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.
Statement) Type-2 ryanodine receptor (RyR2) is responsible for releasing the Ca2+ for contraction of the heart and regulating its rhythm; therefore, dysfunction of RyR2 is implicated in multiple cardiomyopathies including cardiac hypertrophy, failure, and arrhythmia. Mechanistic data on these pathologies primarily comes from transgenic mouse or heterologous cell models, with questionable relevance to human cardiac pathology. In this project, we will study some of these disease-associated mutations of RyR2 in human cardiomyocytes derived from stem cells in the hope of obtaining mechanistic understanding on the molecular regulatory sites of this protein in a human heart relevant model.
