Modeling arrhythmogenic cardiomyopathy fattyfibro pathology through the use of PKP2-deficient human iPSC-derived epicardial cells

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by progressive fatty-fibro replacement of the ventricular myocardium leading to arrhythmias and an increased risk of sudden cardiac death. To date, the cell types and signaling mechanisms involved in fatty-fibro infiltration of the myocardium have yet to be fully resolved. However, given that fatty-fibro replacement is initiated within the subepicardial layer, epicardial cells are predicted to contribute to the development of this pathology. To investigate the epicardial contributions to the development of ACM, we generated human induced pluripotent stem cell (hiPSC) lines from ACM patients harboring either a PKP2 1849C>T or a PKP2 2013delC mutation alongside CRISPR/Cas9 gene-edited isogenic control lines and a PKP2 knockout line. Epicardial cells derived from patient and knockout conditions displayed enhanced epithelial-to-mesenchymal transition characteristics, dysregulated Wnt and interferon signaling, increased lipid accumulation, and an enhanced fibrotic phenotype when converted to epicardial-derived fibroblasts. Transcriptomic analysis revealed upregulation of insulin growth factor 2 (IGF2) in ACM hiPSC-EPCs as well as in human ACM heart tissue. Subsequent treatment with exogenous IGF2 enhanced expression of the adipogenic transcription factors CEBPA and PPARG in hiPSC-EPCs, suggesting insulin growth factor signaling contributes to ACM fatty-fibro remodeling. Overall design: We generated hiPSC lines from two ACM families carrying either a PKP2 1849C>T or PKP2 2013delC mutation, corrected each by CRISPR/Cas9 to create isogenic controls, and produced a PKP2 knockout line. All lines were differentiated into epicardial cells, then assayed for EMT markers, cell migration, lipid accumulation (Nile Red), and fibrotic marker expression. Epicardial-derived fibroblasts versus non-epicardial fibroblasts were compared under adipogenic induction. RNA-seq profiled global transcriptional changes; key findings were functionally validated by IGF2 treatment and IGF-1R inhibition.