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

AIMS: Previous studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought to displace the carboxyl-terminal domain to Ca(2+)-binding, allowing the tryptophan residue (W4645) to regulate Ca(2+) sensitivity of RyR2. To gain insights into regulation of RyR2 Ca(2+)-binding and its interaction with caffeine-binding site, we introduced W4645R-RyR2 point mutation via CRISPR/Cas9 gene-editing in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) and characterized their Ca(2+)-signaling phenotype compared to WT hiPSCCMs. METHODS AND RESULTS: W4645R-RyR2 cardiomyocytes had: (1) no significant change in I(Ca) magnitude or voltage-dependence; (2) slightly reduced CICR; (3) altered relaxation kinetics of Ca(2+)-transients with no change in isoproterenol sensitivity; (4) complete loss of caffeine-triggered Ca(2+) release; (5) larger SR Ca(2+) leak resulting in 40 % lower SR Ca(2+) content, as determined by myocytes' response to 4-CmC; (6) lower incidence of calcium sparks and asynchronous spontaneous SR Ca(2+) releases. CONCLUSIONS: W4645R-RyR2 mutation induces loss of caffeine-triggered SR Ca(2+) release and enhances SR Ca(2+) leak that underlie asynchronous spontaneous Ca(2+) releases, triggering arrhythmia and impairing cardiac function.