Research supplement to promote diversity in Heath-related research

摘要 本研究附录的目的是通过直接检查 形成心肌肌球蛋白的抑制状态，即超松弛状态(SRX)，并考察其对 CMyBP-C和疾病突变对这一关键结构状态的形成/稳定。重要的是 研究表明，cMyBP-C在心肌收缩能力中的作用得到了强调 磷酸化状态在增强收缩能力中起作用，而在心力衰竭时，磷酸化降低 很可能会导致收缩缺陷。CMyBP-C被认为可以影响急性心肌梗死患者心肌肌球蛋白SRx 磷酸化依赖方式。在这项建议中，我们将设计一种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。我们会 还要检查肥厚型心肌病(HCM)已知相互作用的心肌肌球蛋白S2区域的突变 CMyBP-C。在目标1中，我们将通过将FRET信号与其他信号关联来表征FRET生物传感器 SRX的测定，如肌动蛋白激活的ATPase和单周转ATPase的测定。我们会有所不同 离子强度和检测药物Omecamtiv Mercaril以证明FRET传感器可以使用 测定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 突变到S2区，并检测它们对SRX形成/稳定的影响。最后我们会 还要检查cMyBP-C是否可以改变HCM突变体对SRX的形成/稳定的反应。总体而言， Proposal将通过提供对肌球蛋白结构的直接测量来极大地补充母体R01，该结构 将是解释cMyBP-C在肌肉粗丝和细丝调节中作用的研究的关键 收缩。 目的#1.检测人心肌肌球蛋白FRET超松弛状态的形成。 目的#2.检测心肌肌球蛋白S2区HCM突变对功能的影响。

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.