Mechanism of Action Potential Prolongation by RP 58866 and Its Active Enantiomer, Terikalant
Block of the Rapidly Activating Delayed Rectifier K+ Current, IKr
Background The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, are reported to selectively block the inward rectifier K+ current, IK1. These drugs have demonstrated efficacy in animal models of cardiac arrhythmias, suggesting that block of IK1 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 IK1.
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, IKr, with IC50s of 22 and 31 nmol/L, respectively. Block of IK1 was ≈250-fold less potent; IC50s were 8 and 6 μmol/L, respectively. No significant block of the slowly activating delayed rectifier, IKs, was observed at ≤10 μmol/L. The phenotypical IKr 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 IKr. RP 58866 ≤1 μmol/L and dofetilide increased AP duration symmetrically, consistent with selective block of IKr. 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 IK1.
Conclusions These data demonstrate that RP 58866 and terikalant are potent blockers of IKr and prompt a reinterpretation of previous studies that assumed specific block of IK1 by these drugs.
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 IKr and IKs are the primary determinants of early repolarization1 with a potential contribution from the “plateau current,” IKp.2 Because of its anomalous inward rectification, especially at plateau potentials, the IK1 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 IKr.5 Another class III agent, RP 58866, and its active enantiomer, terikalant, have been reported to act by selectively blocking IK1.6 Block of IK1 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 IK1 at and near the potassium equilibrium potential (EK) 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 IK1, 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 IKr 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 IKr. Block of IK1 was observed only at concentrations ≈250-fold higher than those that blocked IKr. Potent block of IKr was corroborated in studies of cultured mouse atrial tumor myocytes (AT-1 cells)12 13 and in Xenopus oocytes expressing HERG.14
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.
Transmembrane potentials were recorded with conventional microelectrodes filled with 3 mol/L KCl (tip resistances, 30 to 50 MΩ) 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.
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 MΩ (average, 5.5±0.3 MΩ). 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 ICaL. Cells were voltage-clamped at a Vh of either −50 or −40 mV to inactivate INa. 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, IK and the inward IK1 were assessed with appropriate voltage step protocols. IKs 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. IKtail amplitude was measured as the difference from the holding current level to the peak IKtail amplitude on return to Vh. IK1 was measured as the absolute current, uncorrected for leak, at the end of 225-ms hyperpolarizing voltage steps from a Vh 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 Iktails and RMPs negative to −35 mV, and did not beat spontaneously. INa and ICaT were inactivated by voltage-clamping the cells to a Vh of −40 mV. ICaL 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.
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).
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 Rhoˆne-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.
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.
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.
In contrast to the effects of RP 58866, dofetilide, a selective blocker of IKr, 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 IKr blockers, whereas at high concentrations, the effects were consistent with additional block of IK1. This hypothesis of a dual effect on IKr and IK1 was tested by measurement of whole-cell currents in voltage-clamp experiments.
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 Vh 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 IK1 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 IKr for dofetilide (References 19 and 20), these data indicate that, at the relatively low concentration of 0.1 μmol/L, RP 58866 selectively blocked IKr with little or no effect on IK1.
Effect on IK1
These experiments were designed to specifically examine the effects of RP 58866 on IK1. Cells were superfused with Ca2+-free HBS and pretreated with 1 μmol/L dofetilide to block IKr. Steady-state IK1 was measured with 225-ms hyperpolarizing voltage steps to Vt between −90 and −40 mV delivered in 10-mV increments from a Vh of −40 mV (Fig 3⇓). As in the initial voltage-ramp experiments (Fig 2⇑), outward IK1 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 IK1 at any other Vt. Higher concentrations (10 and 30 μmol/L) of RP 58866 significantly decreased both inward and outward IK1 (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: IK1, IKr, and IKs (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 IK1, IKr, and IKs. 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 IKr, IK1, and IKs are superimposed in Fig 4B⇓. Terikalant inhibited IKr most potently (IC50≈31 nmol/L) and inhibited IK1 much less potently (IC50≈8 μmol/L). No inhibition of IKs 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 IKr and IK1 of 22 nmol/L and 6 μmol/L, respectively, whereas there was no effect on IKs up to 10 μmol/L (n≥5). Both terikalant and RP 58866 blocked IKr much more potently than IK1. Because of their similar concentration responses and nearly identical selectivity profiles, the compounds were used interchangeably in the remaining experiments.
