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(Circulation. 1997;96:3696-3703.)
© 1997 American Heart Association, Inc.

Articles

Vesnarinone Prolongs Action Potential Duration Without Reverse Frequency Dependence in Rabbit Ventricular Muscle by Blocking the Delayed Rectifier K⁺ Current

Junji Toyama, MD; Kaichiro Kamiya, MD; Jianhua Cheng, MD, PhD; Jong-Kook Lee, MD; Ryoko Suzuki, BSc; ; Itsuo Kodama, MD

From the Department of Circulation (J.T., K.K., J.C., J.K.L.) and the Department of Humoral Regulation (R.S., I.K.), Research Institute of Environmental Medicine, Nagoya (Japan) University.

Correspondence to Kaichiro Kamiya, MD, Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464–01 Japan.

	Abstract

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Background Methanesulfonanilide derivatives, selective inhibitors of the rapidly activating component (I_Kr) of the delayed rectifier potassium current (I_K), prolong action potential duration (APD) of cardiac muscles with reverse frequency dependence, which limits their clinical use because of proarrhythmia. Vesnarinone, a quinolinone derivative developed as a cardiotonic agent, has complex pharmacological properties, but its clinical efficacy is explained in part by I_K reduction. Therefore, we investigated the mode of I_K block by vesnarinone.

Methods and Results I_K of the rabbit ventricular myocyte was activated by voltage-clamp steps applied from a holding potential to various depolarizing levels. The development of I_K block at depolarization (+10 mV) and its recovery process at hyperpolarization (-75 mV) were compared between vesnarinone and E-4031. The I_K block by vesnarinone (3 µmol/L) developed and recovered monoexponentially, with time constants of 361 ms (n=5) and 1.87 seconds (n=4), respectively. I_K block by E-4031 (0.3 µmol/L) developed instantaneously, with no recovery from the block at hyperpolarization. The I_K block by vesnarinone, estimated by I_K tail after a train of depolarizing pulses (for 30 seconds at 0.2 to 2 Hz), was increased with increasing frequency (twofold at 2 from 0.2 Hz), but that by E-4031 was unchanged. In rabbit papillary muscles, vesnarinone (10 µmol/L) prolonged APD at stimulation frequencies >0.2 Hz, whereas E-4031 (0.3 µmol/L) prolonged that in a reverse frequency-dependent manner.

Conclusions Vesnarinone may prolong the repolarization of human cardiac muscle without reverse frequency dependence, because I_Kr is expressed in humans as well as in the rabbit. Thus, this drug may be a model for an ideal class III drug without the risk of proarrhythmia.

Key Words: arrhythmia • potassium channels • action potentials

	Introduction

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Vesnarinone, a quinoline derivative developed as a PDE inhibitor, has been reported to improve both morbidity and mortality rates of patients with chronic CHF.^1–3 However, this drug is effective clinically only at low doses (60 mg/d), below the range at which ventricular contractility and pumping functions are augmented through its PDE inhibitory action.

Recent studies on vesnarinone have clarified that this drug has the pharmacological profile of a blocker of I_K^4–6 and an immunomodulator.^7–10 Therefore, a variety of properties of this drug may be beneficial for patients with chronic CHF. In particular, the I_K blocking action of vesnarinone is effective both for slowing the patient's heart rate,¹¹ which would be accelerated if PDE is inhibited, and for improving the pumping function of failing hearts by causing action potential prolongation, similar to that of the methane sulfonanilide derivatives (pure I_K blockers), which usually inhibit the sinus node activity¹² and augment the contractile force of ventricular muscle.^13–15

However, the class III antiarrhythmic drugs, such as methane sulfonanilide, commonly exhibit a reverse frequency-dependent prolongation of the APD,^16–20 limiting its clinical usefulness because of a risk of proarrhythmia.^21–23

In this study, we compared the effects of vesnarinone with those of the selective I_Kr blocker E-4031 in rabbit myocytes isolated from the apical region of ventricles. We demonstrate that vesnarinone, unlike E-4031, reduces I_K in a use-dependent manner and prolongs APD at relatively higher stimulation frequencies.

