Cardiac Ryanodine Receptor Gene Research Articles

Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterised by adenergically mediated bidirectional and/or polymorphic ventricular tachycardia. CPVT is a significant cause of autopsy-negative sudden death in children and adolescents, although it can also affect adults. It is often caused by pathogenic variants in the cardiac ryanodine receptor gene as well as other rarer genes. Early identification and risk stratification is of major importance. β-blockers are the cornerstone of therapy. Sodium channel blockers, specifically flecainide, have an additive role. Left cardiac sympathetic denervation is playing an increasing role in suppression of arrhythmia and symptoms. Concerns have been raised, however, about the efficacy of implantable cardioverter defibrillator therapy and the risk of catecholamine driven proarrhythmic storms. In this review, we summarise the clinical characteristics, genetics, and diagnostic and therapeutic strategies for CPVT and describe recent advances and challenges.

Characterization of the mechanism by which a nonsense variant in RYR2 leads to disordered calcium handling.

Heterozygous missense variants of the cardiac ryanodine receptor gene (RYR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These missense variants of RYR2 result in a gain of function of the ryanodine receptors, characterized by increased sensitivity to activation by calcium that results in an increased propensity to develop calcium waves and delayed afterdepolarizations. We have recently detected a nonsense variant in RYR2 in a young patient who suffered an unexplained cardiac arrest. To understand the mechanism by which this variant in RYR2, p.(Arg4790Ter), leads to ventricular arrhythmias, human induced pluripotent stem cells (hiPSCs) harboring the novel nonsense variant in RYR2 were generated and differentiated into cardiomyocytes (RYR2‐hiPSC‐CMs) and molecular and calcium handling properties were studied. RYR2‐hiPSC‐CMs displayed significant calcium handling abnormalities at baseline and following treatment with isoproterenol. Treatment with carvedilol and nebivolol resulted in a significant reduction in calcium handling abnormalities in the RYR2‐hiPSC‐CMs. Expression of the mutant RYR2 allele was confirmed at the mRNA level and partial silencing of the mutant allele resulted in a reduction in calcium handling abnormalities at baseline. The nonsense variant behaves similarly to other gain of function variants in RYR2. Carvedilol and nebivolol may be suitable treatments for patients with gain of function RYR2 variants.

Generation of catecholaminergic polymorphic ventricular tachycardia patient-specific induced pluripotent stem cell line

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a genetic disorder characterized by ventricular tachycardia, that can cause the heart to stop beating leading to death. The prevalence is 1/10.000 and in approximately 60% of cases, the syndrome can be due to a mutation of the cardiac ryanodine receptor gene (RyR2). We derived an induced pluripotent stem cell (iPSC) line from an 11-year-old patient blood-cells, carrying a heterozygous missense mutation on the 8th exon of the RyR2 N-terminal part. This reprogramed CPVT line displayed normal karyotype, expressed pluripotent markers and had a capacity to differentiate in trilineage embryonic layers.

Using hiPSC-CMs to Examine Mechanisms of Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac arrhythmia condition, triggered by physical or acute emotional stress, that predominantly expresses early in life. Gain-of-function mutations in the cardiac ryanodine receptor gene (RYR2) account for the majority of CPVT cases, causing substantial disruption of intracellular calcium (Ca2+ ) homeostasis particularly during the periods of β-adrenergic receptor stimulation. However, the highly variable penetrance, patient outcomes, and drug responses observed in clinical practice remain unexplained, even for patients with well-established founder RyR2 mutations. Therefore, investigation of the electrophysiological consequences of CPVT-causing RyR2 mutations is crucial to better understand the pathophysiology of the disease. The development of strategies for reprogramming human somatic cells to human induced pluripotent stem cells (hiPSCs) has provided a unique opportunity to study inherited arrhythmias, due to the ability of hiPSCs to differentiate down a cardiac lineage. Employment of genome editing enables generation of disease-specific cell lines from healthy and diseased patient-derived hiPSCs, which subsequently can be differentiated into cardiomyocytes. This paper describes the means for establishing an hiPSC-based model of CPVT in order to recapitulate the disease phenotype in vitro and investigate underlying pathophysiological mechanisms. The framework of this approach has the potential to contribute to disease modeling and personalized medicine using hiPSC-derived cardiomyocytes. © 2021 Wiley Periodicals LLC.

