Atrial fibrillation (AF) increases mortality, especially in patients with congestive heart failure,1 and it may cause stroke.2 Consequently, AF is responsible for considerable morbidity and medical costs.3 Present therapy has major limitations. Drugs used to terminate AF, such as flecainide,4 sotalol, and dofetilide,5-7 have limited efficacy8 and may induce life-threatening ventricular arrhythmias. One approach to minimize side effects would be the development of drugs that act on potassium channels restricted to the atrium.
It was recently reported that the novel compound AVE0118 significantly prolonged the atrial effective refractory period (AERP) in anesthetized pigs without showing any effect on the QT interval of the ECG.9 Moreover, the drug completely prevented vulnerability to atrial fibrillation induced by an extrastimulus.10 Prolongation of AERP was also observed in conscious goats. Interestingly, this effect was more pronounced in animals with electrically remodelled atria than in animals in sinus rhythm. Again, in the goat model the effect of AVE0118 was atrial selective, because no prolongation of the QT interval occurred.11 Patch-clamp recordings in human atrial myocytes showed that the compound inhibits the transient outward current as well as the sustained outward current,12 and microelectrode studies in human atrial tissues indicated that AVE0118 elevated the plateau potential.13
We subsequently identified a compound with improved pharmacokinetic properties, AVE1231, and performed recordings on ion channels in expression systems and in atrial and ventricular myocytes. In addition, AVE1231 was investigated in 2 large animal models, the anesthetized pig and the instrumented conscious goat. In these animal models, atrial efficacy of AVE1231 was superior to that shown by dofetilide, an established antiarrhythmic agent. Atrial electrical remodeling, the shortening of atrial action potential and refractoriness that is due to the high atrial activation rate during AF, has been shown to modify the efficacy of antiarrhythmic drugs. While the effect of IKr-blockers strongly decreased during electrical remodeling, the effect of the early atrial K+-channel blocker AVE0118 was even increased.11 A preserved or even increased efficacy in electrical remodeling is expected to be highly beneficial for the prevention of recurrence of AF in the first days after cardioversion. Therefore, the atrial efficacy of AVE1231 was investigated both in sinus rhythm and after 72 h of atrial electrical remodeling in the instrumented goat.
Molecular Biology and Cell Culture
Human Kv1.5 cDNA (NM_002234) was subcloned into the eukaryotic expression vectors pcDNA3.1 and pcDNA3.1/zeo (Invitrogen, Groningen, Netherlands). cDNA encoding human Kv4.3 long (Kv4.3l, NM_004980)14 was subcloned into pcDNA3.1, and the cDNA encoding human KChIP2 short (KChIP2.2b, NM_173195)15 was subcloned into pcDNA3.1/zeo expression vector. Chinese hamster ovary (CHO) cells (American Type Culture Collection, Rockville, MD) were transfected with either hKv1.5 or with hKv4.3 and KChIP2.2b expression constructs. Transfection was carried out using lipofectamine (Life Technologies/Gibco BRL, Karlsruhe, Germany) according to the manufacturer's instructions. To boost Kv1.5 channel expression, CHO cells were consecutively transfected with both Kv1.5 expression constructs. Both hKv1.5 and hKv4.3 + hKChIP2.2b were stably expressed in CHO cells, which were maintained in ISCOVE's medium (Biochrom KG, Berlin, Germany), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 350 μg/mL Zeocin (Invitrogen), and 400 μg/mL G418 (PAA Laboratories).
Patch-Clamp Experiments in CHO Cells
Cells expressing hKv1.5 or hKv4.3 plus hKChIP2.2b were assayed using the standard whole-cell patch clamp technique.16 Cells were mechanically removed from the tissue culture flask and placed in a perfusion chamber with a solution containing (in mM): NaCl 140, KCl 4.7, CaCl2 2, MgCl2 1.1, HEPES 10, pH adjusted to 7.4 with NaOH. Patch pipettes were pulled from borosilicate glass capillaries and heat polished. After filling with (in mM) NaCl 10, KCl 120, EGTA 1, HEPES 10, MgCl2 1.1 (pH 7.2 with KOH), pipettes had resistances of 2-3 MΩ. For the recording of hKv1.5 and hKv4.3 + hKChIP2.2b currents, voltage pulses of 500 ms duration were applied from the holding potential of −80 mV to +40 mV at a frequency of 0.1 Hz. Data were recorded with an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany) and the Pulse software (HEKA Elektronik) and stored on a PC for later analysis. Series resistance was in the range of 4 to 9 MΩ and was compensated by 80% by means of the EPC-9's compensation circuit. The experiments were performed under continuous superfusion of the cells with solution heated to 36 ± 1°C.
Isolation of Porcine Atrial Myocytes
All investigations with animals conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-13, revised 1996) and were performed following approval by the Ethical Review Board of the State of Hessen and in accordance with the German animal protection law.
