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CARDIOVASCULAR
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
Received February 3, 2007; accepted March 20, 2007.
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Abstract |
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; Trudeau et al., 1995). Reduction in IKr can cause long QT syndrome (LQTS), a cardiac repolarization disorder that can lead to life-threatening arrhythmias, torsades de pointes, and sudden cardiac death (Keating and Sanguinetti, 2001). Whereas direct blockade of hERG channels by various compounds represents a common mechanism for drug-induced LQTS (Sanguinetti and Tristani-Firouzi, 2006), recent evidence indicates that drug-disrupted hERG trafficking represents another mechanism for drug-induced LQTS (Ficker et al., 2004; Cordes et al., 2005; Kuryshev et al., 2005; Rajamani et al., 2006). Probucol is a cholesterol-lowering drug that has been known for many years to cause LQTS and torsades de pointes arrhythmia in humans and experimental animals (Elharrar et al., 1979; McCaughan, 1982; Jones et al., 1984). In a previous report studying wild-type and M124T mutant hERG channels expressed in Xenopus oocyte, Hayashi et al. (2004) showed that probucol (30 µM) did not affect the hERG current amplitude during depolarization steps but decreased the tail currents as a result of a 
10-mV shift of activation curve to the depolarized direction. It is unknown whether probucol causes similar effects on hERG channels expressed in mammalian cell lines.
Presently, much of the available data of IKr are obtained in hERG channels expressed in mammalian cell lines or Xenopus oocyte. Because the pore-forming subunits of K+ channels incorporate modulatory (
) subunits (Abbott et al., 1999), kinase-anchoring proteins (Gong et al., 1999), cytoskeletal elements, and other proteins, it is necessary to directly study native IKr. However, because of difficulties such as isolating IKr from other cardiac K+ currents, there has been no reported study showing native IKr trafficking at the protein level. In the present study, we identified IKr in neonatal rat ventricular myocytes, which can be recorded at a sufficiently robust level using Cs+ as the charge carrier in whole-cell clamp recordings. Using this native IKr model, we found that clinically relevant concentrations of probucol reduce IKr and prolong the cardiac action potential duration via reducing IKr membrane expression but not via channel blockade. Our study showed that primary culture of neonatal rat cardiomyocytes represents an effective model system for studying cardiac IKr trafficking.
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Materials and Methods |
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). Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Invitrogen, Burlington, OH) with 10% fetal bovine serum. Cardiomyocytes grown on glass coverslips for the electrophysiology study had a "spindle" morphology with a mean capacitance of 13.8 ± 1.3 pF (n = 33).
Molecular Biology. The hERG-human embryonic kidney (HEK) cells (HEK-293 cells stably expressing hERG channels) were a gift from Dr. Craig January (University of Wisconsin-Madison, Madison, WI) (Zhou et al., 1998b). In this cell line, hERG cDNA (Trudeau et al., 1995) was subcloned into BamHI/EcoRI sites of the pcDNA3 vector (Invitrogen). hERG cDNA in pcDNA3 was obtained from Dr. Gail Robertson (University of Wisconsin-Madison) (Trudeau et al., 1995). For immunofluorescence staining of the cell surface hERG channels, a hemagglutinin (HA)-epitope tag of the sequence 436TEEGPPATNSEHYPYDVPDYAVTFEECGY (boldface, insertion; italicized, HA epitope) was inserted into the extracellularly located S1-S2 loop of hERG channels to generate hERG-HAex via polymerase chain reaction using overlap extension method as described previously (Zhang, 2006). The hERG-HAex was transfected to HEK-293 cells, and a stable hERG-HAex cell line (hERG-HAex-HEK) was created using G418. The insertion of HA did not change the electrophysiological and trafficking properties of hERG channels (data not shown), consistent with the results from Ficker et al. (2003).
Patch-Clamp Recording Method. Whole-cell patch-clamp method was used (Hamill et al., 1981). The compositions of pipette and bath solutions for recording various currents are summarized in Table 1. Probucol was dissolved in ethanol to make 10 to 30 mM stock solutions. Procedures of whole-cell patch-clamp method and Cs+-carried IKr recording were performed as described previously (Zhang, 2006). Patch-clamp experiments were performed at room temperature (23 ± 1°C).
