Cardiac Myosin-Binding Protein C: Molecular Mechanisms Governing Cardiac Contractility

心肌肌球蛋白结合蛋白 C：控制心脏收缩力的分子机制

• 批准号: 10624275
• 负责人: Sivaraj Sivaramakrishnan
• 金额: $ 54.49万
• 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/10624275
• 关键词: Ablation; Actins; Actomyosin; Address; Adrenergic Agents; Affect; Atomic Force Microscopy; Binding; Cardiac; Cardiac Myosins; Collaborations; Contractile Proteins; DNA; DNA Sequence Alteration; Defect; Development; Equilibrium; Filament; Generations; Head; Heart; Heart failure; Human; Hypertrophic Cardiomyopathy; Knowledge; Lasers; Location; Mass Spectrum Analysis; Measures; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mosses; Motion; Mutagenesis; Mutation; Myocardium; Myofibrils; Myosin ATPase; N-terminal; Nanotubes; Performance; Periodicity; Phosphorylation; Physiological; Positioning Attribute; Preparation; Proteins; Pump; Regulation; Relaxation; Role; Sarcomeres; Structure; Sudden Death; Surface; Techniques; Testing; Thick; Thick Filament; Thin Filament; Thinness; Time; Transgenic Mice; Vertebral column; beta-Myosin; biophysical techniques; cell motility; citrate carrier; flexibility; improved; in vivo; molecular mechanics; mutant; myosin-binding protein C; nanometer; nanoscale; novel; novel strategies; operation; recruit; response; single molecule; spatial relationship; superresolution imaging; targeted treatment; young adult

Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is essential to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+- dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to β- adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. To address these questions, we propose two specific aims. Aim 1 will test the hypothesis that phosphorylation modulates cMyBP-C's N-terminal domain structure to influence its binding partner interactions (i.e. thin filament and myosin head region). We will use a novel mass-spectrometry technique and atomic force microscopy to characterize the molecular mechanics of cMyBP-C's N terminus that has been structurally altered due to phosphorylation or mutagenesis. The functional impact of these structural perturbations will be characterized in the context of cardiac myofibrils and native thick filaments to determine if cMyBP-C operates only where it exist in the thick filament and whether it can sequester cardiac myosin into a reserve pool of super-relaxed myosin heads. Thus, we will measure the location and time course of fluorescent-ATP turnover in single cardiac myofibrils and the force generated by native thick filaments in the laser trap in preparations from transgenic mice expressing phosphorylation and binding partner ablated mutant cMyBP-C. In Aim 2 we will create DNA-based “designer” thick filament nanotubes to define how the spatial relationships that normally exist in the thick filament between cMyBP-C and its myosin and actin binding partners are critical determinants of cMyBP-C's modes of operation. These DNA-nanotubes will allow exquisite nanometer spatial positioning of expressed cMyBP-C and human β-cardiac myosin on the nanotube surface relative to each other. By this novel approach we can assign cMyBP C's modulation of actomyosin motility to binding of the myosin head and/or thin filament, as assessed by both thin filament motility and force generation using the laser trap. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.