Diagnosis of catecholaminergic polymorphic ventricular tachycardia during late adulthood due to a rare genetic variant in RYR2: a case report

BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe hereditary channelopathy characterized by the presence of ventricular arrhythmias triggered by adrenergic stimuli, usually diagnosed in the first two decades of life. Genetic variants in the cardiac ryanodine receptor gene are the most frequently occurring that cause an increase in intracellular calcium concentration and thus induce ventricular arrhythmias due to a delayed after depolarisation-induced triggered activity.Case presentationWe present the case of a 74-year-old male, a regular athlete with no relevant family history who suffered from sinus dysfunction and frequent premature ventricular complexes with no symptoms. A treadmill test revealed severe polymorphic ventricular arrhythmias which led to the suspicion of CPVT. A genetic study was undertaken, and it identified a rare genetic variant in the RYR2 gene which was possibly associated with its development in heterozygosity: c.14465G > A, p.Arg4822His. While evaluating the co-segregation, we observed that most of his relatives exhibit polymorphic ventricular arrhythmias with exertion without symptoms and carried the same variant.ConclusionsWe described, for the first time, the clinical characteristics and co-segregation of a family diagnosed with CPVT secondary to a little-known genetic variant of the RYR2 gene. It is a variant that, in our case study, suggests an association with a very good prognosis.

Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare congenital arrhythmogenic disorder induced by physical or emotional stress. It mainly affects children and younger adults and is characterized by rapid polymorphic and bidirectional ventricular tachycardia. Symptoms can include dizziness, palpitations, and presyncope, which may progress to syncope, hypotonia, convulsive movements, and sudden cardiac death. CPVT is the result of perturbations in Ca ion handling in the sarcoplasmic reticulum of cardiac myocytes. Mutations in the cardiac ryanodine receptor gene and the calsequestrin isoform 2 gene are most commonly seen in familial CPVT patients. Under catecholaminergic stimulation, either mutation can result in an excess Ca load during diastole resulting in delayed after depolarization and subsequent arrhythmogenesis. The current first-line treatment for CPVT is β-blocker therapy. Other therapeutic interventions that can be used in conjunction with β-blockers include moderate exercise training, flecainide, left cardiac sympathetic denervation, and implantable cardioverter-defibrillators. Several potential therapeutic interventions, including verapamil, dantrolene, JTV519, and gene therapy, are also discussed.

Allele-Specific Silencing of Mutant mRNA Rescues Ultrastructural and Arrhythmic Phenotype in Mice Carriers of the R4496C Mutation in the Ryanodine Receptor Gene (RYR2).

Mutations in the cardiac Ryanodine Receptor gene (RYR2) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise. We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2. We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2-R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2R4496C/+ (mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2-R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities. The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.

Recurrent syncope related to catecholaminergic polymorphic ventricular tachycardia due to de novo RyR2-R2401H mutation

Objective: To explore the clinical and molecular genetic features of a Chinese patient with catecholaminergic polymorphic ventricular tachycardia (CPVT). Methods: Clinical data including resting electrocardiography, echocardiography and treadmill exercise testing of a patient with CPVT admitted to our department in March 2013 were analyzed, and the peripheral venous blood samples of the patient and his family members and 400 ethnicity-matched healthy controls were obtained. All exons and exon-intron boundaries of the six CPVT-related genes including RYR2, CASQ2, TRDN, CALM1, KCNJ2 and ANKB were sequenced to detect the variants related to CPVT. The relationship between the genotypes and phenotypes was analyzed to direct the target therapy. Results: Recurrent syncope induced either by exercise or extreme frightened fear was observed in this patient. There was no positive family history of syncope or sudden death. The resting electrocardiography and echocardiography of the patient were normal, while the exercise testing revealed bidirectional and polymorphic ventricular tachycardia. A cardiac ryanodine receptor gene mutation (R2401H) was identified in this patient, while this mutation was absent in his parents and sister and 400 controls. No variant was detected in the remaining five candidate genes. Treatment with high dose of metoprolol succinate (118.75 mg/d) was effective and patient was free of syncopal attack during the 2 years follow-up. Conclusion: This is the first report on RyR2-R2401H mutation in Chinese patient with CPVT, and high dose of metoptolol is the effective therapy option for CPVT related to RyR2 mutation.