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Western Blot Analysis. Membrane proteins from hERG-HEK cells were isolated using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce Biotechnology, Rockford, IL). Membrane protein (10 µg/lane) was separated on 7% SDS-polyacrylamide electrophoresis gels and transferred onto nitrocellulose membranes. The membranes were blocked using Western Breeze Blocking Reagent (Invitrogen) and incubated with primary goat anti-hERG antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) and secondary anti-goat Western Breeze Chromogenic Detection Kit (Invitrogen). For cleavage of cell surface proteins, cells were washed with phosphate-buffered saline (PBS) and treated with 200 µg/ml proteinase K (Sigma-Aldrich, St. Louis, MO) in a physiological buffer (10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2, pH 7.4) at 37°C. The reaction was terminated by adding ice-cold PBS containing 6 mM phenylmethylsulfonyl fluoride and 25 mM EDTA, and membrane protein was then extracted for Western blot analysis.
To extract membrane proteins from neonatal rat cardiac myocytes, cells from 100-mm plates were rinsed with ice-cold PBS and scraped off into a 1-ml solution containing 200 mM NaCl, 33 mM NaF, 10 mM EDTA, 50 mM HEPES (pH 7.4 with NaOH), plus a protease inhibitor mixture. The cells were homogenized and spun at 500g for 10 min. The membrane fractions were pelleted from the low-speed supernatants by centrifugation at 100,000 rpm for 1 h at 4°C and resuspended in 50 mM Tris-HCl, 15 mM mercaptoethanol, and 1% SDS. The membrane proteins (50 µg/sample) were boiled in sample buffer and electrophoresed on a 7% polyacrylamide SDS gel. The membrane proteins were then electrophoretically transferred onto nitrocellulose membrane using a trans-blot system (Bio-Rad, Hercules, CA). After transfer, the filters were blocked with 5% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline (TBS) for 1 h. The filters were then incubated with goat polyclonal anti-hERG (C-20) antibody at a 1:200 dilution at 4°C overnight. The filters were then washed with TBS/Tween 20 (TBST) solution and incubated with horseradish peroxidase-conjugated donkey anti-goat immunoglobulin diluted 1:60,000 in TBST for 1 h at room temperature. After washing with TBST, bound antibodies were detected with an enhanced chemiluminescence detection kit.
Isolation of Cell Surface Protein with Biotinylating Reagent. A Cell Surface Protein Isolation Kit (Pierce) was used to study the effects of probucol on the cell surface hERG expression. hERG-HEK cells were prepared in 100-mm cell culture plates at 90% confluence. The cells treated with vehicle control (0.3% ethanol) or 100 µM probucol for 48 h were washed twice with ice-cold PBS and labeled with 10 ml of membrane-impermeant biotinylating reagent Sulfo-NHS-SS-Biotin (Pierce) for 30 min at 4°C. The quenching solution (0.5 ml) was then added to quench the reaction. Cells were then lysed with 0.5 ml of lysis buffer with a protease inhibitor mixture. After centrifugation at 10,000g for 2 min at 4°C, the cell lysate was precipitated with Immobilized NeutrAvidin Gel (agarose beads) (Pierce). The bound proteins were released by incubating the resin with SDS-polyacrylamide gel electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 1% SDS, and 10% glycerol) containing 50 mM dithiothreitol. The biotinylated cell surface protein was subjected to 7% SDS-polyacrylamide gel electrophoresis, analyzed using primary goat anti-hERG antibody (C-20, Santa Cruz Biotechnology) and horseradish peroxidase-conjugated donkey anti-goat secondary antibody, and detected with an enhanced chemiluminescence detection kit.
hERG Small Inhibitory RNA Transfection. To inhibit ether-a-go-go-related gene (ERG) mRNA, cultured neonatal rat cardiomyocytes and hERG-HEK cells were transfected with hERG small inhibitory RNA (siRNA) (Santa Cruz Biotechnology) or mouse ERG siRNA (of which one strand targets identical stretches in nucleotide sequence of rat ERG, Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) according to the protocols recommended by the suppliers. Scrambled siRNA (Santa Cruz Biotechnology) was used as a negative control. Experiments were performed 48 h after transfection. For electrophysiology studies, pIRES2-EGFP (Clontech, Palo Alto, CA) was included in the transfection reagent during ERG siRNA transfection, and fluorescent-positive cells were selected for IKr or hERG current recordings.
