REGULATION OF MYOCARDIAL PHOSPHOLIPASES AND LIPASES IN DIABETIC MYOCARDIUM

描述(申请人提供)：糖尿病心肌病是一种复杂的疾病，由于缺乏胰岛素信号，长期和过度使用脂肪酸来刺激糖尿病心肌的收缩功能。然而，在糖尿病心肌中几乎完全使用脂肪酸作为燃料导致广泛的代谢失调，从而导致膜结构和功能的多种有害变化。这些膜介导的糖尿病心肌异常的后果包括血流动力学受损、兴奋收缩偶联缺陷和线粒体功能障碍，这些共同促进了糖尿病患者心力衰竭的进展。此外，糖尿病心肌中底物利用的深刻变化导致了积聚。 导致交织在一起的心肌细胞信号网络中的适应不良改变的多种失调代谢产物。此前，通过遗传学、药理学和化学生物学方法，我们已经鉴定出心肌iPLA2？(PNPLA9)，iPLA2？(PNPLA8)和iPLA2？(PNPLA2；ATGL)可能是糖尿病心肌血流动力学障碍、电生理改变和适应性不良重构的主要介质。最近，我们证明了iPLA2g及其下游信号代谢产物是线粒体通透性转换孔的关键调节因子，线粒体通透性转换孔与糖尿病心肌缺血后的坏死、坏死性下垂和电不稳定性有关。因此，在特定的目标1中，我们将使用我们构建的新的心肌细胞特异性iPLA2g条件性敲除小鼠来确定iPLA2g功能丧失是否可以减轻糖尿病心肌的急性缺血损伤、电生理不稳定性和脂质第二信使的不适应产生。此外，我们还证明了线粒体暴露于钙离子导致iPLA2g激活，导致花生四烯酸、2-花生四烯酸溶血磷脂酰胆碱的释放，从而产生多个下游具有生物活性的脂质第二信使。因此，我们将利用我们与心肌细胞特异性iPLA2g功能丧失小鼠共同开发的整合质谱学平台来检测依赖于iPLA2g的脂质第二信使产生的变化。在具体目标2中，我们将确定酰基辅酶A促进CaMKII磷酸化和iPLA2b激活的分子机制。将对激活的亚磷酸盐(S)进行鉴定和突变，并探讨其在CaMKII介导的iPLA2b激活中的机制作用以及糖尿病心肌缺血。在具体目标3中，将确定iPLA2z(ATGL；PNPLA2)在通过甘油三酯水解、转酰化和酰基转移酶活性催化脂质双向流动中的作用(S)。我们将利用心肌细胞特异性的iPLA2z缺失小鼠来研究iPLA2z参与糖尿病心肌中脂质第二信使的产生，并探讨iPLA2z基因消融对糖尿病状态下心肌功能的影响。总而言之，这些研究是一种多学科的协同方法，以确定糖尿病心肌病的化学机制。

DESCRIPTION (provided by applicant): Diabetic cardiomyopathy is a complex disorder that emanates from the chronic and excessive use of fatty acids to fuel contractile function in diabetic myocardium due to the lack of insulin signaling. However, the nearly exclusive use of fatty acids for fuel in diabetic myocardium results in widespread metabolomic dysregulation that precipitates multiple deleterious alterations in membrane structure and function. Consequences of these membrane-mediated abnormalities in diabetic myocardium include hemodynamic compromise, defective excitation-contraction coupling and mitochondrial dysfunction that collectively conspire to promote the progression of heart failure in diabetic patients. Moreover, the profound alterations in substrate utilization in diabetic myocardium result in the accumulation of multiple dysregulated metabolites that lead to maladaptive alterations in interwoven cardiac myocyte signaling networks. Previously, through genetic, pharmacologic and chemical biological approaches, we have identified three major phospholipases and lipases in myocardium iPLA2? (PNPLA9), iPLA2? (PNPLA8), and iPLA2? (PNPLA2; ATGL) that likely serve as principal mediators of myocardial hemodynamic dysfunction, electrophysiologic alterations and maladaptive remodeling in diabetic myocardium. Recently, we demonstrated that iPLA2g and its downstream signaling metabolites are key regulators of the mitochondrial permeability transition pore which is responsible for necrosis, necroptosis, and electrical instability in diabetic myocardium subjected to ischemia. Accordingly, in Specific Aim 1, we will use the novel cardiac myocyte specific iPLA2g conditional knock out mouse we generated to determine if iPLA2g loss of function attenuates acute ischemic injury, electrophysiologic instability and the maladaptive generation of lipid 2nd messengers in diabetic myocardium. Furthermore, we demonstrated that exposure of mitochondria to calcium ion results in the activation of iPLA2g leading to the release of arachidonic acid, 2-arachidonoyl lysophosphatidylcholine, and the subsequent production of multiple downstream biologically active lipid 2nd messengers. Accordingly, iPLA2g-dependent alterations in lipid 2nd messenger production will be examined employing integrative mass spectrometric platforms we developed in conjunction with the cardiac myocyte specific iPLA2g loss of function mouse. In Specific Aim 2, we will determine the molecular mechanisms through which acyl-CoA facilitates CaMKII phosphorylation and activation of iPLA2b. The activating phosphosite(s) will be identified, mutated and their mechanistic importance in CaMKII-mediated activation of iPLA2b in diabetic myocardium and diabetic myocardium rendered ischemic will be explored. In Specific Aim 3, the role(s) of iPLA2z (ATGL;PNPLA2) in catalyzing the bidirectional flux of lipids through triglyceride hydrolysis, transacylation and acyltransferase activities will e determined. The participation of iPLA2z in generating lipid 2nd messengers in diabetic myocardium will be examined using cardiac myocyte specific iPLA2z null mice and the effects of iPLA2z genetic ablation on myocardial function in the diabetic state will be explored. Collectively, these studies are a synergistic multidisciplinary approach to identify the chemical mechanisms mediating diabetic cardiomyopathy.