Male pigs (German Landrace) weighing 15 to 30 kg were anesthetized with pentobarbital as described previously.17 After a left thoracotomy, the lung was retracted, the pericardium incised, and the heart quickly removed and placed in oxygenated, nominally Ca2+-free Tyrode solution containing (in mM): NaCl 143, KCl 5.4, MgCl2 0.5, NaH2PO4 0.25, HEPES 5, and glucose 10, pH adjusted to 7.2 with NaOH. The hearts were then mounted on a Langendorff apparatus and perfused via the left circumflex coronary artery with Tyrode solution at 37°C with constant pressure (80 cm H2O). All coronary vessels descending to the ventricular walls were ligated, ensuring sufficient perfusion of the left atrium. When the atrium was clear of blood and contraction had ceased (∼5 min), perfusion was continued with the same Tyrode solution, which now contained 0.015 mM CaCl2 and 0.03% collagenase (type CLS II, Biochrom KG, Berlin, Germany), until atrial tissue softened (∼20 min). Thereafter, left atrial tissue was cut into small pieces and mechanically dissociated by trituration. Cells were then washed with storage solution containing (in mM): L-glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, MgCl2 1, glucose 10, HEPES 10, EGTA 2 (pH 7.2 with KOH), and then filtered through a nylon mesh. The isolated cells were kept at room temperature in the storage solution.
Isolation of Guinea Pig Ventricular Myocytes
Ventricular myocytes were isolated by enzymatic digestion as described previously.9 Briefly, Dunkin Hardley Pirbright White guinea pigs (weight, ∼400 g) were sacrificed by concussion and cervical dislocation. Immediately after opening of the thorax, the hearts were removed and placed into ice-cold isotonic saline. The hearts were dissected and perfused retrogradely via the aorta at 37°C with the same solutions as used for isolation of pig atrial myocytes.
Electrophysiological Recordings From Cardiac Myocytes
Whole-cell currents were recorded with an EPC-9 patch clamp amplifier (HEKA Elektronik) as described above for CHO cells. A small aliquot of cell-containing suspension was placed in a perfusion chamber. After a brief period allowing for cell adhesion to the chamber, the cells were perfused with (in mM): NaCl 140, KCl 4.7, CaCl2 1.3, MgCl2 1.0, HEPES 10, glucose 10, pH adjusted to 7.4 with NaOH. Patch pipettes were pulled from borosilicate glass capillaries and heat polished. After filling with (in mM): KCl 130, MgCl2 6, HEPES 10, EGTA 10, MgATP 5, GTP 0.1, and phosphocreatine 5 (pH 7.2 with KOH) pipettes had a resistance of 2 to 3 MΩ. Series resistance was in the range of 6 to 12 MΩ and was compensated by 60% to 70%. Offset voltages generated when the pipette was inserted in NaCl solution (1 to 5 mV) were zeroed before formation of the seal.
For recording the outward current in pig left atrial myocytes, the L-type Ca2+ channels were blocked by adding 0.2 mM CdCl2, and the IKs current was inhibited by adding 1 μM HMR1556 to the bathing solution.18 The current was evoked by applying voltage pulses from the holding potential of −80 mV to +60 mV for 1 s preceded by a pre-pulse to −50 mV for 50 ms in order to inactivate the fast Na+ current.
Effects of AVE1231 on the IKACh were recorded in pig left atrial myocytes by applying voltage pulses from the holding potential of −80 mV to −100 mV for 500 ms; preceded by a pre-pulse to −50 mV for 200 ms at a frequency of 0.2 Hz. The difference current between control and after application of 10 μM carbachol was recorded.
Possible effects of AVE1231 on the currents IK1, IKs and IKr, IKATP, and the L-type Ca2+ current (ILCa) were investigated in guinea pig ventricular myocytes. IK1 currents were recorded by a voltage step from −80 mV to −120 mV lasting 200 ms. When IKs and IKr currents were recorded, 1 μM nisoldipine was added to the bath to block the L-type Ca2+ current. IKs was assessed by voltage pulse to +60 mV for 3 sec, starting from −40 mV. IKr was evaluated as the tail current evoked by a voltage pulse from −10 mV to −40 mV. IKATP was evoked by adding 1 μM rilmakalim19 to the bath and by applying voltage ramps from −80 mV to +80 mV for 500 ms. The IKATP was recorded at the potential 0 mV. For recording the ILCa, KCl in the pipette was replaced by CsCl and voltage pulses of 300 ms duration were applied from the holding potential of -80 mV to 0 mV. All patch-clamp experiments were performed under continuous superfusion of the cells with solution heated to 36 ± 1°C.
Experiments With Guinea Pig Papillary Muscles
Standard microelectrode recordings were performed as described in detail previously.20 The action potential and the upstroke velocity (dV/dtmax) were recorded.