Immunocytochemistry. For immunofluorescent studies, hERG HAex-HEK cells were plated on coverslips for growth under control conditions and in the presence of probucol. Cells were fixed under a nonpermeabilized condition with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. After washing three times with PBS, cells were blocked with PBS containing 5% bovine serum albumin and 2% skim milk. The cells were immunostained with a rabbit anti-HA primary antibody (Anti-HA, 1:500, Sigma-Aldrich) and a green-fluorescent Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary antibody (1:250, Invitrogen). Cells were visualized using a Nikon (Mississauga, ON, Canada) TE2000-U research microscope.
Data are expressed as the mean ± S.E.M. A one-way analysis of variance or Student's t test was used to determine the significance of differences between control and test groups. A p value of 0.05 or less was considered significant.
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). The tail currents were plotted against the depolarizing voltages, and the data were fitted to the Boltzmann equation (Fig. 1B, 
). The half-activation voltage (V1/2) was –6.2 ± 1.7 mV, and the slope factor was 6.9 ± 1.5 mV (n = 4 cells).
E-4031-sensitive IKr is small, and its recording represents a tedious task. Moreover, any alterations of K+ currents during recordings before and after E-4031 will make the subtraction inaccurate. To address this difficulty, we recorded the pure IKr in neonatal rat ventricular myocytes using isotonic Cs+ solutions (135 mM Csi+/135 mM Cso+) (Table 1). We recently showed that hERG and IKr channels display unique Cs+ permeability (Zhang, 2006). Because Cs+ blocks most cardiac K+ channels, Cs+-carried IKr represents a simple and reliable way to directly record IKr (Zhang, 2006). Figure 1C shows a family of Cs+ currents obtained from a single cardiomyocyte. From a holding potential of –80 mV, depolarizations in 10-mV increments to voltages between –70 and +70 mV for 1 s were applied to evoke currents. Depolarizing steps to voltages more positive than 0 mV induced outward currents that inactivated in a voltage-dependent manner. The following tail currents at –80 mV displayed an initial rising phase, which is usually described as a "hook," reflecting the rapid recovery of inactivated channels to the open state before deactivation, and is unique to IKr. Figure 1D shows the I-V relationships of peak currents (
) and currents at the end of 1-s depolarizing steps (). Figure 1E shows the tail current activation curve. The V1/2 and slope factor (k) were –41.7 ± 3.4 and 5.8 ± 0.3 mV, respectively (n = 8 cells). The relatively negative V1/2 was caused by the absence of Ca2+ in the bath solution (Zhang, 2006). The average Cs+ tail current density measured at –80 mV following full channel activation was 17.9 ± 3.3 pA/pF (n = 8 cells).
To further show that the Cs+ current recorded from neonatal rat cardiomyocytes indeed represents the Cs+-carried IKr, the voltage-dependent inactivation and recovery from inactivation, which are unique to IKr, were analyzed. Figure 2A shows the voltage dependence of inactivation of Cs+-carried IKr. The membrane was initially depolarized to +60 mV for 500 ms to inactivate the channels. A 20-ms repolarizing step to –100 mV was applied to recover inactivated channels to the open state. Before deactivation, the membrane was depolarized to various voltages to induce channel inactivation (current decay), which was fitted to a single exponential function to obtain the inactivation time constant (
inact). Figure 2B shows the recovery from inactivation of the Cs+-carried IKr. The channels were activated and inactivated by a 500-ms depolarizing pulse to +60 mV. Repolarizing voltages between –10 and –120 mV were applied to record tail currents. The time constants of recovery from inactivation (rec) were obtained by fitting the rising phase of the tail currents to a single exponential function at different voltages (Sanguinetti et al., 1995). Values of inact (
, n = 6) and rec (, n = 6) are summarized and plotted against the membrane voltages (Fig. 2C). It has been known that extracellular Cs+ slows hERG and IKr inactivation (Zhang, 2006). Consistent with the Cs+ current in neonatal rat ventricular myocytes being Cs+-carried IKr, the Cs+ current inactivation was slowed by Cs+ (Fig. 2, D–F). As well, the Cs+ current was entirely sensitive to hERG/IKr blocker E-4031, with IC50 and Hill coefficient of 2.0 ± 0.4 and 1.2 µM, respectively (Fig. 2, G–I, n = 4).
