Despite improvements in the management of patients with coronary heart disease there is a growing need for the development of new treatments that can be administered not only in response to an acute myocardial infarction, but also prior to surgery. Recently, increased levels of N-acetylglucosamine (0-GlcNAc) on cytoplasmic and nuclear proteins have been linked to improved cell survival in response to stress. Our preliminary data demonstrate for the first time that in the heart, increased protein O-GlcNAcylation resulting from glucosamine treatment is protective in response to ischemia/ reperfusion. We also show that protection associated with hyperglycemia and glutamine may be mediated via the same pathways. Therefore, we propose that increased flux through the hexosamine biosynthesis pathway decreases myocyte injury resulting from ischemia/reperfusion by promoting elevated levels of protein-associated O-GlcNAc thereby leading to improved cellular recovery. Consequently, the aims of this proposal are: 1) Demonstrate that ischemic protection resulting from glucosamine treatment is mediated by protein O- GlcNAcylation; 2) Determine whether other metabolic interventions that lead to ischemic protection do so because of increased protein O-GlcNAcylation; 3) Determine whether signaling pathways such as phosphatidylinositol-3-OH kinase (PI3K), mitogen activated protein kinase (MARK) and protein kinase C (PKC) that have been shown to be modulated in response to increased HBP flux as well as implicated in ischemic cardioprotection, also mediate glucosamine-induced cardioprotection; 4) Identify cardiac proteins that are targets for O-GlcNAcylation resulting from glucosamine treatment that may be involved in glucosamine-induced cardioprotection. Experiments will be performed using the isolated perfused rat heart and cultured neonatal rat ventricular myocytes. We will use NMR isotopomer analysis in combination with cellular, molecular and proteomic techniques to evaluate the effect of acute glucosamine treatment on the heart and to elucidate its cardioprotective mechanisms. The number of people suffering from acute myocardial infarction and its subsequent complications continues to increase. The successful outcome of this proposal will identify novel targets for the development of new therapeutic agents for the treatment and management of patients with ischemic heart disease. ? ? ?
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