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暴露了一种能节能的“超级省力”（SRX）状态，该状态被认为是 收缩的能量和调节的基础。在心肌（包括人）中，SRX状态 通过隔离一部分肌球蛋白远离肌动蛋白，为能源经济做出贡献， 当心脏活动增加时，根据需要。人们认为对SRX状态的病理改变是许多基础 遗传性心肌病和治疗药物似乎通过逆转这些变化而起作用。尽管有它 无处不在和重要性，尚未定义SRX的结构基础，在我们的 了解心脏收缩及其治疗机制。一个广泛认为的是 SRX在结构上与肌肉肌球蛋白的另一个普遍特征相关：“相互作用的基序”（IHM）， 其中肌球蛋白的两个头（被阻塞和自由）相互互动，并与近端肌球蛋白尾巴（S2）， 抑制其活性并保护ATP。但是，最近的研究表明SRX可能是 肌球蛋白自己头部，不需要头部互动。在这笔赠款中，我们将使用单个粒子电子 显微镜（EM），冷冻EM和其他生物物理技术，以定义SRX状态的结构基础和 关键肥厚（HCM）和扩张（DCM）心肌病的突变和治疗的影响 关于IHM结构的药物。 AIM 1将在孤立的心脏肌球蛋白头（S1）和沉重的Meromyosin中定义SRX的基础 （hmm）通过评估是否：（a）SRX直接从肌球蛋白头的特定构象和（b）产生 IHM与SRX状态相关。肌球蛋白结构完成单头（S1）或两个头15 尾巴（15-HEP）的赫普特人将由我们的合作者博士表达和特征。 克里斯托弗·Yengo。控件将具有两个头部，但只有2个尾巴（2-HEP），无法形成完整 ihm。负面染色EM和类平均将揭示S1和IHM构象，而Cryo-EM将显示 这是第一次揭示IHM内部相互作用的近原子分辨率结构 松弛。 AIM 2将通过确定接近 - 从心肌分离的细丝的原子冷冻EM结构。 AIM 3将揭示关键的结构影响 使用冷冻EM，肌球蛋白头和IHM结构上的HCM和DCM诱导突变和治疗药物 和单个粒子成像。 现在，SRX被广泛认为是正常肌肉的基本状态，但其结构性 基础不理解。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 进入心肌放松（舒张）的基本机制，并提高了对 心肌病及其治疗的结构基础。