Experiments in Anesthetized Pigs
Twenty-three male castrated pigs (German Landrace, 21 to 34 kg) were anesthetized with pentobarbital and prepared as described previously.10 Recordings were performed of the atrial effective refractory period (ERP) at different basic cycle lengths (BCL 240/300/400 ms). In brief, a conditioning train of 10 basic stimuli (S1) at twice-diastolic pacing threshold was followed by a diastolic extrastimulus (S2, pulse duration 1 ms) starting about 30 ms below the expected ERP with a 5 ms increment. The longest coupling interval unable to elicit a propagated atrial response was taken as the atrial ERP. During the ERP-measurement procedure the S2-extrastimulus, which followed the 10 conditioning S1 stimuli, frequently triggered runs of atrial tachycardia (paroxysms) in the left atrium as soon as the premature stimulus S2 was captured. The occurrence of S2-induced atrial tachycardias was exploited as a parameter for judging of the antiarrhythmic efficacy (referred to as left atrial vulnerability, LAV). After the first S2 was captured during the ERP-measurement procedure, 4 more S2 were applied while increasing the coupling interval by 5 ms for the subsequent S1-S2 procedure. Thus, the maximum occurrence of arrhythmias was 5 for each BCL. Since ERP-measurements were performed at 3 BCL, the maximum occurrence of arrhythmias was 15 for every time point. AVE1231 was administered to the anesthetized pigs via a gastric tube in a volume of 80 mL dissolved in 10 mL DMSO and 70 mL of warm PEG400.
The effect of AVE1231 on the ECG intervals was investigated in a separate study in 6 pigs because the QT interval in anesthetized pigs is subject to spontaneous changes over several hours. Therefore, a bolus of 3 mg/kg was administered, and the ECG intervals were evaluated 15 min after administration of the compound. Bipolar body surface ECG was recorded using subcutaneous needle electrodes in the classical lead II or lead III arrangement. The QT-interval was corrected for heart rate with Bazett's formula.
Experiments in Conscious Goats
Five female goats (60 ± 5 kg) were used for this study. Instrumentation of the goats was performed as reported previously.21
Atrial refractoriness was measured as described for the pig. The effect of AVE1231 and dofetilide on the LAERP was investigated in sinus rhythm (SR) and also after 72 hours of atrial tachypacing to find out whether electrical remodeling altered the antiarrhythmic efficacy of the compounds.
Electrical remodeling was induced by 72 hours of atrial tachypacing (50 Hz, 2 to 3 V) by an external stimulus unit via the implanted electrodes so that the atrium was fibrillating after 2 minutes. One second of electrical stimulation was followed by a break of 1 second. Ten minutes after the initiation of the tachypacing procedure, the atrium continued to fibrillate in the 1 second break. After stopping atrial tachypacing after 72 hours, sinus rhythm appeared spontaneously within about one minute.
In the 72 hours remodeling protocol, atrial ERP was measured in sinus rhythm before induction of atrial tachypacing and in electrical remodeling after 72 hours of tachypacing, before infusion of the drugs (3 baseline measurements at 20 minute intervals) and during the 1-hour infusion period at 15 minute intervals. In a control experiment using the same protocol, the vehicle had no effect, and there were no spontaneous changes of the ERP during the whole experimental period (no apparent reversal of the electrical remodeling). Vehicle and drugs were studied in the same goats with at least 10 days recovery between the experiments. AVE1231 (3 mg/kg) and dofetilide (10 μg/kg) were infused over 1 hour via a catheter in the jugular vein.
Effect of AVE1231 on Left Ventricular Refractory Period, Monophasic Action Potential Duration, and QT-interval
In a separate group of 6 pigs, the effect of AVE1231 on left ventricular repolarization was investigated as described previously for the Kv1.5-blocker AVE011810 and included the measurement of the QT-interval and left ventricular epicardial monophasic action potential (MAP) at a fixed BCL (about 10 bpm above the sinus rhythm) to avoid correction factors and the determination of the left ventricular refractory periods at 333, 400, and 500 ms BCL.
All averaged data are presented as the mean ± SEM. The Student t test was used to determine the significance of paired or unpaired observations. Differences were considered significant at P < 0.05.
The values for half-maximal inhibition (IC50) and the Hill coefficient were calculated by fitting the data points of the concentration-response curves to the logistic function:
where a represents the plateau-value at low drug concentration and d the plateau-value at high drug concentration; c represents the IC50 value, and n the Hill-coefficient. The curve-fitting and the Student t test were performed with the computer program Sigma-Plot 5.0.
For the investigation of an effect of AVE1231 on the atrial ERP in pigs and goats a 3-way ANOVA with repeated measures was applied. For LAV in the pig, a 2-way ANOVA with repeated measures was applied. For complementary analysis a Dunnett-test was performed.
