PROJECT SUMMARY The death of cardiomyocyte following myocardial infarction (MI) is one of the main causes of heart failure and patient mortality. Our group previously identified MG53, a member of the TRIM family protein (TRIM72), as an essential component of the cell membrane repair machinery. Mice without the mg53 gene develop pathology in the heart and are susceptible to cardiac injury, while transgenic mice with increased levels of MG53 (ctPA-MG53) are resistant to stress-induced MI, supporting the function of MG53 in cardioprotection. Using CRISPR-Cas9 mediated gene knockout and live cell biotin proximity-labeling assay, we identified RIPK1 as a novel molecular partner of MG53 following I/R induced cardiac injury. RIPK1 is a key factor of necroptosis, a programmed necrosis, which hasn’t been extensively studied in MI. In vitro cell culture studies with human induced pluripotent stem cells (hiPSCs) and in vivo murine MI studies demonstrated that necroptosis plays an important role in injury induced cardiac cell death. More importantly, following MI, mg53-/- hearts had higher level of necroptosis, while ctPA-MG53 hearts displayed lower level of necroptosis than those of wild type littermates, indicating MG53 could potentially regulate necroptosis. Indeed, biochemical experiments revealed that wild type MG53 serves as an E3 ligase of RIPK1 following MI, while mutant MG53 without E3 ligase function failed to target and mediate degradation of RIPK1. This research is centered on testing the hypothesis that MG53 plays a critical role in inhibiting necroptosis via directly interacting with and destabilizing RIPK1, targeting this functional interaction could be important for maintaining myocardial homeostasis and developing effective treatments for cardiac diseases”. We will test the hypothesis with two specific aims. Aim 1: Dissect the molecular mechanisms underlying MG53-mediated myocardial protection through regulating RIPK1 or other necroptotic factors. Aim 2: Define the function of MG53-mediated necroptotic inhibition for protection of myocardial injury. Live cell imaging, CRISPR-Cas9 mediated gene editing in hiPSCs, AAV mediated gene delivery and mutagenesis approaches will be utilized to dissect cellular and molecular functions of MG53 on regulation of necroptosis. Overall, knowledge gained from this project will extend our current understanding of MG53 as a membrane repair factor to its function for regulation of cell death following MI, and may have potential translational implications for developing new therapeutic strategies for treating MI.