Mutation in RyR2-FKBP Binding site alters Ca 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 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 imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSCCMs showed: (1) ∼30% suppressed I with no significant changes in the gating kinetics of I; (2) 29% lower SR Ca content and 33% lower RyR2 Ca leak; (3) higher CICR gain and 30-35% increased efficiency of I-triggered Carelease; (4) higher incidence of aberrant SR Ca releases, DADs, and Ca sparks; (5) no change in fractional Ca-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern.

CONCLUSIONS

The more frequent spontaneous Ca releases and longer Ca sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Caleak and SR content result from suppressed Ithat is compensated by higher CICR gain.