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Cardiovascular Research Advance Access first published online on March 26, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn075
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Antiarrhythmic properties of a rapid delayed-rectifier current activator in rabbit models of acquired long QT syndrome

Thomas G. Diness1,2,{dagger}, Yung-Hsin Yeh1,3,{dagger}, Xiao Yan Qi1, Denis Chartier1, Yukiomi Tsuji4, Rie S. Hansen2, Soren-Peter Olesen2, Morten Grunnet2 and Stanley Nattel1,*

1 Department of Medicine and Research Center, Montreal Heart Institute and Université de Montréal, 5000 Belanger Street East, Montreal, Quebec, Canada H1T 1C8
2 NeuroSearch A/S and Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark
3 First Cardiovascular Division, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan, Republic of China
4 Department of Cardiovascular Research, Institute of Environmental Medicine (RIEM), Nagoya University, Nagoya, Japan

* Corresponding author. Tel: +1 514 376 3330 (ext. 3990); fax: +1 514 376 1355. E-mail address: stanley.nattel{at}icm-mhi.org

Received 3 October 2007; revised 3 March 2008; accepted 4 March 2008

Time for primary review: 27 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conclusions
 Funding
 Supplementary material
 References
 
Aims: Impaired repolarization in cardiac myocytes can lead to long QT syndrome (LQTS), with delayed repolarization and increased susceptibility to Torsades de Pointes (TdP) arrhythmias. Current pharmacological treatment of LQTS is often inadequate. This study sought to evaluate the antiarrhythmic effect of a novel compound (NS1643) that activates the rapid delayed-rectifier K+ current, IKr, in two rabbit models of acquired LQTS.

Methods and results: We used two clinically relevant in vivo rabbit models of TdP in which we infused NS1643 or vehicle: (i) three-week atrioventricular block with ventricular bradypacing; (ii) dofetilide-induced IKr inhibition in methoxamine-sensitized rabbits. In addition, we studied effects on ionic currents in cardiomyocytes with IKr suppressed by bradycardia remodelling or dofetilide exposure. Bradypaced rabbits developed QT interval prolongation, spontaneous ventricular ectopy, and TdP. Infusion of NS1643 completely suppressed arrhythmic activity and shortened the QT interval; vehicle had no effect. NS1643 also suppressed ventricular tachyarrhythmias caused by infusion of dofetilide to methoxamine-sensitized rabbits, and reversed dofetilide-induced QT prolongation. NS1643 increased IKr in cardiomyocytes isolated from normal and bradycardia-remodelled rabbits by approximately 75% and 50%, respectively (P < 0.001 for each). Similarly, NS1643 restored IKr suppressed by 5 nmol/L dofetilide (tail current 0.28 ± 0.03 pA/pF pre-dofetilide, 0.20 ± 0.01 pA/pF in the presence of dofetilide, 0.27 ± 0.02 pA/pF after adding NS1643 to dofetilide-containing solution, P < 0.01).

Conclusion: Pharmacological activation of IKr reverses acquired LQTS and TdP caused by bradycardic remodelling and IKr-blocking drugs. IKr-activating drug therapy could be a potentially interesting treatment approach for LQTS.

KEYWORDS Ion channels; Antiarrhythmic agents; Arrhythmia (mechanisms); Long QT syndrome; Ventricular arrhythmias


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Conclusions
 Funding
 Supplementary material
 References
 
Long QT syndrome (LQTS) is caused by impaired ventricular repolarization associated with action potential duration (APD) prolongation, manifesting as an abnormally long QT interval and ventricular arrhythmias. LQTS can be inherited as an autosomal dominant (Romano-Ward syndrome)1,2 or recessive (Jervell and Lange-Nielsen syndrome)3 disorder due to loss-of-function mutations in cardiac K+-channel genes such as HERG, KCNQ1, KCNE1, or KCNE2 or gain-of-function mutations in the cardiac Na+ or Ca2+-channel genes, SCN5A or CACNA1C.4 Often, however, LQTS is acquired upon exposure to drugs that block cardiac K+-channels and/or cardiac disease.5 Delayed repolarization favours the genesis of early after-depolarizations (EADs) and associated polymorphic Torsades de Pointes (TdP) tachyarrhythmias. Repolarization-impairing drugs and mutations amplify the electrical heterogeneities intrinsic to the ventricular myocardium, increasing the dispersion of repolarization, and creating a substrate for reentry.6

