Mutation in RyR2-FKBP Binding site alters Ca(2+) signaling modestly but increases "arrhythmogenesis" in human stem cells derived cardiomyocytes.

AIMS: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca(2+) signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal muscle mutation (N760D-RyR1) associated with central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias. METHODS AND RESULTS: Ca(2+) imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSCCMs showed: (1) ∼30% suppressed I(Ca) with no significant changes in the gating kinetics of I(Ca); (2) 29% lower SR Ca(2+) content and 33% lower RyR2 Ca(2+) leak; (3) higher CICR gain and 30-35% increased efficiency of I(Ca)-triggered Ca(2±)release; (4) higher incidence of aberrant SR Ca(2+) releases, DADs, and Ca(2+) sparks; (5) no change in fractional Ca(2+)-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern. CONCLUSIONS: The more frequent spontaneous Ca(2+) releases and longer Ca(2+) sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Ca(2±)leak and SR content result from suppressed I(Ca)that is compensated by higher CICR gain.