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(Circulation. 1996;94:2938-2946.)
© 1996 American Heart Association, Inc.

Articles

Mechanism of Action Potential Prolongation by RP 58866 and Its Active Enantiomer, Terikalant

Block of the Rapidly Activating Delayed Rectifier K⁺ Current, I_Kr

Nancy K. Jurkiewicz, BS; Jixin Wang, MS; Bernard Fermini, PhD; Michael C. Sanguinetti, PhD; Joseph J. Salata, PhD

the Department of Pharmacology, Merck Research Laboratories, West Point, Pa, and the Cardiology Division and Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City (M.C.S.).

Correspondence to Joseph J. Salata, Department of Pharmacology, Merck Research Laboratories, Sumneytown Pike, PO Box 4, WP46-300, West Point, PA 19486. E-mail joseph_salata@merck.com.

	Abstract

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Background The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, are reported to selectively block the inward rectifier K⁺ current, I_K1. These drugs have demonstrated efficacy in animal models of cardiac arrhythmias, suggesting that block of I_K1 may be a useful antiarrhythmic mechanism. The symmetrical action potential (AP)–prolonging and bradycardic effects of these drugs, however, are inconsistent with a sole effect on I_K1.

Methods and Results We studied the effects of RP 58866 and terikalant on AP and outward K⁺ currents in guinea pig ventricular myocytes. RP 58866 and terikalant potently blocked the rapidly activating delayed rectifier K⁺ current, I_Kr, with IC₅₀s of 22 and 31 nmol/L, respectively. Block of I_K1 was 250-fold less potent; IC₅₀s were 8 and 6 µmol/L, respectively. No significant block of the slowly activating delayed rectifier, I_Ks, was observed at 10 µmol/L. The phenotypical I_Kr currents in mouse AT-1 cells and Xenopus oocytes expressing HERG were also blocked 50% by 200 to 250 nmol/L RP 58866 or terikalant, providing further conclusive evidence for potent block of I_Kr. RP 58866 1 µmol/L and dofetilide increased AP duration symmetrically, consistent with selective block of I_Kr. Only higher concentrations (10 µmol/L) of RP 58866 slowed the rate of AP repolarization and decreased resting membrane potential, consistent with an additional but substantially less potent block of I_K1.

Conclusions These data demonstrate that RP 58866 and terikalant are potent blockers of I_Kr and prompt a reinterpretation of previous studies that assumed specific block of I_K1 by these drugs.

Key Words: action potentials • antiarrhythmia agents • electrophysiology • potassium • dofetilide

	Introduction

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Species and regional differences in the cardiac APD occur largely because of diversity and varying density of repolarizing K⁺ currents. In guinea pig ventricle, the I_Kr and I_Ks are the primary determinants of early repolarization¹ with a potential contribution from the "plateau current," I_Kp.² Because of its anomalous inward rectification, especially at plateau potentials, the I_K1 affects only terminal repolarization and is the dominant current governing the RMP.³

Several newly introduced antiarrhythmic agents, including dofetilide, d-sotalol, E-4031, and MK-499, that selectively prolong APD without affecting conduction velocity (Vaughan-Williams⁴ class III) have been shown to act via specific blockade of I_Kr.⁵ Another class III agent, RP 58866, and its active enantiomer, terikalant, have been reported to act by selectively blocking I_K1.⁶ Block of I_K1 has both theoretical advantages and disadvantages as an antiarrhythmic mechanism and remains controversial.^{7 8} Although increasing APD through deceleration of the final phase of repolarization may produce a beneficial class III effect by prolonging effective refractoriness, it may, on the other hand, extend the phase of relative refractoriness and consequently increase the susceptibility to afterdepolarizations. The high conductance of I_K1 at and near the potassium equilibrium potential (E_K) not only maintains the RMP and blunts diastolic depolarization but also may guard against extraneous weak or undesirable depolarizations, eg, from ectopic foci. RP 58866 and its active enantiomer, terikalant, have been shown to increase APD in vitro without decelerating terminal repolarization or depolarization of the RMP and have displayed antiarrhythmic efficacy in a number of arrhythmia models.^{6 9 10} These agents, however, also cause bradycardia, suggesting an action on a current(s) other than I_K1, which is small or absent in pacemaker cells of the sinoatrial node.¹¹