A variety of clinical approaches have been used to treat LQTS. Therapy of congenital LQTS presently reposes on two modalities: β-adrenoceptor blockers, which are most effective in Type 1-LQTS, and implantable defibrillators.7 Acquired LQTS is treated with intravenous (i.v.) magnesium sulfate, overdrive pacing, and occasionally isoproterenol.8 Certainly, there is room for improvement in treating LQTS: β-blockers and magnesium sulfate are often ineffective, implanted defibrillators can cause a variety of complications and frequent discharges can be a problem. One potentially interesting approach to LQTS therapy would be the use of a drug that directly targets the underlying physiological dysfunction, a deficiency in net repolarizing current. Several K+-channel agonist drugs have been developed recently, among which drugs like NS16439 that increase rapid delayed-rectifier current (IKr) conductance in ERG-expressing cells are of potential interest in LQTS.

Rabbits with complete atrioventricular block (AVB) develop QT prolongation and frequent spontaneous TdP episodes, apparently because of downregulation of ERG/IKr.10 Similarly, IKr blockers cause QT prolongation and TdP in rabbits, particularly in the presence of the {alpha}-adrenoceptor agonist methoxamine, producing a model that shares many features with clinical drug-induced LQTS.11 The present study was designed to investigate the ability of acute NS1643 administration to reverse the electrophysiological and proarrhythmic manifestations of these two models of IKr-related acquired LQTS in vivo, and to relate any actions to changes in the corresponding IKr recordings in vitro.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Conclusions
 Funding
 Supplementary material
 References
 
Animal handling followed the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996); all procedures were approved by the Animal-Experimentation Ethics Committee of the Montreal Heart Institute and University of Copenhagen. Female New Zealand white rabbits were used for all studies.

2.1 Whole-cell patch-clamp
Rabbit ventricular myocytes were obtained and isolated as described previously.10 After cervical dislocation, hearts where quickly excised and perfused in Langendorff mode with oxygenated Ca2+-free Tyrode's solution at 38°C for 5 min, followed by perfusion with Ca2+-free Tyrode's solution containing 0.8% collagenase type 2 (Worthington) for 20–30 min. Tyrode's solution contained (mmol/L): NaCl, 136; KCl, 5.4; MgCl2, 1; CaCl2, 1; NaH2PO4, 0.33; HEPES 5; dextrose 10 (pH 7.35, NaOH). Isolated cardiomyocytes were kept in storage solution containing (mmol/L): KCl, 20; KH2PO4, 10; dextrose, 10; mannitol, 40; L-glutamic acid, 70; β-OH-butyric acid, 10; taurine, 20; EGTA, 10; 0.1% bovine serum albumin (pH 7.3, KOH). Currents were recorded with whole-cell patch-clamp at 36 ± 0.5°C with Na+- and K+-free extracellular solutions containing (mmol/L) N-methyl-D-glucamine (NMDG) chloride, 149; MgCl2, 5; HEPES, 5; CaCl2, 0.9 to eliminate INa and inward-rectifier K+-current (IK1). L-type Ca2+-current was blocked with 10 µmol/L of nifedipine. The pipette solution contained (mmol/L): K+-aspartate, 110; KCl, 20; MgCl2, 1; Mg2+-ATP, 5; Li2+-GTP, 0.1; HEPES, 10; Na+-phosphocreatine, 5; EGTA, 10 (pH 7.3 with KOH). Borosilicate-glass electrodes (tip resistances of 1.5–3.0 M{Omega}) were used to record whole-cell currents. Cell capacitance and series resistance were compensated by approximately 55–70%. IKr was recorded in the presence of the highly selective IKs blocker HMR-1566 (0.5 µmol/L, Sanofi-Aventis, Frankfurt, Germany). Unless otherwise specified, chemicals were obtained from Sigma Chemicals, St Louis, MO.

2.2 Bradypaced atrioventricular block-rabbit model
Rabbits (1.8–2.5 kg) were anaesthetized with intramuscular (i.m.) ketamine (35 mg/kg) and xylazine (10 mg/kg). They were then artificially ventilated with O2-enriched air containing 2% isoflurane. AVB was created and a programmable unipolar right ventricular pacemaker (Vitatron, Arnhem, The Netherlands) was implanted as previously described.10 Rabbits received penicillin-G benzathine 75 000 IU + penicillin-G procaine 75 000 IU i.m. pre-operatively and two days post-operatively, as well as 78 µg of subcutaneous buprenorphine immediately post-operatively and 60 µg the next day.

