Investigating the mechanisms of beta cardiac myosin II during sarcomere formation and function

研究肌节形成和功能过程中 β 心肌肌球蛋白 II 的机制

• 批准号: 9258771
• 负责人: Aidan Mandy Fenix
• 金额: $ 2.87万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258771
• 关键词: Address; Adult; Affect; Arrhythmia; Automobile Driving; Autopsy; Biological Assay; Biology; C-terminal; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cardiomyopathies; Cause of Death; Cells; Contractile System; Data; Development; Dilated Cardiomyopathy; Disease; Electrostatics; FDA approved; Familial Hypertrophic Cardiomyopathy; Filament; Generations; Genetic; Heart; Heart failure; Human; Hypertrophic Cardiomyopathy; Image; Individual; Lead; Life Style; Maintenance; Mesenchymal; Microfilaments; Microscopy; Modeling; Molecular Motors; Motor; Motor Activity; Mus; Muscle; Muscle Contraction; Mutation; Myocardium; Myofibrils; Myosin Type II; Nature; Patients; Pharmaceutical Preparations; Physicians; Process; Protein Isoforms; RNA Interference; Research; Resolution; Role; Running; Sarcomeres; Scheme; Series; Structure; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Work; base; beta-Myosin; disease-causing mutation; effective therapy; experimental study; fluorescence imaging; human stem cells; imaging approach; in vitro activity; in vivo; induced pluripotent stem cell; insight; knock-down; live cell imaging; mutant; non-muscle myosin; repaired; retinal rods; young adult

Project Summary Cardiac sarcomeres represent the basic units of contraction driving the beating of the heart. At their core, cardiac sarcomeres are composed of stacks of beta cardiac myosin II (β CMII) which hydrolyze ATP to generate force by pulling on actin filaments. Mutations in β CMII account for ~40% of all cases of inherited hypertrophic cardiomyopathy (HCM). HCM can lead to arrhythmias, debilitating lifestyle, heart failure, and is the leading cause of death among young adults and athletes. A postmortem hallmark of HCM is sarcomere disarray. Despite their importance, how cardiac sarcomeres are formed and maintained in healthy individuals, and how this is perturbed in disease states is not understood. The lack of a mechanistic understanding of sarcomere formation precludes effective treatment and therapeutics for diseases which affect sarcomere organization, such as HCM. Highlighting this, there are currently no FDA approved drugs that specifically treat HCM. Our lab has recently leveraged super-resolution microscopy to show how non-muscle myosin II (NMII) based contractile systems assemble into large ensembles which resemble cardiac sarcomeres in structure and function (i.e., to generate force). Referred to as NMII stacks, these large ensembles formed via two non- mutually exclusive mechanisms. 1.) NMII stacks grew from an expansion of single NMII filaments via a series of distinct structural steps, and 2.) via concatenation of multiple filaments (i.e., multiple NMII filaments “running into” each other). Due to their structural and functional similarities, I hypothesize that the mechanisms underlying NMII stack formation are conserved in β CMII filaments during sarcomere formation. Expanding upon our previous work in ‘non-muscle’ contractile systems, this project will leverage recent advances in human stem cell technology and super-resolution microscopy techniques to elucidate the mechanisms of cardiac sarcomere formation. Specifically, we will test how β CMII filaments assemble into larger β CMII stacks found at the core of sarcomere structures. Our lab has recently developed a live-cell imaging approach which allows us to observe sarcomere formation in live cells. Fluorescently tagged β CMII will be used in conjunction with this assay to determine if β CMII stacks form via similar mechanisms as NMIIA stacks. We will also utilize genetic mutants to test if mutations in β CMII which lead to HCM affect sarcomere formation and/or maintenance. Having established a model of sarcomere formation, we will test the requirement of NMII isoforms during sarcomere formation, as mice lacking NMIIB fail to develop organized sarcomeres, and NMII isoforms are found in “nascent” sarcomere structures. Together, these experiments will elucidate the mechanisms of cardiac sarcomere formation, and how this is perturbed in disease states which lead to HCM.

项目摘要 心脏肌节代表驱动心脏跳动的基本收缩单位。在其 核心，心脏肌节由β心肌肌球蛋白II（β CMII）堆叠组成，β CMII可水解ATP， 通过拉动肌动蛋白丝产生力。β CMII突变占所有遗传性疾病病例的~40%。 肥厚型心肌病（HCM）。HCM可导致心律失常、衰弱的生活方式、心力衰竭， 是年轻人和运动员死亡的主要原因HCM的死后标志是肌节 混乱尽管它们很重要，但心脏肌节如何在健康个体中形成和维持， 以及在疾病状态下这是如何被扰乱的尚不清楚。缺乏机械的理解， 肌节的形成妨碍了对影响肌节的疾病的有效治疗和疗法 比如HCM。强调这一点，目前没有FDA批准的药物，专门治疗 HCM。 我们的实验室最近利用超分辨率显微镜来显示非肌肉肌球蛋白II（NMII） 基于收缩系统组装成结构上类似于心脏肌节的大集合体， 函数（即，以产生力）。被称为NMII堆栈，这些大型合奏形成通过两个非- 相互排斥的机制1.）的人。NMII堆栈是从单个NMII细丝的扩展通过一系列 不同的结构步骤，和2.）通过多个细丝的连接（即，多根NMII灯丝“运行 “彼此”。由于它们在结构和功能上的相似性，我假设这些机制 在肌节形成过程中，潜在的NM II堆叠形成在β CM II细丝中是保守的。 扩展我们以前在“非肌肉”收缩系统的工作，这个项目将利用最近的 人类干细胞技术和超分辨率显微镜技术的进展，以阐明 心肌肌节形成的机制。具体来说，我们将测试β CMII纤维如何组装成 在肌节结构的核心处发现较大的β CMII堆叠。我们的实验室最近开发了一种活细胞 成像方法，使我们能够观察活细胞中肌节的形成。双标记β CMII 将与本试验结合使用，以确定β CMII堆栈是否通过与NMIIA相似的机制形成 一堆堆。我们还将利用遗传突变体来测试导致HCM的β CMII突变是否影响肌节 形成和/或维持。在建立了肌节形成的模型之后，我们将测试 在肌节形成过程中需要NMII同种型，因为缺乏NMIIB的小鼠不能发育成有组织的 肌节和匪II同种型存在于“新生”肌节结构中。这些实验将 阐明心肌肌节形成的机制，以及在疾病状态下肌节是如何被扰乱的， 导致HCM。