Mechanism of regulation of cardiac contraction by phosphorylation of myosin binding protein C

肌球蛋白结合蛋白C磷酸化调节心脏收缩的机制

• 批准号: 10223413
• 负责人: ROGER W CRAIG
• 金额: $ 68.04万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223413
• 关键词: 3-Dimensional; Acceleration; Actins; Address; Adrenergic Agents; Affect; Antibodies; Binding; Cardiac; Cardiac Myosins; Catheters; Characteristics; Charge; Cryoelectron Microscopy; Data; Defect; Development; Disease; Filament; Functional disorder; Head; Heart; Heart Diseases; Heart failure; Hypertrophic Cardiomyopathy; Image; Inherited; Kinetics; Label; Measures; Microfilaments; Microtomy; Modeling; Molecular; Mosses; Mus; Muscle; Mutate; Mutation; Myocardial; Myocardial Contraction; Myocardium; Myosin ATPase; N-terminal; Phosphorylation; Physiological; Play; Process; Proteins; Pump; Radial; Regulation; Relaxation; Roentgen Rays; Role; Skin; Space Perception; Specificity; Speed; Structural Models; Structure; Surface; Testing; Therapeutic Agents; Thick; Thick Filament; Thin Filament; Transgenic Mice; Tropomyosin; Vertebral column; Work; X ray diffraction analysis; electron tomography; experimental study; heart function; in vivo; insight; muscle physiology; myosin-binding protein C; novel; photolysis; pressure; reconstruction; recruit; three dimensional structure

Project Summary/Abstract Phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates myocardial contraction, but neither the molecular or structural mechanism nor the in vivo significance of these effects is known. Our working model is that phosphorylation regulates twitch kinetics by regulating cross-bridge recruitment to the thin filament. In this dual-PI proposal we will use our complementary expertise in cardiac muscle physiology and myofilament structure to generate an integrated view of cMyBP-C function. This dual approach includes strict cross-checks of data over a range of spatial organization, from isolated filaments to working hearts in vivo. We will test the hypothesis that the mechanism by which phosphorylation of cMyBP-C’s N-terminus enhances contraction involves disruption of its binding to myosin and increased binding to actin, thereby increasing the rate of cross-bridge binding to the thin filament. Aim 1 will test the idea that phosphorylation speeds the cooperative recruitment of cross-bridges in skinned myocardium by measuring rates of force development (kADP) following photolysis of caged ADP, which activates the cooperative recruitment process. N-terminal fragments will be used to determine the roles of charged residues within cMyBP-C’s M-domain in the regulation of force and rate of contraction. Studies will be extended to transgenic mice with the same residues mutated to disrupt cMyBP-C binding to either myosin or actin to determine the effects of these mutations on twitch characteristics in vivo; the possibility that phosphorylation of cTnI also contributes to adrenergic inotropy will be investigated using phosphomimetic cTnI mice. The alternative idea, that phosphorylation accelerates contraction by increasing the rates of cross-bridge transitions, will be investigated by characterizing the steps in the cross-bridge cycle corresponding to force development (Pi release) and relaxation (ADP release). Aim 2 will combine cryo-electron microscopy and 3D reconstruction of thick filaments with X-ray diffraction of myocardium, to test our model structurally. We will determine whether cMyBP-C stabilizes the super-relaxed state of myosin heads on the thick filament, whether cMyBP-C phosphorylation disrupts this, and the role of M-domain charged residues in these effects. We will also test whether phosphorylation releases the cMyBP-C N-terminus from the thick filament backbone, facilitating its binding to actin. Since hypertrophic cardiomyopathies (HCM) due to mutations in cMyBP-C are typically associated with enhanced contraction, Aim 3 explores whether the hypercontractility involves altered interactions of the M- domain, such that binding to myosin is reduced and/or binding to actin is increased, as we propose with phosphorylation. These studies will test the idea that HCM mutations weaken cMyBP-C’s stabilization of myosin heads, accelerating their recruitment to the thin filament. This collaborative project takes advantage of our complementary expertise in myocardial function (Moss) and structure (Craig) and will lead to an in-depth, integrated understanding of cMyBP-C function/dysfunction which would not be possible by either lab alone.