Block of IKr in AT-1 Cells
AT-1 cells express one major outward current that is analogous to IKr of guinea pig myocytes. The similarities include rapid activation, prominent inward rectification, and nanomolar sensitivity to a number of specific IKr 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 IKr in AT-1 cells. Under control conditions during 1-second depolarizations, we recorded time-dependent outward currents that increased as a function of Vt between −30 and +10 mV, then decreased (negative slope conductance) as the Vt was increased further. On return to the Vh of −40 mV, we observed deactivating IKtails that increased sigmoidally with increasing Vt and was fitted to a Boltzmann function. The V1/2 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 IKr were essentially identical to those reported previously.13 18 22
We examined the concentration dependence of the effect of RP 58866 on IKr in AT-1 cells. Current was elicited with a single 1-second depolarizing pulse from a Vh of −40 mV to a Vt of +20 mV applied every 10 seconds. Current amplitude was monitored as IKtail 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 IKtail (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 IC50≈0.21 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 Vt between −30 and +50 mV. RP 58866 decreased IKtail in a slightly voltage-dependent manner. When assessed at a Vt of 0 mV, the IC50 was 0.21±0.01 μmol/L (n=3 to 6). At a Vt 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 IKr in these cells. The V1/2 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 IKr 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 IKtail was plotted to obtain a concentration-response relationship (Fig 6B⇓). Assessed at a Vt of 0 mV, the 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 IKtail amplitude at −60 mV after the 4-second activating pulses (eg, Fig 6A⇑). The normalized IKtails were fit to a Boltzmann function with V1/2 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 Vt 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.
In the present study, we demonstrate that RP 58866 and its active enantiomer, terikalant, are potent blockers of IKr in guinea pig ventricular myocytes. The potency of block of IKr was ≈250-fold greater than block of IK1. Before this study, RP 58866 and terikalant were considered to be relatively selective blockers of IK1. 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 IKr. Furthermore, pretreatment with 1 μmol/L dofetilide precluded the block of IKr by 0.1 μmol/L RP 58866. A similar selective action on IKr over IK1 was observed when voltage-step protocols were used to examine these respective currents. RP 58866 and terikalant blocked IK1 only at substantially higher concentrations (≥1 μmol/L). These compounds also blocked IKr in mouse AT-1 cells and Xenopus oocytes expressing HERG, the α-subunit that forms human IKr channels. The phenotypical IKr 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 IKr. 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 IK1.
Comparison With Previous Studies
RP 58866 and terikalant were originally identified as specific blockers of IK1 in guinea pig ventricular myocytes by Escande et al.6 In their study, RP 58866 and terikalant blocked outward IK1 measured at −40 mV by ≈50% at 10 μmol/L. We observed a comparable degree of block of IK1 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 Vh of −40 to a Vt 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, IK. This voltage-clamp protocol would have activated almost exclusively IKs, which is not blocked by pure class III antiarrhythmic agents such as E-4031 and dofetilide,1 19 25 but only a relatively insignificant IKr. Using similar protocols to record IKs in this study (Fig 4⇑), we also saw no significant effect of RP 58866 and terikalant on IKs at concentrations <100 μmol/L. More importantly, the inclusion of a divalent cation (3 mmol/L Co2+) in the bath solution to block ICaL by Escande et al6 would have blocked IKr as well.26 27 28 29 Thus, the voltage-clamp conditions and antecedent block of IKr by Co2+ reasonably explain why the potent blocking effect on IKr 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 Ito and hasten its inactivation with half-maximal concentrations of 2 and 11 μmol/L, respectively.30 Thus, the potency of effects on Ito is also much less than on IKr.
