MOLECULAR PHYSIOLOGY OF MYOCARDIAL TROPONIN I VARIANTS

描述(申请人逐字描述)：心脏收缩 通过肌丝蛋白的调节相互作用而发生 增加细胞内钙离子浓度。收缩功能障碍 与心肌疾病有关的基因与一种改变的 将蛋白质收缩成钙。肌钙蛋白抑制亚单位肌钙蛋白 I，是一种关键的调节蛋白，它基于其自身的功能调节收缩能力 磷酸化状态。最近，肌钙蛋白I的修饰和突变 与缺血性损伤、心力衰竭和心肌病有关。这个 这项建议的目标是了解疾病相关的改变是如何 肌钙蛋白I修饰其功能并在心肌疾病中发挥中心作用 各州。提出了一种综合的方法来划定这两个 脑血管紧张素转换酶功能改变的病理生理学及分子机制 肌钙蛋白I在心脏中的变体。具体的变种将是 特征是)截短突变体(I-193)，缺失16个氨基酸 来自羧基末端的残基，它概括了截断的形式 在顿抑心肌中由钙依赖的蛋白分解产生，和b) 肌钙蛋白I变异体与蛋白激酶A和蛋白激酶A的定点突变 蛋白激酶C的磷酸化位点来确定这些位点在 内源性收缩、预适应、缺血/再灌注与心脏 失败，最终c)带有单一氨基酸突变的肌钙蛋白I变体 在复制最近发现的突变体的抑制区中 家族性肥厚型心肌病。方法包括测量 转基因小鼠的心室力学研究 电导-微压计导管、稳态力-钙的研究 Fura-2负载小鼠完整骨小梁与体外的关系 利用重组蛋白和合成肽进行的实验 这些肌钙蛋白I变异体的生化特性发生了改变。这项工作将 确定心肌顿抑的分子机制，肌钙蛋白I的作用 体内磷酸化与一种肥厚性疾病的病理生理 心肌病。还预计，获得的信息将 最终为开发新的治疗方法提供合理的基础 心脏功能不全。

DESCRIPTION (the applicant's description verbatim): Contraction of the heart occurs through regulated interactions of the myofilament proteins in response to increasing intercellular calcium concentrations. Contractile dysfunction associated with myocardial disease is linked to an altered response of contractile proteins to calcium. The inhibitory subunit of troponin, troponin I, is a key regulatory protein which modulates contractility based on its phosphorylation status. Recently, modifications and mutants of troponin I have been associated with ischemic injury, heart failure and cardiomyopathy. The goal of this proposal is to understand how disease related alterations to troponin I modify its function and play a central role in myocardial disease states. A comprehensive approach is proposed to delineate the both the pathophysiology and molecular mechanisms of alteration of function produced by troponin I variants in the heart . Specific variants which will be characterized are a) truncated variant (I - 193), a loss of 16 amino acid residues from the carboxy-terminus, which recapitulates the truncated form produced by calcium dependent proteolysis in stunned myocardium, and b) troponin I variants with site-directed mutations in protein kinase A and protein kinase C phosphorylation sites to determine the role of these sites in intrinsic contractility, preconditioning, ischemia/reperfusion and heart failure and finally c) a troponin I variant with a single amino acid mutation in the inhibitory region reproducing a recently described mutant found in a familial hypertrophic cardiomyopathy. Methods include measurements of ventricular mechanics in transgenic mice using a miniaturized conductance-micromanometer catheter, studies of steady state force-calcium relationships in fura-2 loaded intact trabeculae from these mice and in vitro experiments with recombinant protein and synthesized peptides to dissect the altered biochemical properties of these troponin I variants. This work will determine a molecular mechanism of myocardial stunning, the role of troponin I phosphorylation in vivo and the pathophysiology of one form of hypertrophic cardiomyopathy. It is also anticipated that the information gained will ultimately provide a rational basis for the development of novel therapies for cardiac dysfunction.