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(Circulation. 1995;91:1799-1806.)
© 1995 American Heart Association, Inc.

Articles

Ibutilide, a Methanesulfonanilide Antiarrhythmic, Is a Potent Blocker of the Rapidly Activating Delayed Rectifier K⁺ Current (I_Kr) in AT-1 Cells

Concentration-, Time-, Voltage-, and Use-Dependent Effects

Tao Yang, PhD; Dirk J. Snyders, MD; Dan M. Roden, MD

From the Departments of Pharmacology (T.Y., D.J.S., D.M.R.) and Medicine (D.J.S., D.M.R.), Vanderbilt University School of Medicine, Nashville, Tenn.

Correspondence to Dan M. Roden, MD, Division of Clinical Pharmacology, 532 Medical Research Building, Vanderbilt University School of Medicine, Nashville, TN 37232-6602.

	Abstract

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Background Ibutilide is an action potential–prolonging antiarrhythmic currently in clinical trials. The drug shares structural similarities with E-4031 and dofetilide, specific blockers of the rapidly activating delayed rectifier K⁺ current (I_Kr). However, previous in vitro studies in guinea pig myocytes have indicated that ibutilide does not block I_Kr but rather increases a slow inward sodium current.

Methods and Results In this study, we compared the effects of ibutilide with those of dofetilide on outward current in mouse atrial tumor myocytes (AT-1 cells), a preparation in which, unlike guinea pig, a typical I_Kr is the major delayed rectifier and can be readily recorded in isolation from other currents. In AT-1 cells, ibutilide and dofetilide were both potent I_Kr blockers, with EC₅₀ values of 20 (n=12) and 12 (n=8) nmol/L, respectively, at +20 mV. The time and voltage dependence of I_Kr inhibition by the two compounds were virtually identical. The following characteristics were most consistent with open channel block: (1) block increased with depolarizing pulses; (2) block increased with longer pulses; (3) currents deactivated more slowly in the presence of drug, resulting in a "crossover" typical of open channel block; and (4) with repetitive pulsing after drug wash-in, use-dependent block was observed.

Conclusions These data suggest that the clinical actions of ibutilide are mediated at least in part by block of I_Kr; an effect on inward currents is not excluded. AT-1 cells are a useful model system for the study of drug block of this important repolarizing current.

Key Words: ibutilide • dofetilide • antiarrhythmic agents • myocytes

	Introduction

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The delayed rectifier K⁺ current (I_K) plays an important role in controlling cardiac action potential duration, and block of I_K is a major mechanism underlying action potential prolongation by antiarrhythmic drugs.^{1 2 3} A number of drugs with the class III action potential–prolonging attribute are currently in clinical trials. In many mammalian cardiac cells, I_K is now recognized to be made up of at least two components, I_Kr and I_Ks. This distinction was first described in guinea pig cells, where I_Kr was defined as the component of I_K blocked by the methanesulfonanilide antiarrhythmic E-4031.⁴ With the use of a subtraction procedure (ie, examination of the difference between baseline current and current recorded after exposure to a very high concentration of E-4031), it was possible to infer the major physiological features of I_Kr: rapid activation and prominent inward rectification. In other cells (eg, rabbit or cat ventricular myocytes), a prominent I_Kr, blocked by nanomolar concentrations of E-4031 or dofetilide, can be recorded, and no I_Ks is observed.^{5 6}

A prominent I_Kr is also readily recorded and no I_Ks observed in the atria of AT-1 cells.⁷ These cells are derived from mice carrying a transgene in which the atrial natriuretic factor promoter drives expression of the SV40 large T antigen.⁸ AT-1 cells retain morphological, biochemical, and electrophysiological features of cardiac myocytes.^{9 10} In AT-1 cells, I_Kr can be studied directly, without the necessity of a subtraction procedure to eliminate overlapping currents.⁷ I_Kr recorded in AT-1 cells displays features very similar to those deduced in guinea pig cells: it activates rapidly, shows prominent inward rectification, and is blocked by nanomolar concentrations of dofetilide.