	Methods

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Isolation of Single Ventricular Myocytes
Single ventricular myocytes from rabbit hearts were obtained by an enzymatic dissociation procedure as described previously.²⁴ Rabbits of either sex weighing 1.5 to 2.0 kg were anesthetized with thiamylal sodium after being heparinized, and the hearts were rapidly excised and mounted via the aorta on a Langendorff retrograde perfusion apparatus.

Hearts were initially perfused with normal Tyrode's solution (gassed with 100% O₂ at 37°C). When the hearts became clear of blood, perfusion was continued with calcium-free Tyrode's solution until the heart stopped beating and was followed by the same solution containing 0.12 mg/mL collagenase (Yakult) for 15 minutes. Hearts were subsequently washed with high-potassium storage solution (KB solution) for 5 minutes, and the lower third (apical region) of the ventricles was dissected and minced with a pair of surgical scissors. The tissue was then passed though a 200-µm stainless steel mesh. The filtrate was washed twice with KB solution by centrifugation at 170g for 5 minutes. The cells were stored at 4°C in KB solution before use.

Electrophysiological Recording
The single-pipette whole-cell clamp method was used to record the membrane potentials and currents.²⁵ An aliquot of the cell suspension was placed in the recording chamber on the stage of an inverted microscope (Diaphot, Nikon Co). A brief period was allowed for cell adhesion to the coverslip at the bottom of the chamber, and then the cells were superfused with Tyrode's solution at 3 mL/min. The bath temperature in all experiments was maintained at 34°C.

Glass pipettes with a tip diameter of 1 to 2 µm had a resistance of 1 to 3 M after they were filled with pipette solution. The pipettes were connected to a patch-clamp amplifier (List Medical). The series resistance and capacitance were electrically compensated by 70% to 80%. From a holding potential of -60 mV, membrane capacitance was calculated as the area under capacitive transients divided by the amplitude of an applied test pulse (5 mV). The mean capacitance of cells included in this manuscript was 168±15 pF (n=34).

Command potentials were generated by a multichannel stimulator (Nihon-Kohden). When the action potential was elicited, the voltage-clamp mode was switched to the current-clamp mode, and a 5-ms-long rectangular pulse of depolarizing current was injected through the pipette. Voltage records were displayed on an oscilloscope (Tektronix, 5111A) and photographed. Current signals were filtered at 1.5 kHz, digitized at 3 kHz, and stored in a computer (NEC9801DA) by use of an on-line data acquisition system for later analysis. Tyrode's solution used in the isolation of the myocytes and in the experiment was of the following composition (in mmol/L): NaCl 143, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, NaH₂PO₄ 0.25, HEPES 5.0, and glucose 5.6. The calcium-free Tyrode's solution was the same, except that it lacked CaCl₂. The high-potassium storage solution contained (in mmol/L) KOH 70, L-glutamic acid 50, KCl 40, KH₂PO₄ 20, taurine 20, HEPES 10, MgCl₂ 3, glucose 10, and EGTA 0.5. When I_K was measured, the superfusate was changed to NMG solution (in mmol/L: N-methyl-D-glucamine 149, MgCl₂ 5, HEPES 5, and nisoldipine 0.003). In this Na⁺-free, K⁺-free, and nisoldipine (3 µmol/L)-containing external solution, I_Ca, I_K1, the Na⁺-Ca²⁺ exchange current, and the Na-K pump current are negligible.²⁶ The internal pipette solution contained (in mmol/L) KOH 60, KCl 80, L-aspartic acid 40, HEPES 5, EGTA 10, MgATP 5, sodium creatinine phosphate 5, and CaCl₂ 0.65 (pH 7.2; pCa, 8.0).

