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Background-Mutations in the cardiac ryanodine receptor gene (RyR2) underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disease occurring in the structurally intact heart. The proportion of patients with CPVT carrying RyR2 mutations is unknown, and the clinical features of RyR2-CPVT as compared with nongenotyped CPVT are undefined. Methods and Results-Patients with documented polymorphic ventricular arrhythmias occurring during physical or emotional stress with a normal heart entered the study. The clinical phenotype of the 30 probands and of 118 family members was evaluated, and mutation screening on the RyR2 gene was performed. Arrhythmias documented in probands were: 14 of 30 bidirectional ventricular tachycardia, 12 of 30 polymorphic ventricular tachycardia, and 4 of 30 catecholaminergic idiopathic ventricular fibrillation; RyR2 mutations were identified in 14 of 30 probands (36% bidirectional ventricular tachycardia, 58% polymorphic ventricular tachycardia, 50% catecholaminergic idiopathic ventricular fibrillation) and in 9 family members (4 silent gene carriers). Genotype-phenotype analysis showed that patients with RyR2 CPVT have events at a younger age than do patients with nongenotyped CPVT and that male sex is a risk factor for syncope in RyR2-CPVT (relative riskϭ4.2). Conclusions-CPVT is a clinically and genetically heterogeneous disease manifesting beyond pediatric age with a spectrum of polymorphic arrhythmias. ␤-Blockers reduce arrhythmias, but in 30% of patients an implantable defibrillator may be required. Genetic analysis identifies two groups of patients: Patients with nongenotyped CPVT are predominantly women and become symptomatic later in life; patients with RyR2 CPVT become symptomatic earlier, and men are at higher risk of cardiac events. These data provide a rationale for prompt evaluation and treatment of young men with RyR2 mutations. (Circulation. 2002;106:69-74.)

Abstract-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2 R4496Cϩ/Ϫ ). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (nϭ20) in WT but induced DADs in 21 of 33 (63%) RyR2R4496Cϩ/Ϫ myocytes (Pϭ0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2R4496Cϩ/Ϫ myocytes (Pϭ0.001). DADs and triggered activity were abolished by ryanodine (10 mol/L) but not by K201 (1 mol/L or 10 mol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2R4496Cϩ/Ϫ mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6 interaction both in WT and RyR2R4496Cϩ/Ϫ either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2R4496Cϩ/Ϫ myocytes and ventricular arrhythmias in RyR2R4496Cϩ/Ϫ mice; and (3) RyR2-FKBP12.6 interaction in RyR2 R4496Cϩ/Ϫ is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex. Key Words: cardiac electrophysiology Ⅲ ryanodine receptor Ⅲ sudden death Ⅲ transgenic mice Ⅲ ventricular tachycardia C atecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically mediated bidirectional or polymorphic ventricular tachycardia leading to syncope and/or sudden cardiac death in individuals without structural heart disease. 1,2 In 2001, we reported that the autosomal dominant form of CPVT is caused by mutations in the ryanodine receptor gene (RyR2). 3 Based on the evidence that the morphology of ventricular tachycardia observed in CPVT resembles that of digitalis induced ventricular tachycardia (VT), it had been suggested that arrhythmogenesis in CPVT could be mediated by delayed afterdepolarizations (DADs) and triggered activity. Although the discovery that CPVT is caused by mutations in the ryanodine receptor has substantiated this hypothesis, up to now no conclusive demonstration that DADs cause CPVT is available.Furthermore, although several authors have characterized in vitro the functional consequences of RyR2 mutations, 4 -6 the molecular and electrophysiological derangements leading to arrhythmias in CPVT patients are still uncl...

