Marc Pourrier

The new antiarrhythmic drug vernakalant: ex vivo study of human atrial tissue from sinus rhythm and chronic atrial fibrillation

AIMS Vernakalant is a newly developed antiarrhythmic drug against atrial fibrillation (AF). However, its electrophysiological actions on human myocardium are unknown. METHODS AND RESULTS Action potentials (APs) and ion currents were recorded in right atrial trabeculae and cardiomyocytes from patients in sinus rhythm (SR) and chronic AF. Vernakalant prolonged early repolarization in SR and AF, but late only in AF. AP amplitude (APA) and dV/dtmax were reduced in a concentration- and frequency-dependent manner with IC50 < 10 µM at >3 Hz. Effective refractory period was increased more than action potential duration (APD) in SR and AF. INa was blocked with IC50s of 95 and 84 µM for SR and AF, respectively (0.5 Hz). Vernakalant did not reduce outward potassium currents compared with time-matched controls. However, area under the current-time curve was reduced due to acceleration of current decline with IC50s of 19 and 12 µM for SR and AF, respectively. Vernakalant had less effect on APD than the IKr blocker E-4031, blocked IK,ACh, and had a small inhibitory effect on IK1 at 30 µM. L-Type Ca(2+) currents (SR) were reduced with IC50 of 84 µM. CONCLUSION Rate-dependent block of Na(+) channels represents the main antiarrhythmic mechanism of vernakalant in the fibrillating atrium. Open channel block of early transient outward currents and IK,ACh could also contribute.

The molecular basis of high-affinity binding of the antiarrhythmic compound vernakalant (RSD1235) to Kv1.5 channels.

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients (J Am Coll Cardiol 44:2355–2361, 2004). Here, the molecular mechanisms of interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with those of the class IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and vacates the inner vestibule as the channel closes, and thus mutations were made, targeting residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5-selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild type and mutants was assessed by whole-cell patch-clamp experiments in transiently transfected human embryonic kidney 293 cells. The mutational scan identified several highly conserved amino acids, Thr479, Thr480, Ile502, Val505, and Val508, as important residues for affecting block by both compounds. In general, mutations in S6 increased the IC50 for block by vernakalant; I502A caused an extremely local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 predicted that, of these residues, only Thr479, Thr480, Val505, and Val508 are potentially accessible for direct interaction, and that mutation at additional sites studied may therefore affect block through allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel interactions.

Ranolazine improves diastolic function in spontaneously hypertensive rats

Diastolic dysfunction can lead to heart failure with preserved ejection fraction, for which there is no effective therapeutic. Ranolazine has been reported to reduce diastolic dysfunction, but the specific mechanisms of action are unclear. The effect of ranolazine on diastolic function was examined in spontaneously hypertensive rats (SHRs), where left ventricular relaxation is impaired and stiffness increased. The objective of this study was to determine whether ranolazine improves diastolic function in SHRs and identify the mechanism(s) by which improvement is achieved. Specifically, to test the hypothesis that ranolazine, by inhibiting late sodium current, reduces Ca(2+) overload and promotes ventricular relaxation and reduction in diastolic stiffness, the effects of ranolazine or vehicle on heart function and the response to dobutamine challenge were evaluated in aged male SHRs and Wistar-Kyoto rats by echocardiography and pressure-volume loop analysis. The effects of ranolazine and the more specific sodium channel inhibitor tetrodotoxin were determined on the late sodium current, sarcomere length, and intracellular calcium in isolated cardiomyocytes. Ranolazine reduced the end-diastolic pressure-volume relationship slope and improved diastolic function during dobutamine challenge in the SHR. Ranolazine and tetrodotoxin also enhanced cardiomyocyte relaxation and reduced myoplasmic free Ca(2+) during diastole at high-stimulus rates in the SHR. The density of the late sodium current was elevated in SHRs. In conclusion, ranolazine was effective in reducing diastolic dysfunction in the SHR. Its mechanism of action, at least in part, is consistent with inhibition of the increased late sodium current in the SHR leading to reduced Ca(2+) overload.

Atrial selective effects of intravenously administered vernakalant in conscious beagle dogs.

Vernakalant is a relatively atrial-selective antiarrhythmic drug approved for the conversion of recent onset atrial fibrillation in Europe and is under regulatory review in the United States. In this study, we examined the effects of intravenously administered vernakalant (5, 10, and 20 mg/kg) on blood pressure, heart rate, and the electrocardiogram in conscious male beagle dogs and compared them with those of orally administered dl-sotalol (32 mg/kg). Vernakalant had no consistent dose-dependent effects on the heart rate or mean arterial pressure. Although vernakalant inhibits IKr, it tended to decrease the QTc interval but only at the top dose and later time points. The most striking effect of vernakalant on the electrocardiogram was a dose-dependent and selective slowing of atrial conduction (P-wave duration), with no effect on ventricular conduction (QRS duration). In contrast, treatment with dl-sotalol resulted in a marked and statistically significant prolongation of PR and QTc intervals with no effect on QRS or P-wave duration, consistent with its known class II and III antiarrhythmic actions. These results provide further evidence that vernakalant is unlikely to alter ventricular refractoriness or conduction at plasma concentrations in excess of those necessary for conversion of atrial fibrillation to sinus rhythm in patients.

