Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca 2+ storage protein essential for SR Ca 2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca 2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca 2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca 2+ leak, resulting in premature spontaneous SR Ca 2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca 2+ storage, lack of Casq2 also causes increased diastolic SR Ca 2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

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.CPVT is an inherited arrhythmia syndrome characterized by a normal baseline electrocardiogram (ECG), polymorphic ventricular tachycardia induced by adrenergic stress in the absence of structural heart disease, and a high mortality rate in young individuals1. Treatment with β-adrenergic blockers reduces arrhythmia burden and mortality but is not completely effective1, and implantable cardioverter defibrillators (ICDs) are used for the prevention of sudden death. However, painful appropriate or inappropriate defibrillation shocks can trigger further adrenergic stress and arrhythmias, and deaths have occurred despite appropriate ICD shocks2 , 3. In such instances, stellate ganglionectomy4 or even cardiac transplantation5 have been considered. Two CPVT disease-related genes have been identified: RYR2, encoding the cardiac ryanodine receptor Ca 2+ release channel (RyR2), and CASQ2, encoding cardiac calsequestrin 6,7 . Mutations in these genes destabilize the RyR2 Ca 2+ release complex 8,9 . In mice with CPVT-linked mutations, catecholamines cause spontaneous sarcoplasmic reticulum Ca 2+ release resulting in delayed after depolarizations (DADs), and they produce triggered activity in myocytes and polymorphic ventricular tachycardia in ©2009 Nature America, Inc. All rights reserved NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript vivo 10, 11. Here we identify a therapy that directly targets the underlying arrhythmia mechanisms: we found that flecainide, an approved antiarrhythmic drug known to block sodium channels, showed remarkable efficacy in suppressing spontaneous sarcoplasmic reticulum Ca 2+ release by inhibiting RyR2. Flecainide treatment completely prevented adrenergic stress-induced arrhythmias in a mouse model of CPVT and in humans with CASQ2 or RYR2 mutations that are refractory to standard drug treatment.The local anesthetic tetracaine has been used to inhibit RyR2 and suppress spontaneous sarcoplasmic reticulum Ca 2+ release in isolated myocytes 12 . However, tetracaine causes a rebound increase in sarcoplasmic reticulum Ca 2+ release events during prolonged exposure 13 , effective inhibitory concentrations 14 are too high for clinical use, and systemic administration is contraindicated in humans. We searched among clinically available antiarrhythmic drugs for a more useful RyR2 inhibitor and found that flecainide inhibited RyR2 more potently than tetracaine and by a different mechanism. Whereas tetrac...

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 Ca 2+ waves and elementary sarcoplasmic reticulum (SR) Ca 2+ release events, Ca 2+ 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 Ca 2+ leak or SR Ca 2+ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca 2+ leak, resulting in a significantly increased SR Ca 2+ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca 2+ 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 Ca 2+ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca 2+ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca 2+ waves resulting in triggered arrhythmias, such as CPVT.

In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca 2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca 2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca 2+ -sensitizing agent EMD 57033 and prevented by myofilament Ca 2+ desensitization with blebbistatin. Ca 2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca 2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca 2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.

Objective This study evaluated the efficacy and safety of flecainide in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) on top of conventional drug therapy. Background CPVT is an inherited arrhythmia syndrome caused by gene mutations that destabilize cardiac ryanodine receptor Ca2+ release channels. Sudden death is incompletely prevented by conventional drug therapy with β-blockers +/− Ca2+ channel blockers. The anti-arrhythmic agent flecainide directly targets the molecular defect in CPVT by inhibiting premature Ca2+ release and triggered beats in vitro. Methods We collected data from every consecutive genotype-positive CPVT patient started on flecainide at eight international centers before November 13, 2009. The primary outcome measure was the reduction of ventricular arrhythmias during exercise testing. Results Thirty-three patients received flecainide because of exercise-induced ventricular arrhythmias despite conventional, for different reasons not always optimal, therapy (median age of 25 years, range 7 to 68; 70% female). Exercise tests comparing flecainide with conventional therapy alone were available in 29 patients. Twenty-two (76%) patients had either a partial (n=8) or complete (n=14) suppression of exercise-induced ventricular arrhythmias by flecainide (p<0.001). No patient experienced worsening of exercise-induced ventricular arrhythmias. Median daily flecainide dose in responders was 150 mg (range 100 to 300 mg). During a median follow-up of 20 months (range 12 to 40) one patient experienced ICD shocks for polymorphic ventricular arrhythmias, which was associated with low flecainide levels. In one patient, flecainide successfully suppressed exerciseinduced ventricular arrhythmias for 29 years. Conclusions Flecainide reduced exercise-induced ventricular arrhythmias patients with CPVT uncontrolled by conventional drug therapy.