PROJECT SUMMARY While dilated cardiomyopathy (DCM) is one of the most common hereditary heart diseases, we currently lack effective and targeted treatments. Cardiac fibrosis is an important hallmark of DCM and the presence of excessive fibrotic tissues can severely hamper heart function. However, limited information exists on how DCM mutations can induce cardiac fibrosis in familial DCM patients. This study aims to address this significant knowledge gap using patient-specific human induced pluripotent stem cells (iPSCs) generated from common DCM patients. In Aim 1, we will differentiate iPSCs into cardiac fibroblasts (iPSC-CFs) and cardiomyocytes (iPSC-CMs) to study how cardiomyocyte-specific and non-cardiomyocyte-specific mutations can activate cardiac fibroblasts. In Aim 2, we will examine whether iPSC-CFs induce cardiac dysfunction through secretory factors and/or direct interaction with cardiomyocytes using a 3D engineered heart tissues (EHT) platform. In Aim 3, we will identify gene/mutation-specific druggable targets using state-of-the-art CRISPR/dCas9i (inhibitory) and CRISPR/dCas9a (activating) technologies, and validate these targets in an established DCM mouse model. By completing this study, we expect to identify fibrosis-inducing DCM mutations, understand how they activate cardiac fibroblasts, and potentially discover novel druggable mechanism to manage DCM. Collectively, this multi- PI R01 study has tremendous translational value to aid future precision cardiovascular medicine.