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Cardiovascular Pharmacology

Electrophysiological effects of ivabradine in dog and human cardiac preparations:Potential antiarrhythmic actions

István Koncz a, Tamás Szél a, Miklós Bitay e, Elisabetta Cerbai c, Kristian Jaeger a, Ferenc Fülöp d,Norbert Jost b, László Virág a, Péter Orvos f, László Tálosi f, Attila Kristóf b, István Baczkó a,Julius Gy. Papp a,b, András Varró a,b,⁎a Department of Pharmacology & Pharmacotherapy, University of Szeged, Szeged, Hungaryb Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, Szeged, Hungaryc Department of Pharmacology, CIMMBA, University of Florence, Florence, Italyd Institute of Pharmaceutical Chemistry, University of Szeged, Szeged, Hungarye Department of Cardiac Surgery, University of Szeged, Szeged, Hungaryf Rytmion Ltd., Szeged, Hungary

a b s t r a c ta r t i c l e i n f o

Article history:Received 12 October 2010Received in revised form 19 July 2011Accepted 21 July 2011Available online 2 August 2011

Keywords:IvabradinePurkinje fiberDiastolic depolarizationVmax

RepolarizationHuman heart

Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine onmaximum rate of depolarization (Vmax), repolarization and spontaneous depolarization have not yet beenreported in human isolated cardiac preparations. The same applies to large animals close to human in heartsize and spontaneous frequency. Using microelectrode technique action potential characteristics and byapplying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent(0.1–10 μM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneousrate of firing of action potentials and produced a concentration- and frequency-dependent Vmax block in dogPurkinje fibers while action potential duration measured at 50% of repolarization was shortened. In thepresence of ivabradine, at 400 ms cycle length, Vmax block developed with an onset kinetic rate constant of13.9±3.2 beat−1 in dog ventricular muscle. In addition to a fast recovery of Vmax from inactivation (τ=41–46 ms) observed in control, a second slow component for recovery of Vmax was expressed (offset kinetics ofVmax block) having a time constant of 8.76±1.34 s. In dog after attenuation of the repolarization reserveivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation ofrepolarization was only observed at 10 μM ivabradine. Ivabradine in addition to the Class V antiarrhythmiceffect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment ofpatients with ischemic heart disease liable to disturbances of cardiac rhythm.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Alinidine was the first bradycardic agent with known sino-atrialpacemaker current (If) inhibitory property which is an imidazolinecompound derived from clonidine. Further bradycardic agents havebeen derived from the calcium channel inhibitor verapamil: cilobra-dine, zatebradine and ivabradine. Ivabradine (Procoralan®) is an anti-ischemic agent, which inhibits the If current with reasonableselectivity (Vilaine et al., 2003). If plays an important role in regulatingsino-atrial node pacemaker activity (DiFrancesco, 1995). According tothe BEAUTIFUL study, ivabradine can be used to reduce the incidenceof coronary artery disease outcomes in a subgroup of patients who

have heart rates of 70 bpm or greater (Fox et al., 2008). Improvementof regionalmyocardial bloodflowand function and reduction of infarctsize by ivabradine have also been described (Heusch et al., 2008).Ivabradine induces (a marked exponential use-dependent) blockadeof the hyperpolarization activated If current. The IC50 for the blockof If is 2.8 μM (Bois et al., 1996). It has been shown that a highconcentration of ivabradine (10 μM)has no detectable effect on T-typecalcium current and slightly decreases L-type calcium current withoutsignificant use-dependent block. Also it moderately decreases thedelayed outward potassium current in rabbit sino-atrial node cells(Bois et al., 1996). The effect of ivabradine on the action potentialrepolarization was only studied in guinea pig papillary muscle andrabbit sinus node pacemaker cells (Thollon et al., 1994, 1997), i.e. inspecies having relatively small hearts with high frequencies. In thesepreparations ivabradine did not show considerable effect on actionpotential duration at therapeutically relevant concentrations, and athigh concentration (50 μM) it produced frequency-dependent Vmax

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⁎ Corresponding author at: Department of Pharmacology & Pharmacotherapy,Faculty of Medicine, University of Szeged, Dóm tér 12, P.O. Box 427, H-6701 Szeged,Hungary. Tel.: +36 62 545683; fax: +36 62 545680.