AVB-rabbits were paced initially at 130 b.p.m. for three days. The pacing rate was decreased to 90 b.p.m. on day 3 and 60–90 b.p.m. on day 7. If a ventricular escape rhythm faster than 90 b.p.m. was observed, rabbits were demand-paced at 40 b.p.m.

Lead-II echocardiograms (ECGs) were recorded under ketamine (25 mg/kg)/xylazine (5 mg/kg) anaesthesia on days 3, 7, 14, and 21 post-operatively. When recurrent TdP and/or frequent ventricular ectopy were observed, rabbits were re-anaesthetized with i.m. ketamine/xylazine (25/5 mg/kg initially, followed by 17/3 mg/kg every 30 min). After 30 min of stabilization, baseline arrhythmias were recorded for 9 min. Thereafter, NS1643 (1,3-Bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea) synthesized in-house at NeuroSearch A/S, Ballerup, Denmark (1.5 mg/kg/min) or vehicle (50% polyethylene glycol 400/50% isotonic glucose at the equivalent infusion rate, 2 mL/kg/h) was infused for 45 min (Supplementary material online, Figure S1A). At the end of all in vivo non-survival studies, rabbits were euthanized with pentobarbital.

2.3 Dofetilide-induced LQTS
Rabbits (1.8–2.2 kg) were anaesthetized with i.m. ketamine/xylazine (35/10 mg/kg initially, followed by 17/3 mg/kg every 30 min). A first series of rabbits was studied to evaluate effects on dofetilide-induced QT-prolongation in the absence of arrhythmias. After 15 min of baseline ECG recording, dofetilide in acidified saline solution (pH 4) was infused over 4 min at 150 µg/kg/min. Five minutes later, NS1643 (1.5 mg/kg/min) or equivalent volumes of vehicle were infused (Supplementary material online, Figure S1B). QT intervals were measured every third minute, as an average of four successive QT intervals, and corrected for heart rate with the mean of the four corresponding RR intervals. The rate-corrected QT (QTC) was calculated according to Carlsson's formula: QTC = QT – 0.175(RR – 300).11

To study effects on dofetilide-induced arrhythmias, a methoxamine-sensitized rabbit model was adapted from Carlsson et al.12 Rabbits (2.8–3.2 kg) were anaesthetized with i.m. ketamine/xylazine (35/7 mg/kg followed by 17/3 mg/kg every 30 min). Lead-II ECGs were recorded. Methoxamine in isotonic saline solution was infused at 17 µg/kg/min. Simultaneously, vehicle or NS1643 (1.2 mg/kg/min) were infused. Beginning 15 min later, dofetilide in acidified saline solution (pH 4) was co-infused for 35 min at 0.03 mg/kg/min (Supplementary material online, Figure S1C).

2.4 Data analysis
Clampfit 6.0 (Axon Instruments, Foster City, CA) and GraphPad Prism 4 (GraphPad, San Diego, CA) were used for data analysis. All data are expressed as mean ± SEM. A P < 0.05 was considered statistically significant. For I–V curves, two-way repeated measures analysis of variance (ANOVA) was performed using mixed model methodology with NS1643 and membrane potential as main effects. In the case of a significant interaction between the two main effects, Bonferroni-corrected t-tests were used to compare individual mean values. The mixed procedure and multiple comparisons were used to evaluate differences in current amplitudes at different membrane potentials in the absence and presence of NS1643. Current amplitudes in the presence of NS1643 and NS1643/dofetilide were compared with baseline of one-way ANOVA followed by Tukey's post hoc test. One-way ANOVA with repeated measures was used to compare arrhythmias and QT intervals during infusion to baseline values, with Dunnett's post-test. AVB rabbits were evaluated the same way. Non-linear curve fitting and linear regression were performed by least-squares methods. In methoxamine-sensitized rabbits, two-way ANOVA was used to evaluate the effect of dofetilide infusion and of NS1643 on ectopic beat and ventricular tachycardia (VT) prevalence.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Conclusions
 Funding
 Supplementary material
 References
 