Ibutilide, N-[4-[4-(ethylheptylamino)-1-hydroxybutyl]phenyl]-methanesulfonamide, also prolongs cardiac repolarization in vitro and in vivo, including in humans.^{11 12 13} This effect has been attributed to activation of a slow inward current rather than inhibition of outward potassium current¹⁴ ; however, studies characterizing the effects of ibutilide were performed in guinea pig myocytes, in which multiple K⁺ currents are present and subtraction is required to delineate drug effects. Since ibutilide shares the methanesulfonanilide motif (Fig 1) seen in other antiarrhythmics known to block I_Kr (E-4031 and dofetilide), we performed the present study in AT-1 cells to test the hypothesis that ibutilide is a blocker of I_Kr. The characteristics of the effects of ibutilide in this system were compared with those of dofetilide.

View larger version (8K): [in this window] [in a new window]   Figure 1. Structures of ibutilide and dofetilide.

	Methods

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Cell Preparations
The transgenic animals and initial characterization of AT-1 cells have been reported by the Field and Claycomb laboratories (Steinhelper et al⁹ and Delcarpio et al¹⁰ ). We have used these methods to establish a self-sustaining colony of tumor-bearing mice. To isolate the cells, we used procedures described previously.⁷ In brief, tumors were minced and digested with collagenase-containing PC-1 medium. After gentle centrifugation, supernatants were collected, plated, and cultured for at least 1 week at 37°C. In the experiments described here, cells cultured for 7 to 14 days were disaggregated by a brief trypsinization procedure and stored until use (within 48 hours) in culture medium at room temperature (22° to 23°C). Cells that appeared round were then used for electrophysiological study.

Electrophysiological Recording
Recordings were made at room temperature with an Axopatch-1A patch-clamp amplifier (Axon Instruments, Inc) using the whole-cell configuration of the voltage-clamp technique.¹⁵ Microelectrodes were pulled from starbore borosilicate glass (Radnoti Co) and were heat-polished. Ion currents were low pass filtered at 500 Hz (Bessel filter, -3 dB), sampled at 1 kHz, and stored on the hard disk of an IBM PC-AT for subsequent analysis. Data acquisition and command potentials were controlled with a commercial software program (PCLAMP, Axon Instruments). To ensure voltage-clamp quality, microelectrode resistance (R_e) was kept below 3 M. Junction potential was zeroed with the electrode in the bath solution. The microelectrode was then gently lowered onto the cell surface, and gigaohm seal formation was achieved by suction (range, 5 to 50 G). After the seal was ruptured and the whole-cell configuration established, the capacitive transients elicited by 10-mV voltage-clamp steps from -80 to -70 mV were recorded at 50 kHz (filtered at a bandwidth of 10 kHz, -3 dB) for subsequent calculations of capacitive surface area, time constant, and access resistance (R_a). Thereafter, capacitance and series resistance compensation were optimized; approximately 80% compensation was usually obtained.

Solutions and Drugs
The intracellular pipette filling solution contained (mmol/L) KCl 110, K₄BAPTA 5, K₂ATP 5, MgCl₂ 1, and HEPES 10, and the solution was adjusted to pH 7.2 with KOH, yielding a final intracellular K⁺ concentration of approximately 145 mmol/L. The extracellular Tyrode's solution contained (mmol/L) NaCl 130, KCl 4, CaCl₂ 1.8, MgCl₂ 1, HEPES 10, and glucose 10, and the solution was adjusted to pH 7.35 with NaOH.

Ibutilide was provided by Upjohn Pharmaceutical Co. Nisoldipine (to block L-type Ca²⁺ current, I_Ca-L) was obtained from Miles Pharmaceutical, Inc, and dofetilide was provided by Pfizer Central Research. Other chemicals were purchased from Sigma Chemical Co. In the experiments in which the use dependence of drug block was measured, I_Na was eliminated by an Na⁺-free extracellular solution (N-methyl-D-glucamine was substituted for Na_o), and NiCl₂ was used to block T-type Ca⁺ current, I_Ca-T. The final drug concentrations in the bath were obtained by diluting stock solutions into the extracellular solution during experiments.

