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.

项目总结/摘要 心肌肌球蛋白结合蛋白C（cMyBP-C）磷酸化促进心肌收缩， 这些效应的分子或结构机制以及体内意义都尚不清楚。我们 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，我们将使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功能的综合视图。这种双重方法包括 严格的交叉检查数据的范围内的空间组织，从孤立的细丝，以工作的心脏， vivo.我们将检验cMyBP-C N端磷酸化的机制， 增强收缩包括破坏其与肌球蛋白的结合并增加与肌动蛋白的结合，从而 增加了与细丝的交叉桥结合的速率。目标1将验证磷酸化 通过测量力的速率， 的发展（kADP）的笼状ADP的光解，激活合作招聘过程。 N-末端片段将用于确定cMyBP-C的M-结构域内的带电残基在以下中的作用： 收缩力和收缩率的调节。研究将扩展到具有相同基因的转基因小鼠。 突变的残基破坏cMyBP-C与肌球蛋白或肌动蛋白的结合，以确定这些作用。 突变的抽搐特征在体内;磷酸化的cTnI的可能性也有助于 将使用磷酸模拟物cTnI小鼠研究肾上腺素能变力性。另一种观点是， 磷酸化通过增加跨桥转换的速率加速收缩，将被研究 通过表征对应于力发展（Pi释放）的跨桥循环中的步骤， ADP释放（ADP release）目标2将联合收割机结合冷冻电子显微镜和粗纤维的3D重建 用心肌X射线衍射法检测模型的结构。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 磷酸化将cMyBP-C N末端从粗丝骨架中释放出来，促进其与 肌动蛋白。由于cMyBP-C突变引起的肥厚性心肌病（HCM）通常与 增强收缩，目的3探讨是否过度收缩涉及改变的相互作用的M- 结构域，这样与肌球蛋白的结合减少和/或与肌动蛋白的结合增加，正如我们提出的那样， 磷酸化这些研究将验证HCM突变削弱cMyBP-C稳定性的想法。 肌球蛋白头，加速他们的招聘到细丝。这个合作项目利用了 我们在心肌功能（莫斯）和结构（克雷格）方面的互补专业知识，并将导致深入， 对cMyBP-C功能/功能障碍的综合理解，这是任何一个实验室单独都不可能实现的。

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

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

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