Introduction

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Tamoxifen is one of the most effective and frequently prescribed drugs to treat breast cancer (Jordan, 1992). A recent clinical observation indicated that tamoxifen prolongs the QT interval in human subjects (Trump et al., 1992). Prolongation of cardiac repolarization and the QT interval has clinical importance because under certain situations it may confer a class III antiarrhythmic effect; however, it has also been shown to increase the risk of developing a complex form of potentially lethal ventricular arrhythmia known as TdP (Ben-David and Zipes, 1993; Carlsson et al., 1990; Roden et al., 1986).

Crucial to generation of TdP is prolongation of the QT interval and APD that permits EADs to occur (Zeng and Rudy, 1995). Because potassium currents are major determinants of cardiac repolarization and because shortening of the action potential suppresses EADs in isolated myocytes (Bouchard et al., 1995), modulation of cardiac repolarizing potassium channels may be critical in modulating EADs. Clinically, drugs that prolong the QT interval by blocking cardiac potassium channels, especially the rapid component of the delayed rectifier current, I_Kr, are often associated with acquired TdP arrhythmias (Carlsson et al., 1990; Roden et al., 1986; Follmer et al., 1990; Woosley, 1996). In the present study, we evaluated the potential cardiac electrophysiological effects of tamoxifen at clinically relevant concentrations (Murphy et al., 1987) using the whole-cell patch-clamp technique in rabbit ventricular myocytes.

	Methods

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Isolation of ventricular cells. Rabbit (3-4 months old, weight 3-3.5 kg; HRP Inc., Denver, PA) ventricular myocytes were isolated using a modification of the method previously described (Giles and Imaizumi, 1988). Briefly, rabbit hearts were removed and mounted on the Langendorff perfusion system. The following procedure was used: (1) perfusion with normal Tyrode's solution for 10 min; (2) perfusion for ~20 min with a Ca⁺⁺-free Tyrode's solution; (3) perfusion for 30 to 35 min with Tyrode's solution containing 40 U/ml of collagenase II (Worthington Biochemical, Freehold, New Jersey) and 50 µmol/l CaCl_2. The ventricles were then minced and gently stirred in Tyrode's solution containing 100 µmol/l CaCl₂. After stirring for 5 to 10 min, a large number of single ventricular cells was obtained. The resulting cell suspension was then filtered through a nylon mesh. Cells were collected by centrifugation at 50×g and then resuspended in Tyrode's solution containing 250 µmol/l Ca⁺⁺. The cells were again collected by centrifugation, and then resuspended in Tyrode's solution containing 1 mmol/l Ca⁺⁺. After a third centrifugation, the cells were resuspended in DMEM culture medium supplemented with 10% bovine serum (Hyclone Labs, Logon, UT). The myocytes were immediately seeded onto laminin-coated glass microcoverslips at a density of 10⁴ rod-shaped cells/cm² and allowed to attach. The cells were stored in a humidified incubator in 5% CO₂, 95% air at 37°C. Only cells within the first 36 hr after isolation were used.

Whole-cell patch-clamp. The patch-clamp technique was used to record the membrane currents and action potentials in single ventricular myocytes. Command voltage pulses were generated using PCLAMP 6.0.2 Software connected to an interface (Axon Instruments, Foster City, CA), an IBM-compatible Pentium computer, and an Axopatch 200A amplifier. Membrane potentials and current signals were monitored on an oscilloscope (Model 5103, Tektronix, Beaverton, Oregon) and stored in the lab computer. Pipettes with tip resistance of 1 to 4 M were pulled from borosilicate glass (World Precision Instruments, Sarasota, Florida) and filled with an intracellular solution containing (mmol/l) KCl 125, NaCl 10, CaCl₂ 1, Mg-ATP 5, EGTA 14, HEPES 10, cAMP 0.1, adjusted with KOH to pH 7.2. A holding potential of -40 mV was used to inactivate fast sodium and T-type calcium currents. The external solution was Tyrode's solution containing (mmol/l) NaCl 137, KCl 5.4, HEPES 10.0, MgCl₂ 1.0, CaCl₂ 2.0, glucose 10.0, and was adjusted with NaOH to pH 7.4 (NaOH). Cd⁺⁺ (0.2 mmol/l) was used to block the L-type calcium channel (I_Ca) and to shift the I-V relationship of I_to and I_Kr to more positive potentials (Daleau et al., 1997; Agus et al., 1991). This allows 1) separation of I_Kr and the outward portion of I_K1, especially at membrane potentials positive to -30 mV and 2) marked increase of I_to availability at a holding potential of -40 mV. For action potential recording, Cd⁺⁺ was omitted from the extracellular solution and the voltage-clamp mode was switched to current clamp mode.

