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

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，才能保持健康的心跳。 细胞表面的L型钙通道(LTCCs)开放并诱导肌浆网时，收缩开始 网状(SR)钙通道(RyR2)释放更多的钙。这个过程被称为钙诱导的钙释放 (CICR)。有钙处理功能障碍的人会发生心律失常，并有心脏病猝死和 心力衰竭。然而，CICR在健康中的调节及其失调的详细的分子和结构基础 疾病仍然是一个谜。这个项目的目标是利用低温电子的尖端发展。 体层摄影术(冷冻-ET)，相关光学和电子显微镜，人诱导多能干细胞来源 心肌细胞(hiPSC-CMS)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。在本课程的指导阶段 获奖后，该项目将确定CICR蛋白的定位，它们形成的复合体，以及它们 静息状态和β-肾上腺素能受体刺激时的构象状态。在独立的阶段， 获奖后，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，以 剖析了CICR的不稳定性。在该项目的两个阶段，健康细胞将与疾病模型进行比较。这 这项工作将为调节CICR的分子机制以及如何突变提供前所未有的见解 在CICR中，蛋白质会导致心律失常。通过将结构生物学和心血管生物学联系起来，该项目将提供 一种新方法的概念验证，以研究不同的心血管过程并帮助发展 精确的治疗学。它还将为Woldeyes博士提供必要的培训和专业知识，以开始一项学术研究 职业生涯，专注于使用冷冻-ET进行心血管成像。 在这个奖项的培训支持下，以及她的导师、她的顾问委员会成员和 作为合作者，沃尔德耶斯博士将很好地确立她的独立研究生涯。沃尔德耶斯博士的 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。 她是由冷冻电子显微镜/断层扫描领域的领军人物华秋博士和Dr。 约瑟夫·吴是使用患者来源的IPSC-CMS研究心血管疾病的领先者。两个人都有 在指导学员和将学员过渡到独立的学术职业方面有良好的记录。他们的实验室是 为进行拟议的实验提供理想的环境。有了可用的资源和师资， 心血管研究所、斯坦福大学SLAC国家实验室和MOSAIC UE5项目，她将拥有 所需的培训、支持和智力投入，以确保这个研究项目的成功，促进她的事业 发展，并为她过渡到成功的独立研究生涯做好准备。

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.