The goal of this research is to continue developing novel NMR and isotope tracer methods to probe intermediary metabolism in isolated, perfused organs. The five specific aims of this competing renewal application focus on using indirect detect 1H NMR methods to follow I3C-ennchment of metabolites in perfused mouse hearts, novel applications of 2H NMR spectroscopy to address issues of metabolite exchange in mouse hearts, and adenoviral gene transfer techniques to alter intermediary metabolism in vivo. One focus of these studies will be the diabetic heart. Despite clinical evidence that diabetes is associated with an increased risk of mortality due to ischemic heart disease, there is paradoxical evidence that the diabetic heart may be more resistant to ischemic injury. Although hearts from diabetic animals do oxidize more fatty acids and less glucose than control hearts, more recent evidence indicates that palmitate protects the perfused heart against ischemic injury dunng low-flow ischemia. The long-term goal is to better understand the factors that control citric acid cycle flux and substrate utilization in vivo so that interventions may be logically designed to alter substrate handling in post-ischemic hearts, under perfused tissue, and the diabetic heart. NMR is a unique tool in this regard because it offers the possibility of measuring these parameters in intact isolated organs perfused with physiological mixtures of substrates. Ultrasound adenovirus-coated microbubbles will be used to deliver the gene of malonyl-C0A decarboxylase to the myocardium to test the hypothesis that the cardiomyopathy associated with excess fat deposition in hearts of diabetic animals is reversed by lowering plasma levels of free fatty acids. The malic enzyme will also be over-expressed to test the hypothesis that pyruvate recycling is active in heart (like liver) and that it plays a protective role in the diabetic ischemic heart. In addition, several enzymes associated with the apparent 'futile' pyruvate recycling pathways will be over-expressed in rat liver to determine the metabolic impact of this cycle on liver metabolism and a new 13C isotopomer method that is exquisitely sensitive to pyruvate recycling will be developed and tested in two diabetic heart models, hearts from rats treated with streptozotocin (Type I) and from rats fed a prolonged, high-fat diet (Type II).
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