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Am J Physiol Heart Circ Physiol 285: H661-H668, 2003. First published April 17, 2003; doi:10.1152/ajpheart.00686.2002
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Tamoxifen inhibits Na+ and K+ currents in rat ventricular myocytes

Jianying He,1 Margaret E. Kargacin,2 Gary J. Kargacin,2 and Christopher A. Ward1

1Department of Physiology, Queen's University, Kingston, Ontario K7L 3N6; and 2Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Submitted 2 August 2002 ; accepted in final form 11 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. However, tamoxifen has been shown to induce QT prolongation of the electrocardiogram, thereby potentially causing life-threatening polymorphic ventricular arrhythmias. The purpose of the present study was to elucidate the electrophysiological mechanism(s) that underlie the arrhythmogenic effects of tamoxifen. We used standard ruptured whole cell and perforated patch-clamping techniques on rat ventricular myocytes to investigate the effects of tamoxifen on cardiac action potential (AP) waveforms and the underlying K+ currents. Tamoxifen (3 µmol/l) markedly prolonged AP duration, decreased maximal rate of depolarization, and decreased resting membrane potential. At this concentration, tamoxifen significantly depressed the Ca2+-independent transient outward K+ current (Ito), sustained outward delayed rectifier K+ current (Isus), inward rectifier K+ current (IK1), and Na+ current (INa) in the myocytes. Lower concentrations of tamoxifen (1 µmol/l) also decreased the resting membrane potential and significantly depressed IK1 to 79 ± 5% (n = 5; at –120 mV) of pretreatment values. The results of this study indicate that inhibition of Ito, Isus, and IK1 by tamoxifen may underlie AP prolongation in cardiac myocytes and thereby contribute to prolonged QT interval observed in patients.

electrophysiology; arrhythmia; cardiac; torsades de pointes; QT prolongation

THE ESTROGEN RECEPTOR ANTAGONIST tamoxifen has been used for >20 years to actively treat breast cancer and to help reduce the incidence of breast cancer in high-risk women (19). It has also been used to treat cancers of the liver, brain, and pancreas, which indicates that it may have more general application (3). However, increasing evidence suggests that tamoxifen may have untoward cardiovascular effects. Several cases of QT prolongation and ventricular arrhythmias, in particular torsades de pointes, have been reported with tamoxifen (13, 28). These reports prompted the warning of potentially serious adverse cardiac events.

It is likely that tamoxifen promotes QT-segment prolongation by selectively altering ionic currents; however, relatively little work has been done to address this possibility. Tamoxifen was shown to inhibit volume-activated Cl– currents in many cell types and to inhibit a number of voltage- and ligand-gated cation channels primarily in noncardiac tissues (1, 16, 27, 43). As well, tamoxifen has been reported to block neuroblastoma cell proliferation that is mediated by voltage-dependent K+ channels (29). In rat vascular smooth muscle cells, tamoxifen inhibits both voltage-gated Ca2+ currents and contractility (35). Similar effects on Ca2+ currents are reported in a clonal pituitary cell line (32).

There are few published studies that describe the ionic mechanisms by which tamoxifen can initiate arrhythmias. In rabbit ventricular myocytes, Liu et al. (24) reported that tamoxifen inhibits the delayed rectifier K+ current (IKr) and the inward L-type calcium current (ICa,L). They did not, however, detect an effect on either the transient outward K+ current (Ito) or the inwardly rectifying K+ current (IK1). They were also unable to demonstrate that tamoxifen increases action potential (AP) duration, a result that is inconsistent with IKr inhibition and is predicted based on the clinical observations of QT prolongation. Thus further studies are required to evaluate the ionic mechanisms of tamoxifen. Previously, we demonstrated that tamoxifen inhibits canine cardiac muscle sarcoplasmic reticulum (SR) Ca2+ uptake through a mechanism that is most consistent with the inhibition of an SR ion channel (21). We also showed that tamoxifen reduces the amplitude of electrically evoked Ca2+ transients and the amount of Ca2+ that can be released by caffeine in intact isolated rabbit cardiac myocytes. These results are consistent with an effect of tamoxifen on the SR but do not rule out the possibility that tamoxifen also affects plasma membrane ion channels. To test the latter possibility and to elucidate electrophysiological mechanism(s) that may underlie the arrhythmogenic effect of tamoxifen, we examined AP waveforms of rat ventricular myocytes. We determined the concentration-dependent effects of tamoxifen on AP waveforms and the K+ and Na+ currents present in these myocytes. Standard ruptured whole cell and perforated patch-clamping techniques were used for these studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Isolation of myocytes. Right ventricular myocytes were isolated according to methods previously described (42). Briefly, adult male Sprague-Dawley rats (200–225 g body wt) were killed by cervical dislocation. The heart was rapidly removed and mounted on a Langendorff apparatus by aortic cannulation. The heart was initially perfused with Tyrode solution that contained 1 mmol/l Ca2+ at 37°C for 5 min. Perfusion rate was 10 ml/min, and all perfusion solutions were bubbled with 100% O2. The perfusate was then switched to Ca2+-free Tyrode solution for 5 min, followed by perfusion for 7 min with the same solution to which collagenase (0.02 mg/ml; Yakult) and protease (0.004 mg/ml, type XIV; Sigma Chemicals) had been added. After the perfusion, the right ventricular free wall was removed, minced in 10 ml of Tyrode solution that contained collagenase (0.5 mg/ml), protease (0.1 mg/ml), and bovine serum albumin (3 mg/ml; Sigma), and was gently shaken in a water bath (at 37°C) for 20–40 min. Aliquots of suspended cells were removed at 5-min intervals into tubes with Kraftbrühe solution (defined below), and the cells were stored at 4°C until use. Only single rod-shaped cells with clear cross striations and no spontaneous contractions were used for experiments. All the experiments were conducted at room temperature (21–23°C).

