Model Of Catecholaminergic Polymorphic Ventricular Tachycardia Research Articles

Slowed depolarization and irregular repolarization in catecholaminergic polymorphic ventricular tachycardia: a study from cellular Ca2+ transients and action potentials to clinical monophasic action potentials and electrocardiography.

Spontaneous Ca2+ release leads to afterdepolarizations and triggered arrhythmia in catecholaminergic polymorphic ventricular tachycardia (CPVT). Irregular Ca2+ release is hypothesized to manifest as slowed depolarization and irregular repolarization. Our goal was to study depolarization and repolarization abnormalities in CPVT, as they remain largely uninvestigated. We studied intracellular Ca2+ handling and action potentials (APs) in an induced pluripotent stem cell (iPSC) model of CPVT. Induced pluripotent stem cell cardiomyocytes from a RyR2-P2328S patient showed increased non-alternating variability of Ca2+ transients in response to isoproterenol. β-Agonists decreased AP upslope velocity in CPVT cells and in monophasic AP recordings of CPVT patients. We compared 24 h electrocardiograms (ECGs) of 19 CPVT patients carrying RyR2 mutations and 19 healthy controls. Short-term variability (STV) of the QT interval was 6.9 ± 0.5 ms in CPVT patients vs. 5.5 ± 0.4 ms in controls (P < 0.05) and associated with a history of arrhythmic events. Mean T-wave alternans (TWA) was 25 ± 1.4 µV in CPVT patients vs. 31 ± 2.0 µV in controls (P < 0.05). Older CPVT patients showed lower maximal upslope velocity of the ECG R-spike than control patients. Catecholaminergic polymorphic ventricular tachycardia patients show higher STV of repolarization but lower TWA on the 24 h ECG than control patients, which is likely to reflect increased non-alternating variability of Ca2+ release by mutant RyR2s as observed in vitro. β-Agonists slow depolarization in RyR2-mutant cells and in CPVT patients. These findings may constitute a marker of arrhythmogenicity.

Abstract 17874: Aerobic Exercise Training Improves Exercise Capacity, Reduces Arrhythmia Susceptibility but Does Not Normalize Ryanodine Receptor Mediated Aberrant Calcium Release in Catecholaminergic Polymorphic Ventricular Tachycardia

Introduction: Loss of Calsequestrin (CASQ2) promotes abnormal calcium (Ca2+) release events via the cardiac Ryanodine receptor (RyR2) during adrenergic stimulation, which trigger Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Rationale: Since aerobic exercise training (AET) has been shown to normalize Sarcoplasmic reticulum (SR) Ca2+ cycling parameters in diseased hearts, we explored if AET impacts RyR2 dysfunction and CPVT susceptibility in CASQ2-/- mice. Methods and Results: Age matched wildtype (WT) and CASQ2-/- male mice (n=8) were subjected to treadmill running for 6 weeks (16mts/min for 1hr, 5 days/week at 10% incline). Subsequently, a graded exercise test showed that sedentary (Sed) CASQ2-/- mice have a significantly lower exercise capacity relative to SedWT. Compared to trained (Ex) WT mice, AET moderately increased maximal running speed, time, and RER values in ExCASQ2-/- mice, indicating improved aerobic capacity. Electrocardiographic analyses showed that ExCASQ2-/- mice were resistant to triggered arrhythmias compared to their Sed controls. Spectral analyses of heart rate variability indicated that the high frequency band power increased significantly in ExCASQ2-/- mice, especially during Isoproterenol (Iso) challenge compared to ExWT. Despite fewer arrhythmias, confocal Ca2+ imaging revealed that ExCASQ2-/- ventricular cardiomyocytes are prone to spontaneous Ca2+ sparks and waves even at baseline (compared to ExWT) along with a concomitant decrease in Ca2+ transient amplitude and SR Ca2+ load, both at baseline and during Iso challenge. Conclusions: Our results thus far indicate that AET partially improves exercise capacity and aerobic fitness in the CASQ2-/- mouse model of CPVT. Paradoxically, although arrhythmia incidence is reduced, RyR2 mediated dysfunctions in SR Ca2+ cycling are not normalized after 6 weeks of AET. Importantly, the parasympathetic tone is significantly enhanced in the ExCASQ2-/- mice particularly during Iso challenge. Ongoing studies will address mechanisms (SR protein expression, post translational modifications and pharmacological interventions to investigate the observed autonomic imbalance) that could underlie the intriguing effects of exercise in this model of CPVT.

