Novel Protein Kinase C Isoforms in Ventricular Myocytes

描述（由申请人提供）：拟议研究的总体目标是了解哺乳动物心脏中的PKC-e和PKC-S功能，重点是这些钙非依赖性二酰基甘油激活的丝氨酸/苏氨酸激酶如何调节钙处理和收缩特性。待检验的总体假设是，PKC-e和PKC-S可以抑制或刺激心肌收缩力和钙通量，这取决于它们积聚的亚细胞区室。在目的1中，PKC-ε和PKC-S异构体的天然构建体将与荧光蛋白融合并在成年大鼠心室肌细胞中表达，以建立PKC异构体表达水平、易位位点、改变的收缩期钙和正性肌力反应之间的联系。在目的2中，使用显性负性PKC-e和PKC-S构建体将解决涉及对细胞可渗透的PKC激活剂和G蛋白偶联受体激动剂的收缩反应的同种型。在目标3中，将独立于激动剂受体，用光活化笼状化合物控制二酰甘油的亚细胞定位，以确定二酰甘油在成年大鼠肌细胞的表面膜、横小管和核周区域中的功能效应。这项研究的结果将为PKC-ε和PKC-S的作用机制及其通过激动剂如内皮素肽和通过G蛋白偶联受体起作用的其他激动剂的控制提供新的线索。内皮素/二酰基甘油/蛋白激酶C信号传导系统代表哺乳动物心脏中的重要调节轴，其被认为在控制收缩性、细胞内钙、基因表达、生长、细胞死亡和心脏对慢性应激如缺氧/缺血或高血压的反应中起中心作用。越来越多的证据表明，这种信号系统在衰竭的心脏中发生了改变，可能有助于疾病的进展。更好地了解受体和PKC亚型之间的偶联，以及每个亚型调节基本心脏功能的亚细胞区室，最终将为探索各种心脏疾病中的信号传导缺陷和其他心功能障碍机制提供基础。

DESCRIPTION (provided by applicant): The overall aim of the proposed research is to understand PKC-e and PKC-S function in the mammalian heart, with an emphasis on how these calcium-independent diacylglycerol-activated serine/threonine kinases regulate calcium handling and contractile properties. The overall hypothesis to be tested is that PKC-e and PKC-S can inhibit or stimulate cardiac contractility and calcium fluxes depending upon the subcellular compartments in which they accumulate. In Aim 1, native constructs of PKC-e and PKC-S isoforms will be fused with fluorescent proteins and expressed in adult rat ventricular myocytes to establish a link between PKC isoform expression level, sites of translocation, altered systolic calcium and inotropic responses. In Aim 2, use of dominant negative PKC-e and PKC-S constructs will address the isoform(s) involved in contractile responses to cell-permeable PKC activators and to agonists of G-protein coupled receptors. In Aim 3, the subcellular localization of diacylglycerol will be controlled independently of agonist receptors with light-activated caged compounds to determine diacylglycerol's functional effects in surface membranes, transverse-tubules and perinuclear regions of adult rat myocytes. The outcome of this research will shed new light on mechanisms of action of PKC-e and PKC-S and their control by agonists such as the endothelin peptides and other agonists operating through G-protein coupled receptors. The endothelin/diacylglycerol/protein kinase C signaling system represents an important regulatory axis in the mammalian heart which is thought to play a central role in control of contractility, intracellular calcium, gene expression, growth, cell death, and the heart's response to chronic stress such as hypoxia/ischemia or high blood pressure. Evidence is also accumulating that this signaling system is altered in failing hearts and may contribute to disease progression. A better understanding of coupling between receptors and PKC isoforms, and the subcellluar compartments in which each isoform acts to regulate basic cardiac function, will ultimately provide a foundation on which to explore signaling defects and other mechanisms of cardiac dysfunction in various forms of heart disease.