Cardiac ryanodine receptors (RyR2) channels harboring a canonical CPVT mutation increase their arrhythmogenic potential by mixing with a mutation that prevents PKA phosphorylation

VALDIVIA HH; BRADLEY DJ; HERNANDEZ JJ; PALOMEQUE J; ALVARADO FJ; CAMPBELL K; HERRON T; JALIFE J

Boston

Congreso; Heart Rhithm 2015 36th Annual Sientific Sessions; 2015

Introduction:It is unclear whether PKA phosphorylation mitigates or exacerbates the dysfunctional phenotype of mutant RyR2 in catecholaminergic polymorphic ventriculartachycardia (CPVT). We studied a family whose members are heterozygous for 1) a canonical CPVT mutation (RyR2-Y4721C), or 2) a mutation that prevents PKAphosphorylation of RyR2 (RyR2-R2027H), or 3) both mutations.Methods:The clinical history of 9 genotyped family members was examined. RyR2-Y4721C and RyR2-R2028H were generated by site-directed mutagenesis of mouse RYR2and expressed in HEK293 cells. [3H]Ryanodine binding assays were used to test their molecular phenotype. Inducible pluripotent stem cell-derived cardiomyocytes(iPS-CMs) were obtained from skin fibroblasts of patients harboring one or both mutations and their Ca2+ handling properties were examined in iPS-CM monolayers.Results:Father carries RyR2-Y4721C and had exertional syncope as a teen, early-onset atrial fibrillation in his 40s, and has developed dilated cardiomyopathy. Mother carriesRyR2-R2028H and is asymptomatic. Two children with compound heterozygous mutations have a severe phenotype with recurrent resuscitated SCD. The subsequentgeneration, in which there are 4 heterozygotes, 2 of each mutation, are asymptomatic. RyR2-phosphoS2030 antibody reveals PKA phosphorylation of RyR2-WT andRyR2-Y2471C, but not RyR2-R2027H. Ca2+-dependent activation of RyR2-R2028H was indistinguishable from RyR2-WT (EC50 = 618±144 and 728±56 nM,respectively) and lower in RyR2-Y4721C (434±56 nM); however, simultaneous expression of both mutants generates RyR2 channels with increased affinity for Ca2+(250±25 nM, p≤0.05, n=6 for all samples). iPSC-CM monolayers from double mutant, but not from control, generated highly arrhythmogenic multifocal spontaneouscalcium release events.Conclusions:Expression of PKA-insensitive RyR2 subunits (RyR2-R2027H) in the background of a mildly arrhythmogenic CPVT mutation (RyR2-Y4721C) increases Ca2+sensitivity and dramatically increases disease severity. PKA phosphorylation of RyR2 appears to be beneficial, not detrimental, to prevent arrhythmogenesis in CPVT.