Dilated cardiomyopathy (DCM)-associated heart failure is a leading cause of mortality worldwide. About a third of DCM is due to gene variants in a broad range of cardiac muscle proteins. Although this information has improved patient management, it has not yet led to new therapeutics that target the underlying mechanisms of disease. A major roadblock is that the consequences of the DCM-causing mutations are not understood in sufficient detail to identify points of therapeutic intervention. During the first funding cycle of this project, we used large-scale screening of synthetic microRNAs as an entry point to discover genes that, when inhibited, restored contractility of induced pluripotent stem cells (hiPSC-CMs) carrying DCM-causing mutations. We identified two synthetic microRNAs that normalized contractility of DCM cardiomyocytes comparable to CRISPR-correction of the underlying mutation. Neither microRNA affected isogenic, control hiPSC-CMs, indicating that they act on disease-related processes. We biochemically identified their targets, identifying 203 genes, of which individual siRNA-mediated inhibition of 117 restored contractility of TNNT2 mutant DCM mutant hiPSC-CMs from different patient donors. While some of the candidate genes have been tested as therapeutic targets in heart failure, the vast majority represent new therapeutic target space for inherited DCM. This is multi-PI renewal application is to determine the mechanisms of action of these genes and to establish evidence of disease modifying activity using human genetics and a mouse DCM model. The multi-PI and co-I team unites expertise in iPSC and animal models, systems biology, and human genetics that will have a synergistic impact on our long-term goal of defining therapeutic mechanisms for DCM that would not be possible through separate proposals. Given the diverse genetics and clinical presentations of inherited DCM, our overarching hypothesis is that subsets of the candidate genes, and the physiological processes they affect, will revert contractile dysfunction in a DCM mutation-specific manner. Thus, Aim 1 is to define mechanisms that restore contractile function in DCM caused by different gene variants, and associate beneficial molecular genetic and metabolic pathways with particular DCM-causing mutations, Aim 2 is to investigate whether genetic variants linked to each of the miR- target genes associate with human cardiovascular diseases or quantitative traits using existing GWAS studies, and Aim 3 is to test whether targets of a microRNA that selectively ameliorates contractility in TNNT2-mutant hiPSC-CMs converge on ER stress using a mouse knock-in model engineered with the same Tnnt2 mutation as in the patient hiPSCs.