Molecular characterization of the calcium release channel deficiency syndrome

• DOI: 10.1172/jci.insight.135952
• Publication Date: 2020-07-14T16:01:21Z
• Authors: David J. Tester; CS John Kim; Samantha K. Hamrick; Dan Ye; Bailey J. O’Hare; Hannah M. Bombei; Kristi K. Fitzgerald; Carla M. Haglund-...; Dianne L. Atkins; Luis A. Ochoa Nunez; Ian Law; Joel Temple; Michael J. Ackerman
• Keywords: Male; Adolescent; Homozygote; Induced Pluripotent Stem Cells; Cell Differentiation; Ryanodine Receptor Calcium Release Channel; Pedigree; 3. Good health; 03 medical and health sciences; Phenotype; 0302 clinical medicine; Case-Control Studies; Child, Preschool; Gene Duplication; Tachycardia, Ventricular; Humans; Calcium; Female; Myocytes, Cardiac; Child; Research Article

We identified a potentially novel homozygous duplication involving the promoter region and exons 1-4 of the gene encoding type 2 cardiac ryanodine receptor (RYR2) that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2 duplication (RYR2-DUP) induced pluripotent stem cell cardiomyocytes (iPSC-CMs) were generated from 2 unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT-iPSC-CM and RYR2-DUP-iPSC-CM lines. However, compared with WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine-stimulated and isoproterenol-stimulated (ISO-stimulated) Ca2+ transient amplitude, suggesting RyR2 loss of function. There was a greater than 50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared with WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared with WT-iPSC-CMs, there was significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5-fold, 6.8-fold, and 2.4-fold, respectively, from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitated a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss of function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.