Mitochondrial Abnormalities in a Mouse CPVT Model with RyR2 Loss-Of-Function Mutation

Mitochondria exchange matrix content in a unique and dynamic way by one of two mechanisms: a short-range direct interaction via closely spaced kissing junctions or long-range propagation of signal by the extrusion of extended nanotubular extensions (nanotunneling) (Huang et al 2013. Proc Natl Acad Sci U S A, 110(8): 2846-51). Both are normal, but infrequent features of cardiac myocytes. It is interesting to know whether this behavior may be affected by changes in Ca2+ homeostasis. We evaluated the cardiac ultrastructure in 7-month old heterozygous mice with a cardiac ryanodine receptor (RyR2) mutation (RyR2-A4860G) that depresses channel function and leads to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) without hypertrophy (Zhao et al., submitted). The mutation is embryonic lethal in mice homozygous for the mutation. In ventricular myocytes of heterozygotes mice (RyR2-A4860G+/−) it has a dual effect on SR Ca2+ release: an initial reduction due to lower RyR2 activity, linked to random bursts due to SR Ca2+ overload. The most striking and only structural alteration in cardiac myocytes in the mutant left ventricle is a clear increase in the frequency of long thin nano-tunneling mitochondrial extensions. The frequency of nanotunneling was ∼3 fold higher in mutant than in WT mice. Numerous small mitochondrial profiles are more frequent in mutant mice resulting in an average surface area of 0.17± 0.15 µm2 in mutant versus 0.28 ± 0.22 µm2 in WT (p<0.00001). This decrease is mostly due to sections through the thin tunneling extensions. The increased nanotunneling frequency would indicate an enhanced rate of long range intermitochondrial signal transfer. Interestingly, no activation of nanotunneling has been reported by EM in other CPVT models and/or in other alterations affecting proteins of CRU and Ca2+ homeostasis, making this a unique mitochondrial response.

Relative Contribution of Purkinje Fibers to CA2+-Dependent Arrhythmias in a Murine Model of CPVT

Background. Purkinje fibers (PFs) are critical for coordinated electrical excitation of the ventricles and thought to play a key role in abnormal impulse formation and ventricular arrhythmias. However, the arrhythmogenic properties of PFs as compared to ventricular tissue remain to be elucidated and were the focus of the present study.Results. To examine the arrhythmic potential of PFs vs ventricular tissue we performed Ca2+ and membrane potential (MP) imaging in PFs and ventricular tissue preparations derived from both wild-type (WT) mice and genetically modified mice prone to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). PFs and trabeculae dissected from the right ventricles were loaded with the voltage- and Ca2+-sensing dyes RH237 and Rhod-2, respectively. PFs were identified by the characteristic long, narrow shape and lack of T-tubules of Purkinje cells, as evidenced by membrane staining with RH237. In PFs and trabeculae preparations isolated from CPVT but not WT mice, exposure to the beta-adrenergic agonist isoproterenol resulted in frequent diastolic Ca2+ releases both spontaneous and triggered, that were associated with corresponding MP signals. Consistent with their ability to lead to triggered events, diastolic spontaneous Ca2+ releases showed high synchronicity between adjacent cells in both tissues. Overall spontaneous Ca2+ release synchronicity and the rates of occurrence of spontaneous and triggered Ca2+ release events were similar between PFs and trabeculae.Conclusion. These results suggest that the Purkinje system and ventricular tissue possess similar arrhythmic potentials in a setting of CPVT, consistent with a diffuse nature of the genesis of Ca2+-dependent ventricular arrhythmias.

