Loss-of-function W4645R mutation in the RyR2-caffeine binding site: implications for synchrony and arrhythmogenesis

Area of Science:

• Cardiovascular Biology
• Molecular Cardiology
• Calcium Signaling

Background:

• The RyR2 W4645R mutation, located in the caffeine-binding site, is associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1).
• Tryptophan residue W4645 is hypothesized to regulate RyR2 Ca²⁺ sensitivity by influencing the interaction between the caffeine-binding site and the carboxyl-terminal domain.

Purpose of the Study:

• To investigate the regulatory role of RyR2 Ca²⁺-binding and its interaction with the caffeine-binding site.
• To characterize the Ca²⁺-signaling phenotype of W4645R-RyR2 mutation in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs).

Main Methods:

• CRISPR/Cas9 gene-editing was used to introduce the W4645R-RyR2 point mutation in hiPSCCMs.
• Comparison of Ca²⁺-signaling phenotypes between WT and W4645R-RyR2 hiPSCCMs.

Main Results:

• W4645R-RyR2 cardiomyocytes exhibited normal I_Ca but reduced Ca²⁺-induced Ca²⁺ release (CICR) and altered Ca²⁺ transient kinetics.
• A complete loss of caffeine-triggered Ca²⁺ release and a 40% reduction in SR Ca²⁺ content due to enhanced SR Ca²⁺ leak were observed.
• The mutation led to a lower incidence of calcium sparks and asynchronous spontaneous SR Ca²⁺ releases.

Conclusions:

• The W4645R-RyR2 mutation causes a loss of caffeine-gated Ca²⁺ release and increased SR Ca²⁺ leak.
• These alterations contribute to asynchronous spontaneous Ca²⁺ releases, which can trigger arrhythmias and impair cardiac function.