Prevention of Ventricular Arrhythmia and Calcium Dysregulation in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin‐2 Mutation

Arrhythmia Prevention in CPVT . Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium‐regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin‐2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca²⁺ overload and increased Ca²⁺ leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT. Method and results: CPVT mouse models that lack CASQ2 were treated with Ca²⁺‐channel inhibitors, β‐adrenergic inhibitors, or Mg²⁺. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca²⁺ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress‐induced ventricular arrhythmia in mutant mice. The Ca²⁺ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca²⁺ content in mutant myocytes, diminished diastolic Ca²⁺ overload, increased systolic Ca²⁺ amplitude, and prevented Ca²⁺ oscillations in stressed mutant myocytes. Conclusions: Ca²⁺ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil‐induced partial normalization of SR Ca²⁺ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca²⁺ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT . (J Cardiovasc Electrophysiol, Vol. 22, pp. 316‐324, March 2011)     

Mechanism underlying catecholaminergic polymorphic ventricular tachycardia and approaches to therapy

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by VT induced by adrenergic stress in the absence of structural heart disease and high incidence of sudden cardiac death. The diagnosis is made based on reproducible ventricular tachyarrhythmias including bidirectional VT and polymorphic VT during exercise testings. Two causative genes of CPVT have been identified: RYR2, encoding the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel, and CASQ2, encoding cardiac calsequestrin. A mutation in RYR2 or CASQ2 is identified in approximately 60% of patients with CPVT. Mutations in these two genes destabilize the RyR2 Ca²⁺ release channel complex in sarcoplasmic reticulum and result in spontaneous Ca²⁺ release through RyR2 channels leading to delayed after depolarization, triggered activity, and bidirectional/polymorphic VT. Implantable cardioverter defibrillators (ICDs) are recommended for prevention of sudden death in patients with CPVT.1. A.E. Epstein, J.P. DiMarco, K.A. Ellenbogen, et al., ACC/AHA/HRS 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices): developed in collaboration with the American Association for Thoracic Surgery and Society of Thoracic Surgeons. Circulation. 2008;117:e350 However, painful shocks can trigger further adrenergic stress and arrhythmias, and deaths have occurred despite appropriate ICD shocks. Treatment with β-adrenergic blockers reduces arrhythmia burden and mortality, but is not completely effective. The beneficial effects of Ca²⁺ channel blocker verapamil in combination with β-blocker have been reported, but the role of verapamil has not been well assessed. Because Ca²⁺ leakage through ryanodine channel is a common mechanism of CPVT, ryanodine channel block may have a therapeutic effect. We discovered that flecainide directly inhibits RyR2 channels and prevent CPVT. Left cardiac sympathetic denervation may be an effective alternative treatment in combination with ICD, especially for patients whose arrhythmias are not controlled by drug therapies.     

Flecainide inhibits arrhythmogenic Ca waves by open state block of ryanodine receptor Ca release channels and reduction of Ca spark mass

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca²⁺ waves and elementary sarcoplasmic reticulum (SR) Ca²⁺ release events, Ca²⁺ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca²⁺ leak or SR Ca²⁺ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca²⁺ leak, resulting in a significantly increased SR Ca²⁺ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca²⁺ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca²⁺ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca²⁺ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca²⁺ waves resulting in triggered arrhythmias, such as CPVT.     

Mutant ryanodine receptors in catecholaminergic polymorphic ventricular tachycardia generate delayed afterdepolarizations due to increased propensity to Ca2+ waves.