Cardiac Ryanodine Receptor (Ryr2)-mediated Calcium Signals Specifically Promote Glucose Oxidation via Pyruvate Dehydrogenase

Cardiac ryanodine receptor (Ryr2) Ca2+ release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause or consequence of heart failure. Previous in vitro studies revealed that Ca2+ flux into the mitochondria helps pace oxidative metabolism, but there is limited in vivo evidence supporting this concept. Here, we studied heart-specific, inducible Ryr2 haploinsufficient (cRyr2Δ50) mice with a stable 50% reduction in Ryr2 protein. This manipulation decreased the amplitude and frequency of cytosolic and mitochondrial Ca2+ signals in isolated cardiomyocytes, without changes in cardiomyocyte contraction. Remarkably, in the context of well preserved contractile function in perfused hearts, we observed decreased glucose oxidation, but not fat oxidation, with increased glycolysis. cRyr2Δ50 hearts exhibited hyperphosphorylation and inhibition of pyruvate dehydrogenase, the key Ca2+-sensitive gatekeeper to glucose oxidation. Metabolomic, proteomic, and transcriptomic analyses revealed additional functional networks associated with altered metabolism in this model. These results demonstrate that Ryr2 controls mitochondrial Ca2+ dynamics and plays a specific, critical role in promoting glucose oxidation in cardiomyocytes. Our findings indicate that partial RYR2 loss is sufficient to cause metabolic abnormalities seen in heart disease.

Comparison of the in vivo hemodynamic effects of the antiarrhythmic agents vernakalant and flecainide in a rat hindlimb perfusion model.

A series of in vivo experiments were conducted to compare the hemodynamic actions of vernakalant (a novel, relatively atrial selective, antiarrhythmic drug) to flecainide after infusion into the peripheral vasculature. Anesthetized rats were surgically prepared to have an extracorporeal perfusion circuit whereby blood in the abdominal aorta (distal to the renal arteries) was diverted to a constant flow pump and returned to the abdominal aorta at the same level allowing measurement of hindlimb vascular resistance. The effects of cumulative, ascending doses of intravenous vernakalant and flecainide on vascular resistance, after arterial pressures, and heart rate were measured. Blood samples were drawn following each dose to determine drug plasma concentrations. Vernakalant had no significant vasomotor effects on peripheral or systemic vasculature. In contrast, flecainide decreased peripheral vascular resistance (15% at 0.8 μg/mL) and systemic pressures (32% mean arterial pressure at 0.8 μg/mL) in a dose-dependent manner. At therapeutic plasma concentrations, vernakalant (1 μg/mL) had little effect on heart rate (-24 beats/min) or QRS intervals (+3.4 msec), whereas flecainide (0.8 μg/mL) significantly decreased heart rate (55 beats/min) and increased QRS intervals (9.9 msec). In conclusion, vernakalant did not have negative hemodynamic effects at therapeutic plasma concentrations in a rat hindlimb perfusion model.

Rate-Dependent Effects of Vernakalant in the Isolated Non-Remodeled Canine Left Atria Are Primarily Due to Block of the Sodium ChannelClinical Perspective

Background— Several clinical trials have shown that vernakalant is effective in terminating recent onset atrial fibrillation (AF). The electrophysiological actions of vernakalant are not fully understood. Methods and Results— Here we report the results of a blinded study comparing the in vitro canine atrial electrophysiological effects of vernakalant, ranolazine, and dl-sotalol. Action potential durations (APD50,75,90), effective refractory period (ERP), post repolarization refractoriness (PRR), maximum rate of rise of the action potential (AP) upstroke (Vmax), diastolic threshold of excitation (DTE), conduction time (CT), and the shortest S1-S1 permitting 1:1 activation (S1-S1) were measured using standard stimulation and microelectrode recording techniques in isolated normal, non-remodeled canine arterially perfused left atrial preparations. Vernakalant caused variable but slight prolongation of APD90 (P=not significant), but significant prolongation of APD50 at 30 &mgr;mol/L and rapid rates. In contrast, ranolazine and dl-sotalol produced consistent concentration- and reverse rate-dependent prolongation of APD90. Vernakalant and ranolazine caused rate-dependent, whereas dl-sotalol caused reverse rate-dependent, prolongation of ERP. Significant rate-dependent PRR developed with vernakalant and ranolazine, but not with dl-sotalol. Other sodium channel-mediated parameters (ie, Vmax, CT, DTE, and S1-S1) also were depressed significantly by vernakalant and ranolazine, but not by dl-sotalol. Only vernakalant elevated AP plateau voltage, consistent with blockade of ultrarapid delayed rectified potassium current and transient outward potassium current. Conclusions— In isolated canine left atria, the effects of vernakalant and ranolazine were characterized by use-dependent inhibition of sodium channel-mediated parameters, and those of dl-sotalol by reverse rate-dependent prolongation of APD90 and ERP. This suggests that during the rapid activation rates of AF, the INa blocking action of the mixed ion channel blocker vernakalant takes prominence. This mechanism may explain vernakalants anti-AF efficacy.