Post-natal heart adaptation in a knock-in mouse model of calsequestrin 2-linked recessive catecholaminergic polymorphic ventricular tachycardia

Cardiac calsequestrin (CASQ2) contributes to intracellular Ca2+ homeostasis by virtue of its low-affinity/high-capacity Ca2+ binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation–contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2R33Q/R33Q and of CASQ2−/− mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2−/− mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.

Partial and Controlled Depletion of Sarcoplasmic Reticulum (SR) Calcium Prevents Spontaneous Calcium Release and Tachyarrhythmias in a Mouse Model of CPVT

Background SR Ca 2+ ([Ca 2+ ]SR) overload during β-adrenergic stimulation precipitates spontaneous Ca 2+ release (SCR) and tachyarrhythmias in cardiomyocytes harboring catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked RyR2 mutations. We sought to determine whether imperacalcin (IpCa), a cell-permeable high-affinity agonist of RyR2 channels, prevents SCR and tachyarrhythmias in a mouse model of CPVT (RyR2-R4496C +/– ) by partially depleting [Ca 2+ ]SR during β-adrenergic stimulation. Methods Ca 2+ imaging was conducted by confocal microscopy in isolated ventricular myocytes from wild-type (WT) and RyR2-R4496C +/– mice. ECGs and left ventricular (LV) pressure were monitored in Langendorff-perfused beating WT and RyR2-R4496C +/– hearts stimulated with 300 nM isoproterenol (Iso). Cell penetration and topologic localization of IpCa was conducted in rat ventricular myocytes using Alexa546-IpCa. Results Alexa546-IpCa penetrated intact cardiomyocytes and localized in a striated pattern with ~2 μm interspacing, consistent with z-line labeling (n = 6 cells). Rapid perfusion of IpCa (1 µM) onto intact WT cardiomyocytes abruptly increased and then steadily decreased Ca 2+ transient amplitude, reflecting initial opening of RyR2 channels that gradually leads to partial depletion of [Ca 2+ ]SR (caffeine-induced Ca 2+ transient=7.6 ± 1.2 and 4.5 ± 0.82 F/Fo← before and after IpCa, respectively, n=15 cells, P = .001). The frequency of SCR waves in cardiomyocytes subjected to a stress protocol (5-second train of stimulations at 3 Hz under 300 nM Iso) was 3.6 ± 1.4 and 8.6 ± 5.3 waves/min in WT and RyR2-R4496C +/– cells, respectively, but decreased dramatically to 1.1 ± 0.9 and 1.6 ± 1.5 waves/min, respectively, after incubation with 1 µM IpCa (n = 10 cells each, P = 0.001). RyR2-R449C +/– hearts showed 56 ± 39 ectopic beats/min immediately ( Conclusions Partial depletion of [Ca 2+ ]SR with IpCa is a promising therapeutic alternative for Ca 2+ -triggered arrhythmias.

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.

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Abnormal Propagation of Calcium Waves and Ultrastructural Remodeling in Recessive Catecholaminergic Polymorphic Ventricular Tachycardia

