PKC Troponin I phosphorylation & contractile function

PKC 肌钙蛋白 I 磷酸化

• 批准号: 6821334
• 负责人: Margaret V Westfall
• 金额: $ 26.38万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-03 至 2007-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821334
• 关键词: cardiac myocytes; heart contraction; histogenesis; isozymes; laboratory rat; microfilaments; muscle contraction; phosphorylation; protein kinase C; protein structure function; tissue /cell culture; transfection; troponin; western blottings

DESCRIPTION (provided by applicant): Phosphorylation of the cardiac isoform of troponin I (cTnI) in vitro is associated with enhanced contractile function during protein kinase C (PKC) activation in intact myocytes. However, the specific role of cTnI phosphorylation in the acute contractile response to PKC is controversial. The overall objective of this proposal is to understand the contribution of cTnI, as well as regions within cTnI, to the PKC-mediated contractile response in intact cardiac myocytes. The working hypothesis is that cTnI phosphorylation plays a critical role in the relaxation phase of the contractile function response to PKC. This hypothesis will be tested using the potent PKC agonist endothelin-1 (ET), which reproducibly enhances myocyte contractile function in a largely PKC-dependent manner, and is released during several physiological/pathophysiological conditions. The first aim of this proposal is to characterize cTnI phosphorylation and determine its role in the adult rat cardiac myocyte contractile response to acute PKC activation. Initial experiments will evaluate temporal and dose-dependent associations between TnI phosphorylation and the myocyte contractile shortening response to PKC activation by ET. Results from these experiments will lay an essential foundation for subsequently determining the contribution of phosphorylated cTnI to the myocyte shortening response to PKC. This later goal will be achieved using viral-based gene transfer to express unique TnI proteins in adult cardiac myocytes and then comparing PKC-dependent TnI phosphorylation and shortening responses in myocytes. Rapid, specific, and efficient gene transfer, protein expression and myofilament incorporation of delivered TnI genes is achieved in fully differentiated adult myocytes using this powerful approach. The first unique TnI selected for determining the contribution of cTnI phosphorylation to the PKC-mediated shortening response will be a TnI isoform expressed during fetal cardiac development, which lacks at least two putative sites phosphorylated by PKC. Information from these studies will provide a direct understanding of the role phosphorylated cTnI plays in the PKC-mediated myocyte shortening response. Insight also will be gained into differential developmental responses to PKC activation. The second aim will be to identify site(s) within cTnI that are phosphorylated by PKC and determine the importance of each site in the myocyte contractile response to PKC. The relative importance of cTnI site(s) phosphorylated by PKC in the shortening response will be investigated in myocytes expressing mutant TnI containing substitutions in putative phosphorylation sites. A third aim of this proposal is to determine the effect of isoform-specific TnI region(s) on the ability of TnI to be phosphorylated and/or influence the contractile response to PKC. Ultimately, insights gained from the proposed studies may aid in the design and delivery of unique TnI proteins to failing hearts experiencing altered or pathophysiological responses to PKC.

描述（由申请方提供）： 肌钙蛋白I（cTnI）在体外与增强的收缩功能相关 在完整的心肌细胞中蛋白激酶C（PKC）激活期间。但 cTnI磷酸化在PKC急性收缩反应中的特殊作用 是有争议的。本提案的总体目标是了解 cTnI以及cTnI内的区域对PKC介导的 完整心肌细胞的收缩反应。工作假设是， cTnI磷酸化在心肌细胞的舒张期起关键作用， 对PKC的收缩功能反应。这个假设将使用 有效的PKC激动剂内皮素-1（ET），可重复地增强心肌细胞 收缩功能在很大程度上依赖于PKC的方式，并在 几种生理/病理生理条件。第一个目标是 建议是表征cTnI磷酸化并确定其在 成年大鼠心肌细胞对急性PKC激活的收缩反应。初始 实验将评估TnI之间的时间和剂量依赖性关联， 磷酸化与心肌细胞对PKC的收缩缩短反应 激活ET。这些实验的结果将奠定一个基本的 为随后确定磷酸化cTnI的贡献奠定基础 对PKC的缩短反应。这个后来的目标将实现 利用病毒基因转移在成人心脏中表达独特的TnI蛋白 然后比较PKC依赖的TnI磷酸化和缩短 肌细胞的反应。快速、特异、高效的基因转移、蛋白质 表达和肌丝掺入递送的TnI基因， 完全分化的成体心肌细胞。第一 选择独特的TnI用于确定cTnI磷酸化对 PKC介导的缩短反应将是在过程中表达的TnI亚型 胎儿心脏发育，缺乏至少两个推定位点 通过PKC磷酸化。这些研究的信息将提供直接的 了解磷酸化cTnI在PKC介导的心肌细胞 缩短响应。还将深入了解差异化 对PKC激活的发育反应。第二个目标是确定 cTnI内被PKC磷酸化并决定重要性的位点 心肌细胞对PKC的收缩反应中的每个位点。的相对 PKC磷酸化cTnI位点在缩短反应中的重要性 将在表达含有替换的突变TnI的肌细胞中进行研究 在假定的磷酸化位点。本建议的第三个目的是确定 同种型特异性TnI区域对TnI被 磷酸化和/或影响对PKC的收缩反应。最后， 从拟议的研究中获得的见解可能有助于设计和交付 独特的肌钙蛋白I蛋白， 对PKC的病理生理反应。