Björn C. Knollmann

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 Ca2+ 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.

Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia

Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca2+ storage protein essential for SR Ca2+ 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 Ca2+ 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 Ca2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca2+ leak, resulting in premature spontaneous SR Ca2+ 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 Ca2+ storage, lack of Casq2 also causes increased diastolic SR Ca2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

Flecainide therapy reduces exercise-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia

OBJECTIVES This study evaluated the efficacy and safety of flecainide in addition to conventional drug therapy in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT). BACKGROUND CPVT is an inherited arrhythmia syndrome caused by gene mutations that destabilize cardiac ryanodine receptor Ca(2+) release channels. Sudden cardiac death is incompletely prevented by conventional drug therapy with β-blockers with or without Ca(2+) channel blockers. The antiarrhythmic agent flecainide directly targets the molecular defect in CPVT by inhibiting premature Ca(2+) release and triggered beats in vitro. METHODS We collected data from every consecutive genotype-positive CPVT patient started on flecainide at 8 international centers before December 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 25 years; range 7 to 68 years; 73% female). Exercise tests comparing flecainide in addition to conventional therapy with conventional therapy alone were available for 29 patients. Twenty-two patients (76%) had either partial (n = 8) or complete (n = 14) suppression of exercise-induced ventricular arrhythmias with flecainide (p < 0.001). No patient experienced worsening of exercise-induced ventricular arrhythmias. The 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 months), 1 patient experienced implantable cardioverter-defibrillator shocks for polymorphic ventricular arrhythmias, which were associated with a low serum flecainide level. In 1 patient, flecainide successfully suppressed exercise-induced ventricular arrhythmias for 29 years. CONCLUSIONS Flecainide reduced exercise-induced ventricular arrhythmias in patients with CPVT not controlled by conventional drug therapy.

Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange– Nielsen Syndrome

KCNQ1 encodes KCNQ1, which belongs to a family of voltage-dependent K+ ion channel proteins. KCNQ1 associates with a regulatory subunit, KCNE1, to produce the cardiac repolarizing current, IKs. Loss-of-function mutations in the human KCNQ1 gene have been linked to Jervell and Lange–Nielsen Syndrome (JLNS), a disorder characterized by profound bilateral deafness and a cardiac phenotype. To generate a mouse model for JLNS, we created a line of transgenic mice that have a targeted disruption in the Kcnq1 gene. Behavioral analysis revealed that the Kcnq1−/− mice are deaf and exhibit a shaker/waltzer phenotype. Histological analysis of the inner ear structures of Kcnq1−/− mice revealed gross morphological anomalies because of the drastic reduction in the volume of endolymph. ECGs recorded from Kcnq1−/− mice demonstrated abnormal T- and P-wave morphologies and prolongation of the QT and JT intervals when measured in vivo, but not in isolated hearts. These changes are indicative of cardiac repolarization defects that appear to be induced by extracardiac signals. Together, these data suggest that Kcnq1−/− mice are a potentially valuable animal model of JLNS.

Flecainide inhibits arrhythmogenic Ca2+ waves by open state block of ryanodine receptor Ca2+ release channels and reduction of Ca2+ 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 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 flecainides 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.

Myofilament Ca2+ sensitization causes susceptibility to cardiac arrhythmia in mice

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 Ca2+ 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 Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by myofilament Ca2+ desensitization with blebbistatin. Ca2+ 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 Ca2+ 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 Ca2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.

Monophasic action potential recordings from intact mouse heart: validation, regional heterogeneity, and relation to refractoriness.

MAP Recordings from Mouse Heart. Introduction: The monophasic action potential (MAP) technique has been validated in humans and larger animals, but, in mice, MAP recordings available to date show little resemblance to the murine ventricular transmembrane action potential (TAP) measured by conventional microelectrodes. We developed a miniaturized MAP contact electrode technique to establish in isolated mouse hearts: (1) optimal electrode size; (2) validation against TAP; (3) relationship between repolarization and refractoriness; and (4) regional repolarization differences.