E-mail address: a.varro@phcol.szote.u-szeged.hu (A. Varró).

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block in guinea-pig papillary muscle (Pérez et al., 1995). The effects ofivabradine on Vmax, repolarization and spontaneous depolarizationhave not yet been reported in human isolated cardiac preparationsand in large animals close to human in heart size and spontaneousfrequency. Therefore, the aim of our work was to characterize the

cellular electrophysiological effects of ivabradine in human, dog and toa lesser extent in rabbit heart preparations. We found that ivabradineat least at concentrations equal or higher than 1 μM can be consideredas a Na+ channel blocker antiarrhythmic drug with slow kinetic onsetand recovery similar to that of flecainide or propafenone.

Fig. 1. Effect of ivabradine on the spontaneous action potential (AP) firing (A, representative figure) and the amplitude of spontaneous diastolic depolarization (B, results aremean±S.E.M., * = Pb0.05 vs control, n=6, cumulative drug application) in dog Purkinje fiber.

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2. Materials and methods

2.1. Conventional microelectrode technique

2.1.1. Dog tissueAll experiments were carried out in compliance with the Guide

for the Care and Use of Laboratory Animals (USA NIH publicationNo. 85-23, revised 1985). The protocols were approved by the ReviewBoard of the Committee on Animal Research of the University ofSzeged (54/1999 OEj). Ventricular muscles were obtained from theright ventricle, and free-running (false tendons of) Purkinje fiberswere isolated from both ventricles of hearts removed through a rightlateral thoracotomy from anesthetized (thiopental 30 mg/kg i.v.)mongrel dogs of either sex weighing 10–15 kg. The preparationswere placed in a tissue bath and allowed to equilibrate for at least2 h while superfused (flow rate 4–5 ml/min) with Locke's solutioncontaining (in mM): NaCl 120, KCl 4, CaCl2 2, MgCl2 1, NaHCO3 22,and glucose 11. The pH of this solution was 7.40 to 7.45 when gassedwith 95% O2 and 5% CO2 at 37 °C. During the equilibration period,the ventricular muscle tissues were stimulated at a basic cycle lengthof 1000 ms, Purkinje fibers were stimulated at a basic cycle lengthof 500 ms. Electrical pulses of 2 ms in duration and twice diastolicthreshold in intensity (S1) were delivered to the preparationsthrough bipolar platinum electrodes. Transmembrane potentialswere recorded with the use of glass capillary microelectrodes filledwith 3 M KCl (tip resistance: 5 to 15 MΩ). The microelectrodes werecoupled through an Ag–AgCl junction to the input of a high-impedance, capacitance-neutralizing amplifier (Experimetria 2004).Intracellular recordings were displayed on a storage oscilloscope(Hitachi V-555) and led to a computer system (APES) designed for on-line determination of the following parameters: resting membranepotential, action potential amplitude, action potential duration at 50%and 90% repolarization and the maximum rate of rise of the actionpotential upstroke (Vmax). The following types of stimulation wereapplied in the course of the experiments: stimulation with a constantcycle length of 1000 ms (ventricular muscles); stimulation with aconstant cycle length of 500 ms (Purkinje fibers); stimulation withdifferent constant cycle lengths ranging from 300 to 5000 ms (or to2000 ms in the case of Purkinje fibers to prevent spontaneous diastolicdepolarizations at cycle lengths longer than 2000 ms). To determinethe recovery kinetics of Vmax, extra test action potentials were elicitedby using single test pulses (S2) in a preparation driven at a basiccycle length of 400 ms. The S1–S2 coupling interval was increasedprogressively from the end of the refractory period. The effectiverefractory period was defined as the longest S1–S2 interval at which S2failed to elicit a propagated response. The diastolic intervals precedingthe test action potential were measured from the point corresponding

to 90% of repolarization of the preceding basic beat to the upstrokeof the test action potential and were increased progressively. Todetermine the onset kinetics the preparations were continuouslystimulated at cycle length of 1000 ms, the stimulationwas interruptedfor 1 min, and then a train of 40-beat stimuli was applied with a cyclelength of 400 ms. Control recordings were obtained after equilibriumperiod. The effects of ivabradine were determined at the givenconcentrations, with recordings started 25 min after the addition ofeach concentration of the drug in a cumulative manner. For allexperiments ivabradine (Sequoia Research Products Ltd, Pangbourne,United Kingdom) was dissolved in distilled water at stock solutionconcentration of 1 or 10 mM.