Recording Action Potentials in Papillary Muscles
Japanese White rabbits of either sex weighing 1.8 to 2.2 kg were anesthetized by administration of pentobarbitone sodium (30 mg/kg IV), and the right ventricular papillary muscles were removed.²⁷ The muscles (0.4 to 0.6 mm in diameter and 3 to 4 mm in length) were mounted in a tissue bath (0.5 mL) and superfused at 32°C with Krebs-Ringer solution gassed with 95% O₂/5% CO₂. The composition of the solution was (in mmol/L): NaCl 120.3, KCl 4.0, CaCl₂ 1.2, MgSO₄ 1.3, NaHCO₃ 25.2, and glucose 5.5, pH 7.4. The preparation was stimulated by a pair of 1.0-mm platinum wire electrodes placed 1.0 mm apart from both sides of the muscle. Pulses used for stimulation were 2 ms in duration and 20% higher in intensity than the diastolic threshold unless otherwise specified. Transmembrane action potential was recorded through two glass microelectrodes filled with 3 mol/L KCl, one intracellularly and the other extracellularly, placed close together. The electrodes were each connected by Ag/AgCl wire to a high-input impedance buffer amplifier connected to a differential amplifier (MEZ-7101, Nihon Kohden).

Drugs and Data Analysis
Vesnarinone (3,4-dihydro-6[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone, OPC-8212) was obtained from Otsuka Pharmaceutical Co. E-4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methane sulfonamide dihydrochloride dihydrate) was obtained from Eisai Pharmaceutical Co. They were dissolved in distilled water as stock solutions and diluted in superfusates to the desired final concentrations immediately before each experiment.

All animals were treated in accordance with the principles and procedures outlined by the Committee for Animal Experiment of Nagoya University School of Medicine and the Research Institute of Environmental Medicine.

The curve-fitting program Igor (Wave Metrics, Oreg) was used in data analysis. Data in the text are presented as mean±SEM. Statistical analysis in two paired groups was carried out by a paired t test. Group means were compared by post hoc multiple comparisons after ANOVA.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Block of I_K During Activation
Dose-dependent effects of vesnarinone on the I_K activation were examined by application of voltage-clamp steps for 3 seconds from a holding potential of -80 mV to different depolarizing levels up to +50 mV at 0.1 Hz. Representative traces are shown in Fig 1A. Vesnarinone (3 to 30 µmol/L) dose dependently depressed both I_K during the depolarizing pulse and the I_K tail detected on clamping back to the holding potential. Fig 1B shows the dose-response curve of the I_K tail to vesnarinone. The IC₅₀ of the I_K tail current after +10-mV depolarization was calculated to be 5.7±0.2 µmol/L (n=5). Voltage dependence of I_K activation was approximated by the Boltzmann function, and V_0.5, the membrane potential at half-activation, and K, the slope factor, were calculated. Neither V_0.5 nor K was significantly changed between control and drug-treated groups, and these were within the range of 0.6 to 0.8 mV for V_0.5 and 12.1 to 16.5 for K, respectively.

View larger version (29K): [in this window] [in a new window]   Figure 1. Effects of vesnarinone on voltage-dependent activation of IK. A, Typical recordings. IK was elicited by application of voltage-clamp steps from a holding potential of -80 mV to different depolarizing levels between -40 and +50 mV. A time-dependent outward current during depolarization step was followed by outward tail current on repolarization to -50 mV. B, Current-voltage relationship of IK tail and concentration-dependent inhibition by vesnarinone. Tail-current amplitude was plotted as a function of test potential. Vesnarinone inhibited IK with IC50 of 5.7 µmol/L (n=5) at +10 mV level. *P<.05 vs control.

The effects of E-4031 on I_K activation were analyzed by the same protocol. E-4031 (0.3 to 10 µmol/L) also depressed the I_K tail in a dose-dependent manner. IC₅₀ for E-4031 was 0.91±0.3 µmol/L (n=6). The highest concentration of 10 µmol/L E-4031 inhibited I_K by 79.1% (n=6) at +10 mV, and a similar range (76.1% to 82.5%, n=6) of I_K block was observed at voltages ranging from -10 to +50 mV.

