Functional implications of RyR2 mutations in human cardiomyocytes

RyR2 突变对人类心肌细胞的功能影响

基本信息

批准号：
10009812
负责人：
MARTIN MORAD
金额：
$ 60.73万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-15 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10009812
关键词：
Affinity Amino Acids Arrhythmia Back Binding Sites Biological Models Biopsy C-terminal CRISPR/Cas technology Caffeine Calcium Signaling Calmodulin Cardiac Cardiac Myocytes Cardiomyopathies Catecholaminergic Polymorphic Ventricular Tachycardia Catecholamines Cell Line Cell model Cells Characteristics Coupling Cryoelectron Microscopy Data Disease Electrophysiology (science)Evaluation Exertion Exhibits FKBP1B gene Failure Fluorescent Probes Functional disorder Genes Genetic Genetic Engineering Heart Heart Hypertrophy Heart failure Human Ion Channel Link Membrane Missense Mutation Modeling Molecular Mus Muscle Cells Mutagenesis Mutate Mutation Myocardial Myocardial Contraction N-terminal Pathologic Pathology Patients Pharmacotherapy Phenotype Play Prevention Protein Kinase Protein phosphatase Proteins Recombinants Regulation Reporting Research Resolution Role Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Signal Transduction Signaling Protein Site Stem cells Structural Models Structure Structure-Activity Relationship System Time Total Internal Reflection Fluorescent Transgenic Mice base comparative confocal imaging experimental study heart dimension/size induced pluripotent stem cell insight mouse model mutant novel novel strategies patch clamp public health relevance receptor stem cell differentiation

项目摘要

Project Summary/Abstract: Cardiac contractility is regulated by transient release of Ca2+ form the sarcoplasmic reticulum through type-2 ryanodine receptor (RyR2), a tetrameric ~5000 amino acids protein with multiple regulatory domains for Ca2+, Mg2+, protein kinase and phosphatase, and RyR2-stabilizing protein, FKBP12.6. Since a number of RyR2 missense mutations have been reported to associate with lethal cardiomyopathies, a better understanding of regulatory mechanisms of RyR2 is essential for prevention and treatment of these pathologies. Two major research strategies, heterologous cell expression in HEK293 cell lines carrying RyR2 mutations and transgenic mouse models expressing mutant RyR2, have been thus far used to study structure/function relationship of RyR2 and its pathological consequences. These approaches have advanced the understanding of RyR2 regulatory mechanisms, but suffer from inherent drawbacks of cells with non-cardiac genetic backgrounds or size and electrophysiological differences between human and mice. Thus, functional consequences of RyR2 mutagenesis remain to be fully explored in human myocardial model system. In this proposal, we aim to establish a new research platform where RyR2 mutagenesis is carried out in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 gene editing directed to Ca2+ and caffeine binding sites associated with cardiac pathology. Toward this end, we set three specific aims: (1) Establish the RyR2 mutagenesis in hiPSC-derived cardiomyocyte as a reliable platform to reproduce the calcium signaling aberrancies associated with the arrhythmia-linked mutations, by comparing the Ca2+ signaling aberrancies of gene-edited F2483I-RyR2 mutation carrying myocytes with cells derived directly from patient biopsies harboring the same mutations, and previously characterized by us, (2) Characterize functional consequences of mutating the potential Ca2+ and caffeine binding site of RyR2, recently identified in the high resolution cryo-electron microscopy studies, (3) Characterize the Ca2+ signaling phenotypes of RyR2 mutations associated with cardiac pathology in the three structurally distinct domains of RyR2. The membrane currents and global and focal intracellular Ca2+ signals of wild type and mutant hiPSC-derived cardiomyocytes will be quantified in patch-clamped myocytes imaged by confocal/TIRF microscopy using genetically engineered Ca2+ fluorescent probes targeted to various nodes of Ca2+ signaling proteins. This novel approach may enable us to systematically characterize the phenotype of the mutant RyR2 in cells with more relevant genetic background of human cardiac cells in time-effective manner, leading hopefully to better understanding of molecular mechanism of RyR2 regulation and cardiac excitation-contraction coupling based on the near-atomic structural model of RyRs.

项目摘要/摘要：心肌收缩力通过 2 型肌浆网短暂释放 Ca2+ 来调节 ryanodine 受体 (RyR2)，一种四聚体，约 5000 个氨基酸，具有多个 Ca2+ 调节域， Mg2+、蛋白激酶和磷酸酶以及 RyR2 稳定蛋白 FKBP12.6。由于一些 RyR2 据报道，错义突变与致命性心肌病有关，更好地理解 RyR2 的调节机制对于预防和治疗这些病理学至关重要。两大研究策略、携带RyR2突变和转基因的HEK293细胞系中的异源细胞表达表达突变体 RyR2 的小鼠模型迄今为止已被用于研究 RyR2 的结构/功能关系 RyR2 及其病理后果。这些方法增进了对 RyR2 的理解调节机制，但受到非心脏遗传背景细胞的固有缺陷或人类和小鼠之间的体型和电生理差异。因此，RyR2 的功能后果人类心肌模型系统中的诱变仍有待充分探索。在本提案中，我们的目标是建立一个新的研究平台，在心肌细胞中进行 RyR2 诱变使用针对 Ca2+ 的 CRISPR/Cas9 基因编辑从人类诱导多能干细胞 (hiPSC) 中提取和与心脏病相关的咖啡因结合位点。为此，我们制定了三个具体目标： (1) 在 hiPSC 来源的心肌细胞中建立 RyR2 诱变作为复制通过比较 Ca2+ 发现与心律失常相关突变相关的钙信号异常基因编辑的 F2483I-RyR2 突变携带的肌细胞与直接衍生的细胞的信号异常具有相同突变的患者活检，并且之前由我们进行了表征，(2) 表征功能 RyR2 的潜在 Ca2+ 和咖啡因结合位点突变的后果，最近在高高分辨率冷冻电子显微镜研究，(3) 表征 RyR2 突变的 Ca2+ 信号传导表型 RyR2 的三个结构不同的结构域与心脏病理学相关。膜电流野生型和突变型 hiPSC 衍生心肌细胞的整体和局部细胞内 Ca2+ 信号将被使用基因工程 Ca2+ 通过共聚焦/TIRF 显微镜成像的膜片钳肌细胞中进行定量针对 Ca2+ 信号蛋白各个节点的荧光探针。这种新颖的方法可能使我们能够系统地表征具有更相关遗传背景的细胞中突变体 RyR2 的表型以高效的方式对人类心肌细胞进行研究，有望更好地理解分子基于近原子结构的RyR2调节和心脏兴奋-收缩耦合机制 RyR 模型。