Drugs and chemicals. Tamoxifen was purchased from Sigma Chemical (St. Louis, MO), dissolved in ethanol and stored in aliquots at 20°C until used. E-4031 and dofetilide were kindly provided by Eisai Ltd. (Ibaraki, Japan) and Pfizer Central Research (Groten, CN) respectively. All other chemicals were obtained from Sigma Chemical.

Data analysis and statistics. Patch-clamp data were normalized for total cell capacitance to allow comparison between cells of various sizes. The paired Student's t test was used to compare the potassium current amplitude before and after tamoxifen treatment. Data are reported as mean ± S.D., and differences between values were considered statistically significant when P < .05.

	Results

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Effect of E-4031 on I_Kr, I_to and I_K1. I_Kr is one of the major repolarizing currents, and its block has been implicated in TdP (Carlsson et al., 1990; Roden et al., 1986; Follmer and Colatsky, 1990; Woosley, 1996). To evaluate whether tamoxifen affects I_Kr, we first sought to establish the presence of I_Kr in our rabbit ventricular myocytes. Figures 1, A and B, show the membrane currents elicited by a 1.5-sec voltage-clamp step from -40 mV to different test potentials ranging from -10 to +50 mV in the same cell before (A) and after (B) 5-min exposure to 5 µmol/l E-4031, a highly selective I_Kr blocker (Clay et al., 1995; Sanguinetti et al., 1990). Under control conditions, a slowly activating outward current flowed during depolarization, followed by an outward tail current that has been shown to represent the gradual decay of I_K (Follmer et al., 1990; Clay et al., 1995; Sanguinetti et al., 1990). The initial peak in the time-dependent outward current was due to the rapid activation and inactivation of I_to, which is sensitive to 4-aminopyridine (data not shown). E-4031 abolished the tail current on repolarization and also reduced the time-dependent outward current, without affecting the initial peak (I_to) or the holding current (I_K1). Shown in B are the E-4031-sensitive currents obtained by digital subtraction of currents in the bottom tracings from currents in the top tracings in A. Compared with the tail current, the time-dependent current demonstrated marked inward rectification at very positive potentials. I_to was not present in the E-4031-sensitive currents, indicating E-4031 has no effect on I_to at this concentration. Superfusion with 1 to 2.5 µmol/l dofetilide or removal of extracellular K⁺ also abolished the tail current (Liu et al., 1998). These features of the delayed rectifier current (inward rectification of the time-dependent current, complete block of the tail current by E-4031, dofetilide and removal of extracellular K⁺) are consistent with the previous description of I_Kr in rabbit and other species (Clay et al., 1995; Sanguinetti et al., 1990).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Recordings of IKr, Ito (A and B) and IK1 (C) in the same cell before and after 5-min superfusion with 5 µmol/l E-4031. A, IKr and Ito currents before and after superfusion with E-4031. E-4031 abolished the IKr tail current and also reduced the time-dependent IKr current without affecting the transient outward current (Ito) or the holding current. B, E-4031 sensitive currents obtained by digital subtraction of currents after E-4031 from currents before E-4031. Note the inward rectification of the time-dependent IKr currents at very positive potentials compared with the tail currents, C, IK1 current before and after superfusion with E-4031. E-4031 showed little effect on the inward IK1 current recorded at 120 mV. The outward holding currents that represent the amplitude of IK1 at 40 mV are superimposed.

