ANALYSIS OF FUNCTIONAL ROLE OF TROPOMYOSIN IN HEART

心肌收缩活动的调节依赖于 粗丝和细丝肌节间的协同作用 蛋白质。原肌球蛋白(TM)是一种重要的细丝蛋白， 通过钙离子与肌钙蛋白(Tn)相互作用，调节肌肉收缩。 依赖的态度。尽管几项体外研究已经定义了 特定TM区域的作用，这可能不能准确地反映 在活体情况下发生的生物功能。长期的 本提案的目的是理解以下内容的相对重要性 TM在合作相互作用过程中对心脏至关重要 肌肉动力学。转基因小鼠的利用及肌丝的诱变 以蛋白质为工具，我们将检验微扰的中心假说 肌节TM的化学计量学改变心肌收缩能力 它既涉及钙离子对变构的调控，也涉及协同作用 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，我们最近证明了改变 α-TM和β-TM比值导致心肌生理性改变 肌丝的松弛和钙离子处理。我们的基础实验 方法将是在小鼠心脏过表达突变的α-TM，并且 为了确定α-TM的这些变化是否会影响 与肌动蛋白和TN复合体相互作用，从而改变细丝 激活。具体目标涉及以下问题：(1)什么 特定氨基酸决定不同的生理活性 在α-TM和β-TM之间？(2)TM通过什么机制 蛋白质调节细丝激活？以及(3)什么是 转基因动物心血管功能异常的机制 有转基因肌节的老鼠？这份提案明确了 转基因横纹肌的过表达效应 Alpha-TM对心肌功能的分子、生理、 形态和生化水平。 这项建议的具体目的是：(1)划定 分子、生理和形态效应 突变的α-TM蛋白的过表达；以及(2)确定 突变体功能后果的生化和细胞学基础 Alpha-TM蛋白。心肌α-肌球蛋白重链(MHC)启动子 将用于在小鼠心脏表达突变的α-TM cDNA。 这项研究将提供生物学信息以了解 TM在心肌收缩及其与心肌细胞相互作用中的作用 其他细丝蛋白。利用转基因技术进行心血管研究 小鼠模型将提供有关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.