Solutions and chemicals. Tamoxifen (Sigma) was dissolved in ethanol to obtain 1 mmol/l stock solution, and aliquots of this stock solution were stored at –20°C until use. The standard Tyrode solution used for cell isolation and patch-clamp recordings contained (in mmol/l) 140 NaCl, 5.4 KCl, 1 Na2HPO4, 5 HEPES, 10 glucose, and 1 MgCl2, and pH was adjusted to 7.4 with 1 mol/l NaOH. K+ currents [Ito, sustained outward delayed rectifier (Isus), and IK1] were recorded using a standard internal pipette solution that contained (in mmol/l) 20 KCl, 110 K-aspartate, 10 EGTA, 10 HEPES, 1 MgCl2, 5 K2ATP, 1 CaCl2, and 10 NaCl, and pH was adjusted to 7.2 using 1 mol/l KOH. AP waveforms were recorded using solutions identical to those for K+ current recordings with the exception that amphotericin B (300 µg/ml) was added to the pipette solution to perforate the cell membrane. Unless stated, the standard 1 mM CaCl2-containing Tyrode solution was used as the superfusion solution when K+ currents and APs are recorded. Na+ currents were recorded using a pipette solution that contained (in mmol/l) 120 CsF, 10 HEPES, 2 MgCl2, 10 EGTA, 1 CaCl2, and 10 NaCl, and pH was adjusted to 7.2 with 1 mol/l KOH. The superfusion solution for recording Na+ current (INa) contained (in mmol/l) 10 NaCl, 10 HEPES, 5 KCl, 1 MgCl2, 1 CaCl2, 5 CoCl2, 5 CsCl, 10 glucose, and 110 choline chloride, and pH was adjusted to 7.4 with 1 mmol/l NaOH. Modified KB solution for cell storage contained (in mmol/l) 100 K-glutamate, 10 K-aspartate, 25 KCl, 20 glucose, 10 KH2PO4,5 HEPES, 2 MgSO4, 20 taurine, 5 creatine, 0.5 EGTA; plus 0.1% BSA with pH adjusted to 7.2 using 1 mol/l KOH.

Electrophysiological methods. Myocytes were transferred to a small chamber mounted on the stage of an inverted microscope (TE300, Nikon) and allowed to adhere onto the glass bottom of the chamber for 10–15 min. The flow rate through the chamber was 2 ml/min, and solution exchange of the bath was achieved within 30 s. Patch pipettes were pulled from borosilicate glass using a P-97 Puller (Sutter Instruments) and were polished on an MF-200 microforge (World Precision Instruments; Sarasota, FL). When filled with internal solution, the pipette resistance was 2–3 M{Omega}. Whole cell currents and APs were recorded with an Axopatch 1-D amplifier (Axon Instruments; Burlingame, CA) using the pCLAMP 8.0 Digidata 1200 data-acquisition system. We corrected for junction potentials (9–11 mV).

AP waveforms were recorded using the perforated patch-clamp technique. APs were elicited in current-clamp mode by 5-ms, 800-pA current injections at a frequency of 1 Hz. All APs and K+ currents were filtered at 2 kHz and sampled at 5 kHz.

The ruptured patch whole cell configuration was used to measure Ito, Isus, and IK1. Whole cell K+ currents were evoked by 500-ms test pulses ranging between –120 and +50 mV in 10-mV increments from a holding potential of –80 mV. This voltage protocol was repeated with a 100-ms depolarizing step to –40 mV applied immediately before the 500-ms test pulses to voltage-inactive Ito. The currents derived by subtracting these two protocols are the isolated Ito values. Ito amplitude was measured as the peak current amplitude.

To measure IK1, 500-ms test pulses ranging between –120 and +50 mV, in 10-mV increments, from a holding potential of –80 mV, were applied in the presence and absence of 200 µmol/l Ba2+. Ba2+ blocks IK1, whereas it does not inhibit either Ito or Isus (25, 33). Subtraction of the current evoked in the presence and absence of Ba2+ yields the isolated IK1 current. Values obtained at the end of the 500-ms pulse were used for analysis.