AVE1231, HMR1556, and dofetilide were synthesized by the Medicinal Chemistry Department of Sanofi-Aventis Deutschland GmbH. Nisoldipine was purchased from Sigma. For oral application of AVE1231 in pigs, the compound was dissolved in 10 mL dimethyl sulfoxide (DMSO) plus 70 mL of warm polyethylene glycol (PEG400, Sigma). For intravenous application, AVE1231 was dissolved in 1 mL of DMSO, 3 mL of PEG400, and 6 mL of 50% glucose solution per animal. Dofetilide was dissolved in 10 mL of saline per goat.
Inhibition of hKv1.5 Currents in CHO Cells by AVE1231
First, we investigated the effects of AVE1231 on ion channels expressed in CHO cells. Under control conditions (absence of drug), the current activated rapidly and exhibited a marked inactivation. Figure 1 demonstrates that AVE1231 caused a concentration-dependent block of the hKv1.5 channel and induced an apparent increase in the speed of inactivation with only a slight decrease of the current peak amplitude (Figure 1a). Under control conditions, the decay fitted two exponential time constants with τf = 20 ± 2 ms (n = 15) and τs = 241 ± 22 ms (n = 15). In the presence of 3 μM AVE1231, the fast time constant was τf = 14 ± 2 ms (n = 10) and the slow time constant was τf =107 ± 17 ms (n = 10).
To determine the concentration-dependency of inhibition, we used the area under the curve of the total current, as described previously for the transient outward current Ito in rat ventricular myocytes.22 As demonstrated in Figure 1b, the curve fit yielded an IC50 value of 3.6 μM and a Hill-coefficient of 1.1.
Inhibition of hKv4.3 + hKChIP2.2b Currents in CHO Cells by AVE1231
As illustrated in Figure 2a, heterologous expression of the hKv4.3/hKChIP2.2b genes in CHO cells yielded a rapidly activating and inactivating current. In the presence of AVE1231, the speed of inactivation was increased with little effect on the peak amplitude. The concentration response of the area under the curves is shown in Figure 2b. The curve fit yielded a half-maximal inhibition at 5.9 μM and a Hill coefficient of 1.0.
The apparent time-constant of inactivation (τinact) fitted a mono-exponential function and decreased from 11.5 ± 0.9 ms (n = 7, control) to 7.0 ± 0.8 ms (n = 4) at 3 μM AVE1231.
Inhibition of the Outward Current in Pig Left Atrial Myocytes by AVE1231
When applying voltage pulses from the holding potential of -80 mV to +60 mV for 1 sec, a rapidly activating outward current was evoked that decayed to a steady state (Figure 3a). AVE1231 caused a concentration-dependent attenuation both of the peak and steady state current and accelerated the decay time. Under control conditions, the decay fitted 2 exponential time constants with τf = 89 ± 13 ms (n = 7) and τs = 337 ± 46 ms (n = 7). In the presence of 3 μM AVE1231, the fast time constant shortened to τf = 21 ± 2 ms (n = 5), whereas the slow time constant did not change significantly (τf = 392 ± 91 ms, n = 5).
In order to quantify the inhibitory potency of the compound, the area under the curve was evaluated. As demonstrated in Figure 3b, the total current is concentration-dependently blocked. The graph shows that inhibition reached a saturating state and that the highest AVE1231 concentration of 100 μM blocked approximately 34 ± 9% (n = 5) of the sustained current recorded at the end of the voltage pulse. Half-maximal inhibition of the AVE1231 sensitive current can be roughly estimated to be 1 μM.
Inhibition of IKACh Current in Pig Left Atrial Myocytes by AVE1231
We also investigated effects of AVE1231 on the acetylcholine-activated K+ current (IKACh), which was evoked by applying 10 μM carbachol to the bathing solution in pig left atrial myocytes. As demonstrated in Figure 4a, carbachol activated a strong inward current at the voltages below −100 mV. AVE1231 blocked this carbachol-activated current concentration-dependently. This is illustrated in Figure 4b, where the percent inhibition is plotted. The curve fit yielded an IC50 value of 8.4 μM and a Hill coefficient of 0.9. In the absence of carbachol, AVE1231 (10 μM) had no significant effect on the inward current at −100 mV.
Effects of AVE1231 on Other Ion Channels
In order to investigate the selectivity of AVE1231, we tested the compound on several ion channels in guinea pig ventricular myocytes. The IKr current was investigated by evoking steady state currents by voltage pulses from −40 mV to −10 mV for 3 seconds and tail currents by subsequent pulsing to −40 mV. In order to suppress the L-type Ca2+ current, 1 μM nisoldipine was present in the bath. In a separate set of experiments, we showed that 1 μM nisoldipine decreased the IKr tail current by only 12% ± 9% (n = 6). As shown in Table 1, 30 μM AVE1231 blocked the IKr tail current by 50%, so an IC50 close to 30 μM can be estimated.
Next we evoked IKs currents by applying pulses from −40 mV to +60 mV. Again, 1 μM nisoldipine was present, which was shown to suppress the IKs current by 21 ± 4% (n = 6). There was no significant effect on the IKs current at 10 μM and 30 μM AVE1231 (Table 1).