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Western blot analysis was used to identify IKr proteins in neonatal ventricular myocytes. As shown in Fig. 3A, the hERG C-terminal antibody (HERG C-20; Santa Cruz Biotechnology) consistently identified four bands at sizes of approximately 150, 130, 95, and 85 kDa (n = 5 different myocyte isolations). The two higher molecular mass bands (150 and 130 kDa) are similar in size with mature glycosylated and immature core-glycosylated rat ERG1a (Jones et al., 2004). The two lower molecular mass bands are consistent in size with mature glycosylated and core-glycosylated rat ERG1b (Jones et al., 2004). To confirm that the 150- and 95-kDa bands represent the plasma membrane forms of ERG, we cleaved cell surface proteins by treating neonatal cardiomyocytes with proteinase K. As shown in Fig. 3A, proteinase K treatment significantly reduced the 150- and 95-kDa bands, and this was accompanied by the appearance of an additional 60- to 70-kDa band. This treatment did not affect the 130- or 85-kDa bands of the ERG proteins (n = 5). Figure 3B shows Western blot of hERG proteins extracted from hERG-HEK cells (hERG1a). As reported previously (Zhou et al., 1998b; Kuryshev et al., 2005), hERG displayed two bands with molecular masses of 155 and 135 kDa (Fig. 3B), representing the mature fully glycosylated membrane form (155 kDa) and the immature core-glycosylated endoplasmic reticulum (ER) form (135 kDa) (Zhou et al., 1998a,b; Ficker et al., 2004; Kuryshev et al., 2005). The 155-kDa hERG protein is localized on the plasma membrane because it was cleaved by proteinase K treatment (Fig. 3B, n = 3).
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Probucol Reduces ERG Expression. Our results indicate that Cs+-carried IKr in neonatal rat cardiomyocytes can be recorded at a level that is large enough and sufficiently robust to evaluate IKr alterations. Next, we investigated the mechanisms of probucol-induced LQTS by studying the effects of probucol on IKr Cs+ current in neonatal rat ventricular myocytes. Probucol is a cholesterol-lowering drug that causes LQTS in humans (Elharrar et al., 1979; McCaughan, 1982; Jones et al., 1984; Hayashi et al., 2004). We found that acute application of 100 µM probucol had no effect on IKr (Fig. 4A; n = 5). Figure 4A shows Cs+-carried IKr recorded from a cardiomyocyte before and after acute application of 100 µM probucol. The tail current-activation voltage relationships were shown at the bottom of Fig. 4A. The V1/2 and slope factor were –38.5 ± 1.6 and 6.7 ± 0.5 mV, respectively, in the absence of probucol (n = 5 cells). They were –39.5 ± 1.7 and 6.6 ± 0.6 mV in the presence of probucol (n = 5 cells, p > 0.05). Thus, probucol had no acute effect on IKr in neonatal cardiomyocytes. However, chronic treatment of cardiomyocytes with probucol substantially reduced IKr. Figure 4B shows Cs+-carried IKr from cardiomyocytes cultured in the absence (0.3% ethanol vehicle) or presence of 100 µM probucol for 48 h. The tail current-activation voltage relationships were summarized from five cells in the absence of probucol and from seven cells in the presence of 30 µM probucol. The V1/2 and slope factor were –42.2 ± 3.3 and 6.1 ± 0.7 mV, respectively, in control. They were –39.1 ± 2.9 and 6.6 ± 0.5 mV in probucol-treated cells (p > 0.05). Thus, whereas chronic application of probucol reduced IKr amplitude, it did not affect the voltage dependence of activation of IKr. Probucol-induced IKr reduction was concentration-dependent. The bottom of Fig. 4B shows the summarized IKr tail current amplitudes from myocytes treated with various concentrations of probucol for 48 h (n = 5–12 for each concentration). The IC50 and Hill coefficient were 20.6 ± 1.6 µM and 0.9 ± 0.1, respectively.