Isus values were also recorded by 500-ms test pulses ranging between –120 and +50 mV, in 10-mV increments, from a holding potential of –80 mV. A 100-ms prepulse to –40 mV and 200 µmol/l BaCl2 was applied to block Ito and IK1, respectively. Isus amplitude was measured as the residual current at the end of the 500-ms pulses.

Whole cell INa was elicited using 50-ms rectangular voltage steps from a holding potential of –90 mV to a command potential of –30 mV. These data were filtered at 2 kHz and recorded at 20 kHz.

For all experiments, tamoxifen was added to the superfusion solution 10 min before recording. Time-course studies (data not shown) indicated that the maximal effects of tamoxifen can be measured within 8 min of initial exposure.

Data analysis. Ionic currents were corrected for cell capacitance and are expressed in terms of current density (in pA/pF). All averaged and normalized results are presented as means ± SE. Statistical analysis was done using an ANOVA test followed by the Newman-Keuls multiple-comparison test. A P < 0.05 was taken to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of tamoxifen on AP waveforms. Figure 1 illustrates the concentration-dependent effects of tamoxifen on the AP waveforms in rat ventricular myocytes. At all concentrations examined, tamoxifen altered AP waveforms. Typical effects of tamoxifen included reduced resting membrane potential, decreased upstroke velocity, reduced AP amplitude, and broadening of the entire waveform. The concentration-dependent effects of tamoxifen on AP parameters are summarized in Table 1. A complete AP characterization at 10 µmol/l tamoxifen was not possible, because this concentration completely suppressed our ability to stimulate APs. Vehicle control experiments (ethanol alone) did not significantly change the AP waveforms.



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  		Fig. 1. Effects tamoxifen on action potential (AP) waveforms in rat ventricular myocytes. Representative APs recorded in the absence (black traces) and presence (gray traces) of tamoxifen. A: tamoxifen concentration, 1 µmol/l. B: tamoxifen concentration, 3 µmol/l. APs were elicited as described in MATERIALS AND METHODS. Horizontal bar represents 0 mV.

 

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  		Table 1. Concentration-dependent effects of tamoxifen on action potential parameters of isolated rat ventricular myocytes

 

Effects of tamoxifen on Ito. Ito was isolated using techniques outlined in MATERIALS AND METHODS; results are illustrated in Fig. 2D. The effects of tamoxifen at 1, 3, and 10 µmol/l on Ito were assessed with cumulative increasing-concentration applications. The concentration-dependent effects of tamoxifen on Ito are summarized in Fig. 3. Consistent with the AP data, 1 µmol/l tamoxifen had little effect on Ito. In contrast, 3 and 10 µmol/l tamoxifen reduced peak outward current (at +50 mV) to 22.9 ± 1.8 (n = 4; P < 0.05) and 13.9 ± 1.4 pA/pF (n = 4; P < 0.05), respectively, from the pretreatment value of 29.2 ± 3.3 pA/pF. Examination of the current-voltage relationship for Ito demonstrates that 3 and 10 µmol/l tamoxifen significantly attenuate this current at all command potentials positive to –20 mV (Fig. 3D).



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  		Fig. 2. Representative families of current traces to illustrate the methods used to isolate the three primary K+ currents in rat ventricular myocytes. A: all inward and outward whole cell currents recorded by holding the cell to –80 mV and applying a 500-ms command step to potentials ranging from –120 to +50 mV, in 10-mV increments. B: same protocol as in A with the addition of a 100-ms prepulse to –40 mV. C: same protocol as in A recorded in the presence of 200 µM BaCl2. D: isolated transient outward K+ current (Ito) was derived by subtracting the current families of A and B. E: isolated inward rectifier current (IK1) was derived by subtracting C from A. F: isolated sustained delayed rectifier current (Isus) was obtained by using the protocol outlined in B but in the presence of 200 µmol/l BaCl2.

 


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  		Fig. 3. Concentration-dependent effects of tamoxifen on the Ca2+-independent Ito. Representative traces of Ito families are shown with exposure to 10 µmol/l tamoxifen for command potentials of –50, –30, –10, +10, +30, and +50 mV. A: traces before exposure. B: traces after exposure. C: summary of changes of Ito following application of 1, 3, and 10 µmol/l tamoxifen as measured at +50 mV. D: current-voltage relationships for Ito before and after addition of 1 ({blacktriangleup}), 3 ({blacktriangledown}), and 10 ({diamond}) µmol/l tamoxifen. All currents were normalized for cell capacitance and plotted as means ± SE (n = 4); +P < 0.05, data significantly different from pretreatment values; *P < 0.05, command potentials at which data for both 3 and 10 µmol/l groups were significantly different from pretreatment values ({blacksquare}).

