The Function of Slow Skeletal TnT in Muscle Contraction

描述(申请人提供)：拟议实验的总体目标是确定肌钙蛋白T(TnT)调节骨骼肌缓慢收缩的分子机制，并确定TnT的哪些区域对其生理功能重要。为实现这一总体目标，我们将实现两个具体目标： 特定目的1.慢骨骼肌钙蛋白T亚型在肌肉收缩调节中的作用。肌钙蛋白T(TnT)对慢速骨骼肌的调节基本上是未知的，这是本研究的重点，本研究将深入探讨慢速骨骼肌TnT(SSTnT)在横纹肌收缩调节中的作用。这里要检验的主要假设是，不同的N端SSTnT亚型调节钙敏感性和缓慢骨骼肌收缩的松弛特性。 我们将回答以下问题：1)SSTnT的N-或CT端区域是否影响力量发展和/或ATPase活性的钙敏感性？2)SSTnT亚型是否直接影响TNC对钙的亲和力，还是SSTnT对收缩的钙敏感性有间接影响(例如，细丝或交叉桥效应)？3)不同的SSTnT亚型与原肌球蛋白的相互作用是否不同？4)SSTnT亚型是否影响皮肤肌纤维的力的激活和松弛？5)是否有以前未描述的其他SSTnT亚型？这些研究将确定SSTnT的N-末端和C-末端选择性剪接区在骨骼肌缓慢收缩中的作用。特定目的2.慢速骨骼肌钙蛋白T不同区域在肌钙蛋白T生理功能中的作用为了了解TnT不同区域的作用，我们将进行以下工作： 1)对钙非依赖性ATPase活性起重要作用的SSTnT区域的特征。2)SSTnT与SSTn1、cTnC(慢速骨骼肌和心肌中相同的异构体)和原肌球蛋白相互作用的区域(S)的特征。3)探讨SSTn1在SSTnT功能中的重要性。到目前为止，还没有关于SSTnT任何区域的功能研究报道。以上列出的所有特定目的都集中在更详细地了解SSTnT在缓慢骨骼肌收缩中的作用，包括SSTnT激活肌肉收缩的分子机制。

DESCRIPTION (provided by applicant): The overall goal of the proposed experiments is to determine the molecular mechanisms involved in the regulation of slow skeletal muscle contraction by troponin T (TnT) and to determine what regions of TnT are important for its physiological function. To accomplish this overall goal we will carry out two specific aims: SPECIFIC AIM 1. THE ROLE OF SLOW SKELETAL TROPONIN T ISOFORMS IN THE REGULATION OF MUSCLE CONTRACTION. Essentially nothing is known about the regulation of slow skeletal muscle by troponin T (TnT) and this is the prime focus of our study which will thoroughly investigate the function of slow skeletal muscle TnT (SSTnT) in the regulation of striated muscle contraction. The main hypothesis to be tested here is that the various N-terminal SSTnT isoforms modulate Ca2+- sensitivity and the relaxation properties of slow skeletal muscle contraction. We will answer the following questions: 1) Is the Ca2+-sensitivity of force development and/or ATPase activity affected by the N- or Cterminal regions of SSTnT? 2) Do SSTnT isoforms directly affect the affinity of TnC for Ca2+ or is there an indirect effect of SSTnT on the Ca2+ sensitivity of contraction (e.g., thin filament or crossbridge effect)? 3) Do the different SSTnT isoforms interact differently with tropomyosin? 4) Do SSTnT isoforms affect the activation and relaxation of force in skinned muscle fibers? 5) Are there other isoforms of SSTnT that have not been previously described? These studies will determine the role of the N-and C-terminal alternatively spliced regions of SSTnT on slow skeletal muscle contraction. SPECIFIC AIM 2. THE ROLE OF DIFFERENT REGIONS OF SLOW SKELETAL TROPONIN T IN THE PHYSIOLOGICAL FUNCTION OF TROPONIN T. To understand the role of different regions of TnT we will carry out the following: 1) Characterization of the region of SSTnT that is important for Ca2+-independent ATPase activity. 2) Characterization of the region(s) of SSTnT that interacts with SSTnl, CTnC (same isoform in slow skeletal and cardiac muscle) and tropomyosin. 3) Investigation of the importance of SSTnl in the function of SSTnT. No functional studies on any region of SSTnT have so far been reported. All the Specific Aims listed above focus on gaining a more detailed understanding of the role of SSTnT in slow skeletal muscle contraction, including the molecular mechanisms of SSTnT-linked activation of muscle contraction.