The recessive form of catecholaminergic polymorphic ventricular tachycardia is caused by mutations in the cardiac calsequestrin-2 gene; this variant of catecholaminergic polymorphic ventricular tachycardia is less well characterized than the autosomal-dominant form caused by mutations in the ryanodine receptor-2 gene. We characterized the intracellular Ca²⁺ homeostasis, electrophysiological properties, and ultrastructural features of the Ca²⁺ release units in the homozygous calsequestrin 2-R33Q knock-in mouse model (R33Q) R33Q knock-in mouse model. We studied isolated R33Q and wild-type ventricular myocytes and observed properties not previously identified in a catecholaminergic polymorphic ventricular tachycardia model. As compared with wild-type cells, R33Q myocytes (1) show spontaneous Ca²⁺ waves unable to propagate as cell-wide waves; (2) show smaller Ca²⁺sparks with shortened coupling intervals, suggesting a reduced refractoriness of Ca²⁺ release events; (3) have a reduction of the area of membrane contact, of the junctions between junctional sarcoplasmic reticulum and T tubules (couplons), and of junctional sarcoplasmic reticulum volume; (4) have a propensity to develop phase 2 to 4 afterdepolarizations that can elicit triggered beats; and (5) involve viral gene transfer with wild-type cardiac calsequestrin-2 that is able to normalize structural abnormalities and to restore cell-wide calcium wave propagation. Our data show that homozygous cardiac calsequestrin-2-R33Q myocytes develop spontaneous Ca²⁺ release events with a broad range of intervals coupled to preceding beats, leading to the formation of early and delayed afterdepolarizations. They also display a major disruption of the Ca²⁺ release unit architecture that leads to fragmentation of spontaneous Ca²⁺ waves. We propose that these 2 substrates in R33Q myocytes synergize to provide a new arrhythmogenic mechanism for catecholaminergic polymorphic ventricular tachycardia.

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

Calsequestrin-2 (CASQ2) is a Ca(2+) 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. 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. 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.

Calcium Overload Reduces Flecainide Efficacy Against Spontaneous Calcium Release

The antiarrhythmic drug flecainide is effective in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by mutations in RyR2 Ca-release channels or calsequestrin (Casq2). Flecainide inhibits RyR2 channels and blocks spontaneous Ca-release (SCR) in Casq2-knockout (KO) cardiomyocytes, a CPVT model. However, a recent report failed to find flecainide efficacy against SCRs in cardiomyocytes from another CPVT model, RyR2R4496C+/- mice. To address this controversy, we compare the effect of flecainide in Casq2-KO and RyR2-R4496C myocytes under identical conditions. Method/Results: Ventricular myocytes from Casq2-KO and RyR2R4496C+/- mice were loaded with Fura2-AM to measure intracellular [Ca]. After 30min exposure to flecainide (FLEC, 6μM) or vehicle (VEH), myocytes were field-stimulated at 1Hz in presence of isoproterenol (1μM). SCRs were quantified during a 40s pause post-pacing. The flecainide effect was strongly dependent on extracellular [Ca]. At 2mM Ca, only Casq2-KO myocytes exhibited SCRs, which were strongly suppressed by flecainide (Figure). At 3mM Ca, both groups had frequent SCRs. Flecainide had reduced efficacy in Casq2-KO but was highly effective in RyR-R4496C myocytes (Figure). Conclusion: Flecainide inhibits SCRs in all CPVT models tested. However, flecainide efficacy is reduced by Ca overload, which could explain the divergent literature reports.

Abstract 18375: A Luminal Calcium Sensing Deficient Mutation of the Cardiac Ryanodine Receptor Diminishes Stress-Induced Ventricular Tachycardias in Calsequestrin Null Mice

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with syncope and sudden cardiac death. Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) have been linked to CPVT. The sarcoplasmic reticulum (SR) calcium content and the sensitivity of RyR2 to activation by luminal calcium are some of the key factors determining the propensity for Store Overload Induced Calcium Release (SOICR) and SOICR-evoked VTs. We have recently found that the residue E4782 located in the helix bundle crossing (the ion gate) of the RyR2 calcium release channel acts as a SR store calcium sensor responsible for luminal calcium regulation of RyR2 and SOICR. Interestingly, we found that E4782Q mutation completely protected against SOCR-evoked VTs in a CPVT mouse model harboring the disease-causing RyR2 mutation R4496C. In this present study, we determined whether the E4782Q mutation is capable of rescuing the VT phenotype of the CASQ2 null mice, another mouse model of CPVT. We bred E4872Q+/- mice with the CASQ2 null mice to produce CASQ2-/- or CASQ2+/- with or without the heterozygous E4872Q mutation. After the injection of epinephrine (1.6mg/kg) and caffeine (120mg/kg), CASQ2-/- / E4872Q-/- and CASQ2+/- / E4872Q -/- mice showed long lasting VTs during 30-min of ECG recording (83% and 33%, respectively). On the other hand, CASQ2-/- / E4872Q+/- and CASQ2+/- / E4872Q -/- displayed little or no VTs (6.5% and 0.8%, respectively, p &lt; 0.001, when compared with CASQ2-/- / E4872Q -/-, and CASQ2+/- /E4872Q -/-, n=7~9, for each group). Thus, the E4872Q mutation nearly completely rescued the CPVT phenotype in CASQ2 null mice. These observations indicate that targeting the luminal calcium sensor of the RyR2 calcium release channel represents a new and promising therapeutic strategy for treating CPVT and other calcium mediated cardiac arrhythmias.