Effect of tamoxifen on I_Kr, I_to and I_K1. We next tested the effect of tamoxifen on the three major potassium currents using the same protocol as shown in figure 1. Shown in figure 2 are the I_Kr, I_to and I_K1 currents elicited in the same cell before and after 5-min exposure to 10 µmol/l tamoxifen. Similar to E-4031, tamoxifen abolished the I_Kr tail current and also reduced the time-dependent current without affecting I_to or the holding current (fig. 2A). The solvent for tamoxifen, ethanol, had no effect on I_Kr at the concentration (1%, v/v) used to dissolve tamoxifen (data not shown). Figure 2B depicts the tamoxifen sensitive currents obtained by digital subtraction of currents in the bottom tracings from currents in the top tracings in A. There is a striking similarity between the tamoxifen sensitive current and the E-4031-sensitive current shown in figure 1B. In both figures 1B and 2B, the time-dependent current demonstrated strong inward rectification at very positive potentials compared with the tail current, whereas I_to was not present in either the tamoxifen or the E-4031-sensitive current. Figure 2C shows the I_K1 current measured before and after 5-min exposure to 10 µmol/l tamoxifen in the same cell shown in figure 2, A and B. Like E-4031, tamoxifen produced no inhibition of the I_K1 inward current at 120 mV. In fact, I_K1 inward current was even slightly larger after tamoxifen treatment in this cell (fig. 2C). The outward holding currents representing the amplitude of I_K1 at 40 mV were superimposable, indicating that tamoxifen had no effect on the I_K1 outward current.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Recordings of IKr, Ito (A and B) and IK1 (C) in the same cell before and after 5-min superfusion with 10 µmol/l tamoxifen. A, IKr and Ito currents recorded before and after superfusion with tamoxifen. Tamoxifen abolished the IKr tail current and also reduced the time-dependent IKr current, without affecting the transient outward current (Ito). B, Tamoxifen-sensitive currents obtained by digital subtraction of currents before and after tamoxifen superfusion. Note the inward rectification of the time-dependent IKr currents at very positive potentials compared with the tail currents and their similarity to the E-4031 sensitive currents. C, IK1 current recorded before and after superfusion with tamoxifen. Tamoxifen showed no block of IK1 inward current. The outward holding currents that represent the amplitude of IK1 at 40 mV are superimposed.

Time-dependent block of I_Kr by tamoxifen. To further characterize the observed inhibition of I_Kr by tamoxifen, we examined whether this inhibition is time dependent. Figure 3 depicts a typical experiment performed in the same cell before drug administration (control), or after 3-, 5- and 9-min superfusion with 1 µmol/l tamoxifen. As shown in figure 3, block of I_Kr by tamoxifen is time dependent and has a slow onset. Further block can still be observed after superfusion for 5 min. In contrast, I_Kr was readily recorded from control myocytes (no exposure to tamoxifen) for at least 10 min without any sign of run-down. In the absence of drug, the amplitude of I_Kr measured 10 min after membrance rupture was 103.4 ± 6.15% of that measured immediately after membrance rupture (n = 3, P > .05).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Time-dependent block of IKr by tamoxifen. IKr currents were recorded in the same cell before drug admininstration, after 3-, 5- and 9-min superfusion with 1 µmol/l tamoxifen.

Effect of tamoxifen on the I-V relationship of I_Kr. We next evaluated the effect of tamoxifen on the I-V relationship of I_Kr. Figure 4, A and B, demonstrates the effects of 1 and 3.3 µmol/l on the I-V relationship of I_Kr, measured after 5- to 7-min infusion of tamoxifen. Tamoxifen markedly reduced I_Kr current amplitude in a concentration-dependent fashion. A typical voltage-dependent block of I_Kr is shown in figure 4. Note the greater block at more positive potentials. At the test potential of +50 mV, tamoxifen (1 and 3.3 µmol/l) blocked I_Kr by 39.5 ± 1.7% (P < .01) and 84.8 ± 1.3%, respectively (P < .01), whereas no significant block of I_K1 was observed at 3.3 µmol/l (5.5 ± 0.9% test potential = -120 mV, n = 4, P > .05).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Voltage- and concentration-dependent block of IKr by tamoxifen. IKr tail currents were measured after 5- to 7-min superfusion of tamoxifen. A, Effect of 1 µmol/l on IKr I-V relationship. B, Effect of 3.3 µmol/l tamoxifen on IKr I-V relationship. Data are expressed as mean ± S.D., n = 4, *P < .05, **P < .01.