RP 58866 and terikalant have been reported to uniformly prolong APD at concentrations below those expected to block IK1 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 IKr 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 588666 10 increase APD symmetrically without affecting the rate of repolarization or RMP, consistent with a prominent effect on IKr and little or no effect on IK1. The bradycardic effects of the two drugs in rabbit Langendorff-perfused hearts6 is also explained by block of IKr, 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 IKr.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 IK1 in the plateau and repolarization phases of the cardiac AP. Using AP voltage-clamp protocols, they showed that IK1 was the principal current responsible for final repolarization but that its contribution was small during the plateau. In addition, the time courses of IK1 and −dV/dt were closely correlated, indicating that a partial decrease in IK1 slowed the rate of terminal repolarization. A similar slowing of terminal repolarization consequent to a simulated reduction of IK1 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 IK1.38 39 Our AP results are also consistent with experimental studies showing a use-dependent accentuated rectification of IK1 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 IK1 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 IKr or IK1 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 ICaL and ICaT 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 IK1 (Vt, −60 mV) and IKr (Vt, −40 mV) were 595±25 pA (n=29) and 86±7 pA (n=24), respectively. Thus, maximum IK1 appeared to be ≈7-fold greater than IKr. At 1 μmol/L of terikalant, a 10% block of IK1 versus a 100% block of IKr 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 IK1 from 6 to 16 μmol/L (see Fig 3⇑) and significantly reduced the maximum IK1 in control from 595±25 to 472±39 pA (P<.05, n=8), indicating that IKr overlaps with IK1 (see also Fig 2B⇑). Consequently, without prior block of IKr, we actually overestimate the potency of block of outward IK1 by RP 58866 and terikalant because of overlapping block of IKr. Second, IKr appears to play a much larger role than IK1 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 IKr 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 (IC50≈20 to 30 nmol/L). We have observed similar 5- to 10-fold differences in potency between IKr 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 IKr, 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 IKs by azimilide,46 block of Ito by tedisamil,47 and an increase of INa by ibutilide.48 Each of these drugs, however, has now been shown to inhibit IKr more potently and at pharmacologically relevant concentrations16 18 (unpublished observations). It is unknown why IKr 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 Ki values in the nanomolar range49 and further substantiate the potent blocking action on IKr shown in the present study.
We conclude that the class III agents RP 58866 and its active enantiomer, terikalant, accordingly are potent blockers of IKr. These new findings prompt a reinterpretation of previous studies that assumed specific block of IK1 by RP 58866 and terikalant.6 8 9
Selected Abbreviations and Acronyms
|APD||=||action potential duration|
|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|
|INa||=||inward sodium current|
|Ito||=||transient outward current|
|RMP||=||resting membrane potential|
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.
- Copyright © 1996 by American Heart Association
Sanguinetti MC, Jurkiewicz NK. Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol. 1990;96:195-215.
Backx PH, Marban E. Background potassium current active during the plateau of the action potential in guinea pig ventricular myocytes. Circ Res. 1993;72:890-900.
Vaughan-Williams EM. Classification of anti-dysrhythmic drugs. Pharmacol Ther. 1975;1:115-138.
Escande D, Mestre M, Cavero I, Brugada J, Kirchhof C. RP 58866 and its active enantiomer RP 62719 (terikalant): blockers of the inward rectifier K+ current acting as pure class III antiarrhythmic agents. J Cardiovasc Pharmacol. 1992;20:S106-S113.
Opthof T. IK1 blockade is unlikely to be a potentially useful antiarrhythmic mechanism. Cardiovasc Res. 1994;28:420. Controversy.
Rees SA, Curtis MJ. IK1 blockade is a potentially useful antiarrhythmic mechanism. Cardiovasc Res. 1994;28:421. Controversy.
Rees SA, Curtis MJ. Specific IK1 blockade: a new antiarrhythmic mechanism? Effect of RP 58866 on ventricular arrhythmias in rat, rabbit, and primate. Circulation. 1993;87:1979-1989.
Steinhelper ME, Lanson NA Jr, Dresdner KP, Delcarpio JB, Wit AL, Claycomb WC, Field LJ. Proliferation in vivo and in culture of differentiated adult atrial cardiomyocytes from transgenic mice. Am J Physiol. 1990;259:H1826-H1834.
Yang T, Wathen MS, Felipe A, Tamkun MM, Snyders DJ, Roden DM. K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells). Circ Res. 1994;75:870-878.
Salata JJ, Jurkiewicz NK, Wallace AA, Stupienski RF III, Guinosso PJ Jr, Lynch JJ Jr. Cardiac electrophysiologic actions of the histamine H1-receptor antagonists astemizole and terfenadine compared with chlorpheniramine and pyrilamine. J Pharmacol Exp Ther. 1995;76:110-119.
Fermini B, Jurkiewicz NK, Jow B, Guinosso PJ Jr, Baskin EP, Lynch JJ Jr, Salata JJ. Use-dependent effects of the class III antiarrhythmic agent NE-10064 (azimilide) on cardiac repolarization: block of delayed rectifier potassium and L-type calcium currents. J Cardiovasc Pharmacol. 1995;26:259-271.
Delcarpio JB, Lanson NA Jr, Field LJ, Claycomb WC. Morphological characterization of cardiomyocytes isolated from a transplantable cardiac tumor derived from transgenic mouse atria (AT-1 cells). Circ Res. 1991;69:1591-1600.
Yang T, Snyders DJ, Roden DM. Ibutilide, a methanesulfonanilide antiarrhythmic, is a potent blocker of the rapidly activating delayed rectifier K+ current (IKr) in AT-1 cells: concentration-, time-, voltage-, and use-dependent effects. Circulation. 1995;91:1799-1806.