心肌肌球蛋白结合蛋白C（cMyBP-C）是一种肌节粗丝相关蛋白， 正常的心脏结构和功能。cMyBP-C的突变强调了cMyBP-C的重要性 是导致肥厚型心肌病的主要原因尽管是心脏收缩力的关键调节器， cMyBP-C调节肌动球蛋白力和运动产生的分子机制远不是 确定。虽然cMyBP-C的N端结构域可以与肌动蛋白和肌球蛋白头部区域结合，但目前尚不清楚 这些结合伴侣中的哪一个是生理相关的，以及这些结合伴侣相互作用是否 通过直接影响肌动球蛋白发电或间接改变Ca 2 +- 依赖于细丝激活。随着cMyBP-C的N端磷酸化发生在对β- 肾上腺素能刺激，磷酸化可以提供cMyBP-C功能可调性的测量， 增强心脏收缩力。为了解决这些问题，我们提出了两个具体目标。目标1将测试 磷酸化调节cMyBP-C的N-末端结构域结构以影响其结合的假设 伴侣相互作用（即细丝和肌球蛋白头区）。我们将使用一种新的质谱仪 技术和原子力显微镜来表征cMyBP-C的N末端的分子力学， 由于磷酸化或诱变而在结构上发生了改变。这些结构的功能影响 扰动将在心肌肌原纤维和天然粗丝的背景下表征，以确定是否 cMyBP-C仅在其存在于粗肌丝中的地方起作用，以及它是否能将心肌肌球蛋白隔离成 超松弛肌球蛋白头的储备池。我们将以时间和空间为尺度， 单心肌肌原纤维中的荧光-ATP转换和心肌细胞中天然粗丝产生的力 表达磷酸化和结合伴侣消除突变体的转基因小鼠制备物中的激光陷阱 cMyBP-C。在目标2中，我们将创建基于DNA的“设计师”粗丝纳米管，以定义空间 cMyBP-C与其肌球蛋白和肌动蛋白结合之间的关系通常存在于粗丝中 伴侣是cMyBP-C运作模式的关键决定因素。这些DNA纳米管可以让 表达的cMyBP-C和人β-心肌肌球蛋白在纳米管表面的纳米空间定位 相对于彼此。通过这种新的方法，我们可以将cMyBP C对肌动球蛋白运动的调节归因于 肌球蛋白头和/或细丝的结合，通过细丝运动性和力产生评估 使用激光陷阱。随着对cMyBP-C功能的认识和理解， 研究，针对cMyBP-C结合伴侣相互作用的靶向治疗可能有助于 调节和改善衰竭心脏的心脏功能。

项目成果

Cardiac myosin binding protein-C phosphorylation accelerates β-cardiac myosin detachment rate in mouse myocardium

心肌肌球蛋白结合蛋白-C 磷酸化加速小鼠心肌中β-心肌肌球蛋白脱离率

• DOI: 10.1152/ajpheart.00673.2020
• 发表时间: 2021
• 期刊: American Journal of Physiology-Heart and Circulatory Physiology
• 影响因子: 4.8
• 作者: Tanner, Bertrand C.;Previs, Michael J.;Wang, Yuan;Robbins, Jeffrey;Palmer, Bradley M.
• 通讯作者: Palmer, Bradley M.

Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release.

扩张型心肌病β-心肌肌球蛋白突变增强了动力冲程的肌动蛋白激活和磷酸盐释放。

• DOI: 10.1101/2023.11.10.566646
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Bodt,SkylarML;Ge,Jinghua;Ma,Wen;Rasicci,DavidV;Desetty,Rohini;McCammon,JAndrew;Yengo,ChristopherM
• 通讯作者: Yengo,ChristopherM

A dynamic Dab2 keeps myosin VI stably on track.

• DOI: 10.1016/j.jbc.2021.100640
• 发表时间: 2021-01
• 期刊: The Journal of biological chemistry
• 作者: Cirilo JA Jr;Yengo CM
• 通讯作者: Yengo CM

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.

• DOI: 10.7554/elife.77415
• 发表时间: 2022-11-24
• 期刊: eLife
• 影响因子: 7.7
• 作者: Rasicci DV;Tiwari P;Bodt SML;Desetty R;Sadler FR;Sivaramakrishnan S;Craig R;Yengo CM
• 通讯作者: Yengo CM

Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions.

• DOI: 10.1016/j.bpj.2022.05.013
• 发表时间: 2022-06-21
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Touma, Anja M.;Tang, Wanjian;Rasicci, David, V;Vang, Duha;Rai, Ashim;Previs, Samantha B.;Warshaw, David M.;Yengo, Christopher M.;Sivaramakrishnan, Sivaraj
• 通讯作者: Sivaramakrishnan, Sivaraj