Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca ²⁺ handling underlie this disease. Here we analyze the underlying Ca ²⁺ 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. [Ca ²⁺ ] _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), [Ca ²⁺ ] _i transients and sarcoplasmic reticulum Ca ²⁺ load were similar in wild-type and RyR2 ^R4496C cells. However, paced RyR2 ^R4496C ventricular myocytes presented abnormal Ca ²⁺ release during the diastolic period, viewed as Ca ²⁺ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca ²⁺ release was enhanced at faster stimulation rates and by β-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca ²⁺ sparks were more frequent in RyR2 ^R4496C myocytes, indicating increased RyR2 ^R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca ²⁺ ] _i , RyR2 ^R4496C showed a dramatic increase in Ca ²⁺ sensitivity. Isoproterenol increased [Ca ²⁺ ] _i transient amplitude and Ca ²⁺ 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 Ca ²⁺ sensitivity of RyR2 ^R4496C , which induces diastolic Ca ²⁺ release and lowers the threshold for triggered activity.

Activation of Ca²⁺ release channels/ryanodine receptors (RyR) by the inward Ca²⁺ current (I_Ca) gives rise to Ca²⁺-induced Ca²⁺ release (CICR), the amplifying Ca²⁺ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. We reported previously (Lokuta, A. J., Meyers, M. B., Sander, P. R., Fishman, G. I., and Valdivia, H. H. (1997) J. Biol. Chem. 272, 25333–25338) that sorcin, a 22-kDa Ca²⁺-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Here we show that sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca²⁺ sparks) and the I_Ca-triggered activity of RyRs that gives rise to [Ca²⁺]_i transients. Because sorcin decreased the amplitude of the [Ca²⁺]_i transient without affecting the amplitude or kinetics of I_Ca, the overall effect of sorcin was to reduce the “gain” of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca²⁺ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca²⁺] required for the latter process (ED₅₀ = ∼200 μm) appears to be reached only within the dyadic space. Rapid injection of 5 μm sorcin onto the cytosolic face of RyRs reconstituted in lipid bilayers resulted in complete inhibition of channel activity in ≤ 20 ms. Thus, sorcin is a potent inhibitor of both spontaneous and I_Ca-triggered RyR activity and is kinetically capable of playing a role in terminating the positive feedback loop of CICR.

Background— The mineralocorticoid pathway is involved in cardiac arrhythmias associated with heart failure through mechanisms that are incompletely understood. Defective regulation of the cardiac ryanodine receptor (RyR) is an important cause of the initiation of arrhythmias. Here, we examined whether the aldosterone pathway might modulate RyR function. Methods and Results— Using the whole-cell patch clamp method, we observed an increase in the occurrence of delayed afterdepolarizations during action potential recordings in isolated adult rat ventricular myocytes exposed for 48 hours to aldosterone 100 nmol/L, in freshly isolated myocytes from transgenic mice with human mineralocorticoid receptor expression in the heart, and in wild-type littermates treated with aldosterone. Sarcoplasmic reticulum Ca ²⁺ load and RyR expression were not altered; however, RyR activity, visualized in situ by confocal microscopy, was increased in all cells, as evidenced by an increased occurrence and redistribution to long-lasting and broader populations of spontaneous Ca ²⁺ sparks. These changes were associated with downregulation of FK506-binding proteins (FKBP12 and 12.6), regulatory proteins of the RyR macromolecular complex. Conclusions— We suggest that in addition to modulation of Ca ²⁺ influx, overstimulation of the cardiac mineralocorticoid pathway in the heart might be a major upstream factor for aberrant Ca ²⁺ release during diastole, which contributes to cardiac arrhythmia in heart failure.