2.1.2. Human tissueNon-diseased human hearts that were technically unusable for

transplantation (basedon logistical, not patient-related, considerations)were obtained from organ donors. Before cardiac explantation, organdonor patients did not receive medication except dobutamine,furosemide and plasma expanders. The investigations conform to theprinciples outlined in the Declaration of Helsinki of the World MedicalAssociation. All experimental protocols were approved by the Scientificand Research Ethical Committee of the Medical Scientific Board atthe Hungarian Ministry of Health (ETT-TUKEB), under ethical approvalNo. 4991-0/2010-1018EKU (339/PI/010). Human cardiac tissue wasstored in cardioplegic solution at 4 °C for 4–8 h. Papillary muscles wereobtained from the right ventricle. Preparationswere initially stimulatedat a basic cycle length of 1000 ms and allowed at least 1 h to equilibratewhile they were continuously superfused with Locke's solution.Temperature of the superfusate was kept constant at 37 °C.

2.2. Whole cell configuration of the patch-clamp technique

Ventricular myocytes were enzymatically dissociated from heartsof mongrel dogs or rabbits of either sex weighing 10–15 kg and1–2 kg, respectively, as described earlier in detail (Varró et al., 2000;Virág et al., 1998). One drop of cell suspension was placed within atransparent recording chamber mounted on the stage of an invertedmicroscope, and individual myocytes were allowed to settle andadhere to the chamber bottom for at least 5 min before superfusionwas initiated. Only rod shaped cells with clear cross striations wereused. HEPES buffered Tyrode's solution served as the normal super-fusate. This solution contained (in mM): NaCl 144, NaH2PO4 0.33, KCl4.0, CaCl2 1.8, MgCl2 0.53, Glucose 5.5, and HEPES 5.0 at pH of 7.4.Patch-clamp micropipettes were fabricated from borosilicate glasscapillaries using a P-97 Flaming/Brownmicropipette puller (Sutter Co,Novato, CA, USA). These electrodes had resistances between 1.5 and2.5 MΩ when filled with pipette solution containing (in mM): KOH

Table 1The electrophysiological effects of ivabradine in dog Purkinje fibers at basic cycle length of 500 ms.

Parameters MDP (mV) APA (mV) APD50 (ms) APD90 (ms) Vmax (Vs−1)

Baseline −88.3±1.0 129.4±1.9 170.0±9.8 245.7±7.5 640.7±38.4Ivabraine 0.1 μM −88.0±0.9 127.5±1.9 168.8±7.2 248.6±6.2 591.7±36.4Ivabraine 1 μM −88.5±1.5 126.5±2.8 165.8±8.8 252.0±6.8 556.8±42.8a

Ivabraine 10 μM −88.1±1.5 122.6±2.4a 137.7±10.3a 242.8±7.7 471.8±38.1a

MDP, maximum diastolic potential; APA, action potential amplitude; APD50 and APD90, action potential durations at 50% and 90% of repolarization; Vmax, maximum rate ofdepolarization. Results are mean±S.E.M. aPb0.05 vs control, n=8, cumulative drug application.

Time-solvent control in dog Purkinje fibers at basic cycle length of 500 ms.

Parameters MDP (mV) APA (mV) APD50 (ms) APD90 (ms) Vmax (Vs−1)

Baseline −87.8±1.2 130.3±1.1 175.7±13.6 245.1±15.7 730.8±41.2Distilled water 50 μl −87.5±1.2 127.5±1.8 179.9±12.2 250.7±13.3 736.4±39.1Distilled water 5 μl −87.4±0.9 128.6±1.7 183.7±8.9 251.6±12.3 736.8±70.2Distilled water 45 μl −87.0±1.6 120.4±3.4b 170.4±9.6 257.0±13.9 742.2±74.0

MDP, maximum diastolic potential; APA, action potential amplitude; APD50 and APD90, action potential durations at 50% and 90% of repolarization; Vmax, maximum rate ofdepolarization. Results are mean±S.E.M., aPb0.01; bPb0.05 vs control, n=4, cumulative solvent (distilled water) application.