AIMS: Mutations in cardiac ryanodine receptors (RyR2s) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), characterized by risk of polymorphic ventricular tachyarrhythmias and sudden death during exercise. Arrhythmias are caused by gain-of-function defects in RyR2, but cellular arrhythmogenesis remains elusive. METHODS AND RESULTS: We recorded endocardial monophasic action potentials (MAPs) at right ventricular septum in 15 CPVT patients with a RyR2 mutation (P2,328S, Q4,201R, and V4,653F) and in 12 control subjects both at baseline and during epinephrine infusion (0.05 microg/kg/min). At baseline 3 and during epinephrine infusion, four CPVT patients, but none of the control subjects, showed delayed afterdepolarizations (DADs) occasionally coinciding with ventricular premature complexes. In order to study the underlying mechanisms, we expressed two types of mutant RyR2 (P2,328S and V4,653F) causing CPVT as well as wild-type RyR2 in HEK 293 cells. Confocal microscopy of Fluo-3 loaded cells transfected with any of the three RyR2s showed no spontaneous subcellular Ca(2+) release events at baseline. Membrane permeable cAMP analogue (Dioctanoyl-cAMP) triggered subcellular Ca(2+) release events as Ca(2+) sparks and waves. Cells expressing mutant RyR2s showed spontaneous Ca(2+) release events at lower concentrations of cAMP than cells transfected with wild-type RyR2. CONCLUSION: CPVT patients show DADs coinciding with premature action potentials in MAP recordings. Expression studies suggest that DADs are caused by increased propensity of abnormal RyR2s to generate spontaneous Ca(2+) waves in response to cAMP stimulation. Increased sensitivity of mutant RyR2s to cAMP may explain the occurrence of arrhythmias during exercise or emotional stress in CPVT.     

Calcium leak through ryanodine receptors leads to atrial fibrillation in 3 mouse models of catecholaminergic polymorphic ventricular tachycardia

Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca(2+)) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective: In the present study, we sought to determine whether intracellular diastolic SR Ca(2+) leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results: We generated 3 knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca(2+) leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3 CPVT mouse models (RyR2-R2474S(+/-), 70%; RyR2-N2386I(+/-), 60%; RyR2-L433P(+/-), 35.71%) but not in wild-type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca(2+) leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca(2+) leak through the channel. Atrial RyR2 from RyR2-R2474S(+/-) mice were oxidized, and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca(2+) leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. Conclusions: The present study demonstrates that RyR2-mediated diastolic SR Ca(2+) leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca(2+) leak through RyR2 and pacing-induced AF associated with CPVT mutations.     

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca²⁺ release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca²⁺ on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [³H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G^+/−) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G^+/− hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca²⁺ release during systole, gradually overloading the sarcoplasmic reticulum with Ca²⁺. The resultant Ca²⁺ overload then randomly caused bursts of prolonged Ca²⁺ release, activating electrogenic Na⁺-Ca²⁺ exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.In the heart, ryanodine receptor (RyR2) channels release massive amounts of Ca²⁺ from the sarcoplasmic reticulum (SR) in response to membrane depolarization, in turn modulating cardiac excitability and triggering ventricular contractions (1, 2). In their intracellular milieu, RyR2 channels are regulated by a variety of cytosolic and luminal factors so that their output signal (i.e., Ca²⁺) finely grades cardiac contractions (3). However, RyR2 channels operate within a limited margin of safety because conditions that demand higher RyR2 activity (such as sympathetic stimulation) also increase the vulnerability of the heart to life-threatening arrhythmias (4), and this risk is higher in hearts harboring mutant RyR2 channels. Indeed, point mutations in RYR2, the gene encoding for the cardiac RyR channel, are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) (5), a highly arrhythmogenic syndrome triggered by sympathetic stimulation that may lead to sudden cardiac death, especially in children and young adults (6).To date, delayed afterdepolarizations (DADs) triggered by spontaneous Ca²⁺ release stand as the most accepted cellular mechanism to explain cardiac arrhythmias in CPVT. In this scheme, RyR2 channels destabilized by gain-of-function mutations release Ca²⁺ during diastole, generating a depolarizing transient inward current (I_ti) as the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) extrudes the released Ca²⁺. This electrogenic inward current then causes DADs, which, if sufficiently large, reach the threshold to initiate untimely action potentials (APs) and generate triggered activity (6–8). Hence, hyperactive RyR2 channels eager to release Ca²⁺ on their own appear as essential components of this arrhythmogenic scheme. In fact, most RyR2-linked CPVT mutations characterized to date produce hyperactive RyR2 channels (9–12). This scheme therefore appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations (13).How do hypoactive RyR2 channels trigger lethal arrhythmias? Here we studied the RyR2-A4860G mutation, which was initially detected in a young girl presenting idiopathic catecholaminergic ventricular fibrillation (VF) (14). When expressed in HEK293 cells, recombinant RyR2-A4860G channels displayed a dramatic depression of activity, manifested mainly as a loss of luminal Ca²⁺ sensitivity (13). However, this in vitro characterization was insufficient to elucidate the mechanisms by which these hypoactive channels generate cellular substrates favorable for cardiac arrhythmias. We thus generated a mouse model of CPVT harboring the RyR2-A4860G mutation. Inbreeding of mice heterozygous for the mutation (RyR2-A4860G^+/−) yields only WT and heterozygous mice, indicating that the mutation is too strong to be harbored in the two RYR2 alleles. Ventricular myocytes from RyR2-A4860G^+/− mice have constitutively lower Ca²⁺ release than WT littermates, and undergo apparently random episodes of prolonged systolic Ca²⁺ release upon β-adrenergic stimulation, giving rise to early afterdepolarizations (EADs). Thus, this unique RYR2 mutation reveals novel pathways whereby RyR2 channels engage sarcolemmal currents to trigger VF. Although exposed in the setting of CPVT, this mechanism may be extended to a variety of settings, including heart failure, atrial fibrillation, and other cardiomyopathies in which RyR2 down-regulation and posttranslational modifications depress RyR2 function.     

Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor

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 families, to evaluate if the animals would develop a CPVT phenotype and if beta blockers would prevent arrhythmias. Twenty-six mice (12 wild-type (WT) and 14RyR(R4496C)) underwent exercise stress testing followed by epinephrine administration: none of the WT developed ventricular tachycardia (VT) versus 5/14 RyR(R4496C) mice (P=0.02). Twenty-one mice (8 WT, 8 RyR(R4496C), 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 (P=0.02); 4/5 RyR(R4496C) mice pretreated with propranolol developed VT (P=0.56 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.     

Sudden death in a young man with catecholaminergic polymorphic ventricular tachycardia and paroxysmal atrial fibrillation

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial condition that presents with exercise-induced syncope or sudden death in children or young adults. In most cases the disease is caused by a mutation in the cardiac ryanodine receptor (RyR2) gene. Current evidence suggests that primary therapy for CPVT is beta blockade and implantable cardioverter defibrillator (ICD) placement. There is a recent report of a patient with CPVT who died despite appropriate ICD therapies, and we report a similar case. Our patient died after probably initially receiving inappropriate ICD shocks for atrial fibrillation. We recommend that utmost efforts should be made to prevent shocks including repeated exercise testing to confirm suppression of PVT.     

Human cardiac ryanodine receptor mutations in ion channel disorders in Japan

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by adrenergic induced bidirectional or polymorphic ventricular tachycardias. Some of CPVT families were reported to be associated with cardiac ryanodine receptor gene (RyR2) mutations. However, association between RyR2 and other arrhythmogenic disorders is not clarified. In this study, we analyzed 83 Japanese patients including patients with long-QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, arrhythmogenic right ventricular cardiomyopathy and CPVT. Genetic screening of RyR2 revealed 3 distinct mutations among 4 families with CPVT (75% of incidence). However, no mutation was found in other groups. This is the first report to demonstrate prevalence of RyR2 mutations in various arrhythmogenic disorders in Japan. RyR2 mutations were detected frequently in CPVT but not in other diseases.     

