The long-term objectives of this proposal are to elucidate the regulatory mechanism of fatty acid transport and oxidation in the normal and ischemic heart. Plasma long-chain fatty acids are the primary fuel source for energy production in the normal heart. In the ischemic and reperfused myocardium, there is decreased utilization of fatty acids. Furthermore, perfusion with fatty acids results in in situ membrane damage and cardiac dysfunction. Fatty acid-induced injury to ischemic myocardium can be reduced by inhibition of carnitine palmitoyltransferase I (CPTI). Because of its central role in lipid metabolism, CPTI has attracted attention as a potential site for pharmacological intervention in the ischemic heart where elevated levels of acylcarnitines have been associated with arrhythmias; in diabetes mellitus, where fatty acid oxidation is excessive and interferes with glucose oxidation; and in human inherited CPT defects with fatal disturbances in fatty acid oxidation. Therefore, for effective pharmacotherapy of defects in cardiac fatty acid oxidation, it is imperative that we understand the biochemical and molecular mechanisms regulating CPT. The applicant has cloned, sequenced, and expressed human heart CPTI. He now plans to continue these studies with the following specific aims: 1. (a) To map the malonyl CoA and substrate binding sites in human heart CPTI by site-directed mutagenesis and chemical modification studies using residue-specific reagents. (b) To determine the structural basis for the high malonyl CoA sensitivity of human heart CPTI by constructing chimeras between heart and liver CPTI. (2) To prepare milligram quantities of expressed highly purified human heart CPTI and engineered fragments of this enzyme for structural characterization studies. (3) To characterize the promoter region of the gene coding for human heart CPTI, and to study its transcriptional regulation by hormonal, developmental, and dietary factors (a) in cardiac myocytes in vitro and (b) using a transgenic mouse model in vivo.
Showing the most recent 10 out of 20 publications
