instnjctions): Recently, the modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked p-N- acetylglucosamine (0-GlcNAc) has emerged as a novel regulator of the stress response and cell survival. Numerous forms of cellular injury, including cardiac ischemic preconditioning (acute and prolonged), lead to elevated levels of 0-GlcNAc in both in vivo and in vitro models. Elevating 0-GlcNAcylation before, or immediately after, the induction of cellular injury is protective in models of ischemia reperfusion injury, as well as heat stress, oxidative stress, endoplasmic reticulum stress, hypoxia, and trauma hemorrhage. Together, these data suggest that 0-GlcNAc is a novel endogenous cardioprotective agent. However, the molecular mechanisms by which 0-GlcNAc regulates protein function leading to enhanced cell survival and cardioprotection have not been identified. The long term goal of this investigator, is to identify at a molecular level the mechanisms by which 0-GlcNAc promotes cell survival. The objective of this application is to: 1) Define the role(s) of 0-GlcNAc in mediating ischemic preconditioning. In order to characterize the mechanisms by which 0-GlcNAc leads to cardioprotection, proteins dynamically O-GlcNAc modified in response to ischemic-preconditioning will be identified and pathways that lead to enhanced 0-GlcNAcylation will be defined. 2) Elucidate the molecular mechanism(s) by which 0-GlcNAc regulates the process of autophagy leading to cardioprotection. To characterize the molecular mechanisms by which 0-GlcNAc protects cardiomyocytes via autophagy we will define: 1) the role of 0-GlcNAc in inducing autophagy during ischemic preconditioning; 2) if enhanced autophagy is critical for 0- GlcNAc mediated cardioprotection; 3) the identity of proteins involved directly in autophagy (or regulating autophagy) that are modified and regulated by 0-GlcNAc. Together, these studies will characterize a novel endogenous defense mechanism of the heart, highlighting new targets for the development of alternative strategies that enhance the hearts tolerance to ischemia reperfusion injury. RELEVANCE (See instructions): The sugar 0-GlcNAc is a key component of the cellular stress response that enhances the ability of cells and tissues to survive ischemia reperfusion injury (for example, heart attack), but the mechanisms by which O- GlcNAc protects cells are unknown. Our goal is to understand how 0-GlcNAc promotes cell survival in a model of ischemia reperfusion injury at the molecular level, thus identifying new targets for the development of alternative strategies to enhance the heart's tolerance to ischemia reperfusion injury.