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 心肌病是世界范围内最常见的遗传性心血管疾病。心脏的存在 肌钙蛋白变异至少占所有家族性心肌病病例的15%。心肌收缩是 通过两种细丝调节蛋白-原肌球蛋白和 肌钙蛋白复合物Ca 2+与肌钙蛋白结合，从肌球蛋白结合位点置换原肌球蛋白， 肌球蛋白跨桥，其本身有助于细丝激活。肌钙蛋白复合体由 Ca 2+敏感肌钙蛋白C、肌动蛋白结合肌钙蛋白I和Tm结合肌钙蛋白T。几十年来，螺旋方法 到电子显微镜重建的细丝消除了信息的结构的Tn 复杂.因此，细丝的组分之间的复杂相互作用仍然是未知的。我们 开发了cryo-EM非螺旋算法来重建天然心脏细丝，以揭示 生理Ca 2+水平下整个肌钙蛋白复合物的结构。我们表明，细丝是由 具有短程协同性的无钙和钙结合非等效肌钙蛋白复合物阵列 相邻单位之间。与遗传性心肌病相关的肌钙蛋白变体影响细丝Ca 2 +- 依赖性激活我们假设：（1）扩张型（DCM）或肥厚型（HCM）心肌病变异， 肌钙蛋白通过以下方式影响细丝调节：（a）改变游离钙和结合钙的肌钙蛋白之间的平衡 （B）通过Ca 2+敏感肌钙蛋白C单元中的构象变化改变复合物;和/或 通过改变相邻肌钙蛋白之间的通讯来实现无Ca 2+和结合Ca 2+的肌钙蛋白复合物 沿着并穿过细丝。为了验证我们的假设，我们选择了4个战略定位的突变。目标1 我们将利用位于Ca 2+敏感肌钙蛋白N叶远端的肌钙蛋白C的致病性变体来评估 它们如何影响游离钙和结合钙的肌钙蛋白复合物之间的平衡，以及它们是否影响 肌钙蛋白单位之间的通信，可能抑制僵直肌球蛋白-S1的激活作用。在目标2中，我们将专注于 对位于肌钙蛋白T N端的肌钙蛋白T中的高渗透致病性变体，其稳定了 属于相邻肌钙蛋白单位的原肌球蛋白电缆之间的相互作用。我们将评估这些 突变影响相邻肌钙蛋白之间的通讯和肌球蛋白-S1的激活。为了揭示 肌钙蛋白复合体的远端部分（钙敏感肌钙蛋白C和肌钙蛋白T的N末端）进行通讯，我们 将使用钙离子增敏剂，其结合肌钙蛋白C以逆转肌钙蛋白T变体的效应。我们的多学科， 多PI方法与集体专业知识的结构，功能和计算方法将揭示如何 细丝的组分之间的复杂相互作用使心跳成为可能。成功 这些目标的实现可能为开发定制的疗法奠定基础， 细丝的结构来治疗各种形式的心脏病。