PROJECT SUMMARY Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive fibrofatty replacement of the myocardium, arrhythmias, and sudden death. Fibrofatty substitution in arrhythmogenic cardiomyopathy contributes to worsening arrhythmogenesis by creating a non-conductive substrate, and causes ventricular dysfunction leading to heart failure. The mechanisms underlying this disease are still unclear; a better understanding of the pathogenesis is needed to find better options for clinical management. To address this challenge, reliable species-specific models are needed; here we propose to develop a novel human model, that will serve as a system to study the pathogenesis of cardiac fibrofatty infiltration. This study integrates engineering and biomedical sciences, applying tissue engineering, cardiac physiology, bioinformatics and stem cell technologies. Our long-term goal is to provide a model of fibrofatty myocardial infiltration to investigate underlying disease mechanisms, which will lead to the development of greatly needed therapies for patients who suffer from cardiac diseases related to the presence of fibrofatty infiltration. The central objective of this proposal is to demonstrate that fibrofatty infiltration of the myocardium can be replicated in a 3D engineered cardiac tissue, resembling deficient contractility and altered electrophysiological properties that mimic what is observed in patients that suffer from ACM. The molecular signatures of fibrofatty infiltration in the context of our engineered cardiac tissue model will also be analyzed. We will approach this in two aims. In Aim 1 we will develop a 3D engineered cardiac tissue model of fibrofatty infiltration of the myocardium using hiPSCs from patients with ACM. We will combine hiPSC-cardiomyocytes and hiPSC-epicardial cells treated to undergo epithelial-mesenchymal transition; aiming to resemble the ACM functional phenotype. In Aim 2, exploiting the role of the epicardium as source of fibrofatty infiltration; we will develop a 3D engineered cardiac tissue model of myocardial fibrofatty infiltration using hiPSCs from healthy donors. In this study, we propose a strategy based on evidence that fibrofatty infiltration is induced from epicardial activation; hiPSC-derived epicardial cells will be treated to induce their further differentiation into fibroblasts and adipocytes. We will examine functional and structural properties, along with single-cell transcriptomics of the engineered cardiac tissue models. We expect that results from this study will advance our understanding of the contribution of specific cues from ACM-related cells in the pathogenesis of fibrofatty remodeling; our physiologically relevant model will serve to unravel the cell-cell cross-talk and mechanisms responsible for initiation and progression of fibrofatty infiltration of the myocardium. This project will improve the health of patients with ACM by leading the development of a human model of the disea...