Comparison of I_Kr block by tamoxifen and quinidine. Quinidine is a drug that has been frequently associated with TdP (Roden et al., 1986). To compare the block of I_Kr by tamoxifen to that produced by quinidine, we performed the protocol shown in figure 5, using either 10 µmol/l tamoxifen or 10 µmol/l quinidine. Tamoxifen completely blocked the I_Kr tail current with no recovery observed after 5-min washout, whereas the same concentration of quinidine (10 µmol/l) only partially blocked the I_Kr tail currents, with recovery within 3-min washout. In other experiments, complete recovery of I_Kr from quinidine block was usually observed after ~5-min washout, whereas no recovery from tamoxifen could be detected even after 15-min washout (data not shown). Figure 5C compares the percentage inhibition of I_Kr by tamoxifen and quinidine at the same concentration of 3.3 µmol/l. These data show that, at the test potential of +50 mV, tamoxifen produced significantly greater inhibition of I_Kr compared with quinidine (84.8 ± 1.3% vs. 42.5 ± 9.1%, P < .01). Thus, tamoxifen was a more potent and longer lasting blocker of I_Kr than quinidine.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Comparison of IKr block by tamoxifen and quinidine. A, IKr currents recorded from the same cell before drug administration, after 5-min superfusion with 10 µmol/l tamoxifen and after 5-min washout. IKr tail currents were abolished by tamoxifen without recovery. B, IKr currents recorded from another cell before drug administration, after 5-min superfusion with 10 µmol/l quinidine and after 3-min washout. IKr tail currents were reduced but not abolished by quinidine with partial recovery after 3-min washout. C, Inhibition of IKr by 3.3 µmol/l tamoxifen and 3.3 µmol/l quinidine. Data are expressed as mean ± S.D., n = 4, **P < .01.

Effect of tamoxifen on APD and I_Ca. We next examined whether tamoxifen causes prolongation of APD. Figure 6 shows action potentials recorded before and after 4-min exposure to 3.3 µmol/l tamoxifen. Surprisingly, although tamoxifen inhibited I_Kr by ~84.8% at this concentration (fig. 4), no significant prolongation of APD was observed. APD measured at 90% repolarization (APD₉₀) before and after 4- to 5 min superfusion of tamoxifen (3.3 µmol/l) was 341 ± 49 ms and 332 ± 19 ms respectively (n = 16, P > .05). Because under control conditions, no significant shortening of APD was observed in the initial 10 min after cell membrance rupture, the absence of APD prolongation by tamoxifen was not secondary to a "rundown" phenomenon.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of tamoxifen on the APD. Action potentials were elicited by injecting 100 pA depolarizing currents of 2-msec duration at a frequency of 0.45 Hz. Shown in the figure are two superimposed action potential tracings recorded in a single ventricular myocyte before and after 4-min exposure to 3.3 µmol/l tamoxifen. The two traces are averaged traces from 16 trials.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of tamoxifen on the L-type ICa. ICa was recorded in the same cell before tamoxifen administration; after 1-, 2-, 3- and 4-min superfusion of 10 µmol/l tamoxifen; and after 2-, 4-, 8- and 16-min washout. Note the marked inhibition of ICa and partial recovery after washout.

	Discussion

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The major finding of the present study is that the commonly prescribed antiestrogen drug tamoxifen potently blocks the rapid component of the delayed rectifier current, I_Kr, in a voltage-, concentration- and time-dependent fashion. The potassium channel-blocking effect of tamoxifen seems to be specific for I_Kr because no significant effect was observed on I_K1 or I_to at concentrations up to 10 µmol/l (fig. 2). Tamoxifen blocks I_Kr with a potency even greater than quinidine, a drug that has been shown to block I_K and is associated with a high incidence of drug-induced TdP (Roden et al., 1986). This is, to our knowledge, the first study showing that tamoxifen is a potent and selective I_Kr blocker.

Outward potassium channels are the major determinants of the repolarization phase of the cardiac action potential. In the cardiac ventricular myocytes, the transient outward current I_to, the delayed rectifier I_K and the inward rectifier I_K1 mediate different phases of the repolarization process (Giles and Imaizumi, 1988; Sanguinetti et al., 1990; Surawicz, 1992). The contribution of I_K and I_K1 to the APD is well established and reduction of I_K or I_K1 will cause APD and QT prolongation (Giles and Imaizumi, 1988; Sanguinetti and Jurkiewicz, 1990; Surawicz, 1992). On the other hand, although I_to has been shown to play an important role in determining phase 1 repolarization, there is still some uncertainty regarding its contribution to normal APD (Giles and Imaizumi, 1988; Litovsky and Antzelevitch, 1988; Kaab et al., 1996). Interestingly, to our knowledge, all the drugs that are clinically associated with QT prolongation and acquired TdP invariably block I_Kr. The reason why I_Kr is sensitive to block by so many drugs is still not well understood.

