Functional implications of RyR2 mutations in human cardiomyocytes

RyR2 突变对人类心肌细胞的功能影响

• 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
• 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

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.

项目概要/摘要： 心肌收缩力是由肌浆网通过2型钙通道瞬时释放的钙离子调节的 Ryanodine受体（RyR 2）是一种约5000个氨基酸的四聚体蛋白，具有多个Ca 2+调节结构域， Mg 2+，蛋白激酶和磷酸酶，以及RyR 2稳定蛋白，FKBP 12.6。由于许多RyR 2 据报道，错义突变与致命的心肌病有关， RyR 2的调节机制对于预防和治疗这些病理是必不可少的。两大 研究策略，在携带RyR 2突变和转基因的HEK 293细胞系中的异源细胞表达 表达突变体RyR 2的小鼠模型，迄今已被用于研究结构/功能关系， RyR 2及其病理后果。这些方法促进了对RyR 2的理解 调节机制，但遭受具有非心脏遗传背景的细胞的固有缺陷， 大小和电生理差异。因此，RyR 2的功能后果 在人心肌模型系统中诱变仍有待于充分探索。在本建议中，我们的目标是 建立了一个新的研究平台，其中RyR 2诱变在心肌细胞中进行， 使用针对Ca 2+的CRISPR/Cas9基因编辑从人诱导的多能干细胞（hiPSC） 以及与心脏病理学相关的咖啡因结合位点。为此，我们设定了三个具体目标： (1)在hiPSC衍生的心肌细胞中建立RyR 2诱变作为可靠的平台，以再现 钙信号异常与糖尿病相关的突变，通过比较Ca 2 + 携带基因编辑的F2483 I-RyR 2突变的肌细胞与直接来源于 携带相同突变的患者活检，并且先前由我们表征，（2）表征功能性 突变RyR 2的潜在Ca 2+和咖啡因结合位点的后果，最近在高水平的 冷冻电镜研究;（3）RyR 2突变的Ca 2+信号表型 在RyR 2的三个结构不同的结构域中与心脏病理学相关。膜电流 并且野生型和突变型hiPSC衍生的心肌细胞的全局和局灶性细胞内Ca 2+信号将 使用基因工程Ca 2+通过共聚焦/TIRF显微镜成像在膜片钳心肌细胞中定量 荧光探针靶向的各种节点的Ca 2+信号蛋白。这种新方法可以使我们 在具有更相关遗传背景的细胞中系统地表征突变型RyR 2的表型 人类心脏细胞的时间有效的方式，从而有希望更好地了解分子 基于近原子结构的RyR 2调节和心脏兴奋-收缩偶联的机制 RyR模型