3.1 Effects on IKr under conditions causing acquired long QT syndrome
Reductions in IKr due to transcriptional downregulation appear to be an important underlying cause of repolarization and rhythm abnormalities in the AVB-rabbit model.10 To determine whether NS1643 can restore IKr current density in this model, we studied the effect of the drug on ventricular cardiomyocytes. Figure 1A shows IKr recordings before and after 10 µmol/L NS1643. NS1643 increased currents activated by the depolarizing pulse, as well as tail currents recorded upon repolarization. In control rabbits, NS1643 significantly increased the density of IKr tails recorded at –50 mV for all test-pulse voltages between 0 and +50 mV (Figure 1B). For example, for a step to +50 mV, tail currents increased by approximately 75%, from 0.28 ± 0.08 to 0.49 ± 0.12 pA/pF, P < 0.001. NS1643 did not significantly change voltage-dependence of IKr activation (Figure 1C): V1/2 was –4.5 ± 2.3 mV and –7.7 ± 2.6 mV before and after NS1643, respectively (P = NS). Similarly, deactivation time-constants were unaltered: slow and fast time-constants at –50 mV averaged 917 ± 102 and 122 ± 13 ms, respectively, in the absence and 872 ± 159 and 107 ± 5 ms in the presence of NS1643 (n = 5 cells/group, P = NS for both). Qualitatively similar results were obtained in cardiomyocytes from AVB-rabbits (Figure 1D and E). For example, following steps from +50 to –50 mV, the tail current amplitude increased by approximately 50%, from 0.20 ± 0.05 to 0.30 ± 0.05 pA/pF, P < 0.001 (Figure 1D), returning values to the same range as the average control currents in normal-rabbit cardiomyocytes. As in normal-rabbit cells, activation voltage-dependence and deactivation kinetics were not influenced by NS1643. V1/2 averaged –5.0 ± 2.2 mV and –3.2 ± 2.3 mV before and after NS1643, respectively (P = NS for both). Deactivation time-constants averaged: {tau}slow 899 ± 116 vs. 789 ± 103 (n = 6, P = NS); {tau}fast 148 ± 20 vs. 144 ± 27 ms (n = 6/group, P = NS) in the absence and presence of NS1643, respectively.


Figure 1
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  		Figure 1 Effects of NS1643 on IKr in isolated ventricular cardiomyocytes from normal and atrioventricular-blocked (AVB) rabbits. (A) Representative IKr recordings at baseline and after superfusion with 10 µmol/L NS1643. (B) I–V curves for IKr tail currents at baseline and after 10 µmol/L NS1643 in control rabbits. NS1643 significantly increased IKr at all voltages ≥0 mV. *P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline at same voltage. (C) Voltage-dependent activation in control rabbits based on normalized tail currents. Data points were fitted by Boltzmann functions. All data in (B) and (C) are from n = 5 cells isolated from three different control rabbits. (D) IKr tail currents at baseline and after 10 µmol/L NS1643 in AVB-rabbit cardiomyocytes. **P < 0.01, ***P < 0.001 vs. baseline at same voltage. (E) Voltage-dependent activation in AVB-rabbit cells. Boltzmann function fits are shown. All data in (D) and (E) are from n = 5 cells isolated from three different AVB-rabbits.

 
The studies described above indicate that NS1643 can restore IKr that has been reduced by downregulation of IKr expression. We then addressed the issue of whether NS1643 can restore IKr that has been suppressed by pharmacological blockade. Isolated ventricular myocytes were exposed to 5-nmol/L dofetilide and, subsequently, to 10 µmol/L NS1643 in the continued presence of 5 nmol/L dofetilide. Figure 2A shows the time-dependent response in one cardiomyocyte, with corresponding original recordings shown in Figure 2B. Dofetilide reduced IKr, which was then restored by NS1643. Overall, 15 min exposure to dofetilide significantly reduced IKr by approximately 30%, from 0.28 ± 0.03 to 0.20 ± 0.01 pA/pF (Figure 2C). The subsequent co-application of NS1643 increased dofetilide-suppressed IKr by approximately 35%, from 0.20 ± 0.01 to 0.27 ± 0.02 pA/pF.


Figure 2
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  		Figure 2 Effects of NS1643 on dofetilide-suppressed IKr in ventricular cardiomyocytes from normal rabbits. (A) IKr tail current densities at baseline, after superfusion of 5 nmol/L dofetilide, and subsequent co-superfusion of 5 nmol/L dofetilide and 10 µmol/L NS1643 in one representative experiment. (B) Examples of original tail currents recorded at the end of baseline measurements (a), the end of dofetilide-only infusion (b), and at the end of dofetilide plus NS1643 superfusion (c) in the experiment illustrated in (A). (C) Mean ± SEM IKr tail current densities at –50 mV following a depolarizing pulse to +50 mV (**P < 0.01).