I_K has been reported to consist of two components, I_Kr and I_Ks, in guinea pig, dog and human ventricles (Sanguinetti and Jurkiewicz, 1990; Gintant, 1996; Li et al., 1996). In the rabbit ventricular myocytes, I_K has been reported to be absent (Giles and Imaizumi, 1988), consist of only one component (Clay et al., 1995) or consist of two components (Salata et al., 1996). In our experiments, I_K can be consistently recorded with a clear tail in all of the untreated cells we studied. Our previous studies (Liu et al., 1998) have indicated that the major delayed rectifier current that contributes to APD in normal rabbit ventricular myocytes is I_Kr.

Tamoxifen has been shown to prolong the QT interval in human subjects (Trump et al., 1992). The present study suggests that the tamoxifen-induced QT prolongation may be related to a direct block of cardiac I_Kr current. The tamoxifen plasma concentration was greater than 5 µmol/l when QT prolongation was observed clinically by Trump et al. (Trump et al., 1992). As shown in figure 4, tamoxifen was even more potent than quinidine in blocking I_Kr, causing more than 80% block of I_Kr at 3.3 µmol/l. Although no I_Kr recovery from tamoxifen block could be observed even after an extended washout period, the possibility that the block we observed was due to "rundown" of I_Kr can be excluded because block of I_Kr by quinidine can completely recover after 5-min washout.

Interestingly, a recent clinical study using F-18 fluoro-tamoxifen showed significant cardiac uptake of tamoxifen, presumably due to intracellular accumulation of tamoxifen in cardiac myocytes (Inoue et al., 1997). In the present study, I_Kr block by tamoxifen had a slow onset and no recovery was observed after an extended washout period. These results may indicate that tamoxifen blocks I_Kr through an intracellular site. However, further studies are needed to elucidate the exact mechanism of the potent I_Kr block by tamoxifen.

One unexpected finding of this study is that, although we demonstrated a potent inhibition of I_Kr by tamoxifen, it had no significant effect on APD in rabbit ventricular myocytes. This may be due to the fact that tamoxifen is also a potent calcium channel blocker, as documented by Song et al. (1996) and confirmed by our current study (fig. 7). Because blocking of I_Ca will lead to a shortening of the APD, this effect may largely counteract the I_Kr blocking effect of tamoxifen which would otherwise lead to a prolongation of APD in single rabbit cardiomyocyte and prolongation of QT interval in whole heart. The obvious discrepancy between the current study in rabbit (no effect on APD) and the observations in humans (QT prolongation) may result from different relative contributions of I_Kr or I_Ca to the APD and/or different relative potencies of tamoxifen in blocking I_Kr vs. I_Ca in difference species. The net effect of tamoxifen on the APD in a certain species would therefore depend on both the relative contribution of the I_Kr vs. I_Ca to the APD and the relative potency of tamoxifen in blocking I_Kr vs. I_Ca. It is also possible that other ionic currents not examined in this study such as chloride current (Vanderberg et al., 1994), may mediate the differential effects of tamoxifen in different species. Further studies using human venticular myocytes are needed to resolve these issues.

At the present time, there are no clinical data available regarding the risk of developing TdP in patients after tamoxifen administration. Nevertheless, the current finding has important clinical implications. Therapeutic plasma concentrations of tamoxifen (Murphy et al., 1987) in patients are similar to concentrations that produce potent block of I_Kr in isolated rabbit ventricular myocytes (i.e., low micromolar range, see fig. 4). In addition, tamoxifen is frequently used to treat breast cancer in female patients, whereas recent clinical observations and experimental data have indicated that female gender is associated with a higher risk of developing drug-induced TdP (Makkar et al., 1993; Lehmann et al., 1996; Liu et al., 1997). Although the effect of tamoxifen on I_Ca may potentially ameliorate its I_Kr blocking effect, caution should still be taken when administering tamoxifen to patients in situations where other risk factors for TdP also exist, such as hypokalemia, bradycardia, congenital long QT syndrome or coadministration of other drugs that may also delay cardiac repolarization.