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.

Background—: Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored. Methods and Results—: We investigated SAN [Ca ²⁺ ] _i handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation of ryanodine receptor (RyR2 ^R4496C ) and their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2 ^R4496C mice after isoproterenol injection, analogous to what was observed in catecholaminergic polymorphic ventricular tachycardia patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca ²⁺ ] _i transients in SAN cells within the intact SAN by confocal microscopy. RyR2 ^R4496C SAN presented significantly slower pacemaker activity and impaired chronotropic response under β-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca ²⁺ ] _i transients and action potentials) in 75% of the cases. Ca ²⁺ spark frequency was increased by 2-fold in RyR2 ^R4496C SAN. Whole-cell patch-clamp experiments performed on isolated RyR2 ^R4496C SAN cells showed that L-type Ca ²⁺ current ( I _Ca,L ) density was reduced by ≈50%, an effect blunted by internal Ca ²⁺ buffering. Isoproterenol dramatically increased the frequency of Ca ²⁺ sparks and waves by ≈5 and ≈10-fold, respectively. Interestingly, the sarcoplasmic reticulum Ca ²⁺ content was significantly reduced in RyR2 ^R4496C SAN cells in the presence of isoproterenol, which may contribute to stopping the “Ca ²⁺ clock” rhythm generation, originating SAN pauses. Conclusion—: The increased activity of RyR2 ^R4496C in SAN leads to an unanticipated decrease in SAN automaticity by a Ca ²⁺ -dependent decrease of I _Ca,L and sarcoplasmic reticulum Ca ²⁺ depletion during diastole, identifying subcellular pathophysiological alterations contributing to the SAN dysfunction in catecholaminergic polymorphic ventricular tachycardia patients.

Heart Failure (HF) is defined as the inability of the heart to efficiently pump out enough blood to maintain the body's needs, first at exercise and then also at rest. Alterations in Ca²⁺ handling contributes to the diminished contraction and relaxation of the failing heart. While most Ca²⁺ handling protein expression and/or function has been shown to be altered in many models of experimental HF, in this review, we focus in the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the type 2 ryanodine receptor (RyR2). Various modifications of this channel inducing alterations in its function have been reported. The first was the fact that RyR2 is less responsive to activation by Ca²⁺ entry through the L-Type calcium channel, which is the functional result of an ultrastructural remodeling of the ventricular cardiomyocyte, with fewer and disorganized transverse (T) tubules. HF is associated with an elevated sympathetic tone and in an oxidant environment. In this line, enhanced RyR2 phosphorylation and oxidation have been shown in human and experimental HF. After several controversies, it is now generally accepted that phosphorylation of RyR2 at the Calmodulin Kinase II site (S2814) is involved in both the depressed contractile function and the enhanced arrhythmic susceptibility of the failing heart. Diminished expression of the FK506 binding protein, FKBP12.6, may also contribute. While these alterations have been mostly studied in the left ventricle of HF with reduced ejection fraction, recent studies are looking at HF with preserved ejection fraction. Moreover, alterations in the RyR2 in HF may also contribute to supraventricular defects associated with HF such as sinus node dysfunction and atrial fibrillation.