Successful treatment of catecholaminergic polymorphic ventricular tachycardia with flecainide: a case report and review of the current literature

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease that can cause sudden cardiac death due to ventricular fibrillation (VF). While pharmacological therapy with beta-blockers and/or Ca(2)(+) antagonists is often unreliable, a recent study has demonstrated that flecainide can effectively suppress arrhythmia in a murine model of CPVT as well as clinically in two human subjects suffering from CPVT. We here present the case of an 11-year-old boy suffering from CPVT-1 as well as a review of the current relevant literature. After resuscitation due to VF at age 9, an automated implantable cardioverter-defibrillator (ICD) was implanted in 2007. Under beta-blocker therapy, repeated shocks were delivered due to either fast ventricular tachycardia (VT) or VF. This persisted under additional therapy with verapamil. Implantable cardioverter-defibrillator routine interrogations showed frequent non-sustained VT with an average of 8.8 per day. Additionally, the patient suffered from impaired physical performance due to decreased chronotropic competence. In July 2009, flecainide was added to the beta-blocker/verapamil regimen, resulting in a plasma level of 0.20 mg/L. No ICD shock or sustained VT occurred until December 2010. Genetic testing revealed an RyR2 receptor mutation. The case demonstrates the challenge of diagnosis and management of CPVT. It furthermore supports recent experimental evidence that the class 1 antiarrhythmic drug flecainide can suppress CPVT. The presented case supports a novel strategy in treating CPVT with the class I antiarrhythmic agent flecainide.

Inhibition of Cardiac Ca 2+ Release Channels by Class I Anti Arrhythmic Drugs as Therapy for Arrhythmia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia resulting from mutations in either the cardiac Ca2+ release channel (RyR2) or calsequestrin. Flecainide is a class-Ic compound that is highly effective in suppressing arrhythmia in CPVT patients and a calsequestrin-null mouse model of CPVT. Its efficacy is a combination of reduced membrane excitability via its known Na+ channel block and stabilisation of SR Ca2+ release by a recently discovered RyR2 block. Here we report RyR2 blocking kinetics of other class I anti arrhythmic drugs.RyR2 was isolated from sheep and human hearts, incorporated in lipid bilayers and investigated by single-channel recordings in presence of diastolic Ca2+ (100 nM cytoplasmic, 0.1 mM luminal). The class 1c drugs, propafenone, flecainide and encainide decreased RyR open times (IC50 = 17.3 ± 1.6, 16.7 ± 4 and 22.5 ± 1.2 iM respectively) whereas classes Ia and Ib had no significant effect up to 50 µM.Blocking kinetics was also measured in fully activated RyR2 (1 mM Ca2+, 2 mM ATP). All class I drugs tested caused voltage-dependent induction of subconductance states with similar actions from luminal and cytoplasmic sides. However, class Ic drugs produced subconductances with longer duration and lower conductance than classes 1a and 1b. These differences may explain the different efficacy of these drugs to curb Ca2+ release in diastole and prevent arrhythmia.Our results suggest that the efficacy of class I drugs in preventing CPVT depends on a reduction in RyR2 open time with a short blocking duration; kinetics typical of low affinity binding. This may lead to a paradigm shift in drug development process by directing strategies away from discovering high affinity compounds.