Rate-Dependent Effects of Vernakalant in the Isolated Non-Remodeled Canine Left Atria Are Primarily Due to Block of the Sodium ChannelClinical Perspective: Comparison With Ranolazine and dl-Sotalol

Background— Several clinical trials have shown that vernakalant is effective in terminating recent onset atrial fibrillation (AF). The electrophysiological actions of vernakalant are not fully understood. Methods and Results— Here we report the results of a blinded study comparing the in vitro canine atrial electrophysiological effects of vernakalant, ranolazine, and dl-sotalol. Action potential durations (APD50,75,90), effective refractory period (ERP), post repolarization refractoriness (PRR), maximum rate of rise of the action potential (AP) upstroke (Vmax), diastolic threshold of excitation (DTE), conduction time (CT), and the shortest S1-S1 permitting 1:1 activation (S1-S1) were measured using standard stimulation and microelectrode recording techniques in isolated normal, non-remodeled canine arterially perfused left atrial preparations. Vernakalant caused variable but slight prolongation of APD90 (P=not significant), but significant prolongation of APD50 at 30 &mgr;mol/L and rapid rates. In contrast, ranolazine and dl-sotalol produced consistent concentration- and reverse rate-dependent prolongation of APD90. Vernakalant and ranolazine caused rate-dependent, whereas dl-sotalol caused reverse rate-dependent, prolongation of ERP. Significant rate-dependent PRR developed with vernakalant and ranolazine, but not with dl-sotalol. Other sodium channel-mediated parameters (ie, Vmax, CT, DTE, and S1-S1) also were depressed significantly by vernakalant and ranolazine, but not by dl-sotalol. Only vernakalant elevated AP plateau voltage, consistent with blockade of ultrarapid delayed rectified potassium current and transient outward potassium current. Conclusions— In isolated canine left atria, the effects of vernakalant and ranolazine were characterized by use-dependent inhibition of sodium channel-mediated parameters, and those of dl-sotalol by reverse rate-dependent prolongation of APD90 and ERP. This suggests that during the rapid activation rates of AF, the INa blocking action of the mixed ion channel blocker vernakalant takes prominence. This mechanism may explain vernakalants anti-AF efficacy.

Vernakalant Blocks Kv4.3 Channels in The Open State Without Significant Modulation by KChIP2 Subunits

Vernakalant, a relatively atrial selective mixed ion channel blocker, rapidly converts atrial fibrillation to normal sinus rhythm in humans. Previous studies demonstrated that vernakalant blocks Kv4.3 but the state dependence of blockade and influence of KChIP2 were not determined. Kv4.3 ± KChIP2 was transfected in HEK cells and currents were recorded by whole-cell voltage clamp.Measured activation and inactivation kinetics and voltage dependence was consistent with current models of closed, open, open-inactivated, and closed-inactivated states. Vernakalant, with little effect on peak current (τact = 0.39 ± 0.02 ms), induced a very rapid initial decay (τass = 3.90 ± 0.21 ms) followed by the well-described fast (τfast = 39.9 ± 4.1 ms) and slow (τslow= 193 ± 22 ms) components of steady state inactivation. This indicates that vernakalant rapidly associates with the open state causing block (IC50 = 23.0 ± 5.1 μM). Tail currents following short periods of depolarization (+10 mv, 10 ms) insufficient to induce inactivation, initially decayed more rapidly in the presence of vernakalant. However the slow time constant (τdiss = 33.1 ± 5.9 ms, n=2) was much longer than deactivation (τdeact = 14.8 ± 1.8 ms, n=2) leading to crossover of tail currents. Thus, vernakalant rapidly associated with the open state to produce a drug blocked state and less rapidly dissociated back to the open state, which then deactivated to the closed state. Vernakalant did not affect recovery from inactivation. Co-expression with KChIP2 did not affect vernakalants potency (IC50 = 22.3 ± 4.7 μM). This is consistent with previous studies showing that KChIP2 modulates inactivation kinetics with little effect on activation kinetics.In conclusion, vernakalant rapidly blocks Kv4.3 in the open state and KChIP2 does not modulate the Kv4.3 block by vernakalant.