The mechanisms of cardiac thin filament regulation in health and disease.

健康和疾病中心脏细丝调节的机制。

• 批准号: 10343934
• 负责人: P. Bryant Chase
• 金额: $ 59.24万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10343934
• 关键词: 3-Dimensional; Actins; Affect; Algorithms; Bepridil; Binding; Binding Sites; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Cardiovascular Diseases; Communication; Complex; Computing Methodologies; Cryoelectron Microscopy; Data; Development; Disease; Distal; Distant; Electron Microscopy; Equilibrium; Genetic; Goals; Health; Heart Diseases; Hypertrophic Cardiomyopathy; Incubated; Knowledge; Lead; Lobe; Microfilaments; Mission; Modeling; Molecular; Molecular Conformation; Muscle function; Mutation; Myocardium; Myosin ATPase; Pathogenesis; Pathogenicity; Phenotype; Phosphorylation; Physiological; Regulation; Research; Side; Site; Striated Muscles; Structure; Surface; Tail; Testing; Therapeutic Intervention; Thick; Thin Filament; Thinness; Time; Tropomyosin; Troponin; Troponin C; Troponin I; Troponin T; United States National Institutes of Health; Variant; genetic regulatory protein; improved; individualized medicine; inherited cardiomyopathy; innovation; interdisciplinary approach; multidisciplinary; muscle physiology; mutant; outcome prediction; public health relevance; rational design; reconstruction; tool

Project Summary Cardiomyopathies are the most common genetic cardiovascular disease worldwide. The presence of cardiac troponin variants accounts for at least 15% of all familial cardiomyopathy cases. Cardiac muscle contraction is regulated by free intracellular Ca2+ concentration via two thin filament regulatory proteins – tropomyosin and troponin complex. Ca2+ binding to troponin displaces tropomyosin from myosin-binding sites and allows formation of myosin cross-bridges, which on their own contribute to thin filament activation. Troponin complex is composed of Ca2+ sensing troponin C, actin-binding troponin I, and Tm-bound troponin T. For decades, the helical approach to electron microscopy reconstruction of the thin filament eliminated information on the structure of the Tn complex. Hence, the complex interactions among components of the thin filament remained unknown. We developed cryo-EM non-helical algorithm to the reconstruction of native cardiac thin filaments to reveal the structure of the whole troponin complex at physiological Ca2+ levels. We show that the thin filament is comprised of an array of Ca2+-free and Ca2+-bound non-equivalent troponin complexes with short-range cooperativity between adjacent units. Troponin variants associated with inherited cardiomyopathies affect thin filament Ca2+- dependent activation. We hypothesize that: (1) dilated (DCM) or hypertrophic (HCM) cardiomyopathy variants in troponin affect thin filament regulation by: (a) altering the equilibrium between Ca2+-free and Ca2+-bound troponin complexes via conformational changes in Ca2+-sensing troponin C unit; and/or (b) altering the distribution of Ca2+-free and Ca2+-bound troponin complexes by changing communication between the adjacent troponins along and across the thin filament. To test our hypothesis we chose 4 strategically located mutations. In Aim 1 we will utilize pathogenic variants in troponin C located in distal parts of Ca2+-sensing troponin N-lobe to evaluate how they affect the equilibrium between Ca2+-free and Ca2+-bound troponin complexes, and if they affect communication between troponin units that may curb activating effect of rigor myosin-S1. In Aim 2 we will focus on highly penetrant pathogenic variants in troponin T located in N-terminus of troponin T, which stabilizes the interaction between tropomyosin cables belonging to adjacent troponin units. We will evaluate how these mutations affect the communication between neighboring troponins and activation by myosin-S1. To reveal how distal parts of the troponin complex (Ca2+ sensing troponin C and N-terminus of troponin T) communicate, we will use a Ca2+ sensitizer, which binds to troponin C to revert effects of a troponin T variant. Our multidisciplinary, multi-PI approach with collective expertise in structural, functional and computational methods will reveal how the complex interactions between components of the thin filament make heartbeats possible. Successful execution of the aims may set the ground for the development of tailored therapies that could potentially modulate the structure of the thin filament to treat various forms of heart disease.

项目摘要 心肌病是全球最常见的遗传性心血管疾病。心脏的存在 肌钙蛋白变体占所有家庭心肌病病例的15％。心肌收缩是 通过两种细细丝调节蛋白 - 肌球蛋白和 肌钙蛋白复合物。 Ca2+与肌钙蛋白的结合从肌球蛋白结合位点移位，并允许形成 肌球蛋白跨桥的作用，它们自身有助于细丝激活。肌钙蛋白复合物成 Ca2+传感肌钙蛋白C，肌动蛋白结合肌钙蛋白I和TM结合的肌钙蛋白T数十年 薄丝的电子显微镜重建消除了有关TN结构的信息 复杂的。因此，薄丝细丝组成部分之间的复杂相互作用尚不清楚。我们 开发了重建天然心脏薄丝的低温EM非螺旋算法，以揭示 整个肌钙蛋白复合物在物理Ca2+水平上的结构。我们证明细丝已经完成 一系列Ca2+Fre-Fre-Fre-Fre-Fred-bound-BOND非等效的热带蛋白配合物具有短距离配位 在相邻单元之间。与遗传性心肌病有关的肌钙蛋白变体会影响细丝Ca2+ - 依赖激活。我们假设：（1）扩张（DCM）或肥厚（HCM）心肌病变体 肌钙蛋白会通过以下方式影响薄丝调节：（a）更改Ca2+-fre-Fre-Fre-free和Ca2+ - 结合的肌钙蛋白之间的等效物 通过Ca2+传感肌钙蛋白C单元中构象变化的复合物；和/或（b）改变 Ca2+Fre-Fre-Fre和Ca2+ - 结合肌钙蛋白复合物通过改变相邻的肌钙蛋白之间的通信 目标1 我们将利用肌钙蛋白C中的致病变异，位于Ca2+传感肌钙蛋白N-lobe的不同部分 它们如何影响Ca2+-fre-Fre-Fre-Fre-Fred-Bound-Bound Tropicalin复合物之间的等效物，以及它们是否影响 肌钙蛋白单元之间的通信可能会遏制严格肌球蛋白-S1的激活作用。在AIM 2中，我们将集中精力 在位于肌钙蛋白T的N末端的肌钙蛋白T中高度渗透的致病变异上，该变异稳定 属于相邻肌钙蛋白单元的肌动蛋白电缆之间的相互作用。我们将评估这些 突变影响相邻的肌钙蛋白与肌球蛋白S1激活之间的通信。揭示如何 肌钙蛋白复合物的远端部分（Ca2+传感肌钙蛋白C和肌钙蛋白的N末端）to沟 将使用Ca2+传感器，该传感器与肌钙蛋白C结合以恢复肌钙蛋白T变体的影响。我们的多学科， 具有结构，功能和计算方法的集体专业知识的多PI方法将揭示如何 细丝的组成部分之间的复杂相互作用使心跳成为可能。成功的 目标的执行可能为开发可能调节的量身定制疗法奠定基础 细丝的结构以治疗各种形式的心脏病。