 
NS1643 can inhibit currents carried by KCNQ1/KCNE1 co-expressed subunits in Xenopus oocytes.9 Since one of our objectives was to relate in vitro findings to in vivo effects, we determined whether NS1643 alters IKs in rabbit cardiomyocytes. IKs was recorded with the voltage protocol illustrated in Figure 3A. The same solutions were used as for IKr recording, but HMR-1566 was replaced by 1 µmol/L dofetilide (Sequioa Pharmaceuticals, Gaithersburg, MD) in the superfusate. Robust IKs was recorded both before and after NS1643 superfusion (Figure 3A), and neither IKs step (Figure 3B) nor tail (Figure 3C) current densities were affected by NS1643.


Figure 3
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  		Figure 3 Effects of NS1643 on IKs. (A) Examples of IKs recordings (obtained with the voltage protocol shown in the inset) before and after 10 µmol/L NS1643 superfusion. (B) IKs step current and (C) IKs tail current densities (mean ± SEM) obtained from the types of recordings shown in (A). NS1643 had no significant effects on either of the densities. BL = baseline results.

 
3.2 Effects on arrhythmic activity in bradypaced rabbits
The left panel in Figure 4A shows an example of ventricular arrhythmias recorded from a bradypaced rabbit. The right panel depicts superimposed lead-II ECG recordings from an arrhythmic bradypaced rabbit before and after 35 min of infusion of NS1643. Grey and black lines indicate, respectively, ECGs recorded at baseline and during NS1643 infusion. QT-interval abbreviation by NS1643 is evident at the upper left. Prior to drug infusion, ventricular ectopic activity and short runs of TdP arose from the T-wave; no such events were observed in this rabbit after NS1643-administration.


Figure 4
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  		Figure 4 Arrhythmic activity in atrioventricular block bradycardia-remodelled arrhythmic rabbits during infusion of vehicle and NS1643. (A) Left – Continuous lead II ECG recording from a bradycardia-remodelled arrhythmic rabbit. Bigeminal activity is followed by an episode of Torsades de Pointes. Right—two superimposed lead II ECG recordings from an arrhythmic bradycardia-remodelled rabbit. Grey and black recordings indicate, respectively, ECGs at baseline and after 35 min of NS1643. Arrhythmic activity [premature ventricular complexes (PVCs)] is observed at baseline (upper right). The lower panel shows an example of ventricular tachycardia at baseline. (B) Mean (±SEM) PVC frequency in vehicle and NS1643 rabbits. Baseline values were not significantly different between vehicle and NS1643 groups. Vehicle-infused rabbits did not show any significant change in PVC frequency. NS1643 significantly decreased PVC frequency. **P < 0.01 vs. baseline. (C) In four of the six arrhythmic rabbits from each group it was possible to measure QT intervals throughout the experimental period. Baseline QT values were similar for vehicle and NS1643 groups. {dagger}P < 0.05; {dagger}{dagger}P < 0.01 vs. baseline in vehicle rabbits; **P < 0.01 vs. baseline in NS1643 rabbits.

 
Arrhythmic rabbits were randomly assigned to either vehicle or NS1643 groups (n = 6 each). Baseline ectopic-complex frequency (Figure 4B) did not differ between groups. Rabbits receiving vehicle showed no statistically significant time-dependent change. In contrast, the NS1643-infused group displayed a significant decrease in ectopic-complex prevalence beginning 15 min after infusion onset. The antiarrhythmic effect persisted throughout the infusion, with no ectopic complexes observed in the NS1643 treated group from the 24th minute (Figure 4B).

Because of frequent ectopic activity and TdP, it was possible to measure the QT interval throughout the experimental period in only four of the six arrhythmic rabbits in each of the groups. Four successive QT intervals were measured in each of the three-minute intervals during which ectopic beats were counted. Means during the 9 min before infusion-onset were used as baselines. There were no significant differences in baseline QT-values, with a baseline QT interval of 371 ± 11 ms (Figure 4C), slightly longer than the value reported by Tsuji et al.10 of approximately 350 ms. The vehicle-infused group showed an initial small QT-interval increase, with values returning to baseline thereafter. In the NS1643 group, statistically significant QT-interval decreases began 21 min after infusion-onset and progressed throughout the rest of the experiment.

