Project Abstract Profound derangements in pathophysiology occur during heart failure, including alterations in the cardiomyocyte's ability to coordinate metabolic cues. Glucose represents the quintessential substrate for metabolism. Yet, not all glucose is dedicated to the production of ATP; a fraction of intracellular glucose is shunted into several accessory pathways, such as the Hexosamine Biosynthetic Pathway, which forms the monosaccharide donor for the post-translational modification known as O-linked-?-N-acetylglucosamine (O- GlcNAc). The PI's laboratory has identified O-GlcNAcylation as an important event in the failing heart, and O- GlcNAcase (OGA) is a key regulator of O-GlcNAc removal from targets. Despite significant insights related to the phenomenology of O-GlcNAc in acute myocardial ischemia and heart failure, almost nothing is known about the regulation of the O-GlcNAc enzymes (OGA and OGT) in the heart or other tissues. The present proposal will reduce significantly this critical gap in knowledge. To this end, the present proposal will: i) Identify transcriptional and post-transcriptional regulators of OGA expression; again, nothing is known currently regarding such regulation in the cardiomyocyte. In addition to in vivo studies, experiments will be performed in isolated cells and cell lines using common molecular biology techniques (e.g. promoter mutations, transcription factor binding, transduction). ii) Using a loss-of-function approach, the project will determine the impact of OGA suppression during heart failure. Studies will incorporate tissue-specific, inducible, cre-lox-mediated deletion of the OGA gene in cardiomyocytes. The PI will use genetically modified, surgically operated mice coupled with state-of-the-art physiologic assessment of cardiac function. iii) Mechanistic inquiries will focus on mitochondria to understand how OGA suppression attenuates the severity of heart failure. The PI will perform various analyses of mitochondrial function, including bioenergetics assays, along with protein chemistry to determine how suppression of OGA improves mitochondrial function. The overarching hypothesis holds that that OGA suppression attenuates heart failure through preservation of mitochondrial respiratory integrity. Although not the direct, near-time goal of this project, the results could eventually contribute to new potential treatment options for heart failure, and, because of the potential involvement of O-GlcNAcylation in several diseases, will contribute to new directions in studies of diabetes, Alzheimer's, aging, and vascular diseases.