Given these observations and the increasing realization that most class III agents block I_Kr at concentrations lower than those required to block other repolarizing currents in the heart, we reinvestigated the effects of RP 58866 and terikalant on the K⁺ currents in guinea pig isolated ventricular myocytes. Our results indicate that the effects of these agents on AP parameters are consistent with a relatively selective block of I_Kr. Block of I_K1 was observed only at concentrations 250-fold higher than those that blocked I_Kr. Potent block of I_Kr was corroborated in studies of cultured mouse atrial tumor myocytes (AT-1 cells)^{12 13} and in Xenopus oocytes expressing HERG.¹⁴

	Methods

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Isolation of Guinea Pig Ventricular Myocytes
Guinea pig ventricular myocytes were isolated as described previously.^{1 15} After isolation, the cells were stored in HBS containing (in mmol/L) NaCl 132, KCl 4, CaCl₂ 1.8, MgCl₂ 1.2, HEPES 10, and glucose 10, pH 7.2, at 24°C to 26°C until studied, usually within 8 hours after isolation.

AP Studies
Transmembrane potentials were recorded with conventional microelectrodes filled with 3 mol/L KCl (tip resistances, 30 to 50 M) with an Axoclamp 2B amplifier (Axon Instruments) as described in detail previously.^{1 16} Cells were superfused with HBS at a rate of 2 mL/min at 37°C. APs were evoked by passing brief current pulses (1 ms, 1.2 times threshold) at a frequency of 1 Hz through the recording electrode with an active bridge circuit. Only cells showing normal AP configurations and RMP -85 mV were used in this study (see Fig 1). APs were studied after a 5-minute control period and after 5 minutes of superfusion with drug at cumulatively increasing concentrations. For each condition, after a steady-state effect was reached, 20 individual APs were sampled, digitally averaged, and then measured.

View larger version (24K): [in this window] [in a new window]   Figure 1. Comparison of effects of RP 58866 and dofetilide on APs of guinea pig isolated ventricular myocytes. AP recordings are superimposed for control (C) and after 10 minutes of superfusion with either RP 58866 (0.1 and 10 µmol/L, A) or dofetilide (1, 3, and 10 nmol/L, C) during stimulation at 1 Hz. Horizontal dotted lines indicate zero voltage level. Bar graphs show percent changes in APD at 90% repolarization (APD90), maximum rate of repolarization of phase 3 (-dV/dt), and RMP for RP 58866 (B, n=6) and dofetilide (D, n=7 to 11), respectively. Early afterdepolarizations (EAD) were occasionally observed with dofetilide (eg, 3/12 at 10 nmol/L), which precluded measurements and reduced number of cells in some groups (see also Tables 1 and 2). Data are mean±SEM. *P<.05 by Dunnett's t test for ANOVA post hoc comparisons.

Voltage Clamp of Guinea Pig Ventricular Myocytes
Voltage-clamp studies of outward K⁺ currents were performed in the whole-cell recording mode with a List EPC-7 or Axopatch 200 amplifier by techniques described in detail previously.^{1 16} Microelectrodes were made from square-bore (1.0-mm OD) borosilicate capillary tubing (Glass Co of America). These pipettes were filled with either 0.5 mol/L K⁺ gluconate, 25 mmol/L KCl, and 5 mmol/L K₂ATP (solution A, to minimize rundown) and maintained under negative pressure, or with a solution containing (in mmol/L) KCl 110, K-BAPTA 5, K₂ATP 5, MgCl₂ 1, and HEPES 10, pH 7.2 (solution B) and had resistances of 3 to 7 M (average, 5.5±0.3 M). Series resistance was compensated 40% to 70%. Currents were low-pass filtered (-3 dB at 1 kHz) before digitization at 5 kHz.

K⁺ currents were measured during superfusion of the cells at a rate of 2 to 3 mL/min with either Ca²⁺-free or normal HBS (35°C) containing 0.4 to 1 µmol/L nisoldipine to block I_CaL. Cells were voltage-clamped at a V_h of either -50 or -40 mV to inactivate I_Na. The steady-state I-V relationship for K⁺ currents was examined initially by use of hyperpolarizing voltage ramps. Pipette solution B was used for these experiments. In other experiments, I_K and the inward I_K1 were assessed with appropriate voltage step protocols. I_Ks amplitude was measured as the difference from the initial instantaneous current, after the settling of the capacity transient, to the final (steady-state) current level during a depolarizing voltage step to +50 mV. I_Ktail amplitude was measured as the difference from the holding current level to the peak I_Ktail amplitude on return to V_h. I_K1 was measured as the absolute current, uncorrected for leak, at the end of 225-ms hyperpolarizing voltage steps from a V_h of -40 or -50 mV.