The difference in the development of I_K block was compared between these two drugs by measurement of the tail current evoked at the -50-mV step after a depolarizing prepulse to +10 mV with variable durations from 0.05 to 3 seconds (Fig 2). Fig 2A illustrates superimposed current traces before (control) and after vesnarinone (3 µmol/L), with conditioning depolarizing pulses (+10 mV) of 0.1 and 1 second. Reduction of the I_K tail amplitude (I_K block) by vesnarinone is small (25%) at the conditioning pulse of 0.1 second, but the reduction becomes marked (80%) when the conditioning pulse is prolonged to 1 second. This implies that the I_K block by vesnarinone is augmented as the I_K activation proceeds during depolarization.

View larger version (21K): [in this window] [in a new window]   Figure 2. Development of IK channel block during depolarization. A, Pulse protocol and typical recordings. Developments of IK channel block by vesnarinone and E-4031 were observed by application of depolarizing prepulses of variable duration from 0.05 to 3 seconds from the holding potential of -75 mV, and tail currents were evoked at -50 mV step after prepulse. Top, Superimposed tracings of typical recordings in absence (C) and presence of vesnarinone (3 µmol/L) (V) with depolarizing pulse of 0.1 second (left) and 1 second (right). B, IK current amplitude. IK tail-current amplitudes in absence and presence of vesnarinone (3 µmol/L) (n=5) and E-4031 (0.3 µmol/L) (n=7) plotted as a function of pulse duration. C, Ratio of IK tail. Tail-current amplitude of IK in presence of vesnarinone (3 µmol/L) () or E-4031 (0.3 µmol/L) () normalized to untreated control values calculated from data in B and plotted as a function of duration of depolarization (+10 mV). Vesnarinone progressively reduced ratios as clamp duration of prepulse was increased; reduction progressed monoexponentially with a time constant of 361 ms. In presence of E-4031, reduction (40%) in ratios was independent of pulse duration.

Fig 2B compares the summarized data on I_K block by vesnarinone and E-4031, when the depolarizing duration was prolonged from 0.05 to 3 seconds. Fig 2C illustrates the ratio of I_K tails in the presence and absence of either drug that reflects the drug-bound fraction to all the available I_K channels. For vesnarinone, the ratio decreased monoexponentially (with a time constant of 361 ms, n=5) during the depolarization, implying that a first-order reaction proceeds rather slowly between vesnarinone and its specific binding site of the open (or activated)-state I_K channel. However, the ratio of I_K tail for E-4031 (0.3 µmol/L) remained unchanged even when the depolarizing pulse was lengthened to 3.0 seconds. This suggests that the interaction between the I_K channel and E-4031 is either immediate or state-independent.

Unblock of I_K During Deactivation
Modulation of the I_K deactivation process by vesnarinone and E-4031 (Fig 3) was compared between vesnarinone and E-4031. The I_K deactivation process was measured by application of the clamp-pulse protocol indicated in Fig 3A. From a holding potential of -50 mV, the membrane was depolarized to +10 mV for 1 second to activate I_K. The membrane potential was then clamped to -75 mV for a variable duration before a test depolarization to +10 mV for 0.2 second was applied. I_K tails were measured on clamping back to -50 mV after the test pulse.

View larger version (17K): [in this window] [in a new window]   Figure 3. Recovery from IK channel block at hyperpolarization. Recovery of IK channel block at hyperpolarization compatible with physiological resting potential was compared between vesnarinone (3 µmol/L) and E-4031 (0.3 µmol/L). A, Experimental protocol and representative recordings. Hyperpolarizations to -75 mV for variable durations were preceded by a 1-second depolarizing pulse to +10 mV. Then, a test depolarization of 0.2 second to +10 mV was applied and tail current was evoked at -50 mV step. Superimposed tracings in absence (C) and presence of vesnarinone (V) and E-4031 (E) show tail currents after hyperpolarizations for 0.1, 1, and 10 seconds. B, IK channel unblock at hyperpolarization. Ratios of IK tail amplitudes to control are plotted as a function of duration at -75 mV. In presence of vesnarinone (), recovery of the IK tail ratio progressed with a time constant of 1.87 seconds (n=4). As for E-4031 (), no recovery was seen during hyperpolarization (n=4).

