PKC Troponin I phosphorylation & contractile function

描述(由申请人提供)：心脏亚型的磷酸化 体外肌钙蛋白I(CTnI)与增强的收缩功能有关 在完整的心肌细胞中，蛋白激酶C(PKC)被激活。然而， CTnI磷酸化在PKC急性收缩反应中的特殊作用 是有争议的。这项建议的总体目标是了解 CTnI以及cTnI中的区域对PKC介导的 完整心肌细胞的收缩反应。工作假说是 CTnI磷酸化在松弛阶段起着关键作用。 收缩功能对PKC的反应。这一假设将使用 强大的PKC激动剂内皮素-1(ET)，可重复增强心肌细胞 收缩功能在很大程度上依赖于PKC，并在 几种生理/病理生理学状况。这样做的第一个目的是 建议对cTnI磷酸化进行表征，并确定其在 成年大鼠心肌细胞对急性PKC激活的收缩反应。首字母 实验将评估TnI之间的时间和剂量依赖关系 磷酸化与PKC对心肌细胞收缩缩短的反应 由ET激活。这些实验的结果将为 为随后确定磷酸化cTnI的贡献奠定基础 对PKC的心肌细胞缩短反应的影响。这个后面的目标将会实现 利用病毒介导的基因转移技术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 心肌细胞的反应。快速、特异和有效的基因转移，蛋白质 TnI基因的表达和肌丝掺入在 使用这一强大的方法使成年肌细胞完全分化。第一 选择唯一的TnI来确定cTnI磷酸化对 PKC介导的缩短反应将是TnI亚型在 胎儿心脏发育，至少缺少两个假定部位 被PKC磷酸化。来自这些研究的信息将提供直接的 了解磷酸化cTnI在PKC介导的心肌细胞中的作用 缩短反应。洞察力也将从差异中获得 对PKC激活的发育反应。第二个目标将是确定 CTnI中被PKC磷酸化的位点(S)，并决定其重要性 肌细胞对PKC的收缩反应中的每一个部位。相对的 蛋白激酶C磷酸化的cTnI位点(S)在短缩反应中的重要性 将在表达含有取代的突变体TnI的心肌细胞中进行研究 在假定的磷酸化位点。这项提案的第三个目标是确定 异构体特异性TnI区(S)对TnI形成能力的影响 磷酸化和/或影响对PKC的收缩反应。最终， 从拟议研究中获得的见解可能有助于设计和交付 独特的TnI蛋白与衰竭的心脏经历改变或 对PKC的病理生理反应。

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.