AT-1 Cell Preparation and Culture
AT-1 cells were propagated in vivo by subcutaneous injection into syngeneic host mice (female, 2 to 3 months old, B6D2F1/J, Charles River, Wilmington, Mass) as described previously.^{12 17} The original AT-1 mouse colonies were established by Dr Loren Field (Krannert Institute of Cardiology, Indianapolis, Ind), and the lineage at Merck Research Laboratories was established from tumor cells that were kindly provided by Dr Dan Roden (Vanderbilt University, Nashville, Tenn). Isolation and culture of AT-1 cells were conducted as described previously.^{13 18} For voltage-clamp studies, AT-1 cells were trypsinized to be removed from the culture dishes and were stored in PC-1 culture medium (22°C to 24°C). Outward K⁺ currents were studied in normal HBS within 14 hours of isolation at 22°C to 24°C with micropipettes filled with solution B. All cells were round in appearance, had large outward I_ktails and RMPs negative to -35 mV, and did not beat spontaneously. I_Na and I_CaT were inactivated by voltage-clamping the cells to a V_h of -40 mV. I_CaL was blocked with 0.4 to 1 µmol/L nisoldipine.

cRNA Injection and Voltage-Clamp of Oocytes
The HERG cDNA expression construct in the pSP64 transcription vector (Promega), synthesis of cRNA, and the isolation and maintenance of Xenopus oocytes and injection with cRNA have been described previously.¹⁴ Stage V and stage VI oocytes were injected with 50 nL (0.125 ng/nL) of cRNA encoding HERG. Currents were recorded 2 to 4 days after injection with a Dagan TEV-200 amplifier by standard two-microelectrode voltage-clamp techniques. Oocytes were bathed in a solution containing (in mmol/L) NaCl 94, KCl 4, MgCl₂ 2, CaCl₂ 0.1, and HEPES 5, pH 7.6.

General
In all studies, data acquisition and analysis were performed with pClamp software (Axon Instruments) and an IBM-compatible 486 computer. Concentration-response relationships were determined by measurement of AP or currents in each cell under control conditions and during superfusion with successively increasing concentrations of a given drug. Concentration-response curves (Figs 3 through 5) were fit to a logistic equation, y=(a-d)/[1+(x/c)b]+d, by use of a Marquardt-Levenburg algorithm for least-squares nonlinear regression analysis. With this equation, a and d are maximum and minimum responses estimated for infinite and zero concentrations, respectively; c is the inflection point that estimates the 50% effective concentration (IC₅₀); and b is the slope factor (Hill coefficient).

View larger version (14K): [in this window] [in a new window]   Figure 3. Concentration dependence of block of IK1 by RP 58866. Currents were elicited by 225-ms hyperpolarizing steps to Vt between -40 and -90 mV from a Vh of -40 mV. Currents were recorded in the continuous presence of 1 µmol/L dofetilide to block IKr. Example current recordings in one cell are shown at Vt of -40 and -60 mV during control (A) and after addition of RP 58866 at concentrations of 1, 10, and 30 µmol/L in B, C, and D, respectively. Horizontal dotted lines indicate zero current level in this and subsequent figures. E, I-V relationships are plotted for each condition. Absolute currents were measured at end of 225-ms test pulse, uncorrected for leak, and were normalized to cell capacitance. Data are mean±SEM (n=6). *P<.05 by Dunnett's t test.

View larger version (16K): [in this window] [in a new window]   Figure 4. Concentration dependence of block of IK1, IKr, and IKs by terikalant. A, Example current traces (top) from one cell during control (C) and after addition of 1 µmol/L terikalant with separate voltage-step protocols (bottom) from a Vh of -50 mV to selectively elicit IK1, IKr, and IKs. Currents were measured as described in "Methods." B, Data (mean±SEM) are expressed as a percent block of control currents. IC50 values were 0.031±0.009 and 7.9±0.5 µmol/L for IKr and IK1, respectively. Numbers in parentheses indicate number of cells.

View larger version (12K): [in this window] [in a new window]   Figure 5. Concentration dependence of block of IKr recorded in AT-1 cells. A, Example current traces recorded before (control) and after addition of 0.1 and 0.3 µmol/L RP 58866 with a voltage step from Vh of -40 mV to a Vt of +20 mV. IKr was measured as peak IKtail after return to Vh relative to the initial holding current level. B, Data (mean±SEM) are expressed as percent block of control currents. IC50 was 0.21±0.10 µmol/L (n=4 to 7).

