Cellular Mechanism of the QT Prolongation Induced by Sulpiride

Abstract

In this study, the authors investigated the electrophysiological effect of sulpiride on cardiac repolarization using conventional microelectrode recording techniques in isolated canine Purkinje fibers and a whole-cell patch clamp technique in transiently transfected cells with the hERG, KCNQ1/KCNE1, KCNJ2, and SCN5A cDNA and in rat cardiac myocytes for I_Ca. In studies of action potential duration, 10 μM, 100 μM, 300 μM, and 1 mM sulpiride prolonged action potential duration in a concentration-dependent manner. In studies of cardiac ion channels, sulpiride did not significantly affect I_Na, I_Ca, I_Ks, I_K1, except for I_Kr. Sulpiride dose-dependently decreased the hERG tail current. It is considered that the prolonged action potential duration by sulpiride was mainly the result of inhibition of the hERG channel. The data suggest that the clinical use of sulpiride is reasonable within therapeutic plasma concentrations, but all patients taking this drug should be cautiously monitored for clinical signs of long-QT syndrome and severe arrhythmia.

QT interval prolongation and torsade de pointes are well-known drug-induced side effects of commonly used drugs, including antipsychotic drugs. ^1,2 Antipsychotic agents may be divided by chemical class. The phenothiazines are the oldest group and include chlorpromazine (Thorazine), mesoridazine (Serentil), prochlorperazine (Compazine), and thioridazine (Mellaril). These drugs are essentially similar in terms of both activity and adverse effects. Another group of antipsychotic agents is an atypical group, including olanzapine (Zyprexa), risperidone (Risperdal), sulpiride (Dogmatil, Meresa) and amisulpride (Solian). Among these, sulpiride is a substituted benzamide, which is used mainly in the treatment of psychosis and depression. Substituted benzamides such as cisapride, amisulpiride, and metoclopramide commonly evoke cardiotoxic effects. The atypical antipsychotics such as amisulpride, clozapine, flupenthixol, haloperidol, perphenazine, risperidone, thioridazine, and ziprasidone have been reported to be associated with QT prolongation. Although sulpiride reportedly has fewer extrapyramidal side effects than many of the conventional antipsychotics, Sugiyama et al reported that sulpiride induced QT prolongation in halothane-anesthetized dogs and also chronic atrioventricular (AV) block dogs. ³ In addition, Su et al reported that QTc prolongation is associated with the use of sulpiride in schizophrenic patients. ⁴ Recently, Huang et al reported that sulpiride caused torsade de pointes in patients. ⁵ The present study examines the electrophysiological effects of sulpiride using isolated canine ventricular myocytes and whole-cell patch clamp techniques for multicardiac ion channels, including I_Kr, I_Ks, I_K1, I_Na, and I_Ca, to clarify whether there is a relationship between action potentials and each of these channels on QT prolongation.

Methods

Drugs

Sulpiride, 4-aminopyridine (4-AP), BaCl₂, primaquine, nicardipine, and the chemical products used to prepare external and internal solutions were purchased from Sigma-Aldrich Co. (St Louis, MO).

Recording of Action Potentials

This study was conducted in facilities approved by the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International. All procedures were approved by our Institutional Animal Care and Use Committee (IACUC). Purkinje fibers were isolated from the left ventricles of hearts from male beagle dogs killed by overdose with sodium pentobarbitone. Spontaneously beating fibers were mounted in a continuous flow (5 mL/min) and temperature-controlled (37 ± 1 °C)–chamber superfused with normal Tyrode (NT) solution (in mM): 145 NaCl, 5.4 KCl, 5 HEPES, 0.33 NaH₂PO₄, 0.5 MgCl₂, 16.6 glucose, 1.8 CaCl₂. The NT solution was oxygenated with O₂ gas. Throughout the experiment, the preparation was stimulated via silver bipolar electrodes at suprathreshold levels (frequency = 1 Hz, duration = 2 ms, voltage = 1.5–2 V) to evoke cardiac action potentials. Action potentials were recorded using a standard glass microelectrode filled with 3 M KCl (resistance at 10–30 MΩ). Action potentials were amplified using Geneclamp 500B (Molecular Devices Corp, Sunnyvale, CA), and data were stored and analyzed using the Notocord HEM program (NOTOCORD, Croissy Sur Seine, France). Action potential duration at 50% (APD₅₀) and 90% (APD₉₀) of repolarization, total amplitude (TA), resting membrane potential (RMP), and V_max of the phase 0 max depolarization values were determined for each concentration of drug. To examine the drug effects, each drug was allowed to perfuse for 20 min.