 

Effects of tamoxifen on IK1 current. Figure 2, A and C, shows representative current traces of K+ currents before and after exposure to 200 µmol/l Ba2+. The Ba2+-sensitive current, IK1, was obtained by subtracting the current in the presence and absence of Ba2+ as shown by Fig. 2E. Superfusion of myocytes with 1, 3, and 10 µM tamoxifen for 10 min had significant concentration-dependent effects on IK1 (Fig. 4). From a control current density of –13.8 ± 1.4 pA/pF, cumulative additions of tamoxifen decreased IK1, at –120 mV, to –11.0 ± 1.2, –7.0 ± 0.6, and –2.0 ± 0.3 pA/pF (n = 5; P < 0.05 for all concentrations) for 1, 3, and 10 µmol/l, respectively (Fig. 4). The current-voltage relationships for IK1 before and after exposure to tamoxifen are shown in Fig. 4D.



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  		Fig. 4. Concentration-dependent effects of tamoxifen on IK1. Representative traces of IK1 families are shown with exposure to 10 µmol/l tamoxifen for command potentials of –120, –100, –80, –60, –40, and –20 mV. A: before tamoxifen exposure. B: after tamoxifen exposure. C: summary of changes of IK1 following application of 1, 3, and 10 µmol/l tamoxifen as measured at –120 mV. D: current-voltage relationships for IK1 before and after addition of 1 ({blacktriangleup}), 3 ({blacktriangledown}), and 10 ({diamond}) µmol/l tamoxifen. All currents were normalized for cell capacitance and plotted as means ± SE (n = 5); +P < 0.05, data significantly different from pretreatment values; *P < 0.05, command potentials at which data for 1, 3, and 10 µmol/l groups were significantly different from pretreatment values; #P < 0.05, command potentials at which data for both 3 and 10 µmol/l groups were significantly different from pretreatment values ({blacksquare}).

 

Effects of tamoxifen on Isus. Representative tracings of isolated Isus are illustrated in Fig. 2F. Before tamoxifen exposure, peak Isus at +50 mV was determined to be 10.0 ± 0.7 pA/pF (n = 5). Tamoxifen attenuated Isus to 8.5 ± 0.8, 7.4 ± 0.9, and 4.7 ± 0.1 pA/pF (n = 5) for 1, 3, and 10 µmol/l tamoxifen, respectively. These data are summarized in Fig. 5. Although there was a trend for 1 µmol/l tamoxifen to reduce this current, there were no significant differences over the entire voltage range examined (Fig. 5D). In contrast, 3 µmol/l tamoxifen significantly reduced Isus at all command potentials >0 mV, whereas 10 µmol/l significantly reduced Isus at all command potentials greater than –10 mV (Fig. 5D).



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  		Fig. 5. Concentration-dependent effects of tamoxifen on the sustained outward delayed rectifying K+ current (Isus). Representative traces of Isus families are shown with exposure to 10 µmol/l tamoxifen for command potentials of –50, –30, –10, +10, +30, and +50 mV. A: before tamoxifen exposure. B: after tamoxifen exposure. C: summary of changes of Isus following application of 1, 3, and 10 µmol/l tamoxifen as measured at +50 mV. D: current-voltage relationships for Isus before and after addition of 1 ({blacktriangleup}), 3 ({blacktriangledown}), and 10 ({diamond}) µmol/l tamoxifen. All currents were normalized for cell capacitance and plotted as means ± SE (n = 5); +P < 0.05, data significantly different from pretreatment values; *P < 0.05, command potentials at which data for 1, 3, and 10 µmol/l groups were significantly different from pretreatment values; #P < 0.05, command potentials at which data for both 3 and 10 µmol/l groups were significantly different from pretreatment values ({blacksquare}).

 

For all K+ current experiments, the effects of 1 and 3 µmol/l tamoxifen were completely reversible within 10-min washout of the drug. At 10 µmol/l tamoxifen, relatively longer washout periods were required (15–20 min), and occasionally, incomplete reversal was obtained.

Effects of tamoxifen on INa. As noted above and shown in Table 1, tamoxifen at concentrations of 1 µM and greater significantly decreased the maximum upstroke velocity of the AP in rat ventricular myocytes. This suggests that the INa responsible for depolarization of the myocytes is also affected by tamoxifen. To test this possibility, INa was measured in the presence and absence of tamoxifen (Fig. 6A). From a pretreatment value of –37.4 ± 3.5 pA/pF (n = 8), tamoxifen decreased this current to –34.4 ± 3.6, –22.7 ± 2.1, and –7.8 ± 0.9 pA/pF (n = 8) for 1, 3, and 10 µmol/l, respectively. Inhibition by both 3 and 10 µmol/l was statistically significant (P < 0.05). As observed with K+ current recordings, INa was not inhibited in vehicle control experiments (data not shown).