We then sought to determine whether the antiarrhythmic effect of NS1643 was quantitatively related to QT-interval shortening, comparing arrhythmia and QT-interval changes in the four rabbits/group in which they could both be followed for most of the infusion period (Supplementary material online, Figure S2A and B), thus showing that the overall time course of arrhythmia suppression paralleled that of QT-interval changes in these rabbits. NS1643-induced statistically significant effects on ectopic-complex frequency (Supplementary material online, Figure S2A) and QT intervals (Supplementary material online, Figure S2B) began at the same time, the 24th minute of drug infusion. Quantitatively, changes in ectopic-complex frequency appear to be larger over the first 20 min than changes in QT interval. The relationship between QT interval and ectopic-complex frequency changes caused by the drug was pursued (Supplementary material online, Figures S2C and D), in an analysis of ectopic-beat frequency as a function of QT intervals (SEMs are omitted for clarity). Lines connecting points indicate the time-course of infusion. To address the correlation between arrhythmic activity and QT intervals, a log-transformation was performed on the number of ectopic beats (>1) and regression obtained (Supplementary material online, Figure S2D). For the vehicle-group, r2 = 0.13 and the slope did not significantly deviate from 0 (95% confidence interval: –0.003 to 0.014). In the NS1643-group, r2 was 0.90 and the slope (0.023 ± 0.003) significantly deviated from 0 (95% confidence interval 0.015–0.030), indicating a positive relationship between QT interval and ectopic-complex frequency and suggesting that the antiarrhythmic effect was related to QT-shortening.

3.3 Effects on dofetilide-induced QT-prolongation
We next addressed the effects of NS1643 on dofetilide-induced repolarization delays. Dofetilide caused a rapid increase in QT, RR, and QTC intervals (Figure 5A–C). NS1643 infusion progressively decreased QT and QTC intervals, with statistically significant reductions beginning at 30 and 36 min, respectively, after infusion onset (Figure 5A and C). QT and QTC intervals were not significantly altered by vehicle infusion. NS1634 also reversed dofetilide-induced RR-interval changes, but RR-intervals began to increase again after about 70 min (Figure 5B).


Figure 5
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  		Figure 5 Effect of NS1643 on QT, RR, and QTC intervals in dofetilide-induced long QT syndrome. After 15 min of baseline recording, dofetilide was infused over 4 min, followed 5 min later by infusion of either vehicle or NS1643. (A) Dofetilide caused rapid and substantial increases in QT intervals. In the NS1643-infused group, progressive QT-interval shortening began after approximately 20 min of infusion and continued throughout the rest of the experiment. The QT interval remained increased in the vehicle-infused group throughout the experiment. (B) Corresponding RR interval measurements. Dofetilide increased the RR interval. NS1643 reversed dofetilide-induced RR interval increases. (C) QTC intervals obtained with Carlsson's formula. *P < 0.05, **P < 0.01 vs. reference values.

 
3.4 Effects on dofetilide-induced Torsades de Pointes
In preliminary studies, the high mortality rate and variable response to dofetilide of methoxamine-sensitized rabbits proved to be a major obstacle to testing the efficacy of NS1643—many rabbits died before they could receive the compound. We therefore studied the ability of NS1643 to prevent dofetilide-induced TdP. Infusion of NS1643 or vehicle was begun at the same time as methoxamine, with dofetilide infusion started 15 min later. Figure 6 presents the results on a single rabbit (top panels) and group-mean (bottom panels) basis. Dofetilide induced TdP and premature ventricular complexes (PVCs) in all six (100%) of the vehicle-infused rabbits. One of these (Rabbit 4V, Figure 6A) died when TdP degenerated into ventricular fibrillation. Of the rabbits infused with NS1643 prior to and during dofetilide infusion, three of the six were completely devoid of arrhythmias (Rabbits 1–3N, Figure 6B), one had limited ectopy during the first 10 min of the dofetilide-infusion period (Rabbit 6N), one had two episodes of VT during the first 10 min (Rabbit 4N), and one had ectopic activity and VT at a level comparable with the controls (Rabbit 5N). Figure 6C and D shows the mean prevalence of PVCs and VT, respectively. NS1643 significantly reduced the prevalence of PVCs and VT (P < 0.001 for each, n = 6).


