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(Circulation Research. 2009;104:201.)
© 2009 American Heart Association, Inc.

Cellular Biology

Increased Ca²⁺ Sensitivity of the Ryanodine Receptor Mutant RyR2^R4496C Underlies Catecholaminergic Polymorphic Ventricular Tachycardia

María Fernández-Velasco, Angélica Rueda^*, Nicoletta Rizzi^*, Jean-Pierre Benitah, Barbara Colombi, Carlo Napolitano, Silvia G. Priori, Sylvain Richard, Ana María Gómez

From the Institut National de la Santé et de la Recherche Médicale, U637 (M.F.-V., A.R., J.-P.B., S.R., A.M.G.), Université de Montpellier, France; Departamento de Bioquímica (A.R.), Instituto Nacional de Cardiología, México; and Molecular Cardiology (N.R., B.C., C.N., S.G.P.), Fondazione Salvatore Maugeri, Istituto di Ricovero E Cura a Carattere Scientifico, Pavia, Italy.

Correspondence to Ana M. Gómez, Inserm, U637, CHU A de Villeneuve, 34295 Montpellier, France. E-mail ana-maria.gomez{at}inserm.fr

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 β-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 β-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.

Key Words: Ca²⁺ sparks • [Ca²⁺]_i transients • ryanodine receptor • excitation–contraction coupling • CPVT

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