Recording of Ionic Currents

Ionic currrents were recorded in whole-cell configuration using an Axopatch 200B amplifier (Molecular Devices Corp, Sunnyvale, CA). For various aspects of cardiac ion channel study, human embryonic kidney (HEK293) cells were transiently transfected through the lipofectamine method ⁶ for hERG, KCNQ1/KCNE1, KCNJ2, and SCN5A cDNA. The ionic composition of the external solution for recording the I_Kr, I_Ks, and I_Na channel current was as follows (in mM): 143 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 5 HEPES, 0.33 NaH₂PO₃, and 16.6 glucose (pH adjusted to 7.4 with NaOH). The internal (pipette) solution for I_Kr contained the following (in mM): 130 KCl, 5 EGTA, 10 HEPES, 1 MgCl₂, 5 Mg-ATP (pH adjusted 7.25 with KOH), and for I_Ks in the KCNQ1/KCNE1-cotransfected HEK293 cells, contained the following (in mM): 150 KCl, 5 EGTA, 10 HEPES, 2 MgCl₂, 1 CaCl₂, and 5 Na₂-ATP (pH adjusted 7.25 with KOH). The internal (pipette) solution for I_K1 in KCNJ2-transfected HEK293 cells contained (in mM): 130 K-Asp, 15 KCl, 10 HEPES, 1 MgCl₂, 5 Na₂-ATP, 5 EGTA (pH adjusted 7.25 with KOH), and for sodium current in SCN5A-transfected HEK293 cells contained (in mM): 105 CsF, 35 NaCl, 10 EGTA, 10 HEPES (pH adjusted to 7.25 with NaOH). Calcium current was measured in native rat ventricular myocytes, cells were superperfused with an external solution that consisted of (in mM): 137 cholin-Cl, 5 CsCl, 0.5 MgCl₂, 2 4-AP, 10 HEPES, 10 glucose, 1.8 CaCl₂ (pH adjusted to 7.4 with NaOH), whereas the solution used to fill the pipette had the following ionic solution (in mM): 20 CsCl, 100 Cs-aspartate, 10 EGTA, 10 HEPES, 20 TEA-Cl, 5 Mg-ATP (pH adjusted to 7.25 with KOH). I_K1, I_Na, I_Ca, I_Kr, and I_Ks were measured in terms of peak current amplitude. For the hERG tail current, cells were depolarized for 2 s to +20 mV from a holding potential of −80 mV followed by a 3-s repolarization back to −40 mV. Peak inward I_Na was generated by pulses of 20-ms duration to −40 mV from a holding potential of −100 mV delivered at 10-Hz frequency. The peak of I_Ca was induced by a single 500-ms voltage pulse to 0 mV from the holding potential of −80 mV. I_K1 current elicited by the voltage of a 1-step pulse (during 1 s) from −80 mV to −120 mV. For the I_Ks recording, cells were depolarized for 3 s to +60 mV from a holding potential of −80 mV followed by a 3-s repolarization back to −40 mV.

Statistical Methods

Action potential analysis was carried out by comparing the differences between the drug-treated groups and the control group using GraphPad InStat (version 3.05; GraphPad Software Inc, La Jolla, CA). Dunnett’s multiple comparison test was performed and data were considered to be significant when P < .05 or P < .01. With respect to current amplitudes, they were measured before and after application of the respective compound. The relative remaining currents were calculated according to the following equation: Initial current amplitude/Current amplitude in the presence of compound = Relative remaining current.