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.

Computational modelling of the initiation and development of spontaneous intracellular Ca 2+ waves in ventricular myocytes

Intracellular Ca(2+) dynamics provides excitation-contraction coupling in cardiac myocytes. Under pathological conditions, spontaneous Ca(2+) release events can lead to intracellular Ca(2+) travelling waves, which can break, giving transitory or persistent intracellular re-entrant Ca(2+) scroll waves. Intracellular Ca(2+) waves can trigger cellular delayed after-depolarizations of membrane potential, which if they occur in a cluster of a few hundred neighbouring myocytes may lead to cardiac arrhythmia. Quantitative prediction of the initiation and propagation of intracellular Ca(2+) waves requires the dynamics of Ca(2+)-induced Ca(2+) release, and the intracellular spatial distribution of Ca(2+) release units (CRUs). The spatial distribution of ryanodine receptor clusters within a few sarcomeres was reconstructed directly from confocal imaging measurements. It was then embedded into a three-dimensional ventricular cell model, with a resting membrane potential and simple stochastic Ca(2+)-induced Ca(2+) release dynamics. Isotropic global Ca(2+) wave propagation can be produced within the anisotropic intracellular architecture, by isotropic local Ca(2+) diffusion, and the branching Z-disc structure providing inter Z-disc pathways for Ca(2+) propagation. The branching Z-disc provides a broader spatial distribution of ryanodine receptor clusters across Z-discs, which reduces the likelihood of wave initiation by spontaneous Ca(2+) releases. Intracellular Ca(2+) dynamics during catecholaminergic polymorphic ventricular tachycardia (CPVT) was simulated phenomenologically by increasing the Ca(2+) sensitivity factor of the CRU, which results in an increased rate of Ca(2+) release events. Flecainide has been shown to prevent arrhythmias in a murine model of CPVT and in patients. The modelled actions of flecainide on the time course of Ca(2+) release events prevented the initiation of Ca(2+) waves.

Purkinje cell calcium dysregulation is the cellular mechanism that underlies catecholaminergic polymorphic ventricular tachycardia

Inherited arrhythmias can be caused by mutations in the cardiac ryanodine receptor (RyR2). The cellular source of these arrhythmias is unknown. Isolated RyR2(R4496C) mouse ventricular myocytes display arrhythmogenic activity related to spontaneous Ca(2+) release during diastole. On the other hand, recent whole-heart epicardial and endocardial optical mapping data demonstrate that ventricular arrhythmias in the RyR2(R4496C) mouse model of catecholaminergic polymorphic ventricular tachycardia (CPVT) originate in the His-Purkinje system, suggesting that Purkinje cells, and not ventricular myocytes, may be the cellular source of arrhythmogenic activity. The relative effect of the RyR2(R4496C) mutation on calcium homeostasis in ventricular myocytes versus Purkinje cells is unknown. This study sought to determine which cardiac cell type is more severely affected, in terms of calcium handling, by expression of the RyR2(R4496C) mutant channel: the ventricular myocytes or the Purkinje cells. To discriminate Purkinje cells from ventricular myocytes, we crossed the RyR2(R4496C) mouse model of CPVT with the Cx40(EGFP/+) transgenic mouse. This genetic cross yields Purkinje cells that express eGFP, and therefore fluoresce green when excited by the appropriate wavelength; ventricular myocytes, which do not express connexin 40, are not green. Intracellular calcium was measured in each cell type using calcium-sensitive probes. Purkinje cells of the RyR2(R4496C) mouse model of CPVT show an approximately 2x greater rate (P < .05) and approximately 2x to 3x greater amplitude (P < .000001) of spontaneous calcium release events than ventricular myocytes isolated from the same heart. These results demonstrate that focally activated arrhythmias originate in the specialized electrical conducting cells of the His-Purkinje system in the RyR2(R4496C) mouse model of CPVT.

Calcium polymorphic ventricular tachycardia: a new name for CPVT?

