The goal of the work proposed in this application is to determine the overall regulation of fatty acid metabolism in cardiac muscle. Because Coenzyme A (CoA) and carnitine are essential cofactors of fatty acid metabolism, the work has, in recent years, been directed toward determining the regulation of tissue levels and cellular distribution of these cofactors. CoA is synthesized in the cytosol from pantothenic acid which enters the cells by an active-Na+- dependent symport system. Regulation of CoA synthesis occurs at the level of the cystoloic pantothenate kinase. However, about 95% of the total cellular CoA is located in mitochondria. Much of the proposed work relates to understanding the control of this CoA synthetic, cellular distribution pathway. 1. The pantothenate transport system is poorly characterized. We propose to study the regulation of this transporter using perfused hearts, isolated myocytes, purified sarcolemmal membrane vesicles and isolated, reconstituted transporter. Pantothenate transport in heart muscle is decreased by 80-90% while transporter in liver is increased several fold in diabetic animals. We plan to investigate the mechanism of these effects. 2. The decline in pantothenate transport causes intracellular pantothenic acid concentration to decrease and, in chronic diabetes, pantothenic acid concentration may become limiting for CoA synthesis in heart muscle. We plan to determine the physiological consequences of decreased pantothenate transport on CoA synthesis, CoA levels and cellular metabolism. 3. The functional pathway of CoA degradation is not known. We plan to define the pathway and determine its regulation in intact tissue, purified membranes and isolated mitochondria. 4. We will characterize and determine the regulation of the newly discovered system for CoA transport into mitochondria. 5. Carnitine is not synthesized in heart muscle, but is taken up from blood by a Na+- symport system similar to that for pantothenic acid. We plan to begin studies on the isolation and purification of the Na+-symport systems for pantothenic acid and carnitine by first developing assays for the transporters using either specific binding to purified proteins or transport activity in reconstituted lipid vesicles.
