Calcium signaling consequences of RyR2-S4938F mutation expressed in hiPSC-CMs.

Abstract

Ryanodine receptor 2 (RyR2) is the major Ca2+ release channel of cardiac sarcoplasmic reticulum (SR) that regulates the rhythm and strength of the heartbeat, but its malfunction generates arrhythmia leading to heart failure. Here we report on a catecholaminergic polymorphic ventricular tachycardia (CPVT1)-associated RyR2 mutation (S4938F) located in RyR2 carboxyl-terminal that was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying S4938F mutation were studied using total internal fluorescence microscopy (TIRF) and patch clamp technique. ICa densities measured either by depolarizations to zero mV or repolarizations from +100 mV to −50 mV in mutant hiPSC-CMs were comparable to WT cells, but their accompanying Ca2+-transients detected by Fura-2 (cytosolic Ca2+) or ER-GCaMP6 (SR Ca2+ release) were significantly suppressed, suggesting ICa induced Ca2+ release (CICR) was compromised. Even though 5mM caffeine triggered Fura-2 or ER-GCaMP6 signals and the frequency of spontaneously generated calcium transients of mutant cells were comparable to those of WT cells, the 4-CmC triggered cytosolic or SR Ca2+ signals and magnitude of spontaneously occurring Ca2+-transients were significantly suppressed. Spontaneously occurring Ca2+ sparks in S4938F hiPSC-CMs had longer durations but their frequency was comparable to those of WT cells. Isoprenaline increased the beating frequency that led to arrhythmic beating and cessation of excitability. Our data suggests that S4938F-RyR2 mutation does not significantly affect Ca2+content of SR, but decreases its Ca2+ sensitivity resulting in suppressed ICa-gated and spontaneous Ca2+ release from SR. Supported by NIH grant R01HL153504.