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.

Public Health Relevance (Narrative Statement) Ryanodine receptors (RyR2) mediate the release of Ca2+ for cardiac contraction and its dysfunctions underlie many cardiac pathologies, including heart failure and arrhythmia. Mechanistic data on the pathologies of RyR2 have come from transgenic mouse or heterologous cell models, with low relevance to human heart. Here we propose to develop powerful molecular and imaging tools to investigate the RyR2 disease-associated mutations in human stem cell derived cardiomyocytes, and believe that our unique multi-disciplinary approach will enhance the understanding of molecular regulatory sites of this protein and its pathologies.