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。

基本信息

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
项目类别：
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

项目摘要

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.

项目概要心肌病是全世界最常见的遗传性心血管疾病。肌钙蛋白变异占所有家族性心肌病病例的至少 15%。通过两种细丝调节蛋白 - 原肌球蛋白和游离细胞内 Ca2+ 浓度进行调节 Ca2+ 与肌钙蛋白的结合取代了肌球蛋白结合位点的原肌球蛋白并允许其形成。肌球蛋白跨桥，其本身有助于细丝肌钙蛋白复合物的激活。 Ca2+ 感应肌钙蛋白 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，螺旋方法细丝的电子显微镜重建消除了 Tn 结构的信息因此，细丝成分之间复杂的相互作用仍然未知。开发了冷冻电镜非螺旋算法来重建天然心脏细丝，以揭示整个肌钙蛋白复合物在生理 Ca2+ 水平上的结构我们表明细丝是由其组成的。一系列具有短程协同作用的无 Ca2+ 和 Ca2+ 结合的非等价肌钙蛋白复合物与遗传性心肌病相关的肌钙蛋白变异会影响细丝 Ca2+-。我们追求：（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) 改变无 Ca2+ 和 Ca2+ 结合肌钙蛋白之间的平衡通过 Ca2+ 感应肌钙蛋白 C 单元的构象变化和/或 (b) 改变复合物的分布；通过改变相邻肌钙蛋白之间的通讯来形成无 Ca2+ 和 Ca2+ 结合的肌钙蛋白复合物为了检验我们的假设，我们在目标 1 中选择了 4 个战略性定位的突变。我们将利用位于 Ca2+ 感应肌钙蛋白 N 叶远端的肌钙蛋白 C 的致病性变异来评估它们如何影响无 Ca2+ 和 Ca2+ 结合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元之间的通讯可能会抑制僵直肌球蛋白-S1 的激活作用。在目标 2 中，我们将重点关注。位于肌钙蛋白 T N 末端的高渗透致病性变异，可稳定我们将评估属于相邻肌钙蛋白单元的原肌球蛋白电缆之间的相互作用。突变影响邻近肌钙蛋白之间的通讯以及肌球蛋白-S1 的激活。肌钙蛋白复合物的远端部分（Ca2+ 感应肌钙蛋白 C 和肌钙蛋白 T 的 N 末端）进行通信，我们将使用 Ca2+ 敏化剂，它与肌钙蛋白 C 结合以恢复肌钙蛋白 T 变体的作用。具有结构、功能和计算方法方面集体专业知识的多 PI 方法将揭示如何细丝组件之间复杂的相互作用使心跳成功成为可能。这些目标的执行可能为开发定制疗法奠定基础，这些疗法可能会调节细丝的结构可治疗各种形式的心脏病。