This editorial refers to ‘Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse ardiomyocytes with a human CPVT mutation’ by S. Sedej et al ., pp. 50–59, this issue. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by adrenergically mediated ventricular tachyarrhythmia leading to syncope and sudden cardiac death.1 Patients with CPVT show ventricular tachycardia during exercise or emotional stress in the absence of any structural heart disease. Also, any increase in the levels of circulating catecholamines (during stress or exercise, i.e. β-adrenergic stimulation) leads to a bi-directional ventricular tachycardia. Since 2001, more than 70 mutations in either ryanodine receptors (RyR) or an RyR-associated protein [calsequestrin, involved in sarcoplasmic reticulum (SR) Ca2+ buffering] have been identified in CPVT families.2,3 During the last several years, the physiological consequences of such mutations have been mainly investigated in expression systems (e.g. HEK cells expressing mutant RyR).4,5 However, expression systems lack a cardiac intracellular environment (accessory proteins, cell structure, etc.), hampering a complete understanding of how such mutations induce cardiac arrhythmias in native cardiac myocytes. The development of the first knock-in mouse model of human CPVT (mutation in RyR at position R4496C) by Priori's group2 shows that the mouse phenotype has striking similarity with the clinical human CPVT symptoms. Since then, it has been possible to investigate the effect of …

Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in mice and humans

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited arrhythmia syndrome in which drug therapy is often ineffective. We discovered that flecainide prevents arrhythmias in a mouse model of CPVT by inhibiting cardiac ryanodine receptor-mediated Ca(2+) release and thereby directly targeting the underlying molecular defect. Flecainide completely prevented CPVT in two human subjects who had remained highly symptomatic on conventional drug therapy, indicating that this currently available drug is a promising mechanism-based therapy for CPVT.

Flecainide Inhibits Cardiac Ryanodine Channels And Spontaneous Sarcoplasmic Reticulum Calcium Release In Casq2 Null Myocytes

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome due to cardiac Ca2+ release channel (RYR2) or cardiac calsequestrin (CASQ2) mutations. VT is caused by spontaneous Ca2+-release from the sarcoplasmic reticulum (SR) that generates after-depolarizations and triggered beats during catecholamine surge. We recently found that flecainide, a class 1c Na+ channel blocker, suppressed ventricular arrhythmia in Casq2 null (Casq−/−) mice, a model of CPVT. Here, we investigated the effect of flecainide on on cardiac Ca2+ handling in Casq2−/− myocytes loaded with Fura-2 AM, and on sheep RyR2 RyR2 channels reconstituted in lipid bilayers.Results: In isoproterenol-stimulated Casq2−/− myocytes, flecainide (6 μmol/l) reduced triggered beats by over 70% (p<0.001). Unexpected for a Na+ channel blocker, flecainide also reduced SR Ca2+ leak (Ca2+ fluorescence ratio: vehicle: 0.12±0.01 vs. flecainide: 0.08±0.01, n=54 per group, p=0.02) and suppressed the rate of spontaneous Ca2+ releases (SCRs) from the SR (SCRs/min: vehicle: 48±5 vs. flecainide: 29±5, n=45 per group, p=0.006), suggesting flecainide directly inhibits SR Ca2+ release. Lipid bilayer experiments confirmed a direct action of flecainide on RyR2 SR Ca2+ release channels: Flecainide induced brief partial closures of channels to a substate with a conductance equal to 20% of the fully open state. On average, flecainide as low as 5 μmol/L caused a 4-fold increase in the frequency of closed events and caused a significant reduction in the open probability from control levels. The effect of flecainide was concentration dependent (IC50 ∼ 50 μmol/l) and fully-reversible upon washout. Flecainide also inhibited RyR2 channels activated by high luminal Ca2+.Conclusion: We report a heretofore unrecognized inhibitory action of flecainide on RyR2 channels, which together with flecainide's inhibition of Na+ channels may explain flecainide's effectiveness in preventing CPVT.Supported by R01-HL88635.