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Functional implications of CPVT1-associated RyR2 mutations in human cardiomyocytes

人类心肌细胞中 CPVT1 相关 RyR2 突变的功能意义

基本信息

批准号：
10298481
负责人：
MARTIN MORAD
金额：
$ 51.83万
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：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298481
关键词：
Adrenergic Agents Adrenergic beta-Agonists Adult Affinity Amino Acids Arrhythmia Back Binding Binding Sites Biochemical Biological Assay CRISPR/Cas technology Caffeine Cardiac Cardiac Myocytes Cardiomyopathies Catecholaminergic Polymorphic Ventricular Tachycardia Cell model Cells Complement Computer Simulation Coupling Data Disease Dissociation Electrophysiology (science)Exhibits FKBP1B gene Functional disorder Genes Genetic Heart Heart Hypertrophy Heart failure Human Human Engineering Imaging Device Impairment Knock-in Link Maps Mediating Membrane Missense Mutation Modeling Molecular Mus Muscle Cells Mutagenesis Mutate Mutation Myocardial dysfunction Myocardium Pathology Pharmaceutical Preparations Pharmacology Phenotype Phosphoric Monoester Hydrolases Phosphotransferases Protein Kinase Proteins Recombinants Regulation Research Resolution RyR1 Ryanodine Ryanodine Receptor Calcium Release Channel Ryanodine Receptors Sarcoplasmic Reticulum Signal Pathway Signal Transduction Site Specificity Structural Models Structure Structure-Activity Relationship System Tacrolimus Binding Proteins Tail Testing Total Internal Reflection Fluorescent Transgenic Mice alpha helix base confocal imaging human stem cells in vivo induced pluripotent stem cell insight interdisciplinary approach loss of function loss of function mutation molecular imaging mouse model mutant novel novel strategies patch clamp public health relevance response skeletal

项目摘要

Project Summary/Abstract: Cardiac contractility is regulated by Ca2+ release form the sarcoplasmic reticulum through ryanodine receptor (RyR2), a protein with multiple regulatory domains for Ca2+, Mg2+, protein kinase, caffeine and FKBP12.6. Since a number of RyR2 missense mutations associate with lethal cardiomyopathies, a detailed understanding of regulatory mechanisms of RyR2 is essential for treatment of these pathologies. Two strategies of heterologous expression of recombinant RyR2 mutants in HEK293 cells and transgenic mouse models, have been used to study structure/function relationship of RyR2 and the functional consequences of disease-linked RyR2 mutations. Although these approaches have provided new insights into RyR2 regulatory mechanisms, they have inherent drawbacks of cells with non-cardiac genetic background and differences in human and mice hearts. We have therefore established an alternate research platform where RyR2 mutations are introduced in human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes (CMs) using CRISPR/Cas9 gene-editing. Mutant myocytes are then cultured in media that matures them structurally and functionally toward adult cardiomyocyte state. Using this human myocyte platform, we propose to examine molecular mechanisms underlying Ca2+, caffeine, and FKBP regulation of RyR2 associated with CPVT1 pathology. Specifically we aim: 1) To compare Ca2+-signaling consequences of domain specific CPVT1-associated RyR2 mutations expressed in “mature” hiPSC-CMs , rescue their phenotype by back-mutagenesis, and determine their drug specificity; 2) To characterize the functional consequence of mutating the RyR2 Ca2+ and caffeine binding sites, predicted from near atomic structure and determine their interaction; and 3) To characterize mechanisms underlying loss-of-function CPVT1-associated RyR2 mutations and identify the difference between Ca2+ leaky and non-leaky mutations. To accomplish these aims we propose to create multiple mutant lines of our more mature hiPSC-CMs carrying the different RyR2 mutations and examine their Ca2+ signaling aberrancies. Membrane currents and 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 encoded Ca2+ probes targeted to various nodes of Ca2+ signaling pathway. We will also use [3H]ryanodine binding assay, to determine possible alterations in affinities of Ca2+, caffeine and accessory proteins. To assure the reliability of our hiPSC-platform, we will compare the Ca2+ signaling aberrancies of mutagenesis in hiPSC-CMs with in vivo knock-in of RyR2 mutations in mouse models. We hope that our novel approach will make it possible to systematically characterize the phenotype of the CPVT1 mutants, as well as non-CPVT1 mutants with implication to atomic structure of RyR2, in human myocardium, thus providing a novel and synergistic human platform for studies of RyR2 regulation.

项目总结/文摘: