CIC   05421
CENTRO DE INVESTIGACIONES CARDIOVASCULARES "DR. HORACIO EUGENIO CINGOLANI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Cardiac ryanodine receptors (RyR2) channels harboring a canonical CPVT mutation increase their arrhythmogenic potential by mixing with a mutation that prevents PKA phosphorylation
Autor/es:
JULIETA PALOMEQUE; JONATHAN J. HERNÁNDEZ; FRANCISCO J. ALVARADO; ROBERTO RAMOS MONDRAGÓN; XI CHEN; DAVID J. BRADLEY; JOSÉ JALIFE; HÉCTOR VALDIVIA
Lugar:
Portland, ME
Reunión:
Conferencia; Gordon Research Conferences-Excitation contraction coupling; 2015
Institución organizadora:
Gordon Research Conferences
Resumen:
Introduction: Mutations in the cardiac ryanodine receptor channel (RyR2) that are linked to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) are normally ?silent? at baseline, but may trigger catastrophic Ca2+ release under sympathetic stimulation. Adrenergically-induced PKA phosphorylation of RyR2 may be an aggravating factor that breaks the precarious balance on which mutant RyR2s operate, but RyR2 phosphorylation is also known to coordinate Ca2+ release and accelerate RyR2 restitution, enhancing RyR2 function under faster heart rates. Thus, it is unclear whether PKA phosphorylation mitigates or exacerbates the dysfunctional phenotype of mutant RyR2 in CPVT. We studied a family whose members are heterozygous for 1) a canonical CPVT mutation (RyR2-Y4721C), or 2) a mutation that prevents PKA phosphorylation 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 RYR2 and expressed in HEK293 cells. [3H]Ryanodine binding assays were used to test their molecular phenotype. Results: Father carries RyR2-Y4721C and had exceptional syncope as a teen, early-onset atrial fibrillation in his 40s, and has developed dilated cardiomyopathy. Mother carries RyR2-R2028H and is asymptomatic. Two children with compound heterozygous mutations have a severe phenotype with recurrent resuscitated SCD and beta blockers treatment. The subsequentgeneration, in which there are 4 heterozygotes, 2 of each mutation, are asymptomatic. RyR2-phosphoS2030 antibody reveals PKA phosphorylation of RyR2-WT but not RyR2-R2027H. PKA-RyR2 phosphorylation status at S2030 of RyR2-Y2471C and RyR2 double mutant are similar between both but significantly lower than the RyR2-WT. PKA-RyR2 phosphorylation at S2808 are not different between all the proteins studied. Interestingly, only RyR2-Y2471C presents similar basal and PKA-dependent phosphorylation at S2814, a CaMKII proposed RyR2 phosphorylation site. Ca2+-dependent activation of RyR2-R2028H was indistinguishable from RyR2-WT (EC50 = 618±14 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). 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.
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