Results

Effect on Action Potentials

In the first series of experiments, the effect of sulpiride (10, 100, and 300 μM, and 1 mM) was studied on the action potential duration in dog Purkinje fibers. As shown in Figure 1, sulpiride prolonged action potential duration in a concentration-dependent manner. Sulpiride at 100 μM caused significant prolongation of the action potential duration at 90% (APD₉₀) (P < .05) but not 50% (APD₅₀). And 300 μM and 1 mM of sulpiride caused a greater prolongation of the APD₅₀ and APD₉₀ (P < .01) than 100 μM of sulpiride. These effects were statistically significant at the higher concentrations (above 100 μM for APD₉₀ and 300 μM for APD₅₀) than the plasma concentrations (0.395 ± 0.083 μg/mL) in multiple-dose therapy for 4 weeks in the elderly. ⁶ Also the TA of the action potential was significantly decreased by 1 mM sulpiride but the RMP or the V_max were not changed by all of the concentrations tested up to 1 mM (Table 1).

Effect on Cardiac Ionic Currents

Furthermore, we tested various concentrations of sulpiride (10 μM, 100 μM, 300 μM, and 1 mM) on cardiac ion channels. As shown in Figure 2, sulpiride dose-dependently decreased the hERG tail current, such that 10 μM, 100 μM, 300 μM, and 1 mM caused a decrease of the hERG current by 9.4%, 16.9%, 31.2%, and 47.8%, respectively. Maximum inhibition (47.8 ± 4.3%) of the hERG tail current was obtained at the 1 mM concentration within 7 min after exposure to sulpiride. These data indicate that the inhibition of hERG currents by sulpiride occurred in a dose- and time-dependent manner, and this concentration is higher than the therapeutic plasma concentration. ⁷ Although, as seen in Figure 3, the other channel currents (I_Na, I_Ca, I_Ks, and I_K1) except for I_Kr were not significantly modified by 1 mM sulpiride, these currents were instead blocked by specific blockers. We used primaquine, nicardipine, 4-AP, and BaCl₂ as a I_Na, I_Ca, I_Ks, and I_K1 channel blocker. ^8–11 Therefore, it is considered that the prolongation of the action potential duration by sulpiride was mainly the result of inhibition of the hERG channel. Table 2 shows the effect of 1 mM sulpiride on the mean current amplitudes.

Discussion

QT interval prolongation may be induced by disorder of the ventricular cardiac action potentials because of the effect of the active metabolites of drugs, protein interactions, or drug overdose. In particular, blockade of the hERG channel is known to be a major cause of cardiac arrhythmia and sudden death. It is generally accepted that most hERG channel blockers with high potency inhibit I_Kr in the nanomolar range or under, 30-fold greater than the IC₅₀ for the plasma concentration, ^12,13 but there is no agreement on the absolute standard for clinically important hERG channel blockade. The potential effect of QT interval prolongation of sulpiride has been reported in clinical cases and a chronic AV block model. ^3,4 Therefore, we tested the action potential duration in canine Purkinje fibers and blockade with various cardiac ion channels to check the effects through an electrophysiological safety test. Judging from the data obtained, it is considered that sulpiride is safe at the therapeutic plasma concentrations in clinical use. However, particular caution may be necessary in patients who have already presented with acquired long QT syndrome. There is growing evidence that patients who present with acquired long QT syndromes may have overt or cryptic genetic risk factors related to SNPs and mutations. ¹⁴ Thus, genetic background may be associated with the high susceptibility for the acquired long QT syndrome induced by high hERG affinity inhibitors. Therefore, in terms of clinical experience, the risk of an overdose of sulpiride should be avoided in patients at risk for torsade de pointes; the safety of high doses has been demonstrated in clinical studies. In conclusion, to the best of our knowledge, this is the first report on the dose-dependent effects of sulpiride on action potentials and cardiac ion channels. Our data indicate that the clinical use of sulpiride is reasonable within therapeutic plasma concentrations, but all patients taking this drug should be closely and carefully monitored for clinical signs of long-QT syndrome and severe arrhythmias.