Project Summary Heart failure progression is a complex biological process that is precipitated by the maladaptive myocardial response to injury, compounded by failure of the adult heart to replace lost or damaged cardiomyocytes. Our lab has previously outlined the regenerative capacity of the newborn mammalian heart and outlined several mechanisms that regulate this process. Specifically, we demonstrate that the endogenous regenerative capacity of the newborn heart is mediated by proliferation of preexisting cardiomyocytes and is lost when cardiomyocytes exit cell cycle within a few days after birth. We described several fundamental mechanisms that regulate cell cycle exit of cardiomyocytes, including spontaneous DNA damage that occurs as a result of increased mitochondrial oxidative phosphorylation. We also demonstrated that DNA damage activates DNA damage response (DDR), which mediates cell cycle arrest of cardiomyocytes whereby inhibitors of DDR prolong the window of cardiomyocyte proliferation. We also examined other potential consequences of DNA damage, namely the formation of micronuclei, which is free cytoplasmic DNA that often results from DNA damage during mitosis. Our preliminary results indicate that the micronuclei are detected in cardiomyocytes at the time of cell cycle arrest. We also found that the DNA sensing pathway cGAS-STING is induced in the postnatal heart within the same timeframe. Intriguingly, inhibiting cGAS results in prolongation of the postnatal window of cardiomyocyte proliferation. Therefore, this project will focus on the role of intra-cardiomyocyte cGAS-STING in regulation of heart regeneration. We will examine the mechanism of activation of cGAS-STING in cardiomyocytes, the effect of inhibiting cGAS-STING on cardiomyocyte proliferation, and the downstream mechanisms that regulate cGAS function in cardiomyocytes.