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

• 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
• 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

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 是能量学和收缩调节的基础。在心肌（包括人类）中，SRX 状态 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 当心脏活动增加时根据需要。 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，治疗药物似乎可以通过逆转这些变化发挥作用。尽管其 虽然 SRX 无处不在且很重要，但其结构基础尚未定义，这在我们的研究中留下了关键的空白 了解心脏收缩和疾病的机制及其治疗。一个广泛持有的观点是 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， 抑制它们的活性并保存 ATP。然而，最近的研究表明 SRX 可能是 肌球蛋白自己负责，不需要头部相互作用。在这笔赠款中，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 状态的结构基础和 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 SRX 状态相关。肌球蛋白构建体包含单头 (S1) 或具有 15 个头的两个头 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。 克里斯托弗·延戈.对照将有两个头部，但只有 2 个七肽尾部 (2-hep)，不能形成完整的 恕我直言。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 松弛。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。目标 3 将揭示关键的结构性影响 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 基础不明白。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 及其与分子和细丝中 SRX 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。