Cryo-electron tomography to determine crosstalk mechanisms of calcium channels in cardiomyocytes

冷冻电子断层扫描确定心肌细胞钙通道的串扰机制

• 批准号: 10352085
• 负责人: Rahel Asfaw Woldeyes
• 金额: $ 12.58万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10352085
• 关键词: Adopted; Advisory Committees; Arrhythmia; Award; Binding; Biology; Calcium; Calcium Channel; Calcium ion; Calmodulin; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell surface; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Committee Members; Cryo-electron tomography; Cryoelectron Microscopy; Defect; Development; Disease; Disease model; Electric Stimulation; Electron Microscopy; Ensure; Environment; Exercise; Extravasation; Faculty; Functional disorder; Goals; Grant; Health; Heart; Heart Arrest; Heart Contractilities; Heart Rate; Heart failure; Human; Image; Imaging Techniques; In Situ; Institutes; Ions; Laboratories; Lead; Measures; Mediating; Mentors; Methods; Molecular; Molecular Conformation; Mus; Muscle Cells; Muscle relaxation phase; Mutation; Pathologic; Patients; Persons; Phase; Physical activity; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Conformation; Proteins; Quality of life; Records; Refractory; Regulation; Research; Research Project Grants; Research Proposals; Resolution; Resources; Rest; Risk; Role; RyR2; Sarcoplasmic Reticulum; Structure; Testing; Therapeutic; Therapeutic Agents; Time; Training; Training Support; United States National Institutes of Health; Work; adrenergic stress; beta-adrenergic receptor; cardiovascular imaging; career; career development; clinically relevant; experimental study; genome editing; heart cell; high resolution imaging; imaging approach; imaging study; improved; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; insight; light microscopy; millisecond; mutant; nanometer; novel; novel strategies; programs; protein complex; small molecule; small molecule therapeutics; structural biology; success; sudden cardiac death; temporal measurement; time use; tomography; triadin

PROJECT SUMMARY/ABSTRACT Heart cells must precisely control the flow of calcium ions (Ca) within the cell to maintain a healthy heartbeat. Contraction is initiated when L-type Ca channels (LTCCs) on the cell surface open and induce sarcoplasmic reticulum (SR) Ca channels (RyR2) to release more Ca. This process is known as Ca-induced Ca release (CICR). People with Ca handling dysfunctions develop arrhythmia and are at risk for sudden cardiac death and heart failure. Yet, a detailed molecular and structural basis for CICR regulation in health and its dysregulation in disease remains a mystery. The goal of this project is to use cutting-edge developments in cryo-electron tomography (Cryo-ET), correlative light and electron microscopy, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and murine disease models, and CRISPR genome editing to determine the structures and organization of CICR proteins in multiple clinically relevant states. In the mentored phase of this award, the project will determine the localization of CICR proteins, the complexes they form, and their conformational state, both at rest and during β-adrenergic receptor stimulation. In the independent phase of the award, the project will use time-resolved imaging to capture short-lived but functionally important assemblies to dissect CICR refractoriness. At both stages of the project, healthy cells will be compared to disease models. This work will provide unprecedented insight into the molecular mechanisms that regulate CICR and how mutations in CICR proteins lead to arrhythmia. By connecting structural and cardiovascular biology, this project will provide a proof-of-concept for a new approach to study diverse cardiovascular processes and aid the development of precise therapeutics. It will also give Dr. Woldeyes the training and expertise necessary to start an academic career with a focus on using Cryo-ET for cardiovascular imaging. With the training support of this award and guidance from her mentors, her advisory committee members, and collaborators, Dr. Woldeyes will be well positioned to establish her independent research career. Dr. Woldeyes’ long-term goal is to dissect the mechanisms of cardiovascular disease at high spatial and temporal resolution. She is jointly mentored by Dr. Wah Chiu, a leader in the field of cryo-electron microscopy/tomography and Dr. Joseph Wu, a leader in the use of patient-derived iPSC-CMs to study cardiovascular diseases. Both have excellent track records in mentoring and transitioning trainees to independent academic careers. Their labs are an ideal environment for conducting the proposed experiments. With the resources and faculty available at the Cardiovascular Institute, SLAC National Laboratory, Stanford, and the MOSAIC UE5 program, she will have the training, support and intellectual input needed to ensure the success of this research project, enhance her career development, and prepare her for the transition to a successful independent research career.

项目摘要/摘要 心脏细胞必须精确控制细胞内钙离子（CA）的流动，以维持健康的心跳。 当细胞表面上的L型Ca通道（LTCC）打开并诱导肌质时，开始收缩 网状（SR）CA通道（RYR2）释放更多CA。此过程称为CA诱导的CA释放 （CICR）。患有CA处理功能障碍的人会出现心律不齐，并有猝死的风险 心脏衰竭。然而，CICR调节健康及其失调的详细分子和结构基础 疾病仍然是一个谜。该项目的目的是在冷冻电子中使用尖端的发展 断层扫描（冷冻-ET），相关光和电子显微镜，人类诱导的多能干细胞衍生 心肌细胞（HIPSC-CMS）和鼠病模型，以及CRISPR基因组编辑，以确定 在多种临床相关状态下CICR蛋白的结构和组织。在此阶段 奖励，该项目将确定CICR蛋白质的本地化，它们形成的复合物以及其 在静止和β-肾上腺素受体刺激期间，构象状态。在独立阶段 奖励，该项目将使用时间分辨的成像来捕获短暂但功能上重要的组件 剖析CICR磨性。在项目的两个阶段，都将将健康细胞与疾病模型进行比较。这 工作将为调节CICR的分子机制以及如何突变提供前所未有的见解 在CICR蛋白中导致心律不齐。通过连接结构和心血管生物学，该项目将提供 一种新方法来研究潜水员心血管过程的概念证明，并有助于发展 精确理论。它还将为Woldeyes博士提供所需的培训和专业知识，以创建学术 专注于将冷冻ET用于心血管成像的职业。 在该奖项的培训支持和指导下，她的导师，咨询委员会成员以及 合作者Woldeyes博士将在建立她的独立研究生涯中有利。 Woldeyes博士 长期目标是在高空间和临时分辨率下剖析心血管疾病的机制。 她是由低温 - 电子显微镜/断层扫描领域的领导者Wah Chiu博士共同提到的。 约瑟夫·吴（Joseph Wu），使用患者衍生的IPSC-CMS研究心血管疾病的领导者。两者都有 在心理和过渡到独立学术职业方面的出色记录。他们的实验室是 进行提出的实验的理想环境。提供资源和教师 心血管研究所，SLAC国家实验室，斯坦福大学和Mosaic UE5计划，她将拥有 确保该研究项目成功所需的培训，支持和智力投入，增强了她的职业生涯 开发，并准备她过渡到成功的独立研究职业。