Materials
RP 58866, 1-[2-(3,4-dihydro-2H-1-benzopyran-4-yl)ethyl]-4-(3,4-dimethoxyphenyl) piperidine, and its enantiomer, terikalant ([(s)(-)isomer], RP 62719), were gifts from Rhone-Poulenc Rorer. Dofetilide was synthesized by Drs D. Claremon and H. Selnick at Merck Research Laboratories. The purity of the compounds was determined by high-performance liquid chromatography to be 99.7%. The compounds were dissolved in DMSO (myocyte studies) or distilled water (oocyte studies of HERG) at stock concentrations of 1 or 10 mmol/L and diluted directly into test solutions by serial dilutions to achieve the final test concentrations. DMSO at the concentrations used had no significant effect on any of the parameters measured in these studies. Nisoldipine (a gift from Miles Pharmaceuticals) was prepared as a 4-mmol/L stock solution in polyethylene glycol 200 and diluted as needed.

Statistics
Data are expressed as mean±SEM. Concentration-dependent changes in AP parameters and individual ionic currents were assessed by repeated measures ANOVA, and post hoc comparison of the treatment with the control means was made with Dunnett's t test to determine significant concentration-dependent changes. A two-tailed probability of ±5% was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects on APs of Guinea Pig Ventricular Myocytes
RP 58866 caused a concentration-dependent prolongation in APD. Fig 1A shows a representative example of an AP recorded at a stimulus frequency of 1 Hz. At the lower concentrations of 0.1 and 1 µmol/L, RP 58866 significantly increased APD, measured at 50% and 90% of repolarization, in a symmetrical manner without substantially affecting other AP parameters (Fig 1B and Table 1). At 10 and 30 µmol/L, RP 58866 increased APD further, but not simply through a symmetrical or parallel prolongation of the AP. Rather, there was an additional significant decrease in the slope of phase 3 (-dV/dt) of the AP (Fig 1A and 1B, Table 1), and this slowing of repolarization caused substantial delays in the return to the RMP. RP 58866 at 30 µmol/L also significantly decreased the RMP (Fig 1B) and, consequently, the AP amplitude (Table 1). After a brief (5- to 10-minute) washout period, there was a return toward a normal RMP and AP shape, but complete washout was not attained, because APD remained significantly prolonged.

View this table: [in this window] [in a new window]   Table 1. Effect of RP 58866 on AP Parameters of Guinea Pig Isolated Ventricular Myocytes

In contrast to the effects of RP 58866, dofetilide, a selective blocker of I_Kr, increased APD symmetrically throughout the concentration range tested without significantly affecting other AP parameters (Fig 1C and 1D, Table 2). Thus, at relatively low concentrations, RP 58866 had effects on the AP that mimicked I_Kr blockers, whereas at high concentrations, the effects were consistent with additional block of I_K1. This hypothesis of a dual effect on I_Kr and I_K1 was tested by measurement of whole-cell currents in voltage-clamp experiments.

View this table: [in this window] [in a new window]   Table 2. Effect of Dofetilide on AP Parameters of Guinea Pig Isolated Ventricular Myocytes

Voltage-Clamp Studies on Guinea Pig Isolated Ventricular Myocytes: I-V Relationship of Outward K⁺ Currents
The steady-state I-V relationship for K⁺ currents was studied initially with hyperpolarizing voltage ramps to imitate the transmembrane voltage changes that occur during an AP. Beginning from a V_h of -50 mV, a rapid depolarization was applied to +40 mV, followed by a hyperpolarizing ramp to -90 mV at a rate of 0.3 V/s (0.4 second total). During control conditions (Ca²⁺-free HBS), typical N-shaped I-V relationships due to rectification of I_K1 with a reversal potential of -82 mV were obtained (Fig 2). Addition of 0.1 µmol/L of RP 58866 produced a net negative shift in the I-V at voltages between -75 and +40 mV (Fig 2A). In separate experiments, 1 µmol/L dofetilide alone caused qualitatively identical changes in the I-V relation (Fig 2B, Dof), and addition of 0.1 µmol/L RP 58866 produced no further effect on the measured currents (Fig 2B, Dof+RP). Similar results were obtained in three cells for each condition. Assuming specificity of block of I_Kr for dofetilide (References 19 and 20), these data indicate that, at the relatively low concentration of 0.1 µmol/L, RP 58866 selectively blocked I_Kr with little or no effect on I_K1.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effects of RP 58866 and dofetilide on I-V relationships of guinea pig isolated ventricular myocytes. Currents were recorded during hyperpolarizing voltage ramps from +40 to -90 mV at a rate of 0.3 V/s (0.4 second total). Currents were plotted as a function of voltage during the ramp to construct the I-V relationships. A, Traces were recorded during control and after 0.1 µmol/L RP 58866. Cell capacitance, 120 pF. B, In a separate cell, traces were obtained during control and after 1 µmol/L dofetilide (Dof) to block IKr completely. Addition of 0.1 µmol/L RP 58866 (Dof+RP) produced no further change in I-V relationship. Cell capacitance, 121 pF.