Figure 4, C and D, shows the effects of probucol on hERG K+ currents. Acute bath application of 100 µM probucol affected neither the hERG current amplitude nor the voltage dependence of the activation curve of hERG channels (Fig. 4C). The V1/2 and slope factor were –5.1 ± 2.3 and 7.2 ± 0.8 mV, respectively, in control. They were –7.2 ± 2.7 and 7.3 ± 0.7 mV in the presence of probucol (n = 5 cells, p > 0.05). To test whether probucol blocks hERG channels from the internal side of the membrane, we included 100 µM probucol in the pipette solution. We found that the hERG current with 100 µM probucol in the pipette solution was not different from the currents without probucol in the pipette solution (n = 4, data not shown). Thus, probucol does not directly block hERG channels. However, inclusion of probucol in the cell culture medium reduced the hERG current. Figure 4D shows hERG currents from hERG-HEK cells cultured in the control medium (containing 0.3% ethanol vehicle) and in the presence of 30 µM probucol for 48 h. The hERG activation curves under control and probucol-treated conditions are also shown in Fig. 4D. Probucol did not change the voltage dependence of the hERG channel activation (V1/2 =–7.5 ± 1.9 mV, k = 6.6 ± 0.2 mV, n = 14 for control; V1/2 =–3.4 ± 1.7 mV, k = 7.8 ± 0.4 mV, n = 12 for 30 µM probucol, p > 0.05) but significantly reduced the hERG current. To study concentration dependence of probucol effects on hERG current amplitude, tail currents from each probucol-treated cell were normalized to the mean control value (n = 14 cells). The summarized relative tail currents were plotted against probucol concentrations and fitted to Hill equation (n = 9–22 cells at each concentration). The IC50 and Hill coefficient were 10.6 µM and 1.0, respectively (Fig. 4D, bottom).
The chronic nature of probucol-induced hERG and IKr reduction suggests that probucol may decrease ERG membrane expression. To visualize the surface hERG expression, we performed immunofluorescence staining of cell surface hERG-HAex channels using an anti-HA antibody that recognizes the HA-epitope located in the extracellular S1-S2 linker of hERG channels. Because we did not permeabilize hERG-HAex-HEK cells, only the extracellularly exposed hERG was stained. As shown in Fig. 5A, the cell surface staining of hERG is visible throughout the control cell. Treatment of hERG-HAex-HEK cells with 100 µM probucol for 48 h significantly reduced the cell surface hERG staining. Figure 5B shows Western blots of hERG proteins extracted from wild-type hERG-HEK cells cultured in the absence (0.3% ethanol) and presence of 100 µM probucol for 48 h. Probucol essentially eliminated the mature plasma membrane form of hERG channels. Figure 5B, right shows the densities of the hERG 155- and 135-kDa bands after probucol treatment relative to their respective control values. Probucol treatment reduced the density of the 155-kDa band by 88 ± 7% (n = 10, p < 0.01) and increased the density of the 135-kDa band by 13 ± 10% (n = 10, p > 0.05). To confirm the effects of probucol on the surface membrane form of hERG channels, we isolated surface membrane protein using the biotinylation method (Cell Surface Protein Isolation Kit, no. 89881, Pierce). The isolated membrane protein from hERG-HEK cells under control conditions (0.3% ethanol) or treated with 100 µM probucol were analyzed using Western blot with anti-hERG antibody (C-20, Santa Cruz Biotechnology) (Fig. 5C). Probucol treatment reduced surface membrane hERG by 84 ± 9% (n = 4). To examine the effects of probucol on IKr expression, Western blots of ERG proteins extracted from neonatal cardiomyocytes cultured in the absence (0.3% ethanol) or presence of 100 µM probucol for 48 h were compared in Fig. 5D. Probucol treatment significantly reduced the 150- and 95-kDa bands and did not affect the 130- and 85-kDa bands (n = 4). Thus, probucol reduced cell surface form of ERG proteins.