Voltage-Clamp Protocols and Analysis
In this study, I_Na and I_Ca-T were inactivated with a holding potential of -40 mV, and cycle time between pulses was 15 seconds unless indicated otherwise. When drug was added, activating current was monitored by depolarizing pulses every 15 seconds, and "on-drug" data were recorded only when steady state was reached. To obtain current-voltage relations for K⁺ current in AT-1 cells, activating currents were elicited with depolarizing pulses from a holding potential of -40 to +50 mV in 10-mV steps, and deactivating tail currents were recorded on repolarization to -40 mV unless otherwise indicated. As we have previously reported,⁷ activating current elicited under these conditions satisfies an envelope test; ie, it is composed of a single component. Evidence that this current is I_Kr includes the relatively rapid activation of the current (eg, =182±27 milliseconds at +20 mV), its prominent inward rectification, and block by nanomolar concentrations of dofetilide.⁷ AT-1 cells also display I_Na, I_Ca-L, and I_Ca-T, which were eliminated as described above.

As illustrated in Fig 2, activating I_Kr was defined as the difference between initial and steady-state currents obtained at the end of depolarization within a moving window of 10 to 20 milliseconds beyond capacitive transients. The deactivating I_Kr tail was the difference between peak and steady-state currents with repolarizing pulses. Instantaneous current, which in other cell types represents a plateau potassium current or a chloride current,^{16 17} and recovery from fast inactivation (large arrow, Fig 2)¹⁸ were occasionally observed but were not further studied. After linear leak subtraction, macroscopic currents were normalized to cell capacitance.

View larger version (17K): [in this window] [in a new window]   Figure 2. Diagram of data analysis method. Outward current activated by a 5-second depolarizing pulse from -40 to +20 mV is shown. IKr activated by the clamp step is the difference between current recorded just after the beginning of the step and that recorded at the end of the step (dashed lines). After the depolarizing step to +20 mV, the preparation is clamped at -40 mV. Under these conditions, a slowly deactivating "tail" current is observed. The amplitude of the tail current is measured between the peak of the tail current just after the clamp step to -40 mV and the steady-state value at -40 mV (dotted lines). The magnitude of the deactivating tail current is an index of the number of channels activated during the preceding depolarizing pulse. The "instantaneous" current and the "hook" (large arrow) are discussed in the text.

The concentration dependence of drug block of I_Kr was determined by first normalizing on-drug current as I_drug/I_control and then fitting these data to the Hill equation, I_drug/I_control={1+[EC₅₀/(drug)]ⁿ}, where EC₅₀ is the concentration producing 50% block, and n is the Hill coefficient. To analyze the voltage dependence of activation and deactivation, I_drug/I_control versus voltage data were fit to the Boltzmann equation, I_drug/I_control=1/{1+exp[-(E-E_h)k]}, where k represents the slope factor and E_h the voltage at which 50% of the channels are activated or inactivated. The time course of tail currents was fit with sums of multiple exponential terms. All fitting was performed with software developed in our laboratories^{19 20} using nonlinear least-squares fitting procedures.

Results are expressed as mean±SEM. Student's t test was used to compare differences between mean values, with a value of P<.05 considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Concentration Dependence
Activating currents elicited by 1-second pulses to +20 mV and subsequent tail currents at -40 mV are shown in Fig 3. These data demonstrate that ibutilide, like dofetilide, is a potent blocker of I_Kr in AT-1 cells. The concentration dependence of block by the two drugs is presented in Fig 4A. The EC₅₀ for block with 1-second pulses to +20 mV was 20 nmol/L (n=12) for ibutilide and 12 nmol/L (n=8) for dofetilide. Fig 4B shows that the EC₅₀ for drug block during 1-second depolarizing pulses was voltage dependent, with greater drug sensitivity at positive potentials.

View larger version (15K): [in this window] [in a new window]   Figure 3. Tracings show effect of ibutilide and dofetilide on IKr. INa and ICa-T were eliminated by holding at -40 mV, and nisoldipine was added to eliminate ICa-L.

View larger version (19K): [in this window] [in a new window]   Figure 4. A, Line graph shows concentration dependence of IKr block by ibutilide (, n=12) and dofetilide (, n=8). Each data point represents the mean of 4 to 12 experiments. B, Line graph shows voltage dependence of EC50 values determined as in A.

