Increased Ca2+ Sensitivity of the Ryanodine Receptor Mutant RyR2R4496C Underlies Catecholaminergic Polymorphic Ventricular Tachycardia

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca2+ handling underlie this disease. Here we analyze the underlying Ca2+ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2(R4496C) mutation. RyR2(R4496C-/-) littermates (wild type) were used as controls. [Ca2+](i) transients were obtained by field stimulation in fluo-3-loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca2+](i) transients and sarcoplasmic reticulum Ca2+ load were similar in wild-type and RyR2(R4496C) cells. However, paced RyR2(R4496C) ventricular myocytes presented abnormal Ca2+ release during the diastolic period, viewed as Ca2+ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca2+ release was enhanced at faster stimulation rates and by beta-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca2+ sparks were more frequent in RyR2(R4496C) myocytes, indicating increased RyR2(R4496C) activity. When permeabilized cells were exposed to different cytosolic [Ca2+](i), RyR2(R4496C) showed a dramatic increase in Ca2+ sensitivity. Isoproterenol increased [Ca2+](i) transient amplitude and Ca2+ spark frequency to the same extent in wild-type and RyR2(R4496C) cells, indicating that the beta-adrenergic sensitivity of RyR2(R4496C) cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2(R4496C) mutation is attributable to the increased Ca2+ sensitivity of RyR2(R4496C), which induces diastolic Ca2+ release and lowers the threshold for triggered activity. (Circ Res. 2009; 104: 201-209.)