Ventricular arrhythmia and sudden cardiac death (SCD) is a prevalent complication of hypertrophic cardiomyopathy (HCM) especially in young adults. Pathogenic variants of sarcomeric protein genes cause about half of inherited HCM and about a third of sporadic HCM. Little is known, however, about how sarcomeric protein variants lead to arrhythmia and sudden cardiac death. There is a major unmet need for a better understanding of disease mechanisms in order to predict patients at risk for SCD and design mechanism-based therapeutics. To address this gap, we will apply high throughput functional genomics to identify individual protein components of arrhythmogenic signaling, and establish their function using in vitro studies in iPSC- derived cardiomyocytes (iPSC-CMs) and in vivo studies in HCM mutant mice. We will begin by generating functional genomics probes of the intracellular arrhythmogenic signaling in iPSC-derived cardiomyocytes carrying HCM causative variants in MYBPC3, MYH7 and TNNT2. The probes will be identified based on screening synthetic miRNAs (syn-miRs) which collectively suppress nearly all proteins in the cell and therefore make ideal probes of complex biology. Analysis of probe selectivity for gene variants will indicate the existence of common and/or distinct signaling mechanisms. In parallel, we will identify and characterize candidate pathways indicated from analysis of myectomy samples from MYBPC3 mutant HCM patients. Once we have obtained pathway information and probes from these two approaches, we will comprehensively determine the protein mediators by high throughput functional evaluation in the MYBPC3 mutant iPSC-CMs. Based on the effect in the iPSC-CMs, selectivity for MYBPC3 and potential as a drug target, we will prioritize the most promising candidate targets for in vivo evaluation by AAV9 knockdown in HCM transgenic mice carrying a Mybpc3 mutation homologous to that in the iPSC-CMs. We expect that modulating the function of the candidate mediators will suppress arrhythmia and tachycardia in the Mybpc3 mutant mice. In summary, these studies will increase our understanding of the arrhythmogenic signaling caused by HCM mutations and promote the development of improved prognostic and mechanism-based therapeutics for familial HCM patients. It will also increase our understanding of fundamental cardiomyocyte biology that might underlie other cardiac diseases. The Specific Aims are: 1) Determine if discrete signaling mechanisms cause arrhythmic susceptibility across “high” propensity HCM gene variants, and 2) Comprehensively define the proteins that dictate electrical remodeling by functional screening in MYBPC3 mutant iPSC-CMs and test their efficacy as therapeutic targets in an Mybpc3 mutant mouse model of HCM.