Fig 3A shows representative traces of the I_K tails obtained before and after either vesnarinone (3 µmol/L) or E-4031 (0.3 µmol/L). The amplitudes of the I_K tail before drug were decreased gradually as the duration of hyperpolarization was lengthened, whereas those measured after vesnarinone administration were increased with an increase in the duration of hyperpolarization. However, E-4031 did not cause such a time-dependent I_K increase but simply led to a time-independent I_K decrease after hyperpolarization.

Fig 3B relates the ratios of I_K tail amplitudes in the presence and absence of either vesnarinone (3 µmol/L) or E-4031 (0.3 µmol/L) to the duration of hyperpolarization. In the presence of vesnarinone, the ratios were increased monoexponentially, with a time constant of 1.87 seconds as the hyperpolarization pulse was prolonged. This I_K recovery may result from unbinding of the drug from the closed (or deactivated)-state I_K channel. Such I_K recovery during deactivation was also present at a partially hyperpolarized potential level (-50 mV) (data not shown). However, for E-4031, there was no recovery of the I_K tail at the potential level of either -50 (not shown) or -75 mV. This observation supports the concept^21,28 that class III drugs containing methane sulfonanilide bind to the open-state I_K channel and do not unbind from the closed-state channel.

Frequency Dependence of I_K Inhibition
Fig 4 compares frequency dependence of I_K inhibition by vesnarinone (3 µmol/L) and E-4031 (0.3 µmol/L). A train of the depolarizing-clamp pulses of 200 ms to +10 mV to mimic the configuration of ventricular action potential was applied at a rate from 0.2 to 2.0 Hz. After the train of depolarizing pulses for 30 seconds, the I_K tail was measured by a test-clamp pulse. The amplitude of I_K tail before drug was augmented markedly as the pulse rate was increased from 0.2 to 2 Hz. This indicates that decrease of the resting interval at faster rates shortens the time for the I_K channel to deactivate, thus leading to a rate-dependent augmentation of the I_K activation.

View larger version (16K): [in this window] [in a new window]   Figure 4. Frequency dependence of IK inhibition. A, Experimental protocol and representative recordings. A train of repetitive depolarizations (+10 mV for 0.2 second) with a frequency of 0.2 to 2 Hz was applied from holding potential at -75 mV. After train of depolarizing pulses for 30 seconds, IK tail was measured at -50 mV step (top). Representative recordings of IK tail currents are obtained in absence (C) and presence of vesnarinone (V) and E-4031 (E) after pulse train at 0.2 Hz and 2 Hz. B, Frequency-dependent IK inhibition. IK tail ratios in presence of vesnarinone (3 µmol/L) () and E-4031 (0.3 µmol/L) () are plotted as a function of frequency of pulse train.

In the presence of vesnarinone, such rate-dependent augmentation of the I_K tail disappeared, but the I_K tail amplitude decreased at increasing rates (Fig 4A). However, E-4031 reduced the I_K tail markedly at low (0.2-Hz) and high (2-Hz) rates of the stimulation. Fig 4B compares the frequency dependence of I_K inhibition by vesnarinone and E-4031, comparing I_K tails and the ratios of I_K tail amplitudes in the presence and absence of each drug.

In the presence of vesnarinone, the ratio was reduced from 84.3±4.9% to 50.1±1.8% (n=4) as the pulse rate increased from 0.2 to 2.0 Hz. In contrast, there was little or no frequency dependence in I_K inhibition by E-4031.

