RATIONALE: Most cardiac ryanodine receptor (RyR2) mutations associated with 
catecholaminergic polymorphic ventricular tachycardia (CPVT) are postulated to 
cause a distinctive form of Ca(2+) release dysfunction. Considering the spread 
distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity 
also was feasible.
OBJECTIVE: To determine the molecular and cellular mechanisms by which a novel 
RyR2-V2475F mutation associated with CPVT in humans triggers Ca(2+)-dependent 
arrhythmias in whole hearts and intact mice.
METHODS AND RESULTS: Recombinant channels harboring CPVT-linked RyR2 mutations 
were functionally characterized using tritiated ryanodine binding and 
single-channel recordings. Homologous recombination was used to generate a 
knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice 
heterozygous for the mutation (RyR2-V2475F(+/-)) and their wild-type littermates 
were Ca(2+)-imaged by confocal microscopy under conditions that mimic stress. 
The propensity of wild-type and RyR2-V2475F(+/-) mice to have development of 
arrhythmias was tested at the whole heart level and in intact animals. 
Recombinant RyR2-V2475F channels displayed increased cytosolic Ca(2+) 
activation, abnormal protein kinase A phosphorylation, and increased activation 
by luminal Ca(2+). The RyR2-V2475F mutation appears embryonic-lethal in 
homozygous mice, but heterozygous mice have no alterations at baseline. 
Spontaneous Ca(2+) release events were more frequent and had shorter latency in 
isoproterenol-stimulated cardiomyocytes from RyR2-V2475F(+/-) hearts, but their 
threshold was unchanged with respect to wild-type. Adrenergically triggered 
tachyarrhythmias were more frequent in RyR2-V2475F(+/-) mice.
CONCLUSIONS: The mutation RyR2-V2475F is phenotypically strong among other CPVT 
mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living 
mice, this mutation appears too severe to be harbored in all RyR2 channels but 
remains undetected under basal conditions if expressed at relatively low levels. 
β-adrenergic stimulation breaks the delicate Ca(2+) equilibrium of 
RyR2-V2475F(+/-) hearts and triggers life-threatening arrhythmias.