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100, KCl 40, K2ATP 5, MgCl2 5, EGTA 4, CaCl2 1.5 and HEPES 10. ThepH of this solution was adjusted to 7.2 by aspartic acid. MeasuringK+ currents, nisoldipine (1 μM) (gift from Bayer AG, Leverkusen,Germany) was added to the external solution to eliminate inwardL-type Ca2+ current (ICa). The rapid delayed rectifier potassiumcurrent (IKr) was separated from slow IKs component by using theselective IKs blocker HMR-1556 (0.5 μM). Membrane currents wererecorded with Axopatch-200B patch-clamp amplifiers (MolecularDevices–Axon Instruments, Union City, CA, USA) using the whole-cellconfiguration of the patch-clamp technique. After establishing a high(1–10 GΩ) resistance seal by gentle suction, the cell membranebeneath the tip of the electrode was disrupted by suction or byapplication of 1.5 V electrical pulses for 1–5 ms. The series resistancewas typically 4–8 MΩ before compensation (50–80%, depending onthe voltage protocols). Experiments where the series resistance washigh, or substantially increased during measurement, were discarded.Membrane currents were digitized using analog-to-digital converters(Digidata 1200 and Digidata 1322, Molecular Devices–Axon In-struments, Union City, CA, USA) after low-pass filtering at 1 kHzunder software control (pClamp 8.0 Molecular Devices–Axon In-struments, Union City, CA, USA). The same software was used for off-line analysis. All patch-clamp data were collected at 37 °C.

2.3. Statistical analysis

Results were analyzed by using Student's t-test for paired andunpaired data. Differences were considered significant when Pb0.05.Data are expressed as mean±S.E.M. (standard error of the mean).

3. Results

3.1. Effects of ivabradine on transmembrane action potentials

In dog Purkinje strands, stimulation (2 Hz) was terminated toallow development of spontaneous activity. The developed sponta-neous frequency in the control preparations was 0.471±0.06 Hz.Ivabradine concentration-dependently decreased the amplitude ofspontaneous diastolic depolarization (Fig. 1B, n=6) and slowedspontaneous rate of firing (Fig. 1A, n=5) of the Purkinje fibers.Spontaneous activity was completely abolished by 10 μM ivabradinein all preparations. In isolated dog Purkinje fibers ivabradineconcentration- and rate-dependently decreased the maximum rateof rise of the action potential upstroke (Vmax, n=13), and actionpotential amplitude while action potential duration measured at50% of repolarization was shortened in a concentration-dependent

manner (n=8) at pacing with a constant cycle length of 500 ms(Table 1, and Fig. 2; time-solvent group is added).

The depression of Vmax evoked by 1 and 10 μM ivabradine wasstrongly dependent upon stimulation frequency (“use-dependent”);i.e., as pacing cycle length was decreased, the depression of Vmax was

Fig. 3. Onset of frequency-dependent depression of Vmax induced by 10 μM ivabradinein dog ventricular muscle at stimulation cycle length of 400 ms. Mean values are givenfrom 7 experiments (A); effect of 10 μM ivabradine on recovery of Vmax in dogventricular muscle at stimulation cycle length of 400 ms. Mean values are given from8 experiments (B).

Fig. 2. Rate- and dose-dependent effect of ivabradine on the maximum rate of depolarization (Vmax) (A, results are mean±S.E.M., * = Pb0.05, ** = Pb0.01 vs control, n=13,cumulative drug application) and concentration-dependent effect of the drug on the AP waveform/plateau (B, representative figure) in dog Purkinje fiber (recordings originate fromthe same preparation).

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increased. In dog ventricular muscles (n=7) driven at the cyclelength of 400 ms the onset kinetics of Vmax block induced by 10 μMivabradine was fitted to a single exponential, resulting in the onsetrate kinetic constant of 13.9±3.2 beat−1 (Fig. 3A). In dog ventricularmuscles (n=8) at the stimulation cycle length of 400 ms, the

recovery of Vmax during control was best fit to a single exponentialrelation (Fig. 3B). The time constant for recovery of Vmax duringcontrol was fast (τfast=46.2±4.3 ms) and before final repolarizationof the basic action potential, it was almost complete. In the presence of10 μM ivabradine the recovery kinetics of Vmax was best fit with a two

Table 2The electrophysiological effects of ivabradine in dog ventricular muscle preparations at basic cycle length of 1000 ms.

