FATTY ACID OXIDATION IN ISCHEMIC/REPERFUSED AGING HEARTS

我们的长期目标是开发一种策略，控制急性心肌梗死后发生的过量脂肪酸氧化，并利用这些信息造福老年心脏。老年Fisher 344大鼠模型将被用来研究老年心脏缺血再灌流后较成人心脏损伤更大的机制。在基线状态下，老年Fisher 344大鼠只有一组心脏线粒体(纤维间质)出现线粒体电子传递链的老化缺陷。心肌缺血后，非酯化脂肪酸增多，两亲性脂质代谢产物(尤其是长链肉毒碱)积聚，提示脂肪的正常偶联。 酸氧化和心脏功能受到干扰。丙二酸辅酶A对肉碱棕榈酰转移酶-I活性的调节是脂肪酸氧化的控制步骤。我们认为，在缺血/再灌注过程中，由于CPT-I的磷酸化，CPTI对丙二醛-辅酶A抑制的敏感性降低。这种酶的修饰降低了丙二醛-辅酶A的敏感性，同时提高了催化活性。 CPT-I动力学性质的这些变化，再加上组织中丙二醛-辅酶A含量的降低，导致了有毒的长链酰基肉碱。本文描述的五个目标旨在探讨和建立由磷酸化引起的催化活性增加和敏感性降低的机制。目的1测定6月龄和24月龄Fisher 344大鼠心脏CPT-I的磷酸化状态及其与动态变化的关系。目标2和目标3的重点是使用蛋白质组学方法(目标2)鉴定与CPT-I磷酸化有关的蛋白激酶，并根据CPT-I中预测的磷酸化共识序列进行重组实验。目的4研究缺血/再灌注对成年和老年大鼠CPT-I动力学和磷酸化的影响 通过使用特定的蛋白激酶和磷酸酶抑制剂“冷冻夹住”CPT-I。采用脂肪酸灌流的工作心脏模型，从生理、生化和形态水平对其进行表征。目的5测定不同代谢条件下丙二酰辅酶A和棕榈酰辅酶A在细胞内的分布，以及丙二酸辅酶A在不同隔室的浓度变化对线粒体脂肪酸氧化和长链脂肪酸链延长的影响。

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.