Novel Protein Kinase C Isoforms in Ventricular Myocytes

心室肌细胞中的新型蛋白激酶 C 亚型

• 批准号: 7086872
• 负责人: JEFFERY William WALKER
• 金额: $ 31.73万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7086872
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; calcium flux; cardiac myocytes; cell surface receptors; diacylglycerols; endothelin; fluorescence microscopy; fluorescence resonance energy transfer; green fluorescent proteins; heart contraction; laboratory rat; protein isoforms; protein kinase C; receptor coupling

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.

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，重点是这些不依赖于钙的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。要测试的总体假设是 PKC-e 和 PKC-S 可以抑制或刺激心肌收缩力和钙通量，具体取决于它们积聚的亚细胞区室。在目标 1 中，PKC-e 和 PKC-S 亚型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，以在 PKC 亚型表达水平、易位位点、收缩钙改变和正性肌力反应之间建立联系。在目标 2 中，使用显性失活 PKC-e 和 PKC-S 构建体将解决参与细胞渗透性 PKC 激活剂和 G 蛋白偶联受体激动剂收缩反应的亚型。在目标 3 中，将使用光激活的笼状化合物独立于激动剂受体控制二酰基甘油的亚细胞定位，以确定二酰基甘油在成年大鼠肌细胞的表面膜、横管和核周区域中的功能作用。这项研究的结果将为 PKC-e 和 PKC-S 的作用机制及其通过内皮肽等激动剂和其他通过 G 蛋白偶联受体起作用的激动剂的控制提供新的线索。内皮素/二酰甘油/蛋白激酶 C 信号系统代表哺乳动物心脏中的重要调节轴，被认为在控制收缩性、细胞内钙、基因表达、生长、细胞死亡以及心脏对缺氧/缺血或高血压等慢性应激的反应中发挥核心作用。越来越多的证据表明，这种信号系统在衰竭的心脏中发生改变，并可能导致疾病进展。更好地了解受体和 PKC 同工型之间的耦合，以及每种同工型调节基本心脏功能的亚细胞区室，最终将为探索各种形式的心脏病中的信号传导缺陷和心功能障碍的其他机制奠定基础。