Increased Ca ²⁺ Sensitivity of the Ryanodine Receptor Mutant RyR2 ^R4496C Underlies Catecholaminergic Polymorphic Ventricular Tachycardia

Abstract

Abstract-Cardiac] i transients Ⅲ ryanodine receptor Ⅲ excitation-contraction coupling Ⅲ CPVT C atecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by stress-induced, adrenergically mediated bidirectional or polymorphic ventricular tachycardia occurring in structurally normal hearts. 1 During exercise or acute emotions, CPVT patients develop life-threatening ventricular arrhythmias, leading to syncope or sudden death. The first cardiac ryanodine receptor (RyR2) mutation identified in a CPVT family was R4497C. 2 Today, more than 70 RyR2 mutations have been reported (http://www.fsm.it/cardmoc), and they comprise the most common genetic subtype of CPVT, 3-7 although mutations in the calsequestrin gene can also cause CPVT. 8,9 Diverging results and conclusions have been generated from expression studies of RyR2 R4496C in heterologous systems. Jiang et al showed that RyR2 R4496C (the mouse equivalent of the human RyR2 R4497C mutation), when expressed in human embryonic kidney (HEK) cells, exhibits increased basal activity and increased sensitivity to luminal Ca 2ϩ . 10 However, other authors found no difference in the basal activity of RyR2 R4497C but, instead, showed increased activity and gating frequency after protein kinase A phosphorylation 11 or sarcoplasmic reticulum (SR) Ca 2ϩ overload. 12 The expression studies were carried out in a variety of models, which may explain the inhomogeneous findings. Furthermore, heterologous systems lack cardiac intracellular environment with all the RyR2 accessory proteins 13 and most Ca 2ϩ -handling proteins, so analysis in native cardiac myocytes is now critical to elucidate the mechanisms by which the mutation leads to cardiac arrhythmia.Recently, a knock-in mouse model carrier of the RyR2 R4496C mutation was developed. 14 Their phenotype presents extraordinary similarity with the clinical manifestations of patients carrying the RyR2 R4497C mutation, including the development of bidirectional ventricular tachycardia. When exposed to adrenaline and caffeine, the RyR2 R4496C cardiomyocytes develop delayed afterdepolarizations (DADs), 15 suggesting that triggered arrhythmias are elicited by adrenergic activation. 16 Here we demonstrate that untreated RyR2 R4496C myocytes have increased spontaneous Ca 2ϩ release in diastole during electric pacing, because of the enhanced Ca 2ϩ sensitivity of mutant RyR2; this abnormality is further augmented by exposure to isoproterenol and increasing pacing rates. Materials and Methods Ventricular cardiomyocytes from male and female RyR2R4496Cϩ/Ϫ mice (RyR2 R4496C ) and their wild-type (WT) RyR2 R4496CϪ/Ϫ littermates were isolated using a standard enzymatic digestion. 17 [Ca 2ϩ ] i transients and Ca 2ϩ sparks were viewed in isolated myocytes by confocal microscopy and analyzed using homemade routines. All experiments were carried out according to the ethical principles laid down by the French (Ministry of Agriculture) and European Union

RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation induces bradycardia by disturbing the coupled clock pacemaker mechanism

“…We previously analyzed Ca 2+ handling in cardiac myocytes from the RyR 2 R4496C mutation, which is located at the C terminal part of the channel. The defect we identified consisted in enhanced Ca 2+ sensitivity of the channel (16). Other mechanisms have been invoked in mutations located at other hotspots.…”

“…These data point to an intrinsic defect of the RyR 2 R420Q function rather than expression or basal phosphorylation difference. Figure 7A, right), as in some other CPVT1 models (15,16,25). To further examine RyR 2 R420Q function, we analyzed Ca 2+ spark characteristics.…”

“…Approximately 50%-65% of CPVT identified mutations are autosomal dominant mutations in RyR 2 . An impaired β-adrenergic SAN response producing SAN pauses was reported by our group in a RyR 2 -related CPVT mouse model carrying the C-terminal RyR 2 R4496C mutation (15), due to an increased Ca 2+ sensitivity (16). Of note, mutations in other regions of the RyR 2 protein have not been studied with regard to their SAN proarrhythmic potential.…”

Biodegradable polymeric nanocapsules prevent cardiotoxicity of antitrypanosomal lychnopholide

“…Spontaneous Ca 2+ waves during diastolic periods frequently occur as a result of the abnormal opening of the ryanodine receptor 2 (RyR2) [15][16][17][18] . These microscopic Ca 2+ events, referred to as Ca 2+ sparks, can be assessed visually by means of confocal microscopy and the use of the fluorescent Ca 2+ indicator Fluo-4 AM.…”

Molecular and genetic basis of sudden cardiac death

“…CPVT-associated RYR2 mutations sensitize the channel to luminal Ca 2+ , leading to exaggerated spontaneous SR Ca 2+ release (125). The effects of CASQ mutations are more complex and include a loss of SR Ca 2+ buffering, loss of RYR2 regulation by calsequestrin, and remodeling of SR ultrastructure (126)(127)(128).…”