Arrhythmogenesis in Catecholaminergic Polymorphic Ventricular Tachycardia: insights from a RyR2 R4496C knock-in mouse model

Abstract—Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life
threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on
whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased
affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-
RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C
mutation (RyR2R4496C_/_). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n_20) in WT but
induced DADs in 21 of 33 (63%) RyR2R4496C_/_ myocytes (P_0.001). Superfusion with isoproterenol (30 nmol/L)
induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered
activity in 60% of RyR2R4496C_/_ myocytes (P_0.001). DADs and triggered activity were abolished by ryanodine
(10 _mol/L) but not by K201 (1 _mol/L or 10 _mol/L). In vivo administration of K201 failed to prevent induction of
polymorphic ventricular tachycardia (VT) in RyR2R4496C_/_ mice. Measurement of the FKBP12.6/RyR2 ratio in the
heavy sarcoplasmic reticulum membrane showed normal RyR2–FKBP12.6 interaction both in WT and RyR2R4496C_/_
either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely
arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2R4496C_/_ myocytes and ventricular
arrhythmias in RyR2R4496C_/_ mice; and (3) RyR2–FKBP12.6 interaction in RyR2R4496C_/_ is identical to that of WT both
before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with
the RyR2/FKBP12.6 complex