Mechanistic Understanding of Troponin T Role in Cardiac Thin Filament Activation

描述（由申请人提供）：缺乏关于心脏收缩调节蛋白之间相互作用的知识代表了一个重要的问题，因为它不仅限制了我们对这种相互作用如何影响心脏细丝激活的理解，而且还排除了对许多形式心脏病的潜在机制的理解。心肌收缩依赖于收缩调节蛋白之间的协调相互作用，包括心肌肌钙蛋白C （cTnC）、肌钙蛋白T （cTnT）、肌钙蛋白I （cTnI）和原肌球蛋白（Tm）。这些蛋白之间的协调相互作用在通过Ca2+-、强交叉桥(XB)-和肌节长度（SL）介导的细丝激活的收缩过程中起关键作用。然而，关于cTnT如何影响Tm、cTnI和cTnC调节心脏细丝Ca2+-、SL-和XB-活化的作用，我们知之甚少。因此，我们的长期目标是确定收缩调节蛋白的结构差异如何决定Ca2+-、SL-和强xb介导的心肌收缩激活，以及它们在心脏病中如何改变。本提案的总体目标是确定cTnT如何与Tm、cTnI和cTnC相互作用，以调节Ca2+、SL和强XB对心脏细丝的激活。我们的假设是，cTnT的尾部（cT1）和头部（cT2）结构域的结构特征是心脏细丝激活功能特征的关键决定因素。为了验证我们的假设，我们将测量重建心肌纤维的力/ atp酶，张力再开发率和肌纤维动态刚度。此外，互补研究，如Ca2+结合动力学测量和定量数学建模将进行。特异性目的1将确定cT1如何调节心脏细丝激活过程中强XB募集的动态。这一目标将通过确定：影响薄丝激活的cT1的特定区域；连续Tm重叠末端的变化如何影响cT1对心脏细丝激活的影响；以及cT1对细丝活化的影响如何被肌球蛋白同型体修饰。特异性目的2将确定在心脏细丝激活过程中，cTnT如何调节强XB对cTnC构象变化的协同反馈。在这个目的中，我们将确定cT2的关键区域如何调节Ca2+和xb诱导的cTnC结构变化。特异性Aim 3将确定cTnT和cTnI之间的相互作用如何介导强XB对心脏细丝激活的反馈效应。这一目标将通过确定：cT2-cTnI相互作用如何调节心脏细丝激活，以及肌球蛋白异构体的差异如何改变cT2-cTnI对细丝激活的影响来实现。我们的综合和多学科方法的预期结果将产生积极的影响，因为它将显著推进我们对心脏细丝激活的分子机制的理解。我们的研究将为开发旨在改善患病心脏功能的新药理学策略奠定坚实的基础。拟议的研究与公共卫生相关，因为该提案的目标是确定细丝收缩调节蛋白的结构差异如何决定心肌收缩的Ca2+-，强交叉桥-和肌节长度依赖性激活，以及这种相互作用如何在心脏病中改变。拟议的研究是心肌生理学的一个重要领域，因为它有可能为深入了解多种人类心脏病的分子机制做出重大贡献。

DESCRIPTION (provided by applicant): The lack of knowledge about the interactions among cardiac contractile regulatory proteins represents an important problem because it not only limits our understanding of how such interactions affect cardiac thin- filament activation, but it also precludes an understanding of mechanisms underlying many forms of heart disease. Cardiac muscle contraction depends on coordinated interactions among contractile regulatory proteins, which include cardiac troponin C (cTnC), troponin T (cTnT), troponin I (cTnI), and tropomyosin (Tm). Coordinated interactions among these proteins play key roles during contraction via Ca2+-, strong crossbridge (XB)-, and sarcomere length (SL)-mediated activation of thin filaments. However, very little is known about how cTnT influences the actions of Tm, cTnI, and cTnC to modulate Ca2+-, SL-, and XB- activation of cardiac thin filaments. Our long-term goal, therefore, is to determine how structural differences in contractile regulatory proteins determine Ca2+-, SL-, and strong XB-mediated activation of cardiac muscle contraction, and how they are altered in heart disease. The overall objective of this proposal is to determine how cTnT interacts with Tm, cTnI, and cTnC to modulate cardiac thin-filament activation by Ca2+, SL, and strong XB. Our hypothesis is that the structural features of the tail (cT1) and the head (cT2) domains of cTnT are the key determinants of functional features of cardiac thin-filament activation. To test our hypo- thesis, we will measure force/ATPase, rate of tension redevelopment and myofiber dynamic stiffness in re- constituted cardiac muscle fibers. Further, complementary studies such as Ca2+ binding kinetics measurements and quantitative mathematical modeling will be performed. Specific Aim 1 will determine how cT1 modulates the dynamics of strong XB recruitment during cardiac thin-filament activation. This aim will be accomplished by determining: the specific region of cT1 that affects thin-filament activation; how changes in the overlapping ends of contiguous Tm impact cT1 effects on cardiac thin-filament activation; and how cT1 effects on thin-filament activation are modified by myosin isoforms. Specific Aim 2 will determine how cTnT modulates the cooperative feedback of strong XB on conformational changes in cTnC during cardiac thin- filament activation. In this aim, we will determine how key regions of cT2 modulate Ca2+- and XB-induced changes in cTnC structure. Specific Aim 3 will determine how interactions between cTnT and cTnI mediate the feedback effect of strong XB on cardiac thin-filament activation. This aim will be accomplished by determining: how cT2-cTnI interactions modulate cardiac thin-filament activation and how differences in myosin isoforms alter cT2-cTnI effect on thin-filament activation. The expected outcome from our comprehensive and multidisciplinary approach will have a positive impact because it will significantly advance our understanding of the molecular mechanisms underlying cardiac thin-filament activation. Our study will lay a strong foundation for developing novel pharmacological strategies aimed at improving cardiac function in diseased hearts. PUBLIC HEALTH RELEVANCE The proposed research is relevant to public health because the goal of this proposal is to determine how structural differences in thin-filament contractile regulatory proteins determine Ca2+-, strong crossbridge-, and sarcomere length-dependent activation of cardiac muscle contraction, and how such interactions are altered in heart disease. The proposed research is an important area of cardiac muscle physiology because of its potential to contribute significantly to deeper understanding of the molecular mechanisms underlying many forms of human heart disease.