Effect on I_K1
These experiments were designed to specifically examine the effects of RP 58866 on I_K1. Cells were superfused with Ca²⁺-free HBS and pretreated with 1 µmol/L dofetilide to block I_Kr. Steady-state I_K1 was measured with 225-ms hyperpolarizing voltage steps to V_t between -90 and -40 mV delivered in 10-mV increments from a V_h of -40 mV (Fig 3). As in the initial voltage-ramp experiments (Fig 2), outward I_K1 peaked at -60 mV and was 5.4±0.5 pA/pF and 5.2±0.5 pA/pF before and after superfusion with 1 µmol/L RP 58866, respectively (Fig 3E, n=6, P=NS). Likewise, there was no significant effect of 1 µmol/L RP 58866 on outward I_K1 at any other V_t. Higher concentrations (10 and 30 µmol/L) of RP 58866 significantly decreased both inward and outward I_K1 (Fig 3B), as reported previously.⁶

Concentration Dependence of Block of Outward K⁺ Currents
In a separate group of experiments, we examined the concentration dependence of the effects of RP 58866 and its active enantiomer, terikalant, on the three major K⁺ currents in guinea pig ventricular myocytes: I_K1, I_Kr, and I_Ks (Fig 4). As in previous studies,^{15 16 21} we used distinct voltage-step protocols and conditions (Ca²⁺-free HBS) to optimize the separation and measurement of I_K1, I_Kr, and I_Ks. In an initial series of controls, over the experimental time course (4 to 8 minutes), the three K⁺ currents exhibited a rundown of 10%, but this was factored into the fit of the concentration-response curves (see "Methods"). The concentration-response relationships for the block by terikalant of I_Kr, I_K1, and I_Ks are superimposed in Fig 4B. Terikalant inhibited I_Kr most potently (IC₅₀31 nmol/L) and inhibited I_K1 much less potently (IC₅₀8 µmol/L). No inhibition of I_Ks beyond rundown was observed except at the very high concentration of 100 µmol/L (43±7% block, n=3). Similar concentration-response relationships were obtained for RP 58866, which had IC₅₀ values for block of I_Kr and I_K1 of 22 nmol/L and 6 µmol/L, respectively, whereas there was no effect on I_Ks up to 10 µmol/L (n5). Both terikalant and RP 58866 blocked I_Kr much more potently than I_K1. Because of their similar concentration responses and nearly identical selectivity profiles, the compounds were used interchangeably in the remaining experiments.

Block of I_Kr in AT-1 Cells
AT-1 cells express one major outward current that is analogous to I_Kr of guinea pig myocytes. The similarities include rapid activation, prominent inward rectification, and nanomolar sensitivity to a number of specific I_Kr blockers such as dofetilide and E-4031.^{13 18 22} Therefore, to support our findings in guinea pig myocytes, we studied the effects of RP 58866 on I_Kr in AT-1 cells. Under control conditions during 1-second depolarizations, we recorded time-dependent outward currents that increased as a function of V_t between -30 and +10 mV, then decreased (negative slope conductance) as the V_t was increased further. On return to the V_h of -40 mV, we observed deactivating I_Ktails that increased sigmoidally with increasing V_t and was fitted to a Boltzmann function. The V_1/2 was 3.7±0.6 mV, and the slope factor, k, was 10.5±0.4 (n=4). These currents were blocked completely by 1 µmol/L dofetilide. These properties of I_Kr were essentially identical to those reported previously.^{13 18 22}

We examined the concentration dependence of the effect of RP 58866 on I_Kr in AT-1 cells. Current was elicited with a single 1-second depolarizing pulse from a V_h of -40 mV to a V_t of +20 mV applied every 10 seconds. Current amplitude was monitored as I_Ktail on return to -40 mV and was very stable during long (>15-minute) control periods of repetitive pulsing during which little or no rundown occurred. Addition of RP 58866 produced a concentration-dependent block of both time-dependent current and I_Ktail (Fig 5A). Block reached a steady state within 3 minutes of exposure to each cumulative concentration of drug. A maximum of three consecutive concentrations was tested in each cell. The concentration-response curve for RP 58866 is plotted in Fig 5B and had a calculated IC₅₀0.21 µmol/L (n=4 to 7).

