LIPID SECOND MESSENGERS IN DIABETIC CARDIAC DYSFUNCTION

该项目的统一假设是糖酵解改变 糖尿病中的钙流导致钙调节功能失调， 心脏和血管中的独立磷脂酶A2。 这 项目审查的作用，功能失调的监管， 糖酵解偶联产生的脂质第二信使的钙- 非依赖性磷脂酶A2在心肌收缩性心肌病发病机制中的作用 糖尿病性心肌功能障碍和 糖尿病心肌急性缺血。 我们已经证明 糖尿病心肌表现出三个病理改变的标志 脂质代谢包括：1）花生四烯酸的急剧消耗 存在于其内源性磷脂储存库中的酸含量; 2） 钙非依赖性磷脂酶A2水平显著升高 活动;和3）花生四烯酸的异常通量进出 肌细胞磷脂池。 因此，具体目标1将审查 糖酵解通量与花生四烯酸耦合的重要性 释放和溶血磷脂的产生。 的作用 钙非依赖性磷脂酶A2在这一过程中， 通过利用基于机制的 抑制剂（E）-6-（溴亚甲基）-3-（1-萘基）-2-H-四氢吡喃- 2-one（BEL）。 接下来，通过以下方式加速膜水解的作用： 钙非依赖性磷脂酶A2介导的增加 糖尿病心肌对缺血性损伤易感性将是 探讨了 在具体目标2中，花生四烯酸的异质性 糖尿病患者心肌细胞亚细胞膜室消耗 将通过利用功率和灵敏度检查心肌 电喷雾离子化质谱 脂质通量的改变 糖尿病状态下的特定亚细胞膜位点将被 利用定量电子显微镜放射自显影法鉴定 钙非依赖性磷脂酶A2在这一过程中的作用将 使用BEL确定。 在具体目标3中， 钙非依赖性磷脂酶A2的显著增加 糖尿病心肌中的活性将通过分析 钙非依赖性磷脂酶A2亚型质量的改变 在糖尿病患者中的组成和/或翻译后修饰 状态 总的来说，这些研究代表了一个多学科 确定糖酵解偶联重要性的方法 脂质第二信使产生与心功能不全的关系 糖尿病状态

The unifying hypothesis of the program project is that altered glycolytic flux in diabetes results in the dysfunctional regulation of calcium- independent phospholipase A2 in the heart and blood vessels. This project examines the role of the dysfunctional regulation of the glycolytically-coupled generation of lipid second messengers by calcium- independent phospholipase A2 in the pathogenesis of contractile dysfunction in diabetic myocardium and the increased susceptibility of diabetic myocardium to acute ischemia. We have demonstrated that diabetic myocardium exhibits three hallmarks of pathologic alterations in lipid metabolism including: 1) dramatic depletion of the arachidonic acid content present in its endogenous phospholipid storage depots; 2) markedly increased levels of calcium-independent phospholipase A2 activity; and 3) abnormal fluxes of arachidonic acid into and out of myocytic phospholipid pools. Accordingly, Specific Aim 1 will examine the importance of the coupling of glycolytic flux with arachidonic acid release and lysophospholipid generation in diabetic myocardium. The role of calcium-independent phospholipase A2 in this process will be determined by exploiting the specificity inherent in the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran- 2-one(BEL). Next, the role of accelerated membrane hydrolysis by calcium-independent phospholipase A2 in mediating the increased susceptibility of diabetic myocardium to ischemic injury will be explored. In Specific Aim 2, the heterogeneity of arachidonic acid depletion in myocardial subcellular membrane compartments in diabetic myocardium will be examined by exploiting the power and sensitivity of electrospray ionization mass spectrometry. Alterations of lipid flux in specific subcellular membrane loci in the diabetic state will be identified utilizing quantitative electron microscopic autoradiography and the role of calcium-independent phospholipase A2 in this process will be determined utilizing BEL. In Specific Aim 3, the mechanisms underlying the marked increase in calcium-independent phospholipase A2 activity in diabetic myocardium will be identified by analysis of alterations in calcium-independent phospholipase A2 mass, isoform composition and/or post-translational modifications in the diabetic state. Collectively, these studies represent a multidisciplinary approach targeted at identifying the importance of glycolytic coupling of lipid second messenger generation to cardiac dysfunction in the diabetic state.