Calmodulin kinase II inhibition prevents arrhythmias in RyR2(R4496C+/-) 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 Ca(2+) leak from the sarcoplasmic reticulum; however the pathways that mediate the response to adrenergic stimulation have not been defined. In our RyR2(R4496C+/-) knock-in mouse model of CPVT we tested the hypothesis that inhibition of Ca(2+)/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 RyR2(R4496C+/-) mice, while the inactive congener KN-92 had no effect. In ventricular myocytes isolated from the hearts of RyR2(R4496C+/-) 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 Ca(2+)-ATPase (SERCA), increased spontaneous Ca(2+) release and spark frequency. CaMKII inhibition blunted each of these parameters without having an effect on the SR Ca(2+) content. Our data therefore indicate that CaMKII inhibition is an effective intervention to prevent arrhythmogenesis (both in vivo and in vitro) in the RyR2(R4496C+/-) knock-in mouse model of CPVT. Mechanistically, CAMKII inhibition acts on several elements of the EC coupling cascade, including an attenuation of SR Ca(2+) leak and blunting catecholamine-mediated SERCA activation. CaMKII inhibition may therefore represent a novel therapeutic target for patients with CPVT.     

Genotypic heterogeneity and phenotypic mimicry among unrelated patients referred for catecholaminergic polymorphic ventricular tachycardia genetic testing

BACKGROUND: Mutations in the RyR2-encoded cardiac ryanodine receptor/calcium release channel and in CASQ2-encoded calsequestrin cause catecholaminergic polymorphic ventricular tachycardia (CPVT1 and CPVT2, respectively). OBJECTIVES: The purpose of this study was to evaluate the extent of genotypic and phenotypic heterogeneity among referrals for CPVT genetic testing. METHODS: Using denaturing high-performance liquid chromatography and DNA sequencing, mutational analysis of 23 RyR2 exons previously implicated in CPVT1, comprehensive analysis of all translated exons in CASQ2 (CPVT2), KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), KCNE2 (LQT6), and KCNJ2 (Andersen-Tawil syndrome [ATS1], also annotated LQT7), and analysis of 10 ANK2 exons implicated in LQT4 were performed on genomic DNA from 11 unrelated patients (8 females) referred to Mayo Clinic's Sudden Death Genomics Laboratory explicitly for CPVT genetic testing. RESULTS: Overall, putative disease causing mutations were identified in 8 patients (72%). Only 4 patients (3 males) hosted CPVT1-associated RyR2 mutations: P164S, V186M, S3938R, and T4196A. Interestingly, 4 females instead possessed either ATS1- or LQT5-associated mutations. Mutations were absent in >400 reference alleles. CONCLUSION: Putative CPVT1-causing mutations in RyR2 were seen in <40% of unrelated patients referred with a diagnosis of CPVT and preferentially in males. Phenotypic mimicry is evident with the identification of ATS1- and LQT5-associated mutations in females displaying a normal QT interval and exercise-induced bidirectional VT, suggesting that observed exercise-induced polymorphic VT in patients may reflect disorders other than CPVT. Clinical consideration for either Andersen-Tawil syndrome or long QT syndrome and appropriate genetic testing may be warranted for individuals with RyR2 mutation-negative CPVT, particularly females.     

Accelerated Junctional Rhythm and Nonalternans Repolarization Lability Precede Ventricular Tachycardia in Casq2−/− Mice