KATP currents were induced by applying 1 μM of the KATP opener rilmakalim. AVE1231 (30 μM) blocked this current by 35 ± 5% (n = 3). The inward-rectifying current IK1, recorded by a voltage pulse to −120 mV, was not significantly blocked by 10 and 30 μM AVE1231. The L-type Ca2+ current was investigated by applying voltages pulses to 0 mV. AVE1231 (10 μM) blocked this current by 12 ± 2% (n = 7). To record the fast Na+ channel, the extracellular NaCl concentration was reduced to 20 mM, and the experiments were performed at room temperature. Voltage pulses of 15 ms duration were applied from -100 mV to -30 mV at a frequency of 1 Hz. As shown in Table 1, AVE1231 (10 μM) had no effect on the Na+ current.
Effects of AVE1231 on the Action Potential in Guinea Pig Papillary Muscle
Guinea pig papillary muscles were investigated to observe possible effects of AVE1231 on the prolongation of the action potential duration, due to inhibition of the IKr and the IKs current. In addition, possible effects on the upstroke velocity, being a marker of the fast sodium current, can be detected. As demonstrated in Table 1, AVE1231 did not significantly alter the APD90 and the upstroke velocity at the concentrations 10 μM and 30 μM.
Atrial Effects of AVE1231 in Anesthetized Pigs
At oral doses of 0.3, 1, and 3 mg/kg, AVE1231 caused a gradual and dose-dependent increase in atrial refractoriness and a gradual inhibition of left atrial vulnerability as shown for 3 mg/kg in Figure 5 (n = 5 to 7 per dose). Values for the plateau of efficacy at 2.5 hours are shown in Table 2. At the 3 mg/kg dose, the absolute prolongation of atrial ERP at the 3 BCLs was about 30 ms at 2.5 hours after administration, and the inhibition of LAV was about 86% compared with baseline. AVE1231, 3 mg/kg iv had no effect on the ECG intervals including the QTc-interval (Figure 6).
Effect of AVE1231 on the Goat Left Atrial Refractoriness Before and After 72 Hours of Atrial Tachypacing in Comparison With Dofetilide
AVE1231 and dofetilide significantly prolonged the atrial ERP before atrial tachypacing (Figure 7). Atrial tachypacing over 72 hours shortened the atrial ERP (electrical remodeling). Electrical remodeling modified the atrial efficacy of both drugs dramatically in opposite directions. The effect of AVE1231 increased significantly compared with SR (P < 0.05 at 240 and 300 ms BCL), and the effect of dofetilide strongly decreased (P < 0.05 at 300 and 400 ms BCL) (Figure 8). In SR, prolongation of the AERP was 5 to 10 ms greater with AVE1231 compared with dofetilide; after 72 hours of electrical remodeling, the difference increased to 40 ms in favour of AVE1231. AVE1231, 3 mg/kg iv had no effect on the QTc-interval (392 ± 3.1 before vs 387 ± 9.8 ms after AVE1231; n = 5; mean ± SEM) in the goat while dofetilide showed the expected prolongation (383 ± 14.2 vs 468 ± 45.7 ms; n = 4, P < 0.05).
Effect of AVE1231 on Left Ventricular Refractory Period, Monophasic Action Potential Duration, and QT-interval
In a separate group of 6 pigs, the effect of AVE1231 on the QT-interval, the MAP-duration at 50% and 90% repolarization (MAPD50 and MAPD90), and the left ventricular effective refractory period were studied. None of these repolarization parameters changed significantly after 3 mg/kg iv AVE1231 (Figure 9) indicating that it is devoid of an effect on ventricular repolarization.
The present study demonstrated that the novel compound AVE1231 blocked the hKv1.5 and the hKv4.3/KChIP2.2b channels in expression systems. In pig left atrial myocytes, the compound inhibited part of the voltage-dependent outward current with an IC50 of approximately 1 μM. The IKACh current was also inhibited by AE1231 to a lesser extent. It is assumed that it is mainly inhibition of the Kv1.5 channel that causes the prolongation of atrial refractoriness in both pig and goats, as described in the present study.
Inhibition of the human Kv1.5 channel in CHO cells by AVE1231 was accompanied by an acceleration of apparent inactivation. The apparent increase of inactivation is consistent with an open channel block as described for the inhibition of Kv1.5 by S010017623 and AVE0118.9
It was reported that currents through expressed Kv4.3 channels closely resemble the transient outward current24 and that Kv channel-interacting proteins (KChIPs) co-assemble with the Kv4.3 in expression systems and in neurons.25 Moreover, it was shown that KChIP2.2b is the predominant KChIP2 isoform in human heart.15,26 Since it was reported that coexpression of Kv4.3 with KChIP2 resembles more closely the native Ito in human ventricular cells,15 we routinely used this heteromer for studying the inhibition by AVE1231. Under control conditions, the time-constant of current inactivation (τinact) was 9.3 ms, being considerably faster than that observed in Xenopus oocytes (approximately 111 ms).15 This difference is likely due to the lower temperature of 20°C, at which the oocytes were investigated. In human atrial myocytes, it was reported that τinact was around 12 ms at 37°C.27,28 Thus, the obtained value for τinact of hKv4.3 + hKChIP2.2b in CHO cells is nearly identical to that reported for Ito in human atrial myocytes.
