Our long-term goal is to develop a strategy to control the excess fatty acid oxidation that occurs following an acute myocardial infarction and to use that information to the benefit of the elderly heart. The elderly Fisher 344 rat model will be used to study the mechanism for the greater injury in the elderly heart after ischemia and reperfusion compared to the adult heart. At baseline, aging defects in mitochondrial electron transport chain occur in only one population of heart mitochondria (interfibrillar) in elderly Fisher 344 rats. Following ischemia there are increases in non-esterified fatty acids and the accumulation of amphipathic lipid metabolites (long-chain acylcarnitines especially) in the heart, which suggest that the normal coupling of fatty acid oxidation and cardiac performance is disrupted. The regulation of carnitine palmitoyltransferase-I activity by malonyI-CoA is the controlling step in fatty acid oxidation. We propose, that the sensitivity of CPTI to malonyI-CoA inhibition is decreased during ischemia/reperfusion due to phosphorylation of CPT-I. This modification of the enzyme leads to decreased malonyI-CoA sensitivity as well as increased catalytic activity. These changes in CPT-I's kinetic properties, combined with the decreased tissue malonyI-CoA content leads to toxic long-chain acylcarnitines. The five aims described herein are designed to approach and establish the mechanism of increased catalytic activity and decreased sensitivity induced by phosphorylation.
Aim 1 will determine the phosphorylation status of CPT-I in the heart of 6 month and 24 month old Fisher 344 rats and its relation to the observed kinetic changes.
Aim 2 and 3 is focused on the identification of the protein kinases responsible for CPT-I phosphorylation using a proteomics approach (aim 2) and reconstitution experiments based on the predicted phosphorylation consensus sequences present in CPT-I.
Aim 4 will determine the effects of ischemia/reperfusion on CPT-i kinetics and phosphorylation in adult and elderly rats by """"""""freeze-clamping"""""""" CPT-I using specific protein kinase and phosphatase inhibitors. The working heart model with fatty acid perfusion is used and characterized at the physiological, biochemical, and morphological level.
Aim 5 will determine the intracellular distribution of malonyI-CoA and palmitoyI-CoA under different metabolic conditions and how changes of malonyI-CoA concentrations present in different compartments affect mitochondrial fatty acid oxidation and chain-elongation of long-chain fatty acids.
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