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To determine the specificity of the probucol-induced hERG/IKr current reduction, the effects of probucol on Na+ current (INa), transient outward K+ current (Ito), and inward rectifier K+ current (IK1) in neonatal rat cardiomyocytes were examined. Cardiomyocytes were treated with 100 µM probucol for 48 h, and various currents were recorded using specific voltage protocols shown above the corresponding current traces in Fig. 6. The solutions used are summarized in Table 1. Probucol significantly reduced IKr without affecting INa, Ito, or IK1.
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; Gaughan et al., 1998), the action potential in control neonatal rat cardiomyocytes displayed a much more pronounced plateau phase than those in adult rat cardiomyocytes (Fig. 7A). Probucol treatment significantly prolonged action potential duration (Fig. 7B). The action potential durations at 90% repolarization (APD90) under control and probucol-treated conditions are summarized in Fig. 7C. To confirm the role of IKr in action potential duration, the effect of E-4031, a specific IKr blocker, was examined. The action potentials from neonatal cardiomyocytes in the presence of 100 nM E-4031 were compared with those in control. E-4031 (100 nM) significantly prolonged action potential duration (Fig. 7, D–F; n = 12).
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; Jones et al., 2004). The existence of IKr in neonatal rat cardiomyocytes has not been reported. In fact, the systematic electrophysiology data on IKr in adult rats are lacking possibly because of difficulties in isolating IKr from other coexisting K+ currents such as Ito. We have found that Cs+ uniquely permeates hERG, and we have used Cs+ permeation to successfully isolate IKr in rabbit ventricular myocytes (Zhang, 2006). The clone of rat ERG is 96% identical to hERG at the amino acid level (Wymore et al., 1997). In the present study, we have recorded robust Cs+-carried IKr in neonatal rat ventricular myocytes. Because Cs+ slows hERG/IKr inactivation (Zhang, 2006), there are differences in current kinetics between K+- and Cs+-carried IKr (Zhang, 2006). As well, the IC50 for E-4031 to block Cs+-carried IKr is higher than that to block K+-carried IKr (Figs. 1 and 2) (Zhang, 2006). Despite these differences, the Cs+-carried IKr recording represents a simple and reliable way to study IKr density and greatly facilitates studies of drug-induced alterations of IKr expression. Presently, models for studying native IKr trafficking at the protein level are not available. hERG turnover time is approximately 11 h in hERG-HEK cells (Ficker et al., 2003), and IKr turnover time in adult cardiomyocytes is likely to be much slower. Because neonatal cardiomyocytes have a more vigorous metabolism than adult cardiomyocytes, identification of IKr in neonatal cardiomyocyte provides us with a very useful way to analyze the native IKr trafficking. Significantly, using this model, we discovered that probucol reduces functional IKr expression. Previously, pentamidine was reported to disrupt hERG trafficking and cause LQTS (Cordes et al., 2005; Kuryshev et al., 2005). We found that like probucol, pentamidine treatment (10 µM, 48 h) significantly reduced the 150- and 95-kDa bands and did not affect the 130- and 85-kDa bands of ERG of neonatal rat cardiomyocytes (n = 4, data not shown).
Probucol is a cholesterol-lowering drug that has been found to cause long QT syndrome and torsades de pointes arrhythmia in patients and sudden cardiac death in experimental animals (Elharrar et al., 1979; McCaughan, 1982; Browne et al., 1984; Dujovne et al., 1984; Matsuhashi et al., 1989; Tamura et al., 1994; Reinoehl et al., 1996; Hayashi et al., 2004). Our data indicate that chronic probucol exposure reduces the hERG current with an IC50 of 10.6 µM and reduces native IKr with an IC50 of 20.6 µM. We chose 48-h treatment of cells with probucol because the turnover of the hERG channel occurs at a rather slow rate (approximately 11 h) (Ficker et al., 2003). The recommended daily dosage of probucol for human adults is 1 g. In one reported probucol-induced LQTS and torsades de pointes arrhythmia case, the serum probucol concentration measured during the cardiac arrhythmia was 26 µg/ml (Hayashi et al., 2004), which is equivalent to 50.3 µM. In patients who received probucol 1 g daily for periods of 1 to 12 months, the mean plasma levels ranged from 18.2 to 39.2 µg/ml (35.2–75.9 µM) (Heeg and Tachizawa, 1980). Although the drug concentrations that reach cardiac myocytes are unknown, it seems that clinically relevant concentrations of probucol are able to cause IKr reduction and LQTS.
