Structural basis of the super-relaxed state in human cardiac muscle

人体心肌超松弛状态的结构基础

基本信息

批准号：
10502114
负责人：
Raul Padron
金额：
$ 72.28万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
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：
2022-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10502114
关键词：
Accounting Actins Address Animals Cardiac Cardiac Myosins Cardiomyopathies Consumption Cryoelectron Microscopy DNA Sequence Alteration Diastole Dilated Cardiomyopathy Disease Drug usage Electron Microscopy Equilibrium Exhibits Familial Hypertrophic Cardiomyopathy Filament Fishes Goals Grant H-Meromyosin Head Heart Heart Diseases Human Hypertrophic Cardiomyopathy Image Induced Mutation Knowledge Measures Microscope Molecular Conformation Molecular Motors Motor Muscle Muscle function Muscle relaxation phase Mutation Myocardium Myopathy Myosin ATPase Myosin Type II Negative Staining Pathologic Pharmaceutical Preparations Property Proteins Regulation Relaxation Resolution Roentgen Rays Role Savings Slide Structure Tail Testing Therapeutic Thick Filament Time Work biophysical techniques blood pump drug action human disease improved inherited cardiomyopathy insight microscopic imaging molecular dynamics mutant particle repaired

项目摘要

Myosin filaments in muscle exhibit an energy-saving “super-relaxed” (SRX) state that is thought to be fundamental to the energetics and regulation of contraction. In cardiac muscle (including human), the SRX state contributes to energy economy by sequestering a proportion of myosin heads away from actin, to be released as needed when cardiac activity increases. Pathologic alterations to the SRX state are thought to underlie many inherited cardiomyopathies, and therapeutic drugs appear to work by reversing these changes. Despite its ubiquity and importance, the structural basis of the SRX has not been defined, leaving a crucial gap in our understanding of cardiac contraction and the mechanism of disease and its treatment. A widely held view is that SRX is structurally related to another ubiquitous feature of muscle myosin: the “interacting-heads motif” (IHM), in which myosin’s two heads (blocked and free) interact with each other and with the proximal myosin tail (S2), inhibiting their activity and conserving ATP. However, recent studies suggest that SRX may be a property of the myosin heads themselves, and not require head interactions. In this grant we will use single particle electron microscopy (EM), cryo-EM, and other biophysical techniques to define the structural basis of the SRX state and the impact of key hypertrophic (HCM) and dilated (DCM) cardiomyopathy-inducing mutations and therapeutic drugs on the structure of the IHM. Aim 1 will define the basis of SRX in the isolated cardiac myosin head (S1) and heavy meromyosin (HMM) by assessing if: (A) the SRX results directly from a specific conformation of the myosin heads, and (B) the IHM correlates with the SRX state. Myosin constructs comprising single heads (S1) or two heads with 15 heptads of tail (15-hep), enough to form the IHM, will be expressed and characterized by our collaborator, Dr. Christopher Yengo. Controls will have both heads but only 2 heptads of tail (2-hep), which cannot form a full IHM. Negative staining EM and class averaging will reveal S1 and IHM conformations, and cryo-EM will show for the first time the near-atomic resolution structure revealing the interactions within the IHM that underlie cardiac relaxation. Aim 2 will define the structural basis of the SRX in native thick filaments by determining the near- atomic cryo-EM structure of filaments isolated from cardiac muscle. Aim 3 will reveal the structural impact of key HCM- and DCM-inducing mutations and therapeutic drugs on myosin head and IHM structure, using cryo-EM and single particle imaging. The SRX is now widely recognized as a fundamental state of normal relaxed muscle, but its structural basis is not understood. Our high-resolution structural studies will reveal the near-atomic structures of cardiac S1 and the IHM and their relationship to the SRX in both molecules and filaments. This will provide new insights into the fundamental mechanism of relaxation (diastole) in cardiac muscle, and an improved understanding of the structural basis of cardiomyopathies and their treatment.

肌肉中的肌球蛋白丝表现出一种节能的“超松弛”（SRX）状态，这被认为是在心肌（包括人类）中，SRX 状态是能量学和收缩调节的基础。通过将一部分肌球蛋白头与肌动蛋白隔离开来释放能量，从而促进能源经济当心脏活动增加时，SRX 状态的病理改变被认为是许多因素的基础。遗传性心肌病，治疗药物似乎可以逆转这些变化。虽然 SRX 无处不在且很重要，但其结构基础尚未定义，这给我们的研究留下了一个关键的空白对心脏收缩和疾病机制及其治疗的理解是一种广泛的观点。 SRX 在结构上与肌肉肌球蛋白的另一个普遍特征相关：“相互作用头基序”(IHM)，其中肌球蛋白的两个头部（封闭的和自由的）彼此相互作用并与近端肌球蛋白尾部（S2）相互作用，然而，最近的研究表明，SRX 可能是 ATP 的一种特性。肌球蛋白本身有头，不需要头相互作用。在这项资助中，我们将使用单粒子电子。显微镜 (EM)、冷冻电镜和其他生物物理技术来定义 SRX 状态的结构基础关键肥厚型（HCM）和扩张型（DCM）心肌病诱发突变和治疗的影响药物对 IHM 结构的影响。目标 1 将定义离体心肌肌球蛋白头 (S1) 和重粒肌球蛋白中 SRX 的基础 (HMM) 通过评估是否：(A) SRX 直接来自肌球蛋白头的特定构象，以及 (B) IHM 与包含单头 (S1) 或 15 个头的 SRX 状态相关。足以形成 IHM 的尾部七肽 (15-hep) 将由我们的合作者 Dr. 进行表达和表征。 Christopher Yengo。对照将有两个头，但只有 2 个七联体尾部 (2-hep)，无法形成完整的。 IHM 负染色 EM 和类别平均将显示 S1 和 IHM 构象，冷冻电镜将显示首次近原子分辨率结构揭示了心脏基础的 IHM 内的相互作用目标 2 将通过确定近端粗丝来定义 SRX 的结构基础。从心肌中分离出的细丝的原子冷冻电镜结构将揭示关键的结构影响。使用冷冻电镜对肌球蛋白头部和 IHM 结构诱导 HCM 和 DCM 的突变和治疗药物和单粒子成像。 SRX 现在被广泛认为是正常放松肌肉的基本状态，但其结构我们的高分辨率结构研究将揭示心脏的近原子结构。 S1 和 IHM 以及它们与 SRX 在分子和细丝中的关系这将提供新的见解。深入了解心肌舒张（舒张）的基本机制，并加深对心肌病的结构基础及其治疗。