Jurkiewicz NK, Sanguinetti MC. Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III antiarrhythmic agent: specific block of rapidly activating delayed rectifier K+ current by dofetilide. Circ Res. 1993;72:75-83.
Carmeliet E. Voltage- and time-dependent block of the delayed K+ current in cardiac myocytes by dofetilide. J Pharmacol Exp Ther. 1992;262:809-817.
Spector PS, Curran ME, Keating MT, Sanguinetti MC. Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel: open-channel block by methanesulfonanilides. Circ Res. 1996;78:499-503.
Trudeau MC, Warmke JW, Ganetzky B, Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science. 1995;269:92-95.
Liu DW, Antzelevitch C. Characteristics of the delayed rectifier current (IKr and IKs) in canine ventricular epicardial, midmyocardial, and endocardial myocytes: a weaker IKs contributes to the longer action potential of the M cell. Circ Res. 1995;76:351-365.
Balser JR, Bennett PB, Roden DM. Time-dependent outward current in guinea pig ventricular myocytes: gating kinetics of the delayed rectifier. J Gen Physiol. 1990;96:835-863.
Follmer CH, Lodge NJ, Cullinan CA, Colatsky TJ. Modulation of the delayed rectifier, IK, by cadmium in cat ventricular myocytes. Am J Physiol. 1992;262:C75-C83.
Fan Z, Hiraoka M. Depression of delayed outward K+ current by Co2+ in guinea pig ventricular myocytes. Am J Physiol. 1991;261:C23-C31.
Sanguinetti MC, Jurkiewicz NK. Delayed rectifier outward K+ current is composed of two currents in guinea pig atrial cells. Am J Physiol. 1991;260:H393-H399.
McLarnon JG, Xu R. Actions of the benzopyran compound terikalant on macroscopic currents in rat ventricular myocytes. J Pharmacol Exp Ther. 1995;275:389-396.
Veldkamp MW, van Ginneken ACG, Bouman LN. Delayed rectifier channel open probability during diastolic depolarization in sinoatrial node cells. Biophys J. 1996;70:A188. Abstract.
Beregi JP, Escande D, Coudray N, Mery P, Mestre M, Chemla D, LeCarpentier Y. Positive inotropic effects of RP 62719, a new pure class III antiarrhythmic agent, on guinea pig myocardium. J Pharmacol Exp Ther. 1992;263:1369-1376.
Kass RS, Arena JP, Walsh KB. Measurement and block of potassium channel currents in the heart: importance of channel type. Drug Dev Res. 1990;19:115-127.
Nichols CG, Makhina EN, Pearson WL, Sha Q, Lopatin AN. Inward rectification and implications for cardiac excitability. Circ Res. 1996;78:1-7.
Koumi S, Backer CL, Arentzen CE. Characterization of inwardly rectifying K+ channel in human cardiac myocytes: alterations in channel behavior in myocytes isolated from patients with idiopathic dilated cardiomyopathy. Circulation. 1995;92:164-174.
Beuckelmann DJ, Na¨bauer M, Erdmann E. Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res. 1993;73:379-385.
Delmar M, Ibarra J, Davidenko J, Lorente P, Jalife J. Dynamics of the background outward current of single guinea pig ventricular myocytes: ionic mechanisms of hysteresis in cardiac cells. Circ Res. 1991;69:1316-1326.
Kiyosue T, Arita M. Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes. Circ Res. 1989;64:389-397.
Spector PS, Curran ME, Zou A, Keating MT, Sanguinetti MC. Fast inactivation causes rectification of the IKr channel. J Gen Physiol. 1996;107:611-619.
Lynch JJ Jr, Wallace AA, Stupienski RF III, Baskin EP, Beare CM, Appleby SD, Salata JJ, Jurkiewicz NK, Sanguinetti MC, Stein RB, Gehret JR, Kothstein T, Claremon DA, Elliot JM, Butcher JW, Remy DC, Baldwin JJ. Cardiac electrophysiologic and antiarrhythmic actions of two long-acting spirobenzopyran piperidine class III agents, L-702,958 and L-706,000 [MK-499]. J Pharmacol Exp Ther. 1994;269:541-554.
Dukes ID, Cleeman L, Morad M. Tedisamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells. J Pharmacol Exp Ther. 1990;254:560-569.
Lee KS. Ibutilide, a new compound with potent class III antiarrhythmic activity, activates a slow inward Na+ current in guinea pig ventricular cells. J Pharmacol Exp Ther. 1992;262:99-108.