Approximately 200 LQTS-associated hERG mutations have been identified in humans, and missense (single amino acid substitution) mutations represent the dominant predicted protein abnormality (Anderson et al., 2006). Although some patients with hERG mutations may have a prolonged QT interval and clinically be asymptomatic, they are probably more vulnerable to drugs that interact with hERG channels. Previously, Hayashi et al. (2004) reported that hERG M124T mutation caused a mild-to-moderate channel dysfunction but manifested marked QT prolongation or torsade de pointes after taking probucol. They showed that probucol acts to alter hERG function as a result of a 10-mV depolarization shift of activation curve (Hayashi et al., 2004). The authors also reported that acutely applied probucol shifted the reversal potential of hERG channels (Hayashi et al., 2004). Our data obtained in hERG-HEK cells and neonatal rat ventricular myocytes showed that probucol has no acute effect on either hERG or IKr currents. The reason for the discrepancy between our data and those of Hayashi et al. is unknown. The study reported by Hayashi et al. was performed in Xenopus oocytes. However, usually higher concentrations of drugs are needed to block channels expressed in Xenopus oocytes compared with those needed in mammalian cells. There has been no other evidence showing that probucol blocks hERG channels. On the other hand, our finding that probucol disrupts IKr/hERG cell membrane expression provides a plausible explanation for probucol-induced LQTS and torsade de pointes.
hERG proteins are synthesized in the ER and transported to the cell surface via the Golgi apparatus. Misfolded or misassembled proteins are retained in the ER by its quality control mechanism. It is thought that mutations can cause misfolding of hERG proteins, resulting in trafficking defect (Anderson et al., 2006). Because probucol treatment did not reduce the intracellular forms of ERG (135-kDa band of hERG, 130- and 85-kDa bands of ERG in cardiomyocytes) (Fig. 5), the probucol-induced ERG membrane expression does not seem to be a result of inhibition of the channel synthesis. Instead, it could develop from either defective trafficking or accelerated membrane ERG degradation. It has been shown that E-4031, glycerol, or culture in low temperature can rescue some forms of trafficking deficient mutant hERG channels (Zhou et al., 1999). However, we found that none of these manipulations rescued probucol-disrupted hERG surface expression (data not shown). Probucol is a very lipophilic drug that can inhibit the cholesterol synthesis in the cell and may modify the lipid content of the membrane. Whether the lipid content of the cell contributes to hERG/IKr functional expression needs further investigation.
In summary, the present study provides evidence that probucol does not block hERG or IKr channels but disrupts ERG protein trafficking. In drug development, early screening of lead compounds for potential acute hERG channel blockade is becoming a common practice. The finding that probucol reduces hERG and IKr currents by reducing the number of functional ERG channels suggests that further strategies for evaluating the LQTS risk of drugs should be considered.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: hERG, human ether-a-go-go-related gene; IKr, cardiac rapidly activating delayed rectifier K+ current; LQTS, long QT syndrome; probucol, 4,4'-(isopropylidenedithio)-bis-(2,6-di-t-butylphenol); HEK, human embryonic kidney; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TBST, Tris-buffered saline/Tween 20; ERG, ether-a-go-go-related gene; siRNA, small inhibitory RNA; E-4031, 1-[2-(6-methyl-2-pyridyl)ethyl]-4-(methylsulfonyl-aminobenzoyl) piperidine; I-V, current-voltage; ER, endoplasmic reticulum; GFP, green fluorescent protein; HA, hemagglutinin.
Address correspondence to: Shetuan Zhang, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, 351 Tache Avenue, Winnipeg, MB, Canada R2H 2A6. E-mail: szhang{at}sbrc.ca
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) and presence of 135 mM Cs+o (
) and presence of probucol (