In a separate series of experiments, we determined the effects on the I-V relationship and activation parameters by recording currents during 1-second depolarizing steps to V_t between -30 and +50 mV. RP 58866 decreased I_Ktail in a slightly voltage-dependent manner. When assessed at a V_t of 0 mV, the IC₅₀ was 0.21±0.01 µmol/L (n=3 to 6). At a V_t of +20 and +40 mV, the IC₅₀s were decreased to 0.17±0.01 and 0.14±0.01 µmol/L, respectively. However, RP 58866 had no significant effect on the voltage dependence of activation of I_Kr in these cells. The V_1/2 in control was 0.8±0.9 mV, versus -0.2±1.3 mV for 0.1 µmol/L RP 58866. The slope factor in control was 10.0±0.8, versus 14.5±1.2 for drug. (P=NS, n=3).

Block of HERG Currents Expressed in Xenopus Oocytes
HERG is a K⁺ channel gene that when expressed in oocytes induces a current that is essentially identical to I_Kr in cardiac myocytes¹⁴ and that is blocked by the methanesulfonanilide class III antiarrhythmic agents E-4031 and MK-499.^{23 24} We therefore examined the effects of terikalant on HERG expressed in oocytes. Terikalant blocked HERG in a concentration-dependent manner. Block was measured at each concentration of drug by pulsing repetitively with 4-second pulses to 0 mV until a steady state was achieved. The percent inhibition of I_Ktail was plotted to obtain a concentration-response relationship (Fig 6B). Assessed at a V_t of 0 mV, the IC₅₀ was 0.25±0.05 µmol/L (n=3 to 5).

View larger version (16K): [in this window] [in a new window]   Figure 6. Concentration dependence of block of HERG expressed in Xenopus oocytes. A, Example current traces recorded before (control) and after addition of 0.1 and 0.3 µmol/L terikalant. HERG was measured as peak IKtail after return to -60 mV relative to current level during initial prepulse to -60 mV. B, Data (mean±SEM) are expressed as percent block of control currents measured at Vt of 0 mV. IC50 was 0.25±0.05 µmol/L (n=3 to 5).

We also determined the effects of terikalant on the voltage dependence and kinetics of HERG activation. The voltage dependence of activation was measured by normalization of the I_Ktail amplitude at -60 mV after the 4-second activating pulses (eg, Fig 6A). The normalized I_Ktails were fit to a Boltzmann function with V_1/2 values of -28.0±0.2, -27.8±0.6, and -27.7±0.2 mV and slope factors (k) of 9.0±0.2, 10.4±0.6, and 9.4±0.2 (P=NS, n=5) before and after 0.1 and 0.3 µmol/L terikalant, respectively. Thus, terikalant had no significant effect on the voltage dependence of HERG activation. Likewise, there was no significant change in the kinetics of HERG activation. For example, the fast and slow time constants of activation measured at a V_t of 0 mV were 167.6±31.7 and 729.4±115.2 ms versus 188.2±35.3 and 687.8±248.2 ms (P=NS, n=5) before and after 0.3 µmol/L terikalant, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we demonstrate that RP 58866 and its active enantiomer, terikalant, are potent blockers of I_Kr in guinea pig ventricular myocytes. The potency of block of I_Kr was 250-fold greater than block of I_K1. Before this study, RP 58866 and terikalant were considered to be relatively selective blockers of I_K1. During hyperpolarizing voltage ramps, relatively low concentrations of RP 58866 or terikalant (0.1 µmol/L) produced a net inward current shift over plateau potentials. An identical profile of block was observed with dofetilide, which together with its voltage dependence and inward rectification identified this drug-sensitive current as I_Kr. Furthermore, pretreatment with 1 µmol/L dofetilide precluded the block of I_Kr by 0.1 µmol/L RP 58866. A similar selective action on I_Kr over I_K1 was observed when voltage-step protocols were used to examine these respective currents. RP 58866 and terikalant blocked I_K1 only at substantially higher concentrations (1 µmol/L). These compounds also blocked I_Kr in mouse AT-1 cells and Xenopus oocytes expressing HERG, the -subunit that forms human I_Kr channels. The phenotypical I_Kr currents in both these test systems were blocked by submicromolar concentrations of RP 58866 or terikalant. AP studies also supported the selectivity of the effects on I_Kr. A symmetrical increase in APD was produced by dofetilide and low concentrations of RP 58866, whereas only higher concentrations of RP 58866 slowed the rate of terminal repolarization and decreased RMP, consistent with an additional but substantially less potent block of I_K1.

