This competing renewal proposal seeks to extend the use of dynamic mode C13 NMR spectroscopy to monitor regulation of oxidative metabolism. The general goal is to apply C13 NMR observations of subcellular interactions to elucidate mechanisms of metabolic regulation in response to physiologic function. Dynamic models will be explored in addressing the respiratory inefficiency and contractile dysfunction of postischemic hearts. Experimentation will be done with isolated rabbit hearts as well as the cross-perfused canine heart. Five hypotheses will be tested: (1) C13 NMR of metabolite transport across the mitochondrial membrane of normal hearts represents reversible exchange by the a-ketoglutarate-malate carrier; (2) Active exchange, involving the unidirectional glutamate-aspartate carrier, can be recruited by physiologic demand, and this component accounts for delayed C13 enrichment kinetics in postischemic hearts, (3) Regulation of TCA cycle flux by enzyme activation and metabolite influx / efflux can be observed in intact hearts with C13 NMR; (4) Reductions in glutamate C13 enrichment at high workloads in in vivo hearts result from increased TCA cycle enzyme activity which limits transport of labeled intermediates for exchange with glutamate; (5) Transport of long chain free fatty acids into mitochondria is a rate-limiting process for C13 enrichment of glutamate. The long range objective is the ultimate use of C13 NMR to assess the activity and expression of these mitochondrial proteins that regulate metabolic flux in intact and in vivo organs during development and disease.
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