项目概要/摘要 心肌肌球蛋白结合蛋白 C (cMyBP-C) 的磷酸化可加速心肌收缩，但 这些效应的分子或结构机制以及体内意义均未知。我们的 工作模型是磷酸化通过调节跨桥募集来调节抽搐动力学 细丝。在这个双 PI 提案中，我们将利用我们在心肌生理学方面的互补专业知识 和肌丝结构以生成 cMyBP-C 功能的集成视图。这种双重方法包括 对一系列空间组织中的数据进行严格的交叉检查，从孤立的细丝到工作中的心脏 体内。我们将检验以下假设：cMyBP-C N 末端磷酸化的机制 增强收缩涉及破坏其与肌球蛋白的结合并增加与肌动蛋白的结合，从而 增加与细丝的跨桥结合率。目标 1 将检验磷酸化的想法 通过测量力的速率来加速带皮心肌中跨桥的协同募集 笼中 ADP 光解后的发育（kADP），激活合作招募过程。 N 端片段将用于确定 cMyBP-C 的 M 结构域中带电残基的作用 力和收缩率的调节。研究将扩展到具有相同功能的转基因小鼠 残基突变以破坏 cMyBP-C 与肌球蛋白或肌动蛋白的结合，以确定这些的影响 体内抽搐特征的突变； cTnI 的磷酸化也可能有助于 将使用拟磷酸化 cTnI 小鼠研究肾上腺素能正性肌力。另一种想法是 磷酸化通过增加跨桥转换速率来加速收缩，将被研究 通过描述与力发展（Pi 释放）相对应的跨桥循环中的步骤，以及 松弛（ADP 释放）。目标 2 将结合冷冻电子显微镜和粗丝的 3D 重建 通过心肌的 X 射线衍射，从结构上测试我们的模型。我们将确定 cMyBP-C 是否 稳定粗肌丝上肌球蛋白头的超松弛状态，无论 cMyBP-C 磷酸化 破坏了这一点，以及 M 结构域带电残基在这些效应中的作用。我们还将测试是否 磷酸化从粗丝主链中释放 cMyBP-C N 末端，促进其与 肌动蛋白。由于 cMyBP-C 突变导致的肥厚性心肌病 (HCM) 通常与 增强收缩，目标 3 探讨过度收缩是否涉及 M- 相互作用的改变 域，使得与肌球蛋白的结合减少和/或与肌动蛋白的结合增加，正如我们建议的 磷酸化。这些研究将检验 HCM 突变削弱 cMyBP-C 稳定性的观点 肌球蛋白头，加速其募集到细丝。该合作项目利用了 我们在心肌功能（Moss）和结构（Craig）方面的互补专业知识将带来深入的、 对 cMyBP-C 功能/功能障碍的综合理解，这仅靠任何一个实验室都是不可能实现的。

项目成果

Novel Regulatory Elements within Myofilaments of Vertebrate Striated Muscles-Who Knew.

脊椎动物横纹肌肌丝中的新调节元件——谁知道。

• DOI: 10.1016/j.bpj.2018.07.043
• 发表时间: 2018
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Moss,RichardL

Progress on the regulation of myofibrillar function: Part 2.

肌原纤维功能调节的进展：第二部分。

• DOI: 10.1085/jgp.201912380
• 发表时间: 2019
• 期刊: The Journal of general physiology
• 作者: Granzier,HenkL;Moss,RichardL
• 通讯作者: Moss,RichardL

A case for reduced energy utilization associated with spatial disorder of myosin in skeletal muscle.

与骨骼肌肌球蛋白空间紊乱相关的能量利用率降低的案例。

• DOI: 10.1085/jgp.201912444
• 发表时间: 2020
• 期刊: The Journal of general physiology
• 作者: Moss,RichardL

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

• DOI: 10.1085/jgp.202012770
• 发表时间: 2021-07-05
• 期刊: The Journal of general physiology
• 作者: Hanft LM;Fitzsimons DP;Hacker TA;Moss RL;McDonald KS
• 通讯作者: McDonald KS

The central role of the tail in switching off 10S myosin II activity.

尾部在关闭 10S 肌球蛋白 II 活性方面​​发挥着核心作用。

• DOI: 10.1085/jgp.201912431
• 发表时间: 2019
• 期刊: The Journal of general physiology
• 作者: Yang,Shixin;Lee,KyoungHwan;Woodhead,JohnL;Sato,Osamu;Ikebe,Mitsuo;Craig,Roger
• 通讯作者: Craig,Roger