Parameters MDP (mV) APA (mV) APD50 (ms) APD90 (ms) Vmax (Vs−1)

Baseline −86.3±1.2 106.5±1.5 190.1±4.1 223.9±5.9 201.1±17.2Ivabraine 0.1 μM −86.3±1.0 106.7±1.8 195.0±4.7b 229.0±5.7a 205.7±21.6Ivabraine 1 μM −85.4±0.8 107.2±2.1 199.0±5.3b 233.5±6.0a 212.1±20.2Ivabraine 10 μM −87.6±1.1 106.1±2.4 205.0±5.0a 243.8±6.5a 181.8±20.3

MDP, maximum diastolic potential; APA, action potential amplitude; APD50 and APD90, action potential durations at 50% and 90% of repolarization; Vmax, maximum rate ofdepolarization. Results are mean±S.E.M. aPb0.01; bPb0.05 vs control, n=8, cumulative drug application.

Time-solvent control in dog ventricular muscle preparations at basic cycle length of 1000 ms.

Parameters MDP (mV) APA (mV) APD50 (ms) APD90 (ms) Vmax (Vs−1)

Baseline −85.8±0.5 107.5±3.5 181.8±3.8 219.0±4.0 190.9±26.3Distilled water 50 μl −87.9±0.7 107.5±5.4 187.2±6.5 223.0±5.3 185.0±24.4Distilled water 5 μl −86.0±0.8 106.0±3.8 191.7±6.2 228.0±3.6a 182.1±30.2Distilled water 45 μl −84.0±1.1 105.4±3.1 193.8±3.1 230.6±2.7b 195.2±27.6

MDP, maximum diastolic potential; APA, action potential amplitude; APD50 and APD90, action potential durations at 50% and 90% of repolarization; Vmax, maximum rate ofdepolarization. Results are mean±S.E.M., aPb0.01; bPb0.05 vs control, n=5, cumulative solvent (distilled water) application.

Fig. 4. Effect of ivabradine on action potential waveform of dog ventricular muscle with normal and attenuated repolarization reserve at stimulation cycle length of 1000 ms(action potential waveform recordings originate from the same preparation).

Table 3The electrophysiological effects of ivabradine in the dog ventricular muscle after impairing the repolarization reserve at basic cycle length of 1000 ms.

Parameters MDP (mV) APA (mV) APD50 (ms) APD90 (ms) Vmax (Vs−1)

Baseline −83.1±1.5 107.8±2.1 159.2±10.7 200.7±10.9 223.0±26.9BaCl2 30 μM −83.8±3.0 105.9±1.9 171.0±19.0 239.1±18.5a 232.0±26.4Ivabraine 1 μM −84.6±2.7 109.9±2.2 197.1±17.0a 263.3±17.6a 249.8±34.2Ivabraine 10 μM −85.8±2.2 108.9±1.5 230.2±21.7b 306.8±22.0b 230.4±33.0

MDP, maximum diastolic potential; APA, action potential amplitude; APD50 and APD90, action potential durations at 50% and 90% of repolarization; Vmax, maximum rate ofdepolarization. Results are mean±S.E.M. n=5, cumulative drug application.

a Pb0.05 vs control in case of BaCl2 treatment and vs BaCl2 in case of ivabradine application.b Pb0.01.

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exponential relation. In addition to a fast component (τfast=41.2±8.2 ms) which reflects recovery of the drug-free sodium channels(Hondeghem and Katzung, 1984), a slow component (τslow=8.76±1.34 s) of recovery of Vmax was revealed following exposure toivabradine. This second slow component for recovery of Vmax mayreflect effects on drug-affected sodium channels (Hondeghem andKatzung, 1984) (Fig. 3B). In dog ventricular muscle (n=8) at astimulation cycle length of 1000 ms the drug moderately and in aconcentration dependent manner prolonged the action potentialduration (Fig. 4, Table 2, time-solvent group is added), while afterattenuation of the repolarization reserve by inhibition of the inwardrectifier potassium current (n=5) by adding 30 μM BaCl2 it furtherlengthened the ventricular repolarization at concentrations of 1 and10 μM. (Fig. 4, Table 3). In human ventricular muscle preparations(n=6) at a stimulation cycle length of 1000 ms 1 μM ivabradine didnot change ventricular repolarization and a small, but significantprolongation of the action potential repolarization was observed onlyin the presence of high (10 μM) concentration of ivabradine (Fig. 5,Table 4).

