Novel Protein Kinase C Isoforms in Ventricular Myocytes

描述(申请人提供)：拟议研究的总体目标是了解PKC-e和PKC-S在哺乳动物心脏中的功能，重点是这些钙非依赖性的二酰甘油激活的丝氨酸/苏氨酸激酶如何调节钙的处理和收缩特性。有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能力和钙离子流动，这取决于它们聚集在哪个亚细胞间。在目标1中，将天然构建的PKC-e和PKC-S亚型与荧光蛋白融合并在成年大鼠心肌细胞中表达，以建立PKC异构体表达水平、易位位置、收缩期钙变化和变力反应之间的联系。在目标2中，使用显性负性PKC-e和PKC-S结构将解决参与细胞通透性PKC激动剂和G蛋白偶联受体激动剂收缩反应的异构体(S)。在目标3中，二酰甘油的亚细胞定位将独立于激动剂受体和光活化笼状化合物来控制，以确定二酰甘油在成年大鼠心肌细胞表膜、横管和核周区域的功能影响。这一研究结果将为揭示PKC-e和PKC-S的作用机制以及内皮素肽等激动剂对它们的调控提供新的线索。内皮素/甘油二酯/蛋白激酶C信号系统在哺乳动物心脏中是一个重要的调节轴，被认为在控制心脏的收缩能力、细胞内钙离子、基因表达、生长、细胞死亡以及心脏对慢性应激(如缺氧/缺血或高血压)的反应中发挥核心作用。越来越多的证据表明，在衰竭的心脏中，这种信号系统会发生变化，可能会导致疾病的进展。更好地了解受体和PKC亚型之间的偶联，以及每个亚细胞亚室中每个亚型对基本心脏功能的调节，最终将为探索各种形式心脏病中心功能障碍的信号缺陷和其他机制提供基础。

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.