Frequency-Dependent Effects on APD
Effects of vesnarinone and E-4031 on the APD were compared in ventricular papillary muscle preparations when the stimulation rate was increased from 0.1 to 3 Hz. Fig 5 (top) shows examples of the APDs before and after exposure to vesnarinone (10 µmol/L) and E-4031 (0.3 µmol/L), respectively. Vesnarinone lengthened APD moderately at the physiological range (0.5 to 3.0 Hz) of the stimulation but not at a very low range, whereas E-4031 caused a marked, progressively greater APD prolongation as the stimulation was reduced from 3.0 to 0.1 Hz.

View larger version (20K): [in this window] [in a new window]   Figure 5. Frequency dependence of APD prolongation in rabbit papillary muscles. A, Effects of vesnarinone. Top, Pairs of superimposed traces of action potential before and after application of vesnarinone (10 µmol/L) stimulated at 0.1, 0.5, 1, and 3 Hz. Bottom, APDs at -70 mV repolarization plotted against stimulation rates. Vesnarinone prolonged APD significantly at frequencies from 0.5 to 3.3 Hz. *P<.05 vs each control value, n=5. B, Effects of E-4031. Action potentials at top were superimposed before and after application of E-4031 (0.3 µmol/L). Bottom, E-4031 prolonged APD more prominently as stimulation rate was reduced (from 0.1 to 1.0 Hz), giving rise to a marked reverse frequency dependence. *P<.05 vs each control value, n=5.

Fig 5 (bottom) summarizes these results; the APD prolongations by vesnarinone showed maximum (67 ms) at 0.5 Hz but were decreased with either an increase or a decrease in stimulation rate, whereas those by E-4031 were increased simply in inverse proportion to the stimulation rate (1.9 and 6.1 times the control at 1.0 and 0.1 Hz, respectively).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Constituents of I_K in Rabbit Ventricle
It has been established that I_K is composed of I_Kr and I_Ks in the ventricular muscle of guinea pig heart.^6,17,29 In contrast, I_K of rabbit ventricular myocytes in our experiment was reduced to 20% of control at the maximum dose (10 µmol/L) of E-4031, indicating that I_K of rabbit ventricle is composed mainly of I_Kr. However, whether rabbit^6,17,30–32 and human^6,31,33 ventricular muscle may also express solely I_Kr or I_Ks is controversial. Recently, Salata et al³² reported the existence of a large I_Ks and I_Kr in rabbit ventricular myocytes, and more interestingly, Li et al³³ succeeded in recording I_Ks (E-4031–resistant but azimilide- or indapamide-sensitive current) in human ventricular myocytes isolated not by chunk³⁴ but by the perfusion method (arterial perfusion of digesting solution). However, as far as the isolation of rabbit or guinea pig ventricular muscles is concerned, the perfusion method with Langendorff apparatus is usually adopted.^{6,18,26,29,32} Thus, the predominant expression of I_Kr (or E-4031–sensitive current) in the rabbit ventricular muscle is perhaps attributable to species-specific differences in addition to differences in isolation method. Furthermore, I_Kr may be differentially expressed in different regions of the rabbit ventricles. We have recently reported³⁵ that E-4031 (0.1 µmol/L) prolonged epicardial repolarization times more significantly in the apical than in the basal region of Langendorff-perfused rabbit ventricles (61% versus 38% increase in the interval of Q to the peak of the T wave on the ECG, which corresponds to differentiated monophasic potential; n=5, P<05), suggesting that I_Kr is expressed more dominantly in the apex than in the base. Therefore, a predominance of I_Kr expression in our experiment may be partly explained by such regional specificity, because the myocytes used for the experiment were those isolated selectively from the lower thirds (apical region) of the rabbit ventricles.