3.2. Effects of ivabradine on ionic currents

The effects of the drug on rapid delayed rectifier (IKr), the transientoutward (Ito) and the inward rectifier (IK1) potassium currents weremeasured in rabbit and dog ventricular myocytes. 10 μM ivabradine didnot influence IK1 (control: 365±73 pA, drug: 361±83 pA, at −60 mVtest potential, n=5) in rabbit ventricular myocytes (Fig. 6A). The drugdid not affect Ito in rabbit (control: 496±101 pA, drug: 479±76 pA,at 50 mV test potential, Fig. 6B, n=5), and in dog ventricularmyocytes (control: 2592±577 pA, drug: 2573±516 pA, at 50 mVtest potential, Fig. 6 C, n=4). In rabbit ventricular myocytes the

amplitude of IKr was decreased concentration-dependently byivabradine with an estimated IC50 value of 3.5 μM (Fig. 7A, B).

4. Discussion

Some of the results is a confirmation of previous findings in smallanimals at high concentration i.e. ivabradine (S16257) at 50 μMinduces frequency-dependent Vmax block and at 3 and 10 μMprolongsrepolarization in guinea-pig papillary muscles (Pérez et al., 1995;Thollon et al., 1994). The main new finding of the present study isthat ivabradine in large animal (dog) and human cardiac preparations,in addition to decreasing heart rate, inhibits Vmax, IKr, and at a con-centration higher than 1 μM moderately prolongs APD i.e. exertscombined Class I, Class III and Class V antiarrhythmic actions.

4.1. Possible ionic mechanisms

We have examined the specific ionic mechanisms by whichivabradine affects action potential configuration. Repolarizationlengthening could be related to the IKr block found in rabbit isolatedventricular myocytes. Dose- and frequency dependent Vmax block indog Purkinje-fibers could be attributed to the inhibitory effect ofivabradine on the fast Na+ current. Vmax measurements are indicativefor INa function, but cannot be used for quantitative estimation ofsodium channel availability, which could be underestimated by suchmeasurements (Sheets et al., 1998), since Vmax can be regarded as anonlinear indicator of the fast inward sodium current (Cohen et al.,1984). Block of sodium current has been demonstrated to be morepronounced when the resting membrane potential is partly depolar-ized (Pu et al., 1998) e.g. in ischemic tissues. The effects of ivabradineon recovery of Vmax observed in isolated ventricular muscle indicatethat ivabradine resembles kinetically slow antiarrhythmic agents.According to reported data (Bois et al., 1996) at 10 μM concentrationivabradine does not exhibit a 100% blockade of the If current, thereforeadditional factors such as inhibitory effect on sodium channels cannotbe excluded as possible contributor. A slowly activating sodiumcurrent, INa3, has been described by patch-clamp analysis in the latediastolic depolarization of dog single Purkinje cells. This current issensitive to lidocaine and tetrodotoxin, and contributes to attainmentof the threshold for the upstroke in the oscillary range of late diastolicdepolarization (Rota and Vassalle, 2003). Another possible explana-tion might be the functional subunit interspecies and tissue specificdifference of hyperpolarization-activated cyclic nucleotide gated(HCN) channels (Koncz et al., 2011). Inhibition of IKr by ivabradineat higher concentrations might also influence the spontaneousdiastolic depolarization in Purkinje fibers owing to the “K+ currentdecay hypothesis” (Vassalle et al., 1995). Effect on the plateau slopemight be related to the inhibitory effect of ivabradine on thepersistent,‘window’ Na+ current. Block of this Na+ current mightalso have an additional therapeutic value (Saint, 2008) and wouldalso limit action potential prolongation at slow rate due to the IKrinhibition of the drug at higher concentration. In this context it has tobe mentioned that similar relatively high (2.8 μM) IC50 of ivabradinewas reported on If in rabbit sinus node cells (Bois et al., 1996).

Fig. 5. Effect of ivabradine on action potential waveform of undiseased humanventricular muscle at stimulation cycle length of 1000 ms (recordings originate fromthe same preparation).

Table 4The electrophysiological effects of ivabradine in nondiseased human ventricular muscle preparations at basic cycle length of 1000 ms.