Figure 6
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  		Figure 6 Effect of NS1643 in methoxamine-sensitized rabbit model of Torsades de Pointes. Infusions of NS1643 or vehicle were begun before a continuous infusion of dofetilide. (A) Ectopic-complex prevalence in each rabbit (numbered IV–6V) of the group infused with the vehicle for NS1643. Rabbit 4V died from ventricular fibrillation at the eleventh minute. (B) Ectopic-complex prevalence for each rabbit (numbered 1N–6N) in the NS1643-infused group. (C) Mean ± SEM premature ventricular complex (PVC) prevalence. NS1643-treated rabbits showed significantly reduced PVC prevalence relative to vehicle-treated animals (P < 0.001). (D) Mean ± SEM ventricular tachycardia (VT) prevalence. NS1643 treatment significantly reduced VT prevalence relative to vehicle-treated animals (P < 0.001).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Conclusions
 Funding
 Supplementary material
 References
 
In this study, we investigated the antiarrhythmic effect of pharmacological activation of IKr in vivo. Two rabbit models representing clinically distinct forms of acquired LQTS, bradycardia-induced remodelling and selective pharmacological block of IKr, were studied. In bradypaced rabbits, NS1643 suppressed arrhythmic activity and normalized the QT interval. In rabbits at risk of arrhythmias caused by dofetilide-induced IKr block, NS1643 prevented the emergence of arrhythmic activity, and in rabbits with important dofetilide-induced QT-prolongation, NS1643 substantially decreased the QT interval. Consistent with its in vivo effects, NS1643 increased IKr in cardiomyocytes from bradycardia-remodelled rabbits and in cells exposed to dofetilide.

4.1 Relationship to previous studies of IKr activators
To date, descriptions of five ERG-channel activators have been published.9,13–16 A variety of mechanisms are responsible for increasing ERG current: RPR260243 and mallotoxin slow ERG-current deactivation,13,16 whereas NS1643 and NS3623 accelerate recovery from inactivation and positively shift the voltage-dependence of inactivation.9,15,17 NS3623 also slows inactivation. Four of the ERG current activators have been shown to shorten APD in guinea-pig ventricular cardiomyocytes.9,13–15 ECG waveforms recorded from Langendorff-perfused guinea-pig hearts and arterially perfused rabbit ventricular wedges show QT-interval shortening, both in the absence and presence of dofetilide.13,14 Our results add to the literature by demonstrating the ability of an IKr activator to suppress QT-prolongation and ventricular arrhythmias in vivo in two clinically relevant arrhythmia models. Furthermore, we demonstrate restoration of IKr in corresponding cardiomyocyte models, confirming the underlying ionic mechanism of action.

Activators of a variety of other cardiac K+-channels have previously been studied, including KATP and KCNQ1 activators.18,19 Both types of K+-channel openers decrease APD in cardiac myocytes.19,20 Cardiomyocytes isolated from rabbits with hypertensive left ventricular hypertrophy develop APD prolongation and EADs upon exposure to dofetilide: superfusion with a benzodiazepine IKs activator reverses APD prolongation and suppresses EADs.21 KATP channel activators suppress EADs induced in cardiac Purkinje fibres by caesium, quinidine, and sematilide, and prevent caesium-induced ventricular-tachyarrhythmia induction in rabbits.22 Nicorandil, a KATP channel opener and antianginal drug, suppresses repolarization abnormalities induced by phenylephrine infusion to LQT1 patients.23 The clinical use of KATP channel openers has been limited by risks of vasodilation-induced hypotension.

4.2 Novel findings and potential significance
This is to our knowledge the first study of an IKr-activating drug in vivo, as well as the first examination of effects of such drugs on native IKr currents in either normal or LQTS model cardiomyocytes. LQTS is a significant cause of sudden cardiac death. In addition to congenital LQTS, which can be caused by mutations of at least 10 different genes24 and the well-recognized acquired form caused by IKr-blocking drugs;25 similar pathophysiological mechanisms may contribute to sudden cardiac death in other contexts, including congestive heart failure,26 diabetic cardiomyopathy,27 and hypertrophic heart disease.28 The only widely used drug therapy for congenital LQTS is β-blockers, which can reduce the overall risk of syncope in LQTS patients.29,30 β-Blockers are most effective for LQT1 patients, with a significantly higher failure rate in LQT2 and LQT3 syndromes.30 In LQT3 patients, Na+-channel blockers like mexiletine may be useful by selectively blocking plateau Na+-current through abnormally inactivating Na+-channels.31 Defibrillators are often used for LQTS patients and have a low failure rate, but have a wide range of potential complications, and in some patients frequent recurrent arrhythmic events requiring shocks are problematic. Emergency therapy of drug-induced TdP is limited, with i.v. magnesium sulfate considered the treatment of choice but with variable efficacy and risk of neuromuscular depression with excessive doses. Our demonstration of the efficacy of an IKr-enhancing drug in two clinically relevant experimental paradigms suggest that this type of approach could be an interesting potential addition to the clinical armamentarium for LQTS, particularly for patients with an inadequate response to conventional therapy, and one that warrants further investigation.

