Molecular characterization of the calcium release channel deficiency syndrome
David J. Tester, … , Joel Temple, Michael J. Ackerman
David J. Tester, … , Joel Temple, Michael J. Ackerman
Published July 14, 2020
Citation Information: JCI Insight. 2020;5(15):e135952. https://doi.org/10.1172/jci.insight.135952.
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Research Article Cardiology Genetics

Molecular characterization of the calcium release channel deficiency syndrome

Abstract

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.

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-Turnquist, Dianne L. Atkins, Luis A. Ochoa Nunez, Ian Law, Joel Temple, Michael J. Ackerman

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Figure 6

Field potential recording–based arrhythmic activity measurement.

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Field potential recording–based arrhythmic activity measurement. (A) Rep...
(A) Representative field potential (FP) recordings from WT (WT1) and the homozygous RYR2 duplication iPSC-CMs for both patients (RYR2 Dup 1 and RYR2 Dup 2) at baseline (top) and following ISO (100 nM) treatment (bottom). (B) A bar graph summary showing the erratic beating frequency (i.e., arrhythmic events) present at baseline and following ISO treatment in WT1 iPSC-CMs compared with RYR2 duplication iPSC-CMs for both patients. WT1-iPSC-CM baseline (n = 158, SEM = 1.25), WT-iPSC-CM ISO (n = 160, SEM = 1.5), RYR2 Dup 1-c1-iPSC-CM baseline (n = 165, SEM = 1.9), RYR2 Dup 1-c1-iPSC-CM ISO (n = 419, SEM = 1.7), RYR2 Dup 2-c1-iPSC-CM baseline (n = 129, SEM = 2.9), RYR2 Dup 2-c1-iPSC-CM ISO (n = 100, SEM = 3.8). (C) Representative FP recordings from WT1 control and RYR2 duplication iPSC-CMs from patient 2 (RYR2 Dup 2 clone 1) at baseline, following ISO (100 nM) treatment alone, and following ISO with nadolol (10 μM). (D) A bar graph summary of the erratic beating frequency (i.e., arrhythmic events) present in WT1 iPSC-CMs compared with RYR2 duplication iPSC-CMs from patient 2 (RYR2 Dup 2 clone 1) at baseline, following ISO (100 nM) and in response to pharmacotherapies (nadolol at 10 μM, propranolol at 1 μM, and flecainide at 6 μM). Data are shown as number of experiments, where each experiment includes data acquired from 250–500 electrode recordings each. WT1-iPSC-CM baseline (n = 4, SEM = 1.5), ISO (n = 12, SEM = 0.4), ISO + nadolol (n = 12, SEM = 0.20), ISO + propranolol (n = 12, SEM = 0.20), and ISO + flecainide (n = 12, SEM = 1.7). RYR2 Dup 2-c1-iPSC-CM baseline (n = 4, SEM = 3.9), ISO (n = 12, SEM = 1.5), ISO + nadolol (n = 12, SEM = 1.1), ISO + propranolol (n = 12, SEM = 0.72), and ISO + flecainide (n = 12, SEM = 1.2). Data are presented as mean ± SEM. The symbol *** represents P < 0.0001. A 1-way ANOVA with Tukey’s test was performed to determine statistical significance between multiple groups. P < 0.05 was considered significant.