Parameters MDP (mV) APA (mV) APD50 (ms) APD90 (ms) Vmax (Vs−1)

Control −85.4±0.7 106.1±4.0 181.1±15.8 243.3±15.7 278.4±24.3Ivabradine 1 μM −85.5±0.5 104.5±4.3 187.7±15.9 252.0±15.8 266.3±32.4Ivabradine 10 μM −86.0±0.9 102.4±4.0 191.8±16.5 269.8±15.5a 224.5±35.4a

MDP, maximum diastolic potential; APA, action potential amplitude; APD50 and APD90, action potential durations at 50% and 90% of repolarization; Vmax, maximum rate ofdepolarization. Results are mean±S.E.M.

a Pb0.05 vs control, n=6.

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Therefore, the possible decrease of INa by the drug may contribute tothe inhibition of the pacemaker function.

4.2. Possible clinical implications

As the present study shows, ivabradine, in addition to blocking thepacemaker current, at micromolar concentration ranges also inhibitsthe rapid component of the delayed rectifier potassium current,

and presumably, the sodium channels. Though therapeutic plasmaconcentrations of ivabradine are about 0.04–0.07 μM, the drug hasbeen tested at higher (0.17 μM) concentration in healthy volunteers(Joannides et al., 2005), therefore the (0.1–1–10 μM) concentrationsapplied in our experiments may be relevant, since the tissueconcentration can be expected to be higher than that of the plasma,in accordance with the high volume of distribution (close to 100 L)value of the drug. In this context, the relatively low therapeutic

Fig. 6. Effect of ivabradine on IK1 in rabbit ventricular myocyte (A, n=5); effect of ivabradine on Ito in rabbit ventricular myocyte (B, n=5); effect of ivabradine on Ito in dogventricular myocyte (C, n=4).

Fig. 7. Effect of ivabradine on IKr in rabbit ventricular myocyte (A, n=5); concentration-dependent effect of ivabradine on IKr in rabbit ventricular myocyte (B, n=3–5).

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plasma concentration might be the result of possibly meaningfultissue appearance of ivabradine, therefore, we cannot underestimatethe probable INa and the proven IKr blocking ability of ivabradine athigher concentrations. Mostly, because CHF (congestive heart failure)-induced impairments (e.g. deterioration in Vmax) in dog Purkinjetissue conduction have been lately reported (Maguy et al., 2009). Ithas to be emphasized that the drug so far has proved to be safe andfree from any proarrhythmic events in clinical trials (Camm andSavelieva, 2008; Tardif and Borer, 2010). However, considering thebradycardic action of the drug, possible very rare proarrhythmicepisodes cannot be completely ruled out in certain, special casesduring application of ivabradine, especially in some pathologicalconditions, e.g. in case of drug accumulation or intoxication, or in caseof attenuated repolarization reserve like in heart failure, diabetes or inLQT. On the other hand, the mild sodium channel blocking ability onPurkinje fibers might be considered as an antiarrhythmic property andmight suppress the initiation of an extrasystole, limit any repolariza-tion lengthening andmost importantly, it could decrease dispersion ofrepolarization. The frequency-dependent Vmax block might diminishconduction of early extrasystoles or bursts with fast rates whichpotentially elicit cardiac arrhythmias in the presence of an ischemiccardiac substrate. Therefore, the most conspicious finding was thatthis bradycardic drug might possess subsidiary antiarrhythmicactions, which can be utilized in the therapy.

5. Conclusion

Based on the cellular cardiac electrophysiological properties ofivabradine it can be concluded that the drug, in addition to its wellestablished bradycardic effect, also shows Class I and III antiarrhyth-mic properties which can be advantageous to treat patients withischemic heart disease, heart failure (Tavazzi andMugelli, 2006) liableto disturbances of cardiac rhythm.

Acknowledgments

This work was supported by grants from the Hungarian ScientificResearch Fund (OTKA CNK-77855, OTKA K-82079); HungarianMinistry of Health (ETT 302-03/2009 and ETT 306-03/2009); theNational Office for Research and Technology — National TechnologyProgramme (TECH_08_A1_CARDIO08); National Development Agency(TÁMOP-4.2.2-08/1-2008-0013); European Community (EU FP7grant ICT-2008-224381, preDiCT); European Union (NORMACOR); theHungarian Academy of Sciences; and by the János Bolyai ResearchScholarship (to N.J.).

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