Application of AVE1231 caused a pronounced acceleration of the apparent time-constant of channel inactivation. An increase in τinact was previously observed for inhibition of the human atrial Ito by flecainide, quinidine, and 4-aminopyridine.28 As discussed by the authors, an acceleration of inactivation can be the result of rapid block of open channels; however, blockade of the inactivated channel cannot be excluded. The present data do not allow one to discriminate between the 2 possibilities. Further studies on the mechanism of channel blockade are necessary to elucidate the mechanism of Ito inhibition.
The effect of AVE1231 on the IKACh was recorded in pig atrial myocytes. Application of 10 μM carbachol induced a pronounced inward current at voltage steps from the holding potential of -80 mV to -100 mV. (Figure 4a). AVE1231 inhibited this current with an IC50 value of 8.4 μM (Figure 4b). Inhibition of the IKACh was previously observed with dronedarone and amiodarone in guinea pig atrial myocytes, where IC50 values of ca. 10 nM and 1 μM, respectively, were observed.29
When voltage pulses were applied from the holding potential of -80 mV to +60 mV in pig left atrial myocytes, an outward current occurred that activated rapidly and decayed to a steady state (Figure 3a). The decay of the current could be best fitted by 2 exponential time constants. Compared with the fast time constant obtained for hKv1.5 and hKv4.3/KChIP2.2b channels in CHO cells, the fast time constant in pig atrial cells was considerably longer. On the other hand, it was recently shown that the pharmacological properties of the transient outward current in pig atrial myocytes are consistent with Kv1.5 channel characteristics.30 Therefore, we calculated the area under the curve of the total outward current and estimated its inhibition by AVE1231. As demonstrated in Figure 3, high concentrations of the compound inhibited the transient component nearly completely, whereas the current at the end of the 1 s voltage pulse was only partly (by 34% of control) blocked. This indicates that pig atrial myocytes possess a background current that is unaffected by the Kv1.5 channel blocker AVE1231. A similar current, the nature of which is still undefined, was previously described in human atrial myocytes.27
The present data demonstrate that AVE1231 potently blocks the outward current in pig atrial myocytes, which is likely to explain the prolongation of the effective refractory period observed in pig left atrium.
As demonstrated in this study, 10 μM AVE1231 had only mild inhibitory effects on the L-type Ca2+ channel and the ATP-dependent K+ channel IKATP, whereas no significant effects were observed on the inwardly rectifying channel IK1, the slow delayed outward rectifying K+ current IKs, and the fast Na+ channel. However, the fast delayed outward rectifying K+ current IKr was significantly blocked at higher concentrations with an estimated IC50 value of 30 μM. On the other hand, in pigs and goats there was a complete absence of effects on ECG intervals, ventricular MAP, and ventricular refractoriness, indicating that inhibition of the IKr is too weak and therefore not relevant at physiological concentrations.
In pigs and goats, AVE1231 induced a strong dose-dependent prolongation of the atrial refractoriness with no prolongation of the QTc interval, indicating that it is devoid of an effect on ventricular repolarization. In the pig, the self-limiting paroxysms of AF induced by a premature beat (left atrial vulnerability) were nearly totally suppressed at 1 and 3 mg/kg. Such a strong inhibition of left atrial vulnerability without any effect on ventricular repolarization has only been observed previously with the biphenyl derivative AVE0118.10,11 As shown previously, dofetilide did not significantly inhibit left atrial vulnerability.31 Atrial fibrillation may have its origins mainly in the left atrium and is initiated by premature beats from the pulmonary veins.32,33 The response of the atrium (occurrence of paroxysms of AF) to a premature beat (S2) in our porcine model is a valuable measure of atrial vulnerability and a relevant antiarrhythmic parameter for a drug that is intended for the prevention of the recurrence of AF.
In the goat in sinus rhythm, both AVE1231 and dofetilide showed a clear prolongation of atrial refractoriness. Electrical remodeling strongly modified the efficacy of both compounds in opposite directions. During the remodeling process, the atrial action potential and refractoriness is shortened mainly via downregulation of the calcium channel. As a consequence, the contribution of the later activating IKr and IKS decreases, whereas the relative importance of the early activating potassium currents Ito and IKur increases.34,35 As previously reported, the IKr-blocker dofetilide lost most of its ERP-prolonging effect after 48 hours of electrical remodeling.11 By contrast, electrical remodeling for 72 hours increased the effect of AVE1231 significantly, in agreement with a similar effect observed with AVE011811 and suggesting that this is a class effect that applies to early atrial K+-channel blockers. Since, in the first days after cardioversion of AF, the atria are still in an electrically remodelled state,36,37,21 an antiarrhythmic drug that prolongs atrial refractoriness even more than in sinus rhythm may be particularly efficacious against the early recurrence of AF.
