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.

项目概要/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。 当细胞表面的 L 型 Ca 通道 (LTCC) 打开并诱导肌浆收缩时，就会启动收缩。 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) 患有钙处理功能障碍的人会出现心律失常，并有心源性猝死的风险。 然而，健康中 CICR 调节及其失调的详细分子和结构基础。 该项目的目标是利用冷冻电子技术的尖端发展。 断层扫描 (Cryo-ET)、相关光学和电子显微镜、人类诱导多能干细胞衍生 心肌细胞 (hiPSC-CM) 和小鼠疾病模型，以及 CRISPR 基因组编辑以确定 CICR 蛋白在多种临床相关状态下的结构和组织。 该项目将确定 CICR 蛋白的定位、它们形成的复合物以及它们的作用 构象状态，无论是在休息时还是在β-肾上腺素受体刺激期间。 该项目将使用时间分辨成像来捕获短暂但功能重要的组件 剖析 CICR 不应性 在项目的两个阶段，健康细胞将与疾病模型进行比较。 这项工作将为调控 CICR 的分子机制以及突变如何发挥作用提供前所未有的见解。 通过连接结构生物学和心血管生物学，该项目将提供 CICR 蛋白导致心律失常的研究。 研究不同心血管过程并帮助开发新方法的概念验证 它还将为 Woldeyes 博士提供开展学术所需的培训和专业知识。 职业生涯的重点是使用 Cryo-ET 进行心血管成像。 在该奖项的培训支持和导师、顾问委员会成员的指导下， 合作者，Woldeyes 博士将有能力建立她的独立研究生涯。 长期目标是以高空间和时间分辨率剖析心血管疾病的机制。 她由冷冻电子显微镜/断层扫描领域的领军人物Wah Chiu博士和Dr. Joseph Wu，使用源自患者的 iPSC-CM 研究心血管疾病的领导者。 他们的实验室在指导和引导学员走向独立学术生涯方面有着出色的记录。 拥有进行拟议实验的理想环境。 心血管研究所、斯坦福SLAC国家实验室和MOSAIC UE5项目，她将拥有 确保该研究项目成功、提升她的职业生涯所需的培训、支持和智力投入 发展，并为她过渡到成功的独立研究生涯做好准备。