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  		Fig. 6. Concentration-dependent effects of tamoxifen on Na+ currents. A: representative traces of INa inhibition by tamoxifen. Dashed line, zero-current level. B: summary of changes of INa following application of 1, 3, and 10 µmol/l tamoxifen measured at –30 mV from a holding potential of –90 mV. All currents were normalized for cell capacitance and are plotted as means + SE (n = 8); *P < 0.05, data significantly different from pretreatment values.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Tamoxifen is the leading antisteroidal substance available to treat breast cancer and acts as a weak estrogen by competing for estrogen receptors much like phytoestrogens. Currently, tamoxifen is prescribed to more women with breast cancer than any other drug. Nevertheless, evidence has accrued that tamoxifen may prolong cardiac repolarization and hence also prolong the QT interval of the surface ECG. Potentially life-threatening ventricular arrhythmias (e.g., torsades de pointes) may occur especially in cases of overdosage or pharmacokinetic interactions (13). The fact that a number of drugs that block cardiac K+ channels and prolong AP duration can cause long-QT syndrome and induce ventricular arrhythmia (4) considered with the demonstrated ability of tamoxifen to block some types of ion channels motivated us to examine the effects of tamoxifen on cardiac K+ channels. The roles that K+ channels are known to play in the repolarization phase of the cardiac AP are consistent with the mechanisms thought to be involved in mediating acquired forms of long-QT syndrome (prolonged repolarization that leads to early afterdepolarizations and triggered arrhythmias; Ref. 36).

In the present study, we used standard whole cell and perforated patch-clamping techniques to investigate the effects of tamoxifen on cardiac AP waveforms and select K+ currents (Ito, Isus, and IK1) and INa in isolated rat ventricular myocytes (2, 20, 34, 42). The rat model was chosen because the electrophysiological properties of these cells have been widely characterized and provide a better representation of human myocytes compared with those isolated from rabbits. Significant alterations of AP waveforms by tamoxifen were observed. All three K+ currents and INa were significantly and reversibly depressed by tamoxifen at 3 and 10 µmol/l concentrations. The marked reductions of these currents can explain in part the altered AP waveforms induced by tamoxifen in our experiments and also suggest ionic mechanisms that may contribute to the prolonged QT interval of the electrocardiogram that has been described in patients.

The contributions of individual K+ currents to the overall AP waveform have been extensively characterized. The Ca2+-independent Ito is present throughout the hearts of many species and is largely responsible for the early repolarization phase of the cardiac AP (6). Inhibition of this current would be expected to delay this repolarization phase and would contribute primarily to the increase in AP duration at 50% repolarization (ADP50) that was seen in our experiments. IK1 is a constitutively active, strong inward rectifier that determines the basic electrophysiological properties of both ventricular and atrial myocytes. It contributes to the acceleration of the final phase of phase III repolarization of the cardiac AP and stabilization of the resting membrane potential (5, 34). Inhibition of this current could therefore be responsible in part for the change in resting membrane potential and the increase in AP duration at 90% repolarization (ADP90) seen in our experiments. The rapidly activating and noninactivating current, Isus, also contributes to the overall repolarization process. Thus all three of these currents contribute to membrane repolarization, and their inhibition is consistent with a prolongation the AP waveform. This result is also consistent with studies in which some drugs (such as dofetilide and azimilide) reported to significantly prolong the QT interval in a clinical setting were shown to inhibit the rapid component of the IKr in electrophysiological studies and to block K+ channels encoded by the human ether-à-go-go gene (13).

In contrast with the tamoxifen-induced K+ current effects reported in our present study, it has been suggested that tamoxifen selectively blocks IKr with little effect on IK1, Ito, and AP duration in rabbit ventricular myocytes (24). These differences in the inhibitory effects of tamoxifen between our study in rat ventricular myocytes and the previous observations in rabbit ventricular myocytes are reasonable, because different compositions of K+ currents and/or different inhibitory effects of tamoxifen are potentially present when these different species are compared. For example, the delayed, time-dependent current IKr is either absent or insignificant in rat ventricle (7), whereas it is prominent in rabbit ventricular myocytes (37). Also of interest are the differences of effects on IK1. Of the three K+ currents that we have examined, it is apparent that IK1 is more sensitive to tamoxifen than either Ito or Isus. This finding is consistent with our AP data obtained in the presence of 1 µM tamoxifen in which we observe a depolarization of the resting membrane potential while other AP waveform parameters are not altered; this effect is characteristic of selective IK1 inhibition. Inhibition of IK1 is in direct contrast to the study of Liu et al. (24) and requires additional study. However, to fully elucidate the ability of tamoxifen to inhibit these currents in human tissues, further studies are required.