Repolarization Lability in Casq2?/? Mice . Background: Calsequestrin‐2 (CASQ2) is a Ca²⁺ buffering protein of myocardial sarcoplasmic reticulum. CASQ2 mutations underlie a form of catecholaminergic polymorphic ventricular tachycardia (CPVT). The CPVT phenotype is recapitulated in Casq2 ?/? mice. Repolarization lability (RL)—beat‐to‐beat variability in the T wave morphology—has been reported in long‐QT syndrome, but has not been evaluated in CPVT. Methods and Results: ECG from Casq2 ?/? mice was evaluated with respect to heart rate (HR) and RL changes prior to onset of ventricular tachycardia (VT) to gain insight into arrhythmogenesis in CPVT. Telemetry from unrestrained mice (3‐month‐old males, 5 animals of each genotype) and ECG before and after isoproterenol administration in anesthetized mice was analyzed. Average HR in sinus rhythm (SR), occurrence of nonsinus rhythm and RL were quantified. HR was slower in Casq2 ?/? animals. Accelerated junctional rhythm (JR) occurred more frequently in Casq2 ?/? mice and often preceded VT. In Casq2 ?/? mice, HR increased prior to VT onset, prior to onset of JR and on transition from JR to VT. RL increased during progression from SR to VT and after isoproterenol administration in Casq2 ?/?, but not in Casq2+/+ animals. Isoproterenol did not increase repolarization alternans in either genotype. Conclusions: Accelerated JR, likely caused by triggered activity in His/Purkinje system, occurs frequently in Casq2 ?/? mice. The absence of CASQ2 results in increased RL. The increase in HR and in RL precede onset of arrhythmias in this CPVT model. Nonalternans RL precedes ventricular arrhythmia in wider range of conditions than previously appreciated. (J Cardiovasc Electrophysiol, Vol. 23, pp. 1355‐1363, December 2012)     

A novel RYR2 loss-of-function mutation (I4855M) is associated with left ventricular non-compaction and atypical catecholaminergic polymorphic ventricular tachycardia

Background

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy usually caused by gain-of-function mutations ryanodine receptor type-2 (RyR2). Left ventricular non-compaction (LVNC) is an often genetic cardiomyopathy. A rare LVNC-CPVT overlap syndrome may be caused by exon 3 deletion in RyR2. We sought to characterize the phenotypic spectrum and molecular basis of a novel RyR2 mutation identified in a family with both conditions.

A novel mutation in FKBP12.6 binding region of the human cardiac ryanodine receptor gene (R2401H) in a Japanese patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an autosomal dominant inherited disorder characterized by adrenergic induced polymorphic ventricular tachycardias and associated with sudden cardiac death. The human cardiac ryanodine receptor gene (RyR2) was linked to CPVT. A 20-year-old male was referred to our hospital because of recurrent syncope after physical and emotional stress. Routine cardiac examinations including catheterization revealed no structural abnormality. Exercise on treadmill induced premature ventricular contraction in bigeminy and bidirectional ventricular tachycardia was induced during isoproterenol infusion. Beta-blocking drug was effective in suppressing the arrhythmias. We performed genetic screening by PCR-SSCP method followed by DNA sequencing, and a novel missense mutation R2401H in RyR2 located in FKBP12.6 binding region was identified. This mutation was not detected in 190 healthy controls. Since FKBP12.6 plays a critical role in Ca channel gating, the R2401H mutation can be expected to alter Ca-induced Ca release and E-C coupling resulting in CPVT. This is the first report of RyR2 mutation in CPVT patient from Asia including Japan.     

Catecholaminergic Polymorphic Ventricular Tachycardia from Bedside to Bench and Beyond

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a primary electrical myocardial disease characterized by exercise- and stress-related ventricular tachycardia manifested as syncope and sudden death. The disease has a heterogeneous genetic basis, with mutations in the cardiac Ryanodine Receptor channel (RyR2) gene accounting for an autosomal-dominant form (CPVT1) in approximately 50% and mutations in the cardiac calsequestrin gene (CASQ2) accounting for an autosomal-recessive form (CPVT2) in up to 2% of CPVT cases. Both RyR2 and calsequestrin are important participants in the cardiac cellular calcium homeostasis.We review the physiology of the cardiac calcium homeostasis, including the cardiac excitation contraction coupling and myocyte calcium cycling. The pathophysiology of cardiac arrhythmias related to myocyte calcium handling and the effects of different modulators are discussed.The putative derangements in myocyte calcium homeostasis responsible for CPVT, as well as the clinical manifestations and therapeutic options available, are described.     

Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model

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(R4496C+/-)). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2(R4496C+/-) myocytes (P=0.001). 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 RyR2(R4496C+/-) myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 micromol/L) but not by K201 (1 micromol/L or 10 micromol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2(R4496C+/-) mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6 interaction both in WT and RyR2(R4496C+/-) 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 RyR2(R4496C+/-) myocytes and ventricular arrhythmias in RyR2(R4496C+/-) mice; and (3) RyR2-FKBP12.6 interaction in RyR2(R4496C+/-) 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.     