A novel heterozygous mutation in cardiac calsequestrin causes autosomal dominant catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal inherited arrhythmia syndrome characterized by adrenergically stimulated ventricular tachycardia. Mutations in the cardiac ryanodine receptor gene (RYR2) cause an autosomal dominant form of CPVT, while mutations in the cardiac calsequestrin 2 gene (CASQ2) cause an autosomal recessive form. The aim of this study was to clinically and genetically evaluate a large family with severe autosomal dominant CPVT. Clinical evaluation of family members was performed, including detailed history, physical examination, electrocardiogram, exercise stress test, and autopsy review of decedents. We performed genome-wide linkage analysis in 12 family members and exome sequencing in 2 affected family members. In silico models of mouse and rabbit myocyte electrophysiology were used to predict potential disease mechanisms. Severe CPVT with dominant inheritance in 6 members was diagnosed in a large family with 2 sudden deaths, 2 resuscitated cardiac arrests, and multiple appropriate implantable cardioverter-defibrillator shocks. A comprehensive analysis of cardiac arrhythmia genes did not reveal a pathogenic variant. Exome sequencing identified a novel heterozygous missense variant in CASQ2 (Lys180Arg) affecting a highly conserved residue, which cosegregated with disease and was absent in unaffected family members. Genome-wide linkage analysis confirmed a single linkage peak at the CASQ2 locus (logarithm of odds ratio score 3.01; θ = 0). Computer simulations predicted that haploinsufficiency was unlikely to cause the severe CPVT phenotype and suggested a dominant negative mechanism. We show for the first time that a variant in CASQ2 causes autosomal dominant CPVT. Genetic testing in dominant CPVT should include screening for heterozygous CASQ2 variants.

A knock-in mouse model of N-terminal R420W mutation of cardiac ryanodine receptor exhibits arrhythmogenesis with abnormal calcium dynamics in cardiomyocytes

Cardiac ryanodine receptor gene (RyR2) mutations cause fatal arrhythmogenic diseases such as catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy. The N-terminal region of RyR2 is one of the hot spots for mutations. In this study, we investigated cardiac phenotypes of a knock-in mouse model carrying R420W mutation of RyR2. The N-terminal R420W mutation has already been found in juvenile sudden death cadavers of unrelated families. The depolarization-induced Ca2+ transient amplitude was significantly lower in cardiomyocytes from RyR2R420W/R420W mice compared with wild-type mice. The time to peak of the Ca2+ transient was significantly increased in RyR2R420W/R420W mice. Furthermore, the prolonged decay time from the peak of the Ca2+ transient was detected in RyR2R420W/R420W mice. ECG telemetry revealed that various types of arrhythmias were induced in RyR2R420W/R420W mice in response to administration of caffeine and adrenaline. The mutant mice showed high occurrences of arrhythmias in response to heart stimulants compared with wild-type mice. These findings suggest that R420W mutation impairs depolarization-induced Ca2+ oscillation in cardiomyocytes, which possibly results in sudden death due to stress-induced arrhythmias.

Hypertrophy of lymphoid organs is a possible phenotypic characteristic of R420W mutation of the cardiac ryanodine receptor gene: a study using a knock-in mouse model.

Cardiac ryanodine receptor gene (RyR2) mutations sometimes result in sudden cardiac death due to fatal arrhythmias. N-terminal R420W mutation of RyR2 is known to show similar phenotypes to arrhythmogenic right ventricular cardiomyopathy and to cause juvenile sudden death. We previously reported two sudden death cases with the same R420W mutation. Interestingly, the cases showed hypertrophy of lymphoid organs such as the thymus and mesenteric lymph nodes. The present study examined whether R420W mutation of RYR2 causes hypertrophy of lymphoid organs by generating a mouse model carrying the mutation. Homozygous (RyR2(R420W/R420W)) mice showed significant increases in thymus and spleen weights but not in kidney, heart, and brain weights compared with wild-type mice. The mice also showed remarkable hypertrophy of mesenteric lymph nodes. Immunohistochemical study revealed that RyR2 protein was prominently expressed in epithelial cells of the thymic medulla in the thymus. These findings show that mice with R420W mutation of RyR2 exhibit hypertrophy of lymphoid organs. Sudden unexplained death cases with the mutation may display such findings at autopsy.

