Research supplement to promote diversity in Heath-related research

研究补充以促进健康相关研究的多样性

• 批准号: 10221154
• 负责人: Sivaraj Sivaramakrishnan
• 金额: $ 3.8万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221154
• 关键词: ATP phosphohydrolase; Biological Assay; Biosensor; Cardiac; Cardiac Myosins; Cardiomyopathies; Complement; Contractile Proteins; Defect; Disease; Fluorescence Resonance Energy Transfer; Goals; Heart failure; Human; Hypertrophic Cardiomyopathy; Impairment; Ionic Strengths; Measurement; Measures; Mole the mammal; Molecular Conformation; Muscle Contraction; Mutation; Myocardium; Myosin ATPase; Parents; Pharmaceutical Preparations; Phosphorylation; Play; Property; Regulation; Research; Role; Sarcomeres; Signal Transduction; Structure; Thick Filament; Thin Filament; Work; design; disease-causing mutation; insight; mutant; myosin-binding protein C; response; sensor; treatment strategy

Abstract The goal of this research supplement is to complement the aims of the parent R01 by directly examining the formation of the inhibited state of cardiac myosin, super-relaxed state (SRX), and to examine the influence of cMyBP-C and disease mutations on the formation/stabilization of this key structural state. The importance of understanding the role of cMyBP-C in cardiac contractility is highlighted by work demonstrating that its phosphorylation state plays a role in enhancing contractility and during heart failure decreased phosphorylation likely contributes to contractile defects. cMyBP-C is proposed to influence the cardiac myosin SRX in a phosphorylation dependent manner. In this proposal we will design a FRET biosensor of the SRX which will allow direct examination of the influence of cMyBP-C on this crucially important conformation of myosin. We will also examine hypertrophic cardiomyopathy (HCM) mutations in the cardiac myosin S2 region known to interact with cMyBP-C. In Aim 1 we will characterize the FRET biosensor by correlating the FRET signal with other measurements of the SRX, such as actin-activated ATPase and single turnover ATPase assays. We will vary the ionic strength and examine the drug Omecamtiv Mercarbil to demonstrate that the FRET sensor can be used to measure the mole fraction of cardiac myosin in the SRX conformation. In Aim 2 we will introduce HCM mutations into the S2 region and examine their impact on the formation/stabilization of the SRX. Finally we will also examine if cMyBP-C can alter the HCM mutants response to formation/stabilization of the SRX. Overall, the proposal will greatly complement the parent R01 by providing direct measurements of myosin structure which will be crucial for interpreting the studies of cMyBP-C role in thick and thin filament regulation of muscle contraction. AIM #1. Examine the formation of the super-relaxed state (SRX) by FRET in human cardiac myosin. AIM #2. Examine the functional impact of HCM mutations in the S2 region of cardiac myosin.

抽象的 本研究补充的目标是通过直接检查 心肌肌球蛋白抑制状态、超松弛状态（SRX）的形成，并考察 cMyBP-C 和疾病突变对这一关键结构状态的形成/稳定的影响。的重要性 研究表明，cMyBP-C 在心肌收缩力中的作用强调了对它的理解 磷酸化状态在增强收缩力方面发挥作用，在心力衰竭期间磷酸化降低 可能导致收缩缺陷。 cMyBP-C 被提议影响心肌肌球蛋白 SRX 磷酸化依赖方式。在本提案中，我们将设计 SRX 的 FRET 生物传感器，它将 允许直接检查 cMyBP-C 对肌球蛋白这一至关重要的构象的影响。我们将 还检查已知相互作用的心肌肌球蛋白 S2 区域的肥厚型心肌病 (HCM) 突变 与 cMyBP-C。在目标 1 中，我们将通过将 FRET 信号与其他信号相关联来表征 FRET 生物传感器。 SRX 测量，例如肌动蛋白激活 ATP 酶和单周转 ATP 酶测定。我们会有所不同 离子强度并检查药物 Omecamtiv Mercarbil 以证明可以使用 FRET 传感器 测量 SRX 构象中心肌肌球蛋白的摩尔分数。在目标 2 中，我们将介绍 HCM S2 区域的突变并检查它们对 SRX 形成/稳定的影响。最后我们将 还检查 cMyBP-C 是否可以改变 HCM 突变体对 SRX 形成/稳定的反应。总体而言， 该提案将通过提供肌球蛋白结构的直接测量来极大地补充母体 R01， 对于解释 cMyBP-C 在肌肉粗丝和细丝调节中的作用研究至关重要 收缩。 目标#1。通过 FRET 检查人心肌肌球蛋白中超松弛状态 (SRX) 的形成。 目标#2。检查心肌肌球蛋白 S2 区域 HCM 突变的功能影响。