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 这项研究补充的目的是通过直接检查父母R01的目标 抑制心脏肌球蛋白，超级释放状态（SRX）的形成，并检查 CMYBP-C和有关该关键结构状态形成/稳定的疾病突变。重要性 通过工作表明它 磷酸化状态在增强收缩力和心力衰竭期间起作用，磷酸化降低 可能导致收缩缺陷。提议CMYBP-C影响心脏肌球蛋白SRX 磷酸化依赖性方式。在此提案中，我们将设计一个SRX的Fret生物传感器 直接检查CMYBP-C对肌球蛋白至关重要的构象的影响。我们将 还检查心脏肌球蛋白S2区域中已知相互作用的肥厚性心肌病（HCM）突变 与CMYBP-C。在AIM 1中，我们将通过将FRET信号与其他相关性来表征FRET生物传感器 SRX的测量，例如肌动蛋白激活的ATPase和单个周转ATPase分析。我们会有所不同 离子强度并检查药物的Omecamtiv杂物，以证明可以使用FRET传感器 测量SRX构象中心脏肌球蛋白的摩尔分数。在AIM 2中，我们将介绍HCM 突变进入S2区域并检查其对SRX形成/稳定的影响。最后我们会的 还要检查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， 提案将通过提供直接测量肌球蛋白结构来大大补充父母R01 对于解释CMYBP-C在肌肉厚和薄丝调节中的作用的研究至关重要 收缩。 目标＃1。检查人类心脏肌球蛋白中FRET的超浮肿状态（SRX）的形成。 目标＃2。检查HCM突变在心脏肌球蛋白S2区域的功能影响。