-inflammatory signaling originates from epicardium-derived fibroblasts in a PKP2 deficient model of arrhythmogenic cardiomyopathy

ABSTRACT: Introduction: Arrhythmogenic Cardiomyopathy (AC) is an inherited disease that is caused by desmosome protein mutation with plakophilin 2 (PKP2) mutation being most common. AC is known to cause sporadic ventricular arrhythmias, myocyte damage, and subepicardial fibrosis. Given the origin of fibrosis from epicardium, we wanted to understand how epicardium-derived cells (EDCs) contribute to the AC pathogenesis. Hypothesis: EDCs propagate the pro-inflammatory signaling that contributes to AC pathogenesis. Methods: We developed transgenic mice that lack PKP2 in cardiomyocytes (PKP2-cKO) or in both cardiomyocyte and epicardial cells (PKP2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Tamoxifen injected mice expressing Cre in cardiomyocytes, but with no floxed PKP2 gene, was used as a control. EDCs were traced with Cre-dependent Green Fluorescence Protein (GFP). Non-myocyte populations were isolated 21 days post-tamoxifen injection for single cell RNA-sequencing (scRNA-seq) using 10X Genomics platform. Perfused hearts were separated into left and right ventricle for qRT-PCR. Immunohistochemistry was used for cardiac structure and cellular composition. Echocardiography was performed to measure cardiac physiology. Results: The scRNA-seq analysis identified an epicardium-derived fibroblast population that secretes pro-inflammatory cytokines, including CCL2, CCL7, Thbs1, and PTX3. While pro-inflammatory EDCs are found in both PKP2-cKO and PKP2-ceKO mice, they were most numerous in PKP2-ceKO mice. Importantly, macrophages and B cells accumulated in both PKP2-cKO and PKP2-ceKO mice compared to controls. qRT-PCR confirmed that the expression of genes encoding fibrosis markers (POSTN, Col1A1, Col1A2, and Col3A1), pro-inflammatory fibroblast markers (CCL2, CCL7, Thbs1, PTX3), macrophage markers (F4/80, Cx3cr1, Spp1, Trem2, and CCR2), and B cell markers (CD19 and CD79b) were significantly enriched in both PKP2-cKO and PKP2-ceKO mice compared to control. Our expression and histologic data also revealed an exaggerated pro-inflammatory response in PKP2-ceKO mice, that progresses from the right to bi-ventricular predominance. However, echocardiography showed no significant difference in cardiac function between PKP2-cKO and PKP2-ceKO, and B cell depletion did not alter disease progression. Conclusion: A subset of epicardium-derived fibroblasts in PKP2 KO mice secretes pro-inflammatory cytokines that contributes to cardiomyocyte damage and cardiac fibrosis in AC.

ORGANISM(S): Mus musculus

PROVIDER: GSE300141 | GEO | 2026/02/12

REPOSITORIES: GEO

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Project description:Rationale: The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium â?? endothelium - epicardium). We previously described that Id1â??/â??Id3â??/â?? double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early demise precluded the studies of the roles of Id in the adult mice. Objective: To elucidate postnatal roles of Id genes in the heart. Methods and Results: We ablated Id1 gene in the vasculature and Id3 gene globally to generate Tie2Cre+Id1F/â??Id3â??/â?? and Tie2Cre+Id1F/FId3â??/â?? conditional KO (Id cKO) embryos. Half of the Id cKO mice die at birth. Postnatal demise was associated with cardiac underdevelopment, enlargement, muscular ventricular septal and endothelial defects. Surviving Id cKO mice exhibited dilated, fibrotic cardiomyopathy associated with defects in the vasculature. The adult cardiac phenotype progressed into heart failure and resembled endomyocardial fibroelastosis. An abnormal vascular response was also observed in the healing process of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Conclusions: Conditional ablation of Id genes in the vasculature leads to dilated fibrotic cardiomyopathy. The findings could reveal important insights into the role(s) of the endocardial network of the endothelial lineage to the development of dilated fibrotic cardiomyopathy and identify a potential therapeutic target, IGFbp3, in its treatment. Total RNA from heart tissue was isolated (RNeasy, QIAGEN) from P180 WT, Id control, Id cKO, IGFbp3 incubated Id cKO, and control incubated Id cKO. RNA was converted to cDNA, cRNA, and hybridized to DNA sequences contained in the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Information from at least duplicate samples was compared and filtered by fold change >2 (Id cKO vs WT, Id control vs WT, IGFbp3 incubated Id cKO vs. control incubated Id cKO) and statistical p-value<0.001.

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Pro-inflammatory signaling originates from epicardium-derived fibroblasts in a PKP2 deficient model of arrhythmogenic cardiomyopathy

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