Comparison With Previous Studies
RP 58866 and terikalant were originally identified as specific blockers of I_K1 in guinea pig ventricular myocytes by Escande et al.⁶ In their study, RP 58866 and terikalant blocked outward I_K1 measured at -40 mV by 50% at 10 µmol/L. We observed a comparable degree of block of I_K1 at -60 mV in this study with an IC₅₀ value of 6 µmol/L. On the basis of the lack of an effect on the time-dependent current during 10-second depolarizing voltage steps from a V_h of -40 to a V_t of +40 mV, Escande et al concluded that neither RP 58866 nor terikalant at concentrations as high as 50 µmol/L had any significant effect on the delayed rectifier K⁺ current, I_K. This voltage-clamp protocol would have activated almost exclusively I_Ks, which is not blocked by pure class III antiarrhythmic agents such as E-4031 and dofetilide,^{1 19 25} but only a relatively insignificant I_Kr. Using similar protocols to record I_Ks in this study (Fig 4), we also saw no significant effect of RP 58866 and terikalant on I_Ks at concentrations <100 µmol/L. More importantly, the inclusion of a divalent cation (3 mmol/L Co²⁺) in the bath solution to block I_CaL by Escande et al⁶ would have blocked I_Kr as well.^{26 27 28 29} Thus, the voltage-clamp conditions and antecedent block of I_Kr by Co²⁺ reasonably explain why the potent blocking effect on I_Kr might have been precluded or overlooked previously. Indeed, under analogous conditions in this study, ie, during pretreatment with dofetilide, we saw a similar lack of effect with RP 58866 and terikalant at concentrations 1 µmol/L (Figs 2 and 3). Terikalant has been shown to inhibit peak I_to and hasten its inactivation with half-maximal concentrations of 2 and 11 µmol/L, respectively.³⁰ Thus, the potency of effects on I_to is also much less than on I_Kr.

RP 58866 and terikalant have been reported to uniformly prolong APD at concentrations below those expected to block I_K1 in other studies.^{6 10} RP 58866 at concentrations between 0.1 and 10 µmol/L significantly increased APD₅₀ and APD₉₀ of guinea pig papillary muscles.¹⁰ RP 58866 at 0.1 µmol/L and terikalant at 0.3 µmol/L increased APD₉₀ by an average of 19%¹⁰ and 21%,⁶ respectively. In the present study, we observed slightly greater but qualitatively similar increases in APD₉₀ at 0.1 and 1.0 µmol/L RP 58866 (Fig 1). A more potent prolongation of APD in isolated myocytes compared with papillary muscles is a typical finding for other I_Kr blockers; eg, 10 nmol/L dofetilide increased APD₉₀ by 42% in ventricular myocytes (this study) versus 20% in papillary muscles.³¹ More importantly, we and others have shown that both dofetilide³¹ and low concentrations of RP 58866^{6 10} increase APD symmetrically without affecting the rate of repolarization or RMP, consistent with a prominent effect on I_Kr and little or no effect on I_K1. The bradycardic effects of the two drugs in rabbit Langendorff-perfused hearts⁶ is also explained by block of I_Kr, which has recently been shown to play an important role in the diastolic depolarization of the sinoatrial node.³² The moderate positive inotropic and lusitropic effects of terikalant³³ have likewise been observed with other selective blockers of I_Kr.³⁴

Only at concentrations of RP 58866 10 µmol/L was there a significant slowing of the rate of terminal repolarization, whereas 30 µmol/L was required to reduce the RMP. Shimoni et al³⁵ clearly defined the role of I_K1 in the plateau and repolarization phases of the cardiac AP. Using AP voltage-clamp protocols, they showed that I_K1 was the principal current responsible for final repolarization but that its contribution was small during the plateau. In addition, the time courses of I_K1 and -dV/dt were closely correlated, indicating that a partial decrease in I_K1 slowed the rate of terminal repolarization. A similar slowing of terminal repolarization consequent to a simulated reduction of I_K1 has been predicted in computer models of the AP.^{36 37} Clinical investigations likewise have shown that ventricular myocytes isolated from patients with dilated cardiomyopathy exhibit prolonged AP and slowing of phase 3 repolarization due to a reduction in the amplitude and/or slope conductance of I_K1.^{38 39} Our AP results are also consistent with experimental studies showing a use-dependent accentuated rectification of I_K1 that minimizes its contribution during the AP plateau.^{35 40 41} RP 58866 caused a significant decline in the RMP only during substantial inhibition (>50%) of I_K1 at a concentration of 30 µmol/L (Figs 1 and 3), in agreement with previous reports.¹⁰