Abstract-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by adrenergically mediated polymorphic ventricular tachycardia leading to syncope and sudden cardiac death. The autosomal dominant form of CPVT is caused by mutations in the RyR2 gene encoding the cardiac isoform of the ryanodine receptor. In vitro functional characterization of mutant RyR2 channels showed altered behavior on adrenergic stimulation and caffeine administration with enhanced calcium release from the sarcoplasmic reticulum. As of today no experimental evidence is available to demonstrate that RyR2 mutations can reproduce the arrhythmias observed in CPVT patients. We developed a conditional knock-in mouse model carrier of the R4496C mutation, the mouse equivalent to the R4497C mutations identified in CPVT , and 5 RyR R4496C pretreated with beta-blockers) received epinephrine and caffeine: 4/8 (50%) RyR R4496C mice but none of the WT developed VT (Pϭ0.02); 4/5 RyR R4496C mice pretreated with propranolol developed VT (Pϭ0.56 nonsignificant versus RyR R4496C mice). These data provide the first experimental demonstration that the R4496C RyR2 mutation predisposes the murine heart to VT and VF in response caffeine and/or adrenergic stimulation. Furthermore, the results show that analogous to what is observed in patients, beta adrenergic stimulation seems ineffective in preventing life-threatening arrhythmias. (Circ Res. 2005;96:e77-e82.) Key Words: arrhythmias Ⅲ genetics Ⅲ ion channels Ⅲ transgenic mice Ⅲ calcium Ⅲ catecholamine C atecholaminergic polymorphic ventricular tachycardia (CPVT; OMIM: 604772) is a highly malignant cardiac disease manifesting in childhood and adolescence. It is characterized by adrenergically-mediated bidirectional or polymorphic ventricular tachycardia leading to syncope and/or sudden cardiac death. 1,2 Based on previously reported linkage data that had mapped the disease to chromosome 1q42-43, 3 we reported that the gene for the autosomal dominant variant of CPVT was RyR2; ie, the gene encoding for the cardiac isoform of the ryanodine receptor. 4 The first family in which a RyR2 mutation was identified was affected by a highly malignant form of the disease that was resistant to beta blockers; the mutation present in the family (R4497C) is a hot spot that we subsequently identified in other CPVT patients unrelated to the first kindred. The R4497C mutation has been extensively investigated in different in vitro models that demonstrated that it enhances the release of calcium from the sarcoplasmic reticulum. [5][6][7][8] It has therefore been inferred that arrhythmias may develop as a consequence of abnormal calcium release from intracellular stores. However, experimental evidence linking this mutation to the development of life-threatening arrhythmias is still lacking. Here we report on a conditional knock-in mouse-model carrier of the R4496C mutation that is the mouse equivalent of the human mutation R4497C. The objectives of the present study were to evaluate if the R449...

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

Abstract-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disordercharacterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2 R33Q/R33Q mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2R33Q/R33Q mice develop bidirectional VT on exposure to environmental stress whereas CASQ2 R33Q/R33Q myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2 R33Q/R33Q hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2 R33Q/R33Q CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) 4 The identification of the genes underlying CPVT has had implications that extend beyond those impacting clinical management of patients inasmuch as it stimulated fundamental research targeted to understand the links between intracellular calcium regulation and arrhythmogenesis. We recently developed 5 a knock-in mouse model carrying the R4496C RyR2 mutation identified in the first genotyped CPVT family and demonstrated that the RyR2 R4496C mice develop bidirectional and polymorphic VT similar to those observed in patients. In this model we also demonstrated 6 the occurrence of delayed after depolarizations (DADs) induced by adrenergic stimulation in isolated myocytes from the heart of heterozygous mice, suggesting that arrhythmias are elicited by triggered activity. Recently 2 mutants CASQ2 knock-in mice models were developed by Song et al 7 : the first strain carries the homozygous point mutation discovered by Lahat et al 3 in the first recessive CPVT family (D307H), and the second strain carries a homozygous deletion ⌬E9/⌬E9; in analogy with RyR2 mice, 5 both models develop bidirectional-polymorphic Materials and MethodsDetailed methods for mouse generation, electrophysiological measurements, immunofluorescence, real-time PCR, microarray, protein and electron microscopy analysis are reported in the online supplement (available online at http://circres.ahajournals.org). Generation of Knock-In of R33Q CASQ2 in Mouse ModelThe kno...

The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS •+ ) assay was adapted to a flow injection (FI) system to obtain a sensitive and rapid technique for the monitoring of antioxidant activity of pure compounds and complex matrixes, such as beverages and food extracts. The FI system includes a HPLC pump that flows the mobile phase (a solution of ABTS •+ in ethanol) through a 20 µL loop injector, a single bead string reactor filled with acid-washed silanized beads, a delay coil and a photodiode array UV-visible detector. The technique was very sensitive, with limits of detection and of quantification of 4.14 and 9.29 µmol of Trolox/L, respectively, and demonstrated high repeatability and reproducibility. The proposed technique was then applied to the evaluation of the antioxidant activity of some pure compounds, demonstrating good agreement with published data obtained by the original spectrophotometric ABTS •+ assay. Finally, the total antioxidant activity of 10 beverages was determined by both the proposed and the original method. The values ranged from 0.09 mmol L -1 for cola to 49.24 mmol L -1 for espresso coffee and did not result significantly different from those obtained by the original spectrophotometric ABTS •+ assay (Student's paired t-test: t ) 1.4074, p ) 0.1929). In conclusion, the proposed FI technique seems suitable for the direct, rapid and reliable monitoring of total antioxidant activity of pure compounds and beverages and, due to the ability to operate in continuous, it allows the analysis of about 30 samples h -1 making the assay particularly suitable for large screening of total antioxidant activity in food samples.

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17-40 years), adult (41-65 years) and aged (66-91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66-91 year-old vs. the 17-40 year-old men; MnSOD activity was significantly greater in 66-91 year-old vs. 17-40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41-65 and 66-91 year-old vs. the 17-40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.