The distribution of tamoxifen into tissues and the availability of the drug and active metabolites to target cells are obvious determinants of tamoxifen effects. The apparent volume of distribution for tamoxifen is high (50–60 l/kg; Ref. 22), which suggests extensive distribution into peripheral tissues and the presence of only a minor portion of the drug in the serum. In humans, levels were 10- to 60-fold higher in tissues than in serum (23). Tamoxifen chronically administered for breast cancer at therapeutic doses (40 mg/day) reaches plasma concentrations of 0.75 µmol/l (30). Even higher doses such as 200 mg/day have been used for patients in clinical trials to test the efficacy of tamoxifen in the treatment of gliomas and in reverting multidrug resistance (8, 38). Therefore, the concentrations used in the present study could be clinically relevant. To more definitively explore this possibility requires additional information. Although the serum and tissue concentrations of tamoxifen can be quite high, the free concentration in extracellular fluid or in the blood is a more relevant parameter if tamoxifen acts directly on the ion channel proteins (see the discussion below). The solubility of tamoxifen also prevented us from determining its effects over a broader range of concentrations. As noted previously (21), tamoxifen at concentrations above ~15 µM precipitates in the buffers used in our experiments. This has prevented us from determining whether additional inhibition is observed at higher concentrations and, consequently, we have not been able to determine more complete dose-response curves for its action on the cardiac ion currents. Tamoxifen is extensively metabolized by cytochrome P-450 in humans and produces active estrogen receptor-binding metabolites. In addition to tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4-hydroxy-N-desmethyltamoxifen, and N-desdimethyltamoxifen were abundant in most tissues (23). It is most likely that tamoxifen can modulate cardiac muscle K+-channel function by at least two different pathways: directly by affecting ion channels and indirectly through conversion to a variety of metabolites. Additional studies to correlate ion-current blockade of metabolites of tamoxifen associated with arrhythmias would be beneficial.

Our results showing that tamoxifen inhibits Ito, IK1, and Isus can explain some but not all of the alterations in the AP waveform that we observed in this study. On the basis of our data, we suggest that the observed decreases of Ito, IK1, and Isus density by tamoxifen underlie the prolongation of the AP that we observed in rat ventricular myocytes. In the intact heart, this could contribute to prolongation of the QT interval and arrhythmogenesis. From our AP data (see Table 1), however, we would also predict that INa is inhibited, because the AP upstroke velocity was significantly depressed in the presence of tamoxifen. Also consistent with strong INa inhibition is our inability to stimulate APs in the presence of 10 umol/l tamoxifen. The effects of tamoxifen on INa were verified by direct measurement of peak inward INa. Our results demonstrating that tamoxifen inhibits INa are consistent with our observed decreases in AP upstroke velocity and further demonstrate the nonselectivity of tamoxifen as an ionic channel inhibitor. As we have shown, at least four cardiac ion currents appear to be affected by tamoxifen. Tamoxifen is also known to inhibit volume-regulated Cl– currents in cardiac muscle (15) and L-type Ca2+ channels (24) and to block cell-to-cell communication in neonatal cardiac myocytes (24). That tamoxifen appears to affect so many cardiac ion channels suggests that its actions are nonspecific. As discussed, there are, however, a number of ion channels that are not affected by tamoxifen (14, 24).

The mechanisms by which tamoxifen interacts with ion channels and other proteins have not been well determined. Tamoxifen and related compounds are highly lipophilic and readily insert into lipid bilayers (9–11, 18, 26). It has therefore been suggested that tamoxifen alters protein-lipid interactions and consequently exerts its effects on channels or other proteins indirectly (discussed in Refs. 10, 11, 35, 39, 40). It is also possible that tamoxifen interacts directly with proteins (see Refs. 12, 17, 35, 41). Sahebgharani et al. (31) showed recently that volume-activated Cl– currents were inhibited by tamoxifen but not by a membrane-impermeant quaternary derivative of tamoxifen (ethylbromide tamoxifen). This indicates that tamoxifen must be in the lipid bilayer to affect these channels; however, whether the channel inhibition involves a direct interaction between tamoxifen and one of the transmembrane portions of the Cl– channel protein or is mediated by tamoxifen-induced changes in lipid structure or lipid-protein interactions has not yet been established.

