This proposal will study the cellular regulation of fatty acid oxidation (FAO) in ischemic hearts and cultured neonatal rat cardiac myocytes. The role of the cardiac isoform of acetyl-CoA carboxylase (C-ACC) in the cytoplasmic synthesis of malonyl-CoA and the subsequent inhibition of carnitine palmitoyltransferase I (CPT-I) in unknown. Lactate may affect the expression of C-ACC to regulate FAO during ischemia and early reperfusion, conditions in which tissue lactate is elevated. Transient activation of C-ACC by te production of lactate during ischemia may shift oxidative metabolism to the use of carbohydrate when these conditions prevail during early reperfusion. Recovery of fatty acid as an energy fuel should parallel a reduction in C-ACC activity. How C-ACC activity is regulated by phosphorylation will be correlated with tissue levels of lactate and CAMP, and with its enzyme product, malonyl-CoA. How malonyl-CoA modulates CPT-I activity will be investigated by examining the kinetics of substrate and inhibitor interaction and CPT-I sensitivity to malonyl-CoA in control and hypoxic cardiac myocytes in culture. A malonyl-CoA-sensitive isoform of CPT located in the sarcoplasmic reticulum (SR) will be isolated, cloned and expressed in yeast. Intact tissue, cultured cardiac cells, and isolated organelles will be analyzed immunologically and biochemically to examine cardiac metabolism in control and ischemic injured tissue. The presence and location of different isoforms of CPT-I also will be determined using the same techniques together with cell biology and immunoelectron microscopy. These experiments are important for defining the subcellular mechanisms that regulate FAO in the normal heart. No information is available suggesting that C-ACC plays a role in the regulation of FAO by malonyl- CoA in the heart, nor are there any data for cardiac muscles as for liver that describe a role for an adaptive response of CPT-I to physiological and pathological stimuli. Determining the changes in these regulatory mechanisms in the metabolic and functional responses of the ischemic, reperfused heart will provide information relevant to the therapeutic management of the stunned myocardium, and about the energetics of recovery following ischemia.
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