Relation of APD Prolongation and I_Kr Block
Vesnarinone (3 µmol/L) prolonged APDs of rabbit ventricular muscle by 20% to 30% in the physiological range of the stimulation rate, like amiodarone,²⁷ but its APD-prolonging effect was minimal or negligible at very-low-frequency ranges (0.1 to 0.2 Hz). In contrast, E-4031 prolonged APD in a reverse frequency-dependent manner (APD prolongation becomes greater in inverse proportion to the stimulation rate), like other methane sulfonanilide class III agents,^{13,16,19,36,37} (d-sotalol, sematilide, dofetilide, ambasilide, and almokalant) and class I agents.^37–40

The mechanisms^{21,37,41–43} by which such reverse frequency-dependent APD prolongation by methane sulfonamides occurs are explained as follows: (1) the drug binds to the open (or activated) state of the I_Kr channel with a high affinity (open-state channel block); (2) the drug is kept bound to the closed (or deactivated)-state channel; (3) its blocking effect is therefore accumulated by repetition of the procedure to make the channel open; and (4) after equilibrium is attained, the drug action becomes independent of time and channel states (tonic block); thus, the smaller the slope (sum of ionic currents) of the plateau potential associated with reduced firing rates, the more augmented the APD prolongation resulting from I_Kr reduction.

Actually, the mode of I_Kr inhibition by E-4031 (ratio of the I_K tail in the presence and absence of the drug) appeared to be tonic (Fig 2, bottom), because the block was independent of time length of depolarization (or activation), duration of repolarization (or deactivation), or frequency of stimulation. Conversely, I_K inhibition by vesnarinone (3 µmol/L) proceeded monoexponentially with a time constant (0.4 second; see Fig 2) as small as that calculated from the time course of [³H]dofetilide binding to high-affinity sites on guinea pig cardiac myocytes.⁴⁴

There are many reports^{19,21,37,41,42} on modes of I_K channel block by class I and III antiarrhythmic drugs. Vesnarinone is the first drug that unblocks I_K channels almost completely (80% recovery) at the physiological resting membrane potential (-75 mV). Carmeliet¹⁸ already reported that almokalant unblocked the I_K channel of rabbit ventricular muscle very slowly (with a time constant of 10 seconds) only at a partially depolarized potential level (>90% recovery at -50 mV) but very incompletely at full repolarization (20% recovery at -75 mV) and suggested that unblocking of this drug was practically absent in the physiological condition.

Vesnarinone blocked I_K channel frequency-dependently (or use-dependently), whereas E-4031 blocked it frequency-independently (see Fig 4). This difference in frequency dependence of I_K block is attributed to the presence and absence of I_K channel unblock during the resting interval; the I_K channel unblock by vesnarinone proceeds at a rate of 53% recovery per second, the reciprocal of the time constant (1.87 seconds) for the I_K recovery in Fig 3. As a result, the I_K block augmented at high rates is attenuated in the low range of the stimulation (see Fig 4), whereas E-4031 did not unblock the I_K during the resting interval, thus leading to frequency-independent I_K block.

Effects of Vesnarinone on Ionic Currents Other Than I_Kr
Recent progress in molecular mechanisms of long QT syndrome^45,46 suggests that the human I_K is also composed of I_Kr and I_Ks; this speculation was confirmed by Li et al.³³ Similarly, I_K of the rabbit ventricular muscle was recently reported to consist of I_Kr and I_Ks.³² More interestingly, the contributions of the two components to APD may be different in different regions of the ventricles.^47,24 Also, APD may be greatly affected by adrenergic signaling, because I_Ks is very sensitive to isoproterenol⁴⁸ compared with I_Kr.

The effects of vesnarinone on I_Ks are complicated because vesnarinone may augment I_Ks indirectly through its PDE inhibitory action in addition to its direct action. However, as far as I_Ks (the E-4031–resistant component of I_K) of the myocyte isolated from the basal region of the rabbit ventricles was tested in our preliminary study, it remained unchanged (or increased minimally) in the presence of low concentrations of vesnarinone (3 to 30 µmol/L) (data not shown).

