ANALYSIS OF FUNCTIONAL ROLE OF TROPOMYOSIN IN HEART

心肌收缩活动的调节依赖于 粗细丝肌节之间的协同作用 proteins. 原肌球蛋白（TM），一种必需的细丝蛋白， 与肌钙蛋白（Tn）相互作用，并在Ca 2 +- 依赖的方式。 尽管一些体外研究已经确定了 特定TM区域的作用，这可能无法准确反映 在体内情况下发生的生物学功能。 长期 本建议的目的是理解以下方面的相对重要性： TM在协同作用过程中对心脏至关重要， 肌肉动力学 利用转基因小鼠和肌丝突变 蛋白质作为工具，我们将测试的核心假设，扰动 肌节TM的化学计量改变心肌收缩力 这涉及钙离子的变构控制和协同作用， 所有肌丝蛋白之间的相互作用。 使用我们的beta-TM 转基因小鼠模型，我们最近已经证明，改变 α-和β-TM的比例导致心肌细胞的生理变化， 肌丝的松弛和Ca 2+处理。 我们的基本实验 方法将是在鼠心脏中过表达突变体α-TM， 以确定这些alpha-TM的改变是否影响 与肌动蛋白和Tn复合物相互作用，从而改变细丝 activation. 具体目标涉及以下问题：（1） 决定不同生理活性的特殊氨基酸 在alpha-和beta-TM之间？(2)TM是通过什么机制 蛋白质调节细丝激活？（3）什么是 转基因小鼠心血管功能异常的机制 有转基因肌节的老鼠 该提案定义了 转基因横纹肌过表达的影响 α-TM对心肌功能的分子，生理， 形态和生化水平。 本建议的具体目标是：（1）界定 分子、生理和形态学效应， 突变体α-TM蛋白的过表达;和（2）确定突变体α-TM蛋白的过表达。 突变体的功能后果的生物化学和细胞基础 α-TM蛋白。 心肌α-肌球蛋白重链（MHC）启动子 将用于在鼠心脏中表达突变的α-TM cDNA。 本研究将为了解这一现象提供生物学信息。 TM在心肌收缩中的作用及其与 其他细丝蛋白。 使用转基因的心血管研究 小鼠模型将提供关于心脏功能的明确信息 在正常和疾病状态下。

Regulation of contractile activity in cardiac muscle is dependent upon a cooperative interaction between thick and thin filament sarcomeric proteins. Tropomyosin (TM), an essential thin filament protein, interacts with troponin (Tn) and regulates muscle contraction in a Ca2+- dependent manner. Although several in vitro studies have defined the role of specific TM regions, this may not accurately reflect the biological functions that occur in the in vivo situation. The long-term objective of this proposal is to comprehend the relative importance of TM in the cooperative interaction process that is essential for cardiac muscle dynamics. Using transgenic mice and mutagenesis of myofilament proteins as tools, we will test the central hypothesis that perturbation of stoichiometry of sarcomeric TM modifies cardiac muscle contractility which involves both allosteric control by Ca2+ and cooperative interactions among all of myofilament proteins. Using our beta-TM transgenic mouse model, we have recently demonstrated that altering the ratio of alpha- and beta-TM leads to physiological changes in myocardial relaxation and Ca2+ handling of myofilaments. Our basic experimental approach will be to overexpress mutant alpha-TM in the murine heart, and to determine whether these alterations in alpha-TM affect the interactions to actin and Tn complex, thereby changing thin filament activation. The specific aims address the following questions: (1) what specific amino acids determine differential physiological activity between alpha- and beta-TM?; (2) what is the mechanism by which TM protein modulates thin filament activation?; and (3) what is the mechanism that causes abnormal cardiovascular function in transgenic mice that have a genetically modified sarcomere? This proposal defines the effect of overexpression of genetically modified striated muscle alpha-TM on cardiac muscle function at molecular, physiological, morphological and biochemical levels. The specific aims of this proposal are: (1) delineate the molecular, physiological, and morphological effects which result from overexpression of mutant alpha-TM protein; and (2) determine the biochemical and cellular basis of functional consequences of the mutant alpha-TM protein. The cardiac alpha-myosin heavy chain (MHC) promoter will be used to express the mutant alpha-TM cDNAs in the murine heart. This study will provide biological information for understanding the role of TM in cardiac muscle contraction and in its interaction with other thin filament proteins. Cardiovascular studies using transgenic mouse model will provide explicit information on the heart function during both normal and diseased states.