Voltage and Time Dependence
Block of activating and deactivating outward current is shown in Figs 5 and 6. Fig 5 shows the very prominent inward rectification of activating I_Kr and demonstrates that virtually all activating current was inhibited by 1 µmol/L of either drug. Similarly (Fig 6), deactivating tails were abolished by 1 µmol/L of either drug. Concentrations near the EC₅₀ (Figs 6 and 7) were used to assess the voltage dependence of tail current block. Block by both drugs was most prominent after pulses to positive potentials, consistent with the lower EC₅₀ values calculated at positive potentials (Fig 4B).

View larger version (20K): [in this window] [in a new window]   Figure 5. Line graphs show current-voltage relations for activating current in the absence () and presence () of 1 µmol/L of ibutilide or dofetilide. Drug-sensitive current is shown ().

View larger version (19K): [in this window] [in a new window]   Figure 6. Line graphs show current-voltage relations for deactivating tail currents in the absence () and presence (, 10 nmol/L; , 1 µmol/L) of ibutilide and dofetilide.

View larger version (18K): [in this window] [in a new window]   Figure 7. Line graphs show block of IKr tail current by drug concentrations near the EC50. Block is voltage dependent.

The data with intermediate drug concentrations (10 to 100 nmol/L) (Fig 3) suggest that the time course of activation was altered by the drugs: the depression of activating current became more prominent as the pulse duration increased. Envelope of tails tests (Fig 8) were also consistent with this finding. In the absence of drug, the time course of peak currents paralleled that of activating current with a long pulse; ie, the envelope of tails test was satisfied. When either drug was added, two effects became apparent. First, block of activating current was much more prominent than block of tails, and second, the time course of activating current now displayed slow inactivation-like behavior, again suggesting drug block of open channels. Fractional block of deactivating tail current as a function of activating pulse duration developed with a time constant of 289 milliseconds at +20 mV (Fig 9). These data indicate that block increased with increasing pulse duration, strongly suggesting block of open channels.

View larger version (23K): [in this window] [in a new window]   Figure 8. Tracings show envelope of tails test in the absence (top) and presence (bottom) of drug.

View larger version (22K): [in this window] [in a new window]   Figure 9. Line graph shows time dependence of block by ibutilide obtained by comparison of data acquired during envelope tests (Fig 8).

State Dependence
Further evidence in favor of open channel block is presented in Fig 10. Note that in these experiments, nisoldipine was omitted from the extracellular solution, and an inward I_Ca-L was recorded and unaffected by either drug. With 2-second depolarizing pulses to +20 mV, the evidence of open channel block during the depolarizing pulse described above was again observed. With repolarization to -20 mV, the magnitude of the deactivating tails was reduced, but their time courses appeared similar to those recorded before drug. At -20 mV, deactivation was well characterized by a monoexponential function, and the time constants were very similar before and during drug (Table). However, with subsequent repolarization to -50 mV, deactivation was slower in the presence of drug than in control, resulting in a tail current "crossover" that, as discussed below, has been taken as indicating open channel unblocking. At -50 mV, deactivation was fit with a biexponential function, and both time constants were prolonged by the drugs (Table). In addition, whereas I_Kr was reduced 41±6% by ibutilide at -20 mV, block was much less prominent at -50 mV (22±5%, n=4), consistent with the voltage dependence shown in Fig 7.

View larger version (13K): [in this window] [in a new window]   Figure 10. Tracings show time and voltage dependence of drug block. See text for details. Note that in these experiments, nisoldipine was omitted, and neither drug affected the initial inward current.