Effects of vesnarinone on L-type Ca current (I_Ca)⁴⁹ are more complicated than those on I_Ks; vesnarinone inhibited the peak of I_Ca of the rabbit ventricular myocytes mildly (10%) in low concentrations of the drug, but it augmented the I_Ca dose-dependently in concentrations >30 µmol/L through its PDE inhibitory action. Thus, vesnarinone appears to affect both the I_Ks and I_Ca of the rabbit ventricular muscle minimally within the range of its clinical concentrations⁶ (10 µmol/L).

As for other ionic currents that contribute to the plateau of action potential, Lathrop et al⁶ reported that neither Na⁺, inward rectifier K⁺, nor transient outward current was affected by a lower concentration of vesnarinone (12 µmol/L). Therefore, vesnarinone may prolong APD mainly through its I_Kr channel blocking action, but further experiments are necessary to clarify the effects of this drug on cAMP-sensitive (Na-Ca exchanger, Cl^- channel) and ATP-sensitive (Na-K pump, ATP-sensitive K⁺ channel) currents.

We measured the effects of vesnarinone on the frequency-dependent change of APD in rabbit papillary muscle preparations. These preparations showed consistent data on APD changes in response to both change of stimulation rates and application of drugs for several hours.²⁷ A marked E-4031–sensitive prolongation of APD indicates that ventricular papillary muscle expressed I_Kr as dominantly as the apical ventricular muscle. In fact, the main part of the papillary muscle mass was included in the apical region dissected from the digested rabbit heart. Therefore, it is reasonable to discuss APD prolongations of the ventricular papillary muscle induced by E-4031 or vesnarinone in terms of I_Kr inhibition of the apical ventricular myocyte by the drug.

Lathrop et al⁵⁰ reported that vesnarinone, like sotalol, prolonged the APD of canine Purkinje fiber with a reverse frequency dependence. The ionic currents that contribute to the plateau potential of Purkinje fiber⁵¹ may be greatly different from those that contribute to ventricular muscle,⁵² possibly leading to contradictory results.

Limitations
APD is determined essentially by a sophisticated balance of inward (Na⁺, Ca²⁺, and Na⁺-Ca²⁺ exchange) and outward (K⁺, Cl^-, and Na⁺-K⁺ pump) currents at the plateau phase of action potential.⁵² But the balance of these contributing currents may be different in either species^53,54 of animal or regions⁵¹ of the heart, and furthermore, it may be modulated by intracellular substances such as Ca²⁺, H⁺, and cAMP.^51,55

Therefore, the frequency-dependent APD changes in the presence of vesnarinone should be examined further from the viewpoints of modulation of intracellular Ca²⁺ and cAMP signaling as well as state-dependent block of I_K.

Clinical Significance
Class III drugs have been aimed at terminating reentrant ventricular tachyarrhythmias with a excitable gap^56,57 by prolongation of the ventricular repolarization, but they carry a risk of provoking ventricular arrhythmias such as torsade de pointes^22,23 if the APD prolongation is augmented with a reverse frequency dependence. Thus, the property of I_Kr unblock at the resting potential level is essential for the ideal class III drug, because the I_Kr block accumulated during the systolic phase can be attenuated by the unblocking process during the diastolic phase, leading to limited excess of APD prolongation at low heart rate. Thus, vesnarinone may be a model for development of ideal class III drugs; furthermore, this drug may benefit CHF through the mechanisms of opposing the neurohumorally augmented sinus node activity^12,11 and improvement of the pumping function of failing hearts with a minimal risk of proarrhythmia, like amiodarone.^58,59

	Selected Abbreviations and Acronyms

 

APD = action potential duration CHF = congestive heart failure IK = delayed rectifier potassium current IKr = rapidly activating component of IK IKs = slowly activating component of IK IK1 = inward rectifier potassium current PDE = phosphodiesterase

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid for Scientific Research (07670774) from the Ministry of Education, Science, Sports, and Culture, Japan. The authors gratefully acknowledge the helpful comments and suggestions by Dr James N. Weiss.

Received March 18, 1997; revision received July 28, 1997; accepted August 5, 1997.

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