In summary, we have shown that tamoxifen has inhibitory effects on K+ and Na+ currents. These findings provide insight into the cellular mechanisms by which tamoxifen prolongs AP waveforms and potentially contributes to the induction of arrhythmias.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by the Heart and Stroke Foundation of Ontario (Grant T4180 to C. A. Ward), the Heart and Stroke Foundation of Alberta, the American Heart Association, and the Canadian Institutes for Health Research. G. J. Kargacin is an Alberta Heritage Foundation for Medical Research Senior Scholar. C. A. Ward is a Heart and Stroke Foundation of Canada Research Scholar.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Ward, Dept. of Physiology, Queen's Univ., Kingston, Ontario K7L 3N6, Canada (E-mail: wardc{at}post.queensu.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Allen MC, Newland C, Valverde MA, and Hardy SP. Inhibition of ligand-gated cation-selective channels by tamoxifen. Eur J Pharmacol 354: 261–269, 1998.[ISI][Medline]
  2. Apkon M and Nerbonne JM. Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes. J Gen Physiol 97: 973–1011, 1991.[Abstract/Free Full Text]
  3. Butta A, Maclennan K, Flanders KC, Sacks NP, Smith I, McKinna A, Dowsett M, Wakefield LM, Sporn MB, and Baum M. Induction of transforming growth factor beta 1 in human breast cancer in vivo following tamoxifen treatment. Cancer Res 52: 4261–4264, 1992.[Abstract/Free Full Text]
  4. Camm AJ, Janse MJ, Roden DM, Rosen MR, Cinca J, and Cobbe SM. Congenital and acquired long QT syndrome. Eur Heart J 21: 1232–1237, 2000.[Free Full Text]
  5. Carmeliet E. K+ channels in cardiac cells: mechanisms of activation, inactivation, rectification and K+ sensitivity. Pflügers Arch 414, Suppl 1: S88–S92, 1989.
  6. Carmeliet E. K+ channels and control of ventricular repolarization in the heart. Fundam Clin Pharmacol 7: 19–28, 1993.[ISI][Medline]
  7. Chadwick CC, Ezrin AM, O'Connor B, Volberg WA, Smith DI, Wedge KJ, Hill RJ, Briggs GM, Pagani ED, and Silver PJ. Identification of a specific radioligand for the cardiac rapidly activating delayed rectifier K+ channel. Circ Res 72: 707–714, 1993.[Abstract/Free Full Text]
  8. Couldwell WT, Weiss MH, DeGiorgio CM, Weiner LP, Hinton DR, Ehresmann GR, Conti PS, and Apuzzo ML. Clinical and radiographic response in a minority of patients with recurrent malignant gliomas treated with high-dose tamoxifen. Neurosurgery 32: 485–489, 1993.[ISI][Medline]
  9. Custódio JBA, Almeida LM, and Madeira VMC. A reliable and rapid procedure to estimate drug partitioning in biomembranes. Biochem Biophys Res Commun 176: 1079–1085, 1991.[ISI][Medline]
  10. Custódio JBA, Almeida LM, and Madeira VMC. The anti-cancer drug tamoxifen induces changes in the physical properties of model and native membranes. Biochim Biophys Acta 1150: 123–129, 1993.[Medline]
  11. Custódio JBA, Almeida LM, and Madeira VMC. The active metabolite hydroxytamoxifen of the anticancer drug tamoxifen induces structural changes in membranes. Biochim Biophys Acta 1153: 308–314, 1993.[Medline]
  12. Custódio JBA, Almeida LM, and Madeira VMC. The effect of the anticancer drugs tamoxifen and hydroxytamoxifen on the calcium pump of isolated sarcoplasmic reticulum vesicles. Toxicol In Vitro 10: 523–531, 1996.
  13. De Ponti F, Poluzzi E, and Montanaro N. QT-interval prolongation by non-cardiac drugs: lessons to be learned from recent experience. Eur J Clin Pharmacol 56: 1–18, 2000.[ISI][Medline]
  14. Dick GM, Kong ID, and Sanders KM. Effects of anion channel antagonists in canine colonic myocytes: comparative pharmacology of Cl–, Ca2+, and K+ currents. Br J Pharmacol 127: 1819–1831, 1999.[ISI][Medline]
  15. Duan D, Hume JR, and Natel S. Evidence that outwardly rectifying Cl– channels underlie volume-regulated Cl– currents in heart. Circ Res 80: 103–113, 1997.[Abstract/Free Full Text]
  16. Hardy SP, deFelipe C, and Valverde MA. Inhibition of voltage-gated cationic channels in rat embryonic hypothalamic neurones and C1300 neuroblastoma cells by triphenylethylene antioestrogens. FEBS Lett 434: 236–240, 1998.[ISI][Medline]
  17. Hardy SP and Valverde MA. Novel plasma membrane action of estrogen and antiestrogens revealed by their regulation of a large conductance chloride channel. FASEB J 8: 760–765, 1994.[Abstract]
  18. Jordan VC. Biochemical pharmacology of antiestrogen action. Pharmacol Rev 36: 245–276, 1984.[ISI][Medline]
  19. Jordan VC. Tamoxifen treatment for breast cancer: concept to gold standard. Oncology (Huntingt) 11: 7–13, 1997.
  20. Josephson IR, Sanchez-Chapula J, and Brown AM. Early outward current in rat single ventricular cells. Circ Res 54: 157–162, 1984.[Abstract/Free Full Text]
  21. Kargacin ME, Ali Z, Ward CA, Pollock NS, and Kargacin GJ. Tamoxifen inhibits Ca2+ uptake by the cardiac sarcoplasmic reticulum. Pflügers Arch 440: 573–579, 2000.[ISI][Medline]