View this table: [in this window] [in a new window]   Table 1. Deactivation Time Constants

Pulses at frequencies of 0.2 to 5.0 Hz in the presence of either drug did not reveal any steady-state frequency dependence of block (Fig 11). However, when preparations were studied after prolonged quiescence while drug was washed in, further evidence for activation-dependent block was observed (Figs 12 and 13). With pulsing after 5 minutes of quiescence in control, tail currents following successive depolarizations at 0.66 Hz were superimposable (Figs 12A and 13A). However, when the same protocol was repeated after drug wash-in, two effects were observed. First, block was use dependent, supporting the likelihood of open channel block. The time constant for onset of drug block was 3.8±0.2 pulses with ibutilide (Fig 12D) and 2.8±0.3 pulses with dofetilide (Fig 13D). Second, even with the first pulse, tail current was reduced 30±3% with ibutilide and 32±3% with dofetilide. This is consistent with rest-state block or with development of open-state block during the first pulse. However, activating outward current during the first pulse was reduced 24±5% at 20 mV with ibutilide and 26±4% at 20 mV with dofetilide (Figs 12C and 13C), suggesting that rest-state block need not be involved to explain the reduction in the first tail current during these trains. Block by ibutilide or dofetilide was not reversible with washout.

View larger version (17K): [in this window] [in a new window]   Figure 11. Graph shows that trains of 20 pulses at varying frequencies during exposure to 10 nmol/L ibutilide showed no evidence of frequency-dependent block.

View larger version (22K): [in this window] [in a new window]   Figure 12. Graphs show use-dependent block of IKr by ibutilide. In this experiment, activating and deactivating currents were measured repetitively during the pulse trains shown (at 0.66 Hz) after 5 minutes of quiescence. First, the experiment was performed in the control (drug-free) state (A) and then again after 5 minutes of quiescence during wash-in of 10 nmol/L ibutilide (B). IKr activated during pulse 1 of each train is shown in C, and fractional block of tail currents as a function of pulse number is shown in D (n=3). INa was eliminated by substituting N-methyl-D-glucamine for Nao; ICa-T was eliminated by 100 µmol/L Ni2+ and ICa-L by 1 µmol/L nisoldipine.

View larger version (22K): [in this window] [in a new window]   Figure 13. Graphs show use-dependent block of IKr by 10 nmol/L dofetilide (n=3). See Fig 12 legend for protocol.

	Discussion

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A challenge to contemporary electrophysiology is to determine the relation between specific ion channel blocking effects and drug actions in the whole heart.^{12 21 22} Thus, the initial description that ibutilide appeared to exert a novel ion channel–activating behavior has led to its being used as a probe to determine the effect of this drug action in cells and in the intact heart.²² However, the data we present here demonstrate convincingly that ibutilide does block I_Kr and that the block is qualitatively very similar to that produced by dofetilide. Moreover, ibutilide block occurs at relatively low concentrations, raising the possibility that block of I_Kr is one mechanism whereby the drug exerts its in vivo effects. In previous experiments in guinea pig myocytes, Lee¹⁴ reported that ibutilide activated a slow inward current with a K_d of 1 to 2 nmol/L; the concentrations during treatment in humans have not yet been reported.

The methanesulfonanilide moiety is common to a number of compounds thought to prolong cardiac action potentials by blocking I_Kr. These include not only dofetilide^{6 23} but also E-4031,^{5 24} sotalol,²⁴ sematilide,²¹ and L691,121.²⁵ Although some of these (eg, sotalol²⁶ ) may exert other actions, I_Kr block appears to be an important component of their clinical actions. In the present study, we did not undertake a comprehensive reevaluation of the effect of ibutilide on other ion currents, including the slow inward sodium current described by Lee.¹⁴ Thus, we cannot rule out the possibility that ibutilide exerts additional effects that will contribute to its clinical actions.

Prolongation of cardiac action potentials, by specific I_Kr block or by nonspecific effects, appears to exert a number of potentially important and desirable effects on whole heart electrophysiology. These include facilitated defibrillation²⁷ and efficacy in animal models of ischemic ventricular fibrillation (in contrast to sodium channel block).^{28 29} On the other hand, marked QT prolongation and torsades de pointes have occurred with many new I_Kr blockers (not all of which have the methanesulfonanilide structure), including dofetilide,³⁰ almokalant,³¹ sotalol,³² sematilide,³³ and ibutilide.¹³ However, many of the reported episodes have occurred with intravenous administration during initial dose-finding studies, so the overall incidence of this problem during treatment with ibutilide or many other new I_Kr blockers remains to be determined. The initial clinical data do suggest that at least some torsades de pointes during treatment with this group of drugs is concentration dependent, in contrast to the well-recognized "idiopathic" nature of the syndrome with quinidine, a drug with a multiplicity of electrophysiological effects.