Generation and characterization of a mouse model harboring the exon-3 deletion in the cardiac ryanodine receptor.

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/− mice are not susceptible to CPVT. Ex3-del+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.

CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro ‘patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after β-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca2+/calmodulin-dependent serine–threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.

Familial Evaluation in Catecholaminergic Polymorphic Ventricular Tachycardia

Background— Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with mutations in the cardiac ryanodine receptor gene ( Ryr2 ) in the majority of patients. Previous studies of CPVT patients mainly involved probands, so current insight into disease penetrance, expression, genotype-phenotype correlations, and arrhythmic event rates in relatives carrying the Ryr2 mutation is limited. Methods and Results— One-hundred sixteen relatives carrying the Ryr2 mutation from 15 families who were identified by cascade screening of the Ryr2 mutation causing CPVT in the proband were clinically characterized, including 61 relatives from 1 family. Fifty-four of 108 antiarrhythmic drug-free relatives (50%) had a CPVT phenotype at the first cardiological examination, including 27 (25%) with nonsustained ventricular tachycardia. Relatives carrying a Ryr2 mutation in the C-terminal channel-forming domain showed an increased odds of nonsustained ventricular tachycardia (odds ratio, 4.1; 95% CI, 1.5–11.5; P =0.007, compared with N-terminal domain) compared with N-terminal domain. Sinus bradycardia was observed in 19% of relatives, whereas other supraventricular dysrhythmias were present in 16%. Ninety-eight (most actively treated) relatives (84%) were followed up for a median of 4.7 years (range, 0.3–19.0 years). During follow-up, 2 asymptomatic relatives experienced exercise-induced syncope. One relative was not being treated, whereas the other was noncompliant. None of the 116 relatives died of CPVT during a 6.7-year follow-up (range, 1.4–20.9 years). Conclusions— Relatives carrying an Ryr2 mutation show a marked phenotypic diversity. The vast majority do not have signs of supraventricular disease manifestations. Mutation location may be associated with severity of the phenotype. The arrhythmic event rate during follow-up was low.

Guidelines for the diagnosis and management of Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited arrhythmia syndrome, characterised by polymorphic ventricular tachycardia induced by adrenergic stress. CPVT can be caused by mutations the cardiac ryanodine receptor gene (RYR2) or mutations in the cardiac calsequestrin gene CASQ2. Structural heart disease is usually absent and the baseline ECG is usually normal. Patients with CPVT often present with exercise- or emotion induced syncope, the first presentation can also be sudden cardiac death. Besides removal of triggers treatment with beta blockers is currently a class I indication in clinically diagnosed patients. Beta blockage should be titrated up to an effective level. The addition of flecainide seems to be a promising approach in patients where arrhythmias are not completely suppressed by beta blockers. A cardioverter-defibrillator (ICD) or left cervical sympathetic denervation might be considered under special circumstances. Genetic counselling is recommended and all first degree relatives should be properly evaluated.

Defining the Disconnect Between In Vitro Models and Human Arrhythmogenic Disease

Understanding the pathogenetic chain of events between genotype and phenotype is critical to the appropriate diagnosis and treatment of heritable diseases. Although our knowledge of the molecular substrate of many diseases continues to increase, identifying the mechanistic links between a susceptibility variant and disease expression remains a major challenge. A minority of mendelian diseases display complete clinical penetrance in individuals harboring known disease-causing mutations, with rare examples such as achondroplasia and Huntington disease. Even in the most well-characterized hereditary arrhythmia syndrome, the long-QT syndrome (LQTS), in which genotype-phenotype relationships and gene-specific management strategies1 exist for the most common molecular subtypes (LQTS 1 to 3), the reasons for incomplete penetrance and variable expression in this disease remain largely unknown. In fact, LQTS has relatively low penetrance; however, in those LQTS genotype-positive individuals who do exhibit symptoms, the initial presentation can be catastrophic, and all too often, families come to clinical attention as a result of aborted cardiac arrest or sudden cardiac death. Therefore, identification of disease-associated mutations in individuals at risk for heritable arrhythmogenic disorders is imperative for subsequent risk reduction in their family members and them. Article see p 1001 Over the past 10 to 15 years, it has been shown that mutations in SCN5A , the gene encoding the α subunit of the human cardiac sodium channel (Na v 1.5), have pleiotropic effects, including risk for type 3 LQTS, Brugada syndrome, progressive cardiac conduction disease, atrial fibrillation, sick sinus syndrome, sudden infant death syndrome, and dilated cardiomyopathy. This phenotypic plasticity is not unique to SCN5A , but is also observed with other cardiovascular disease–associated genes. For example, mutations in the cardiac ryanodine receptor gene, RYR2 , are associated with catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular dysplasia/cardiomyopathy. In the current issue of Circulation , Watanabe et al …