Remodelling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin

1 Overexpression of cardiac calsequestrin (CSQ) impairs Ca2+ signalling in murine myocytes, leading to marked cardiac hypertrophy. Here we report on contractile, histological and electrophysiological changes accompanying the development of cardiac hypertrophy and failure in CSQ‐overexpressing mice. 2 CSQ mice developed contractile dysfunction after 60 days of age, with only 40% survival at 6 months. Four‐ to 6‐month‐old CSQ mice revealed biventricular dilatation, cardiomyocyte hypertrophy, patchy interstitial fibrosis and tissue calcifications. 3 Cardiac hypertrophy of CSQ mice was accompanied by progressive P‐R and Q‐T interval prolongation, conduction blocks, 2‐fold prolongation of the ventricular action potential and increased cellular membrane capacitance. 4 Remodelling of ionic currents included marked reduction of both density and absolute magnitude of transient outward (Ito) and inward rectifying (IK1) K+ currents. The density, but not the absolute magnitude, of basal and isoproterenol (isoprenaline)‐stimulated Ca2+ current (ICa) was decreased by 42% and the inactivation kinetics of ICa were significantly slowed. Na+ current density was suppressed by 50%, but its steady‐state activation and inactivation were shifted to more positive potentials. The density of Na+‐Ca2+ exchange current was increased by 35%. 5 In CSQ but not in control myocytes dialysed with cAMP, isoproterenol continued to enhance ICa. This apparent lower responsiveness of ICa to cAMP could be reversed by the non‐hydrolysable cAMP analogue 8‐Br‐cAMP or the phosphodiesterase inhibitor IBMX, suggesting high phosphodiesterase activity of CSQ myocytes. 6 In young CSQ mice (< 60 days) with compensated cardiac hypertrophy, only Ito was significantly suppressed. All other currents remained relatively intact. 7 An increase in cardiac Ca2+‐storage capability by overexpression of CSQ results in a dilated cardiomyopathy with tissue fibrosis, calcifications, impaired β‐adrenergic signalling and progressive remodelling of ionic currents. The extent of the changes in ionic currents was age dependent.

Inhibition of Cardiac Ca2+ Release Channels (RyR2) Determines Efficacy of Class I Antiarrhythmic Drugs in Catecholaminergic Polymorphic Ventricular Tachycardia

Background— Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin (Casq2) and can be difficult to treat. The class Ic antiarrhythmic drug flecainide blocks RyR2 channels and prevents CPVT in mice and humans. It is not known whether other class I antiarrhythmic drugs also block RyR2 channels and to what extent RyR2 channel inhibition contributes to antiarrhythmic efficacy in CPVT. Methods and Results— We first measured the effect of all class I antiarrhythmic drugs marketed in the United States (quinidine, procainamide, disopyramide, lidocaine, mexiletine, flecainide, and propafenone) on single RyR2 channels incorporated into lipid bilayers. Only flecainide and propafenone inhibited RyR2 channels, with the S-enantiomer of propafenone having a significantly lower potency than R-propafenone or flecainide. In Casq2−/− myocytes, the propafenone enantiomers and flecainide significantly reduced arrhythmogenic Ca2+ waves at clinically relevant concentrations, whereas Na+ channel inhibitors without RyR2 blocking properties did not. In Casq2−/− mice, 5 mg/kg R-propafenone or 20 mg/kg S-propafenone prevented exercise-induced CPVT, whereas procainamide (20 mg/kg) or lidocaine (20 mg/kg) were ineffective (n=5 to 9 mice, P<0.05). QRS duration was not significantly different, indicating a similar degree of Na+ channel inhibition. Clinically, propafenone (900 mg/d) prevented ICD shocks in a 22-year-old CPVT patient who had been refractory to maximal standard drug therapy and bilateral stellate ganglionectomy. Conclusions— RyR2 cardiac Ca2+ release channel inhibition appears to determine efficacy of class I drugs for the prevention of CPVT in Casq2−/− mice. Propafenone may be an alternative to flecainide for CPVT patients symptomatic on &bgr;-blockers.

Ablation of triadin causes loss of cardiac Ca2+ release units, impaired excitation–contraction coupling, and cardiac arrhythmias

Heart muscle excitation–contraction (E-C) coupling is governed by Ca2+ release units (CRUs) whereby Ca2+ influx via L-type Ca2+ channels (Cav1.2) triggers Ca2+ release from juxtaposed Ca2+ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn−/−). The structure and protein composition of the cardiac CRU is significantly altered in Trdn−/− hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn−/− hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn−/− myocytes. CRU function is impaired in Trdn−/− myocytes, with reduced SR Ca2+ release and impaired negative feedback of SR Ca2+ release on Cav1.2 Ca2+ currents (ICa). Uninhibited Ca2+ influx via ICa likely contributes to Ca2+ overload and results in spontaneous SR Ca2+ releases upon β-adrenergic receptor stimulation with isoproterenol in Trdn−/− myocytes, and ventricular arrhythmias in Trdn−/− mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.