In four of six cells, RP 58866 at concentrations 10 µmol/L depressed the AP plateau (eg, Fig 1A). This result is inconsistent with block of either I_Kr or I_K1 observed in this study. Additional undefined actions of the drug, such as block of an inward current, may account for this effect. Although block of both I_CaL and I_CaT appears to be unlikely, terikalant has been shown to decrease peak sodium current.^{6 30} Thus, a reduction in the late sodium current could account for the decline in the AP plateau.⁴²

When appropriate voltage-clamp protocols and conditions were used to maximize the measurement of each current during determination of the concentration-response relationships (Fig 4), the control amplitudes of outward I_K1 (V_t, -60 mV) and I_Kr (V_t, -40 mV) were 595±25 pA (n=29) and 86±7 pA (n=24), respectively. Thus, maximum I_K1 appeared to be 7-fold greater than I_Kr. At 1 µmol/L of terikalant, a 10% block of I_K1 versus a 100% block of I_Kr translates into comparable absolute reductions in outward current of 60 versus 86 pA, respectively, which arguably could have similar impact on the APD. However, this may not be an accurate representation, for the following reasons. First, pretreatment with dofetilide substantially increased the IC₅₀ for RP 58866 block of I_K1 from 6 to 16 µmol/L (see Fig 3) and significantly reduced the maximum I_K1 in control from 595±25 to 472±39 pA (P<.05, n=8), indicating that I_Kr overlaps with I_K1 (see also Fig 2B). Consequently, without prior block of I_Kr, we actually overestimate the potency of block of outward I_K1 by RP 58866 and terikalant because of overlapping block of I_Kr. Second, I_Kr appears to play a much larger role than I_K1 in early repolarization because it exhibits less rectification during the AP plateau (see Fig 2B), perhaps as a consequence of differing mechanisms of rectification and their timing.^{37 41 43 44}

RP 58866 and/or terikalant also blocked I_Kr in mouse AT-1 cells and Xenopus oocytes expressing HERG with similar IC₅₀ values of 210 and 250 nmol/L, respectively. Thus, the blocking concentrations in these two test systems were 10-fold higher than in guinea pig myocytes (IC₅₀20 to 30 nmol/L). We have observed similar 5- to 10-fold differences in potency between I_Kr in myocytes⁴⁵ versus AT-1 cells (unpublished observations) and HERG expressed in oocytes²³ with MK-499, for instance.

Most of the newly introduced class III antiarrhythmic agents act via specific blockade of I_Kr, including dofetilide, sematilide, d-sotalol, E-4031, and MK-499.⁵ Alternative mechanisms have been proposed to account for the AP-prolonging effects of several other class III agents, eg, block of I_Ks by azimilide,⁴⁶ block of I_to by tedisamil,⁴⁷ and an increase of I_Na by ibutilide.⁴⁸ Each of these drugs, however, has now been shown to inhibit I_Kr more potently and at pharmacologically relevant concentrations^{16 18} (unpublished observations). It is unknown why I_Kr is blocked by so many drugs or whether all drugs act at a common site on the channel. Binding studies in guinea pig myocytes, however, indicate that many class III agents, including RP 58866, act at a common high-affinity site with K_i values in the nanomolar range⁴⁹ and further substantiate the potent blocking action on I_Kr shown in the present study.

We conclude that the class III agents RP 58866 and its active enantiomer, terikalant, accordingly are potent blockers of I_Kr. These new findings prompt a reinterpretation of previous studies that assumed specific block of I_K1 by RP 58866 and terikalant.^{6 8 9}

	Selected Abbreviations and Acronyms

 

AP = action potential APD = action potential duration HBS = HEPES-buffered saline I-V = current-voltage ICaL = L-type calcium current ICaT = T-type calcium current IK = time-dependent delayed rectifier K+ current IK1 = inward rectifier K+ current IKr = time-dependent rapidly activating component of IK IKs = time-dependent slowly activating component of IK IKtail = tail current INa = inward sodium current Ito = transient outward current RMP = resting membrane potential Vh = holding potential Vt = test potential

	Acknowledgments

 
This study was supported in part by US Public Health Service grant RO1-HL-55236. We thank Dr Mark Keating for providing the HERG clone. We also thank Holly Waldrop and Terry Schwegel for their expert technical assistance in obtaining and establishing the AT-1 cell line.

Received May 5, 1996; revision received June 25, 1996; accepted July 5, 1996.

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