Kinetics of Cardiac Myofilament Activation

心肌丝激活动力学

• 批准号: 6905197
• 负责人: WEN-JI DONG
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6905197
• 关键词: actins; binding sites; biological signal transduction; biophysics; calcium binding protein; calcium transporting ATPase; cell adhesion; chemical kinetics; conformation; fluorescence resonance energy transfer; fluorescence spectrometry; heart contraction; heart function; laboratory rabbit; microfilaments; muscle contraction; myosins; phosphorylation; protein binding; protein isoforms; protein kinase C; protein structure function; recombinant proteins; sarcomeres; thermodynamics; troponin

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.

描述（由申请人提供）：横纹肌收缩期间的力量发展是由 Ca2+ 与肌钙蛋白 C (TnC) 的特定位点结合引发的，引发细丝内的一系列功能性结构变化，包括 N 结构域的开放TnC 的变化、肌钙蛋白 I (Tnl) 抑制区的构象变化，以及将 Tnl 和肌动蛋白之间的相互作用转换为 Tnl 和 TnC，最终导致肌动蛋白之间的循环相互作用和肌球蛋白形成强大的产生力的跨桥。完全的肌肉激活需要 Ca2+ 结合和跨桥循环的反馈调节。在心肌中，它也受到蛋白质磷酸化的调节，蛋白质磷酸化在心力衰竭/肥厚过程中发挥重要作用。要充分了解肌肉调节机制，需要有关力量发展过程中每个结构转变的结构、热力学和动力学信息。我的长期研究目标是阐明薄鳍在收缩/松弛极端之间的运动动力学，并了解它们如何通过跨桥循环和磷酸化进行修改。为了实现这一目标，该提案解决了以下三个问题：（1）细丝的每个单独激活/失活过程的动力学是什么？ (2) 过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？新设计的基于福斯特共振能量转移的构象标记物可监测这些结构转变，将用于不同活化条件下的停流动力学和 Ca2+ 滴定测量，以获得所需的信息。这些标记物将与/不含有磷酸化蛋白质一起并入重建的细丝、肌原纤维和带皮纤维中，以指定细丝响应 Ca2+ 的特定域运动的时间依赖性变化。这项研究的结果将增强我们对 Ca2+ 响应细丝激活的分子机制以及蛋白质磷酸化在心力衰竭中的作用的理解。