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 心脏肉瘤代表了促使心脏节拍的合同的基本单位。在他们的 核心，心脏肉瘤由β心脏肌球蛋白II（βCMII）组成，将其水解为ATP 通过拉动肌动蛋白丝来产生力。 βCMII中的突变占所有遗传病例的40％ 肥厚性心肌病（HCM）。 HCM会导致心律不齐，使生活方式衰弱，心力衰竭，并且是 年轻人和运动员死亡的主要原因。 HCM的验尸标志是Saromere 混乱。尽管它们的重要性，但如何在健康个体中形成和维持心脏肉瘤， 以及如何在疾病状态下扰动这一点。缺乏对机械的理解 肌节的形成排除了影响肉瘤的疾病的有效治疗和治疗 组织，例如HCM。强调这一点，目前尚无FDA批准的药物专门治疗 HCM。 我们的实验室最近利用了超分辨率显微镜，以显示非肌肉肌球蛋白II（NMII）如何 基于收缩系统组装成大型集合，类似于心脏肉瘤的结构和 功能（即产生力）。这些大型合奏被称为NMII堆栈，通过两个非 - 相互排斥的机制。 1.）NMII堆栈从单个NMII丝的扩展中生长 由不同的结构步骤和2.）通过多个细丝的串联（即多个NMII丝” 我彼此之间的结构和功能相似性，我假设这些机制 在肌节形成期间，基本的NMII堆栈形成在βCMII丝中是保守的。 扩展我们以前在“非肌肉”收缩系统中的工作，该项目将利用最新的 人类干细胞技术和超分辨率显微镜技术的进步，以阐明 心脏肌节形成的机制。具体而言，我们将测试βCMII丝如何组装成 在肌节结构的核心中发现了较大的βCMII堆栈。我们的实验室最近开发了一个现场电池 成像方法，使我们能够观察活细胞中的肌节形成。荧光标记为βCMII 将与该测定法结合使用，以确定是否通过与NMIIA相似的机制形成βCMII堆栈 堆栈。我们还将利用遗传突变体来测试导致HCM的βCMII中是否影响肌节的突变 编队和/或维护。建立了肌节形成模型后，我们将测试 肌膜形成过程中NMII同工型的需求，因为缺乏NMIIB的小鼠无法组织起来 肉瘤和NMII同工型在“新生”的肌节结构中发现。这些实验将在一起 阐明心脏肌节形成的机制，以及如何在疾病状态下扰动这一点 导致HCM。