Kinetics of Cardiac Myofilament Activation

描述（由申请人提供）：横纹肌收缩过程中的力发展是由Ca2+结合到肌钙蛋白C （TnC）的特定位点，引发细丝内一系列功能性结构变化，包括TnC的n结构域的打开，肌钙蛋白I （Tnl）抑制区域的构象变化，以及Tnl和肌动蛋白之间的相互作用转换为Tnl和TnC。最终导致肌动蛋白和肌凝蛋白之间的循环相互作用，形成强大的产生力的交叉桥。肌肉的完全激活需要Ca2+结合和跨桥循环的反馈调制。在心肌中，它也受到蛋白磷酸化的调节，蛋白磷酸化在心力衰竭/肥厚过程中起重要作用。为了充分理解肌肉的调节机制，需要在力发展过程中每一个结构转变的结构、热力学和动力学信息。我的长期研究目标是阐明在收缩/松弛极端之间的薄尾纤运动动力学，并了解它们是如何被过桥循环和磷酸化修饰的。为了实现这一目标，本提案解决了以下三个问题：(1)细丝的每个单独的活化/失活过程的动力学是什么？(2)跨桥循环如何影响这些动力学过程？(3)磷酸化在调节这些转变中的作用是什么？新设计的构象标记基于福斯特共振能量转移来监测这些结构转变，将用于不同激活条件下的停流动力学和Ca2+滴定测量，以获得所需的信息。这些标记将与/不含磷酸化蛋白的重组细丝、肌原纤维和剥皮纤维结合，以指定细丝响应Ca2+时特定结构域运动的时间依赖性变化。本研究结果将加深我们对Ca2+激活的分子机制和蛋白磷酸化在心力衰竭中的作用的理解。

DESCRIPTION (provided by applicant): Force development during striated muscle contraction is initiated by the binding of Ca2+ to the specific sites in troponin C (TnC), triggering a series of functional structural changes within the thin filament, including opening of the N-domain of TnC, conformational change of the inhibitory region of troponin I (Tnl), and switching interaction between Tnl and actin to Tnl and TnC, which ultimately lead to a cyclic interaction between actin and myosin to form strong force-generating cross-bridges. Full muscle activation requires both Ca2+ binding and feed back modulation of cross-bridge cycling. In cardiac muscle it is also modulated by protein phosphorylation which plays important roles in heart failing/hypertrophic process. To fully understand muscle regulatory mechanism requires structural, thermodynamic and kinetic information on each of these structural transitions during force development. My long-term research goal is to elucidate the kinetics of movements of the thin finlament betweem extremes of contraction/relaxation, and understand how they are modified by cross-bridge cycling and phosphorylation. To achieve the goal, this proposal addresses the following three issues: (1) What is the kinetics of each individual activation/deactivation process of the thin filament? (2) How does the cross-bridge cycling affect these kinetic processes? And (3) what is the role of phsophorylation in modulating these transitions? Newly designed conformational markers based on Forster resonance energy transfer to monitor these structural transitions will be used for stopped-flow kinetic and Ca2+ titration measurements at different activation conditions to acquire the desired information. These markers will be incorporated into reconstituted thin filament, myofibrils and skinned fibers along with/without phosphotylated proteins to specify the time-dependent changes of specific domain movements of the thin filament in response to Ca2+. Results of this study will enhance our understanding of molecular mechanisms of thin filament activation in response of Ca2+ and the role of protein phosphorylation in heart failure.