PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) affects 1:500 of the population and is the leading cause of sudden cardiac death in young people. Clinical presentation of HCM includes thickening of the left ventricular wall, diastolic dysfunction, and fibrosis. Tissue remodeling from fibrosis replaces 30 to 50% of the myocardium in end-stage HCM and is a key determinant in patient outcome. Mutations in numerous sarcomeric proteins that regulate cardiac contractility have been identified as causes of HCM, about 30% of which are in located β-myosin heavy chain (MYH7), but it remains unclear how the intrinsic changes in contractility of cardiomyocytes lead to fibrotic remodeling. While previous studies have provided important insight into fibrosis, limitations in experimental models, such as limited patient samples, limited ability to study human cardiomyocytes ex vivo, and species variances in cardiovascular biology, have made it difficult to determine a mechanism of fibrosis preceding stromal activation. Advancements in human induced pluripotent stem cell (hiPSC) and CRISPR/Cas9 technology have allowed investigators to model and study inherited cardiac diseases compared to a healthy isogenic background in vitro. While hiPSC-CMs have provided important insight into functional changes in diseased cardiomyocytes, a multicellular 3D model is needed to study the pathological remodeling in fibrosis. Cardiac microtissue (CMT) platforms offer a unique tool to study the effects of cardiomyocytes on stromal cells and the microenvironment. The overall hypothesis of this proposal is that MYH7-variant hiPSC-CMs pathogenically activate stromal cells through paracrine signaling, leading to a fibrotic phenotype. This proposal will determine targets to attenuate a fibrotic phenotype in the following Specific Aims. Aim 1. To model stromal activation in MYH7-variant hiPSC-CM in vitro models of HCM. Aim 2. To determine key paracrine factor signaling from pathogenic MYH7 variants that leads to a fibrotic response in stromal cells. Aim 3. To target paracrine factor receptors in stromal cells to decrease fibrotic development in MYH7-variant CMTs. The in vitro model of stromal activation will be characterized and validated with the quantification of collagen deposition, stiffness, gene expression, and contractility. The paracrine signaling from MYH7-variant hiPSC-CM leading to these changes will be identified using a combination of conditioned media experiments, phosphoproteomics, and RNA-sequencing. Key signaling pathways will be targeted with small-molecule inhibitors, and the attenuation of stromal activation will be confirmed through the quantification of collagen deposition, stiffness, gene expression, and contractility. The results of this study will provide new insights into the disease pathology of HCM and will provide potential therapeutic targets to attenuate this pathology, and thus improve clinical outcomes.