Since AVE1231 did not prolong ventricular repolarization, as indicated by the lack of an effect on the ventricular effective refractory period and MAP duration in pigs and on the QT interval in pigs and goats at strong atrial ERP prolongation, it is expected to be devoid of ventricular proarrhythmic effects (early afterdepolarizations, Torsades de Pointes arrhythmias).
AVE1231 has similar properties as the previously described compound AVE0118.9,10,11 The drawback of AVE0118 is a short half life, being 0.2 to 0.4 hours in dogs and pigs (unpublished data of Sanofi-Aventis). Therefore, this compound is not suited for the prevention of recurrence of AF through the oral route. By contrast, AVE1231 has favourable pharmacokinetic properties and seems appropriate for an oral development.
Although several companies are developing Kv1.5 channel blockers for the therapy of atrial fibrillation, published data are scarce. Most advanced in clinical development is RSD1235, which showed positive results in termination of recent onset AF.38 This compound blocks hKv1.5 channels with an IC50 value of 13 μM and, in addition, exerts a frequency-dependent inhibition of the fast Na+ channel Nav1.5.39 In human atrial cells the inhibition of the Na+ current was reported to be more potent than inhibition of the IKur.40 In contrast, AVE1231 is devoid of effects on the fast Na+ channel.
Another compound with properties resembling AVE1231 is NIP-142. This compound inhibits IKur, and Ito in human atrial myocytes41 and was effective in a dog model of vagally-induced AF.42 However, no further information about the development of NIP-142 was reported thus far.
The present study demonstrates that the novel compound AVE1231 preferentially inhibits the IKur (Kv1.5), the Ito (Kv4.3/KChIP2.2) and to a lesser extend the IKACh. Inhibition of these K+ channels is likely to explain the selective prolongation of atrial refractoriness in goats and pigs and the pronounced inhibition of atrial arrhythmias in the pig in the absence of any effect on ventricular repolarization. Based on its strong antiarrhythmic efficacy in large animals in sinus rhythm and a further gain in efficacy in electrical remodeling, AVE1231 could be suited for the prevention of atrial fibrillation.
We thank S. O'Connor for valuable discussion and M. Allessie and A. van Hunnik, University of Maastricht, for their support in setting up the goat model in our laboratory, and G. Itter and F. Afkham for instrumenting the goats.
1. Middlekauf HR, Stevenson WG, Stevenson LW. Prognostic Significance of atrial fibrillation in advanced heart failure. A study of 390 patients. Circulation
2. Hart RG, Halpertin JL. Atrial fibrillation and Stroke. Concepts and controversies. Stroke
3. Kannel WB, Abbot RD, Savage DD, et al. Epidemiologic features of chronic atrial fibrillation. The Framingham Study. N Engl J Med
4. Falk RH. Flecainide-induced ventricular tachycardia and fibrillationin patients treated for atrial fibrillation. Ann Intern Med
5. Waldo AL, Camm AJ, deRuyter H, et al. Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. The SWORD investigation. Survival with oral d-sotalol. Lancet
6. Torp-Pedersen C, Moller M, Bloch-Thomsen PE, et al. Dofetilide in patients with congestive heart failure and left ventricular dysfunction. Danish Investigations of Arrhythmia and Mortality on Dofetilide Study Group. N Engl J Med
7. Singh S, Zoble RG, Yellen L, et al. Efficacy and safety of oral dofetilide in converting to and maintaining sinus rhythm in patients with chronic atrial fibrillation or atrial flutter - the Symptomatic Atrial Fibrillation Investigative Research on Dofetilide (SAFIRE-D) study. Circulation
8. Feld GK. Atrial fibrillation: is there a safe and highly effective pharmacological treatment? Circulation
9. Gögelein H, Brendel J, Steinmeyer K, et al. Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels. Naunyn Schmiedebergs Arch. Pharmacol
10. Wirth KJ, Paehler T, Rosenstein B, et al. Atrial effects of the novel K+
-channel-blocker AVE0118 in anesthetized pigs. Cardiovasc Res
11. Blaauw Y, Gögelein H, Tieleman RG, et al. “Early” class III drugs for the treatment of atrial fibrillation - Efficacy and atrial selectivity of AVE0118 in remodeled atria of the goat
12. Bosch R, Lazlo R, Schneck AC, et al. AVE0118, an antiarrhythmic drug with novel mechanism of action - Block of IKur and Ito potassium currents in human atrial myocytes. Circulation
13. Wettwer E, Hala O, Heubach JF, et al. Human atrial action potentials from patients in sinus rhythm and atrial fibrillation are differently affected by inhibitors of the ultra rapidly activating potassium current IKur.Naunyn-Schmiedeberg's Arch Pharmacol
. 2003;367:R 99.