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value. This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers. The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2. Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes. M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.

A novel mutation in the cardiac ryanodine receptor gene (RyR2) in a patient with an unequivocal LQTS

A novel mutation in the cardiac ryanodine receptor gene (RyR2) in a patient with an unequivocal LQTS

Calmodulin kinase II inhibition prevents arrhythmias in RyR2R4496C+/− mice with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by life-threatening arrhythmias elicited by adrenergic activation. CPVT is caused by mutations in the cardiac ryanodine receptor gene (RyR2). In vitro studies demonstrated that RyR2 mutations respond to sympathetic activation with an abnormal diastolic Ca2+ leak from the sarcoplasmic reticulum; however the pathways that mediate the response to adrenergic stimulation have not been defined. In our RyR2R4496C+/− knock-in mouse model of CPVT we tested the hypothesis that inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) counteracts the effects of adrenergic stimulation resulting in an antiarrhythmic activity. CaMKII inhibition with KN-93 completely prevented catecholamine-induced sustained ventricular tachyarrhythmia in RyR2R4496C+/− mice, while the inactive congener KN-92 had no effect. In ventricular myocytes isolated from the hearts of RyR2R4496C+/− mice, CaMKII inhibition with an autocamtide-2 related inhibitory peptide or with KN-93 blunted triggered activity and transient inward currents induced by isoproterenol. Isoproterenol also enhanced the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA), increased spontaneous Ca2+ release and spark frequency. CaMKII inhibition blunted each of these parameters without having an effect on the SR Ca2+ content. Our data therefore indicate that CaMKII inhibition is an effective intervention to prevent arrhythmogenesis (both in vivo and in vitro) in the RyR2R4496C+/− knock-in mouse model of CPVT. Mechanistically, CAMKII inhibition acts on several elements of the EC coupling cascade, including an attenuation of SR Ca2+ leak and blunting catecholamine-mediated SERCA activation. CaMKII inhibition may therefore represent a novel therapeutic target for patients with CPVT.

Sudden Infant Death Syndrome in Mice With an Inherited Mutation in RyR2

Mutations in the cardiac ryanodine receptor gene (RyR2) have been recently identified in victims of sudden infant death syndrome. The aim of this study was to determine whether a gain-of-function mutation in RyR2 increases the propensity to cardiac arrhythmias and sudden death in young mice. Incidence of sudden death was monitored prospectively in heterozygous knock-in mice with mutation R176Q in RyR2 (R176Q/+). Young R176Q/+ mice exhibited a higher incidence of sudden death compared with wild-type littermates. Optical mapping of membrane potentials and intracellular calcium in 1- to 7-day-old R176Q/+ and wild-type mice revealed an increased incidence of ventricular ectopy and spontaneous calcium releases in neonatal R176Q/+ mice. Surface ECGs in 3- to 10-day-old mice showed that R176Q/+ mice developed more ventricular arrhythmias after provocation with epinephrine and caffeine. Intracardiac pacing studies in 12- to 18-day-old mice revealed the presence of an arrhythmogenic substrate in R176Q/+ compared with wild-type mice. Reverse transcription-polymerase chain reaction and Western blotting showed that expression levels of other calcium handling proteins were unaltered, suggesting that calcium leak through mutant RyR2 underlies arrhythmogenesis and sudden death in young R176Q/+ mice. Our findings demonstrate that a gain-of-function mutation in RyR2 confers an increased risk of cardiac arrhythmias and sudden death in young mice and that young R176Q/+ mice may be used as a model for elucidating the complex interplay between genetic and environmental risk factors associated with sudden infant death syndrome.