Regulation of Myocardial Phospholipases and Lipases in Diabetic Myocardium

摘要 心脏病是工业化国家最常见的死亡原因。潜在糖尿病的存在 是心脏病发展的最大危险因素。在当前的授予间隔期间，我们有 发现了以前未知的脂质代谢途径和信号分子， 类花生酸溶血磷脂值得注意的是，心肌中绝大多数类花生酸被发现是 酯化到溶血磷脂的甘油骨架上。此外，在野生型小鼠中诱导I型糖尿病， 小鼠或分离的野生型小鼠心脏的缺血性损伤导致促炎性细胞因子显著增加， 类花生酸溶血磷脂。这类新的磷脂作为炎症介质，通过诱导 从单核细胞或巨噬细胞释放TNF α。重要的是，iPLA 2g（PNPLA 8）的基因消融 显著降低糖尿病状态下心肌中类花生酸溶血磷脂的水平， 在糖尿病心肌缺血时，心肌缺血和协同减少它们的合成。 因此，我们认为iPLA 2g在糖尿病心脏的病理生理发展中起着重要作用 糖尿病性心肌病是糖尿病性心肌病的一种严重并发症。在具体目标1中，我们将利用稳定 从基因工程心肌细胞特异性分离的灌注小鼠心脏的同位素标记 条件性iPLA 2g敲除小鼠。这些研究将调查iPLA 2g在 代谢通量：1）非酯化和酯化类二十烷酸; 2）类二十烷酸-溶血磷脂;和3）其他 突出的氧化磷脂。稳定同位素脉冲追踪实验，随后进行穿透生物信息学 分析将确定通过这些新发现的途径的代谢流率。翻译，我们 将探讨2型糖尿病对心肌缺血性损伤的影响以及缺血性心肌损伤的潜在补救措施。 心肌细胞特异性iPLA 2g KO小鼠的心肌。分析终点包括梗死 大小、血流动力学性能和iPLA 2g的翻译后修饰。在具体目标2中，我们将利用 我们已经产生了心肌细胞特异性iPLA 2b KO小鼠，以探索iPLA 2b在促进心肌细胞凋亡中的作用。 II型糖尿病背景下WT与iPLA 2b KO小鼠的心肌缺血性损伤和心律失常。接下来， 我们将确定iPLA 2b催化酰基转移酶或转酰基酶介导的 类花生酸-溶血磷脂产生氧化磷脂， 相关的分子模式。在具体目标3中，高脂肪饮食诱导 将研究伴随炎症和线粒体功能障碍的类二十烷酸-溶血脂质合成。的 溶血磷脂酶在调节类花生酸-溶血磷脂水平中的作用和 将检查iPLA 2g。总的来说，拟议的研究将确定iPLA 2g和iPLA 2b的意义 介导新鉴定的类花生酸-溶血磷脂合成和代谢途径， 确定它们对糖尿病心肌病和糖尿病心脏急性缺血性损伤的影响。

ABSTRACT Heart disease is the most common cause of death in industrialized nations. The presence of underlying diabetes is the greatest risk factor for the progression of heart disease. During the current grant interval, we have discovered previously unknown lipid metabolic pathways and signaling molecules which lead to the generation of eicosanoid-lysophospholipids. Remarkably, the vast majority of eicosanoids in myocardium were found to be esterified to the glycerol backbone of lysophospholipids. In addition, induction of Type I diabetes in wild-type mice or ischemic damage in isolated wild-type mouse hearts resulted in dramatic increases in pro-inflammatory eicosanoid-lysophospholipids. This new class of phospholipids serve as inflammatory mediators by inducing the release of TNFa from monocytes or macrophages. Importantly, genetic ablation of iPLA2g (PNPLA8) substantially decreased the levels of eicosanoid-lysophospholipids in myocardium in the diabetic state, during myocardial ischemia and synergistically decreased their synthesis in diabetic myocardium rendered ischemic. Accordingly, we propose that iPLA2g plays a central role in the pathophysiologic development of diabetic heart disease and promotes the lethal sequelae of diabetic cardiomyopathy. In Specific Aim 1, we will utilize stable isotope labeling of isolated perfused mouse hearts from genetically engineered cardiac myocyte-specific conditional iPLA2g knockout mice we have generated. These studies will investigate the roles of iPLA2g in the metabolic flux of: 1) non-esterified and esterified eicosanoids; 2) eicosanoid-lysophospholipids; and 3) other salient oxidized phospholipids. Stable isotope pulse-chase experiments followed by penetrating bioinformatic analyses will determine rates of metabolic flux through these newly discovered pathways. Translationally, we will explore the impact of Type 2 diabetes on myocardial ischemic damage and the potential salvage of ischemic myocardium in cardiac myocyte-specific iPLA2g KO mice we engineered. Endpoints of analysis include infarct size, hemodynamic performance, and post-translational modifications of iPLA2g. In Specific Aim 2, we will utilize cardiac myocyte-specific iPLA2b KO mice we have generated to explore the role of iPLA2b in promoting myocardial ischemic damage and arrhythmias in WT vs. iPLA2b KO mice in the context of Type II diabetes. Next, we will determine the ability of iPLA2b to catalyze acyltransferase or transacylase mediated re-esterification of eicosanoid-lysophospholipids to generate oxidized phospholipids which have been implicated in damage associated molecular patterns. In Specific Aim 3, the mechanisms through which a high fat diet induces eicosanoid-lysolipid synthesis accompanied by inflammation and mitochondrial dysfunction will be studied. The roles of lysophospholipases in modulating eicosanoid-lysophospholipid levels and activation mechanisms for iPLA2g will be examined. Collectively, the proposed studies will establish the significance of iPLA2g and iPLA2b in mediating the newly identified pathways of eicosanoid-lysophospholipid synthesis and metabolism and determine their impact on diabetic cardiomyopathy and acute ischemic damage in diabetic hearts.