儿茶酚胺敏感性多形性室速的基因诊治进展

儿茶酚胺敏感性多形性室速(CPVT)是具有较高猝死风险的罕见单基因遗传病.已知多种CPVT基因突变可通过影响肌浆网钙通道蛋白RyR2的功能,破坏细胞内钙稳态,触发室性心律失常,而依靠腺相关病毒载体(AAVs)及CRISPR/Cas9技术进行基因层面的干预有望为CPVT的治疗提供新思路.本文就其遗传特征及基因干预等领域的研究现状作一总结.     

Bidirectional Ventricular Tachycardia Due to Coronary Allograft Vasculopathy a Unique Presentation

Bidirectional ventricular tachycardia (BVT) is an uncommon type of polymorphic ventricular tachycardia (PVT) with alternating polarity of the QRS complex most commonly described digitalis toxicity. Recent data has demonstrated the possible molecular basis of this electrocardiographic phenomenon. To our knowledge this is the first reported case of BVT in a patient with orthotopic cardiac transplantation and coronary allograft vasculopathy.     

Intravenous epinephrine infusion test in diagnosis of catecholaminergic polymorphic ventricular tachycardia

Epinephrine Infusion in Diagnosis of CPVT. Introduction: A test involving intravenous infusion of epinephrine has been proposed as a method alternative to exercise stress test in diagnosis of catecholaminergic polymorphic ventricular tachycardia (CPVT). We aimed at estimating the predictive value of intravenous epinephrine administration in CPVT patients with frequent exercise‐induced ventricular ectopy. Methods and Results: We recruited 81 subjects, including 25 CPVT‐linked ryanodine receptor 2 (RYR2) mutation carriers, 11 genetically undefined CPVT patients, and 45 unaffected family members. All subjects underwent a maximal exercise stress test and an intravenous epinephrine infusion test. Exercise stress test was positive in 25 (31%) patients including 14 of 25 (56%) established RYR2‐mutation carriers and all 11 (100%) genetically undefined CPVT patients. Epinephrine infusion induced arrhythmias in 3 (12%) RYR2‐mutation carriers, 4 (36%) genetically undefined CPVT patients, and 1 (2%) unaffected family member. A total of 18 exercise stress test positive patients did not respond to intravenous epinephrine administration, whereas only 1 epinephrine test responder had a normal exercise stress test. Thus, if exercise stress test is used as a standard, the sensitivity of the epinephrine infusion test is 28% and specificity is 98%. Conclusions: Intravenous epinephrine infusion has low sensitivity and may not be considered as an alternative method for a maximal exercise stress test in diagnosis of CPVT. (J Cardiovasc Electrophysiol, Vol. 23, pp. 194‐199, February 2012)     

S4153R Is a Gain-of-Function Mutation in the Cardiac Ca Release Channel Ryanodine Receptor Associated With Catecholaminergic Polymorphic Ventricular Tachycardia and Paroxysmal Atrial Fibrillation

Mutations in ryanodine receptor 2 (RYR2) gene can cause catecholaminergic polymorphic ventricular tachycardia (CPVT). The novel RYR2-S4153R mutation has been implicated as a cause of CPVT and atrial fibrillation. The mutation has been functionally characterized via store-overload-induced Ca²⁺ release (SOICR) and tritium-labelled ryanodine ([³H]ryanodine) binding assays. The S4153R mutation enhanced propensity for spontaneous Ca²⁺ release and reduced SOICR threshold but did not alter Ca²⁺ activation of [³H]ryanodine binding, a common feature of other CPVT gain-of-function RYR2 mutations. We conclude that the S4153R mutation is a gain-of-function RYR2 mutation associated with a clinical phenotype characterized by both CPVT and atrial fibrillation.