  22. Lien EA, Solheim E, Lea OA, Lundgren S, Kvinnsland S, and Ueland PM. Distribution of 4-hydroxy-N-desmethyltamoxifen and other tamoxifen metabolites in human biological fluids during tamoxifen treatment. Cancer Res 49: 2175–2183, 1989.[Abstract/Free Full Text]
  23. Lien EA, Solheim E, and Ueland PM. Distribution of tamoxifen and its metabolites in rat and human tissues during steady-state treatment. Cancer Res 51: 4837–4844, 1991.[Abstract/Free Full Text]
  24. Liu XK, Katchman A, Ebert SN, and Woosley RL. The antiestrogen tamoxifen blocks the delayed rectifier potassium current, IKr, in rabbit ventricular myocytes. J Pharmacol Exp Ther 287: 877–883, 1998.[Abstract/Free Full Text]
  25. Nichols CG and Lopatin AN. Inward rectifier potassium channels. Annu Rev Physiol 59: 171–191, 1997.[ISI][Medline]
  26. Pazoutová S, Kren V, Rezanka T, and Sajdl P. Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing claviceps purpurea strains. Biochem Biophys Res Commun 152: 190–196, 1988.[ISI][Medline]
  27. Phillis JW, Song D, and O'Regan MH. Tamoxifen, a chloride channel blocker, reduces glutamate and aspartate release from the ischemic cerebral cortex. Brain Res 780: 352–355, 1998.[ISI][Medline]
  28. Pollack IF, DaRosso RC, Robertson PL, Jakacki RL, Mirro JR, Blatt J, Nicholson S, Packer RJ, Allen JC, Cisneros A, and Jordan VC. A phase I study of high-dose tamoxifen for the treatment of refractory malignant gliomas of childhood. Clin Cancer Res 3: 1109–1115, 1997.[Abstract]
  29. Rouzaire-Dubois B and Dubois JM. Tamoxifen blocks both proliferation and voltage-dependent K+ channels of neuroblastoma cells. Cell Signal 2: 387–393, 1990.[ISI][Medline]
  30. Rutqvist LE and Mattsson A. Cardiac and thromboembolic morbidity among postmenopausal women with early-stage breast cancer in a randomized trial of adjuvant tamoxifen. The Stockholm Breast Cancer Study Group. J Natl Cancer Inst 85: 1398–1406, 1993.[Abstract/Free Full Text]
  31. Sahebgharani M, Hardy SP, Lloyd AW, Hunter AC, and Allen MC. Volume-activated chloride currents in HeLa cells are blocked by tamoxifen but not by a membrane impermeant quaternary analogue. Cell Physiol Biochem 11: 99–104, 2001.[ISI][Medline]
  32. Sartor P, Vacher P, Mollard P, and Dufy B. Tamoxifen reduces calcium currents in a clonal pituitary cell line. Endocrinology 123: 534–540, 1988.[Abstract]
  33. Shieh RC, Chang JC, and Arreola J. Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes. Biophys J 75: 2313–2322, 1998.[Medline]
  34. Shimoni Y, Clark RB, and Giles WR. Role of an inwardly rectifying potassium current in rabbit ventricular action potential. J Physiol 448: 709–727, 1992.[Abstract/Free Full Text]
  35. Song J, Standley PR, Zhang F, Joshi D, Gappy S, Sowers JR, and Ram JL. Tamoxifen (estrogen antagonist) inhibits voltage-gated calcium current and contractility in vascular smooth muscle from rats. J Pharmacol Exp Ther 277: 1444–1453, 1996.[Abstract/Free Full Text]
  36. Surawicz B. Electrophysiologic substrate of torsade de pointes: dispersion of repolarization or early afterdepolarizations? J Am Coll Cardiol 14: 172–184, 1989.[Abstract]
  37. Tande PM, Bjornstad H, Yang T, and Refsum H. Rate-dependent class III antiarrhythmic action, negative chronotropy, and positive inotropy of a novel IK blocking drug, UK-68,798: potent in guinea pig but no effect in rat myocardium. J Cardiovasc Pharmacol 16: 401–410, 1990.[ISI][Medline]
  38. Trump DL, Smith DC, Ellis PG, Rogers MP, Schold SC, Winer EP, Panella TJ, Jordan VC, and Fine RL. High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine. J Natl Cancer Inst 84: 1811–1816, 1992.[Abstract/Free Full Text]
  39. Verrecchia F and Hervé J. Reversible inhibition of gap junctional communication by tamoxifen in cultured cardiac myocytes. Pflügers Arch 434: 113–116, 1997.[ISI][Medline]
  40. Verrecchia F and Hervé J. Reversible inhibition of gap junctional communication elicited by several classes of lipophilic compounds in cultured rat cardiomyocytes. Can J Cardiol 13: 1093–1100, 1997.[ISI][Medline]
  41. Waldegger S, Lang U, Herzer T, Suessbrich H, Binder K, Lepple-Wienhues A, Nagl U, Paulmichl M, Franz HBG, Kiesl L, Lang F, and Busch AE. Inhibition of minK protein induced K+ channels in Xenopus oocytes by estrogens. Naunyn Schmiedebergs Arch Pharmacol 354: 698–702, 1996.[ISI][Medline]
  42. Ward CA and Giles WR. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J Physiol 500: 631–642, 1997.[Abstract/Free Full Text]
  43. Zhang JJ, Jacob TJ, Valverde MA, Hardy SP, Mintenig GM, Sepulveda FV, Gill DR, Hyde SC, Trezise AE, and Higgins CF. Tamoxifen blocks chloride channels. A possible mechanism for cataract formation. J Clin Invest 94: 1690–1697, 1994.[ISI][Medline]



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