What is the clinical evidence from this study?

Study design: Other. Population: Calcium release channel deficiency syndrome (RYR2 duplication) (n=4). Intervention: Homozygous RYR2 duplication vs. Wild-type (WT) control iPSC-CMs. Primary outcome: RYR2 transcript and protein expression, and calcium transient amplitude response to isoproterenol and caffeine.

July 14, 2020Open Access

Molecular characterization of the calcium release channel deficiency syndrome

DTDavid J. TesterElectrophysiology CKCS John KimMayo Clinic SHSamantha K. HamrickMayo Clinic

Key Result

Homozygous RYR2 duplication caused a >50% reduction in RYR2 expression and a loss of function in calcium handling, rendering the calcium-induced calcium release apparatus insensitive to catecholamines and caffeine.

Structured PICO

Population

Patient-specific induced pluripotent stem cell cardiomyocytes derived from 2 children (ages 10 and 15) with a homozygous RYR2 duplication and exertion-related sudden cardiac arrest, compared to 2 healthy controls.

Exposure

In vitro treatment with isoproterenol (100 nM), caffeine (10 mM), nadolol, propranolol, and flecainide.

Comparator

Wild-type (WT) iPSC-CMs from healthy controls and baseline untreated conditions.

Outcome

Calcium handling measurements (Ca2+ transient amplitude, decay time, upstroke time), RYR2 transcript/protein expression, and arrhythmic activity (delayed afterdepolarizations, erratic beating).surrogate

A homozygous multiexon duplication in RYR2 causes a loss-of-function calcium release channel deficiency syndrome, leading to catecholamine-insensitive calcium handling but increased arrhythmic activity that is responsive to beta-blockers and flecainide.

Limitations

In vitro iPSC-CM model may not fully replicate in vivo whole-heart physiology
Small sample size of patient lines (n=2)
The exact molecular mechanism by which the duplication reduces expression remains to be fully elucidated

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.

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