I_Kr can be dissected from I_Ks in guinea pig myocytes by using pharmacological probes such as E-4031, dofetilide, or lanthanum.^{4 19 23 34} However, for the study of time or voltage dependence of block, multiple subtraction procedures (eg, [control versus high concentration] versus low concentration) are required. Moreover, if I_Kr happens to be absent from a particular cell or isolation, erroneous conclusions may be drawn regarding the presence or mechanism of drug block. Thus, the study of drug block of ion channels requires systems in which specific currents can be readily isolated from potentially overlapping components. For I_Ks, guinea pig myocytes may still be appropriate. For I_Kr, on the other hand, more suitable preparations are those in which I_Ks is absent, allowing I_Kr to be studied in isolation; these include cat⁵ and rabbit⁶ myocytes as well as AT-1 cells. AT-1 cells are derived from an atrial tumor in a transgenic animal and so might not truly represent normal cardiac cells. However, they do retain morphological, biochemical, and electrophysiological characteristics of atrial myocytes,^{7 9 10} and in particular the I_Kr seen in this preparation shares all the important characteristics for I_Kr in guinea pig: inward rectification (Fig 5), rapid activation and deactivation (eg, Figs 3 and 8), and block by dofetilide, which is thought to be I_Kr specific. Thus, AT-1 cells are well suited for the further study of I_Kr physiology and pharmacology.

The characteristics of I_Kr block by dofetilide in rabbit myocytes have been reported previously.⁶ The data were quite similar to those we observed here, and the EC₅₀ for dofetilide block found here (12 nmol/L) is well within the range of other reports (3 to 39 nmol/L). The voltage dependence (Figs 6 and 7) and time dependence (Figs 8 through 10) of block are similar to those observed previously and argue for open channel block. The tail current "crossover" presented in Fig 10 also supports this idea. A crossover has also been observed with quinidine block of I_Ks,³⁵ quinidine block of the cloned channel Kv1.5,²⁰ and E-4031 block of I_Kr in cat myocytes.⁵ The interpretation is that as the drug-blocked channel protein deactivates, it shuttles transiently into a conducting state before closing: drug · openopenclosed. If drug unblocking is slow compared with channel closing, macroscopic deactivation slows and a crossover occurs. The lack of steady-state frequency-dependent block (Fig 11) has been observed by another researcher⁶ and is still consistent with very slow recovery from open channel block. Indeed, with specifically designed protocols, strong evidence for open channel block was observed. Drug effects could not be washed out. However, a simple time-dependent change in I_Kr cannot account for the observed drug effects, particularly since I_Kr consistently remains stable for more than 60 minutes in the absence of drug intervention. There is a paradox in these data: the crossover phenomenon implies relatively rapid unblocking, but we are unable to even partially wash out the effect of either drug, even after holding at -40 or -80 mV for 5 minutes. This suggests to us that ibutilide and dofetilide block I_Kr through at least two mechanisms: one a readily reversible one that could account for the crossover phenomenon, and one in which the channel is trapped in a drug-associated, nonconducting state from which recovery is very slow. The latter could account for the apparent frequency independence of block (Fig 11).

Block of I_Kr is under active clinical investigation as an antiarrhythmic mode of action. The greatest obstacle to the widespread use of this strategy appears to be the occasional development of torsades de pointes.^{1 2 3} AT-1 cells are a convenient model system in which factors that modulate drug block of I_Kr can be determined. The further use of this or similar preparations may now allow a systematic evaluation of the characteristics of I_Kr block by individual drugs. In this way, features of block that maximize efficacy and minimize risk may be identified.

	Acknowledgments

 
This study was supported in part by grants from the US Public Health Service (HL-46681, HL-47599, and HL-49989). Dr Roden is the holder of the William Stokes Chair in Experimental Therapeutics, a gift from the Daiichi Corp. The authors wish to thank Holly Waldrop for maintaining the AT-1 cells and Patricia James for preparing the manuscript.

	Footnotes

 
Previously presented in abstract form (Biophys J. 1994;66:A83).

Received August 25, 1994; accepted October 31, 1994.

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