14. Dilks D, Ling H-P, Cockett M, et al. Cloning and expression of the human Kv4.3 potassium channel. J Neurophysiol
15. Decher N, Uyguner O, Scherer CR, et al. hKChIP2 is a functional modifier of hKv4.3 potassium channels: Cloning and expression of a short hKChIP2 splice variant. Cardiovasc Res
16. Hamill OP, Marty M, Neher E, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch
17. Wirth KJ, Knobloch K. Differential effects of dofetilide, amiodarone, and class Ic drugs on left and right atrial refractoriness and left atrial vulnerability in pigs. Naunyn Schmiedebergs Arch Pharmacol
18. Gögelein H, Brüggemann A, Gerlach U, et al. Inhibition of IKs
channels by HMR 1556. Naunyn Schmiedebergs Arch. Pharmacol
19. Krause E, Englert H, Gögelein H. Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim. Pflugers Arch
20. Gögelein H, Hartung J, Englert HC, et al. HMR 1883, a novel cardioselective inhibitor of the ATP-sensitive potassium channel. Part I: Effects on cardiomyocytes, coronary flow and pancreatic b-cells. J Pharmacol Exp Ther
21. Wijffels MC, Kirchhof CJ, Dorland R, et al. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation
22. Dukes ID, Cleemann L, Morad M. Tedisamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells. J Pharmacol Exp Ther
23. Decher N, Pirard B, Bundis F, et al. Molecular basis for Kv1.5 channel block - Conservation of drug binding sites among voltage-gated K+
channels. J Biol Chem
24. Dixon JE, Shi W, Wang H-S, et al. Role of the Kv4.3 K+
channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res
25. An WF, Bowlby MR, Betty M, et al. Modulation of A-type potassium channels by a family of clacium sensors. Nature
26. Ohya S, Morohashi Y, Muraki K, et al. Molecular cloning and expression of the novel splice variants of K+
channel-interacting protein 2. Biochem Biophys Res Commun
27. Amos GJ, Wettwer E, Metzger F, et al. Differences between outward currents of human atrial and subepicardial ventricular myocytes. J Physiol (Lond)
28. Wang Z, Fermini B, Nattel S. Effects of flecainide, quinidine, and 4-aminopyridine on transient outward and ultrarapid delayed rectifier currents in human atrial myocytes. J Pharmacol Exp Ther
29. Guillemare E, Marion A, Nisato D, et al. Inhibitory effects of dronedarone on muscarinic K+
current in guinea pig atrial cells. J Cardiovasc Pharmacol
30. Ehrlich JR, Hoche C, Coutu P, et al. Properties of a time-dependent potassium current in pig atrium - evidence for a role of Kv1.5 in repolarization. J Pharmacol Exp Ther
31. Knobloch K, Brendel J, Peukert S, et al. Electrophysiological and antiarrhythmic effects of the novel I(Kur) channel blockers, S9947 and S20951, on left vs right pig atrium in vivo in comparison with the I(Kr) blockers dofetilide, azimilide, d,l-sotalol and ibutilide. Naunyn Schmiedebergs Arch Pharmacol
32. Bennett MA, Pentecost BL. The pattern of onset and spontaneous cessation of atrial fibrillation in man. Circulation
33. Haissaguerre M, Jais P, Shah DC, et al. Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins. N Engl J Med
34. Yue L, Feng J, Gaspo R, et al. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res
35. Courtemanche M, Ramirez RJ, Nattel S. Ionic targets for drug therapy and atrial fibrillation-induced electrical remodeling: insights from a mathematical model. Cardiovasc Res
36. Raitt MH, Kusumoto W, Giraud G, et al. Reversal of electrical remodeling after cardioversion of persistent atrial fibrillation. J Cardiovasc Electrophysiol
37. Franz MR, Karasik PL, Li C, et al. Electrical remodeling of the human atrium: similar effects in patients with chronic atrial fibrillation and atrial flutter. J Am Coll Cardiol
38. Roy D, Rowe BH, Stiell IG, et al. A randomized, controlled trial of RSD1235, a novel anti-arrhythmic agent, in the treatment of recent onset atrial fibrillation. J Am Coll Cardiol
39. Fedida D, Orth PM, Chen JY, et al. The mechanism of atrial antiarrhythmic action of RSD1235. J Cardiovasc. Electrophysiol
40. Beatch GN, Lin S, Hesketh C, et al. Electrophysiological mechanism of RSD1235, a new atrial fibrillation converting drug. Circulation
. 2003; 108:IV-85.
41. Seki A, Hagiwara N, Kasanuki H. Effects of NIP-141 on K currents in human atrial myocytes. J Cardiovasc Pharmacol
42. Nagasawa H, Fujiki A, Fujikura N, et al. Effects of a novel class III antiarrhythmic agent, NIP-142, on canine atrial fibrillation and flutter. Circ J