The LQTS subtype-specificity of IKr-enhancing compounds remains to be determined. In the present study, we have used NS1643 in models of LQTS associated with IKr dysfunction. It is most likely that NS1643 acts by altering the biophysical properties of the remaining functional IKr-channels to acutely enhance their function. In unpublished studies in heterologous expression systems, we have found that NS1643 fails to rescue trafficking-deficient HERG mutants and that complete IHERG block by very high dofetilide concentrations is not reversed by NS1643. These results suggest that NS1643 efficacy in LQTS2 or acquired LQTS related to IKr-deficiency may be limited to cases with residual IKr-function. On the other hand, NS1643 might also be effective in LQTS1 and LQTS3 patients, as an agent to increase repolarization reserve through functional IKr channels.

4.3 Potential limitations
IKr-encoding mRNA is present in the sinus node of rabbits32 and blocking IKr suppresses the firing rate.33 We did observe significant heart-rate acceleration with NS1643, which could contribute to acceleration of repolarization but which could also lead to adverse effects. In addition, excess IKr agonism could be potentially arrhythmogenic if repolarization is excessively accelerated. Vigilance will be needed in clinical trials to detect such complications, if they arise.

The ideal IKr agonist for use in LQTS syndrome would be devoid of other actions. Potential effects of NS1643 have been tested on heterologously expressed KCNQ1, KCNQ1/KCNE1, Kv1.5, and Kv4.3 channels, as well as L-type Ca2+ and Na+ currents from guinea-pig ventricular myocytes.9 NS1643 has a minor blocking effect on KCNQ1 and KCNQ1/KCNE1 currents in Xenopus oocytes. In addition, NS1643 also slightly reduces Kv4.3 current. However, these effects are small and occur at higher concentrations than those that enhance ERG current. It is unlikely that these collateral actions contributed to the antiarrhythmic effect of NS1643 that we observed, since if anything they would be expected to delay repolarization. Nevertheless, if further investigation supports the potential value of IKr agonism for the treatment of arrhythmias related to delayed repolarization, chemical development to create more potent and selective IKr agonists might be of potential interest.

NS1643 had significant (albeit short-lasting) effects on heart rate (Figure 5B), transiently reversing the heart-rate slowing caused by dofetilide. The effects of NS1643 on heart rate in the absence of IKr blockers, the effects of chronic NS1643 (rather than simply acute administration as performed in this study), and potential interactions with medications, like β-blockers, frequently administered to LQTS patients, remain to be assessed in future work.

In addition to the absolute changes in repolarization delay, variations in spatial dispersion of repolarization may be a very important determinant of TdP occurrence. Changes in other indices, like repolarization dispersion, may contribute to the apparently larger alterations in ectopic-complex frequency vs. QT interval for the first 20 min after drug administration (Supplementary material online, Figure S2), although the lack of statistically significant change in either variable over this time period makes it difficult to draw conclusions. Technical limitations prevented us from measuring reliable indices of repolarization dispersion in the present study, but this will be an important issue to address in future work.


   Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conclusions
Funding
 Supplementary material
 References
 
We have shown that acute exposure to an IKr-enhancing drug abbreviates the QT interval and suppresses TdP arrhythmias in two clinically relevant animal models. Patch-clamp studies confirmed the ability of the drug to reverse IKr suppression in corresponding bradycardia-remodelled and dofetilide-exposed cardiomyocytes. Thus, pharmacological enhancement of IKr may be an interesting potential approach for patients with arrhythmic conditions caused by impaired cardiac repolarization.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conclusions
 Funding
Supplementary material
 References
 
Supported by the Canadian Institutes of Health Research, the Quebec Heart and Stroke Foundation, and the Danish National Research Foundation.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conclusions
 Funding
 Supplementary material
References
 
Supplementary Material is available at Cardiovascular Research Online.


   Acknowledgements
 
The authors thank Chantal St-Cyr for excellent technical help and France Thériault for secretarial help with the manuscript.

Conflict of interest. Thomas Goldin Diness, Rie Schultz Hansen, and Morten Grunnet have minor-equity holdings in NeuroSearch A/S and Soren-Peter Olesen is a consultant of NeuroSearch A/S, which has a patent on NS1643. The other authors have no potential conflicts of interest.


   Notes
 
{dagger} These authors contributed equally and should be considered to share first authorship. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conclusions
 Funding
 Supplementary material
 References
 

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