Introduction

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Malaria remains one of the most important and widespread diseases in the world. Chloroquine is one of the drugs of first choice for treatment of malaria. Chloroquine is also used as an anti-inflammatory agent in rheumatoid arthritis and in lupus erythematosus (Webster, 1992). However, the use of chloroquine has been associated with toxic cardiovascular effects, including a fall in blood pressure (Olatunde, 1970) and rhythm abnormalities (Williams, 1966; Guedira et al., 1998). Prolonged therapy can lead to cardiac failure (Hughes et al., 1971) and electrocardiographic changes, including T-wave depression or inversion, and prolonged QRS and QTc intervals (Sanghvi and Mathur, 1965; Bustos et al., 1994). Chloroquine has also been reported to induce torsade de pointes (Harris et al., 1988; Fauchier et al., 1993), a tachyarrhythmia associated with medications that block repolarizing cardiac K⁺ currents. Acute poisoning by chloroquine can cause death by failure of myocardial contraction and cardiac arrest (Don-Michael and Aiwazzadeh, 1970).

The proarrhythmic effects of chloroquine are well documented; however, there are only two published studies describing the cellular electrophysiological effects of this drug. Harris et al. (1988) used microelectrode techniques and multicellular preparations (sheep ventricular muscle and Purkinje fibers) to demonstrate that 5 to 50 µM chloroquine produced significant reduction in the maximum upstroke velocity (V_max) of the action potential, an indirect measure of peak sodium channel conductance. Harris et al. (1988) also demonstrated that chloroquine prolonged action potential duration and refractory period, effects usually attributed to block of K⁺ currents. Recently, Benavides-Haro and Sanchez-Chapula (2000) demonstrated that chloroquine blocked inward rectifier current (I_K1) and the acetylcholine K⁺ current, I_K(Ach), in guinea pig atrial and ventricular myocytes.

A slowing in ventricular conduction and an excessive lengthening in the QT interval have been proposed as the mechanisms of the proarrhythmic effects of chloroquine (Harris et al., 1988; Bustos et al., 1994; Guedira et al., 1998). Blockade of inward sodium current (I_Na) has been suggested as the principal cause of impaired ventricular conduction, and acquired long QT syndrome is usually caused by blockade of one or more potassium currents (Nattel, 1998). In the present study, we investigated the effects of chloroquine on action potentials and the major ionic currents contributing to the shape of the action potential in isolated feline ventricular myocytes. Chloroquine lengthened action potentials of cat Purkinje fibers, and increased automaticity. Standard voltage-clamp techniques were used to record I_Na and L-type calcium current (I_Ca-L), and four potassium currents, including the I_K1, the transient outward current (I_to), and the rapid and slow delayed rectifier outward currents (I_Kr and I_Ks). Chloroquine blocked four of these currents: I_K1, I_Kr, I_Na, and I_Ca-L. These findings provide the cellular mechanism for the prolonged action potentials and reduction in V_max of cardiac action potentials previously reported by Harris et al. (1988), and insights into the mechanism of induction of arrhythmias.

	Materials and Methods

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Standard Microelectrode Technique. Adult cats (2-4 kg) were anesthetized with sodium pentobarbital (35 mg/kg) and heparinized (1000 U/kg). Free-running Purkinje strands were obtained from the left ventricle of the cat hearts. The Purkinje strands were fixed to the Sylgard (Dow Corning Co., Midland, MI)-coated bottom of a Plexiglas chamber (2-ml volume) with micropins. The preparations were superfused with a solution containing 125 mM NaCl, 24 mM NaHCO_3, 0.43 mM NaH₂PO₄, 4 mM KCl, 1.8 mM CaCl₂, 1.05 mM MgCl₂, and 11 mM glucose. The solution was equilibrated with 95% O₂, 5% CO₂ (pH 7.4). Temperature was kept constant at 35°C. The preparations were allowed to equilibrate for 60 min before experimental protocols were performed. During this time the preparations were stimulated at a frequency of 1 Hz with rectangular stimuli (3-ms duration, 1.5 times diastolic threshold intensity) delivered by insulated (except at the tips) silver bipolar electrodes. Action potentials were recorded by using glass microelectrodes filled with 3 M KCl (resistance 10-15 M) coupled to the input of a high-impedance preamplifier (World Precision Instruments, New Haven, CT). Action potential signals were digitized at a sampling rate of 10 kHz by use of an analog-to-digital converter (Digidata 1200 interface; Axon Instruments, Burlingame, CA) and stored on a hard disk, Axotape data-acquisition software (Axon Instruments), and a 486DX2 computer. Data analysis was performed using pClamp software (version 6.0.4; Axon Instruments). We have performed experiments recording action potentials in Purkinje fibers for up to 8 h under control conditions. We have not observed significant changes in action potential parameters or spontaneous firing frequency.

Cell Preparation. Single ventricular myocytes were obtained from the right ventricular free wall of adult cats as previously described (Sanchez-Chapula, 1996). The hearts were mounted on a Langendorff apparatus and perfused for 5 min with normal Tyrode's solution, and then switched to a nominally calcium-free solution for an additional 5 min. Afterward, the hearts were perfused for 30 min with a zero-calcium solution containing 1 mg/ml type I collagenase (Sigma Chemical Co., St. Louis, MO) and 0.05 mg/ml protease XIV (Sigma Chemical Co.). The enzymes were washed out by perfusion with a high-potassium, low-chloride saline (KB medium; Isenberg and Klöckner, 1982) for 5 min. The free wall of the right ventricle was dissected away from the rest of the heart and cut into small pieces. Single cells were maintained in a high-potassium, low-chloride solution at 4°C for up to 10 h before use in electrophysiological experiments.

Electrical Recordings. A few drops of the cell suspension were placed in a chamber (0.5-ml volume) mounted on a modified stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan). The chamber was superfused at a rate of 0.5 ml/min with normal external solution. Action potential experiments in isolated ventricular myocytes were performed at 35°C, using the whole-cell "perforated patch" current clamp technique. Sodium, calcium, and potassium currents were recorded using the whole-cell standard patch-clamp method (Hamill et al., 1981) and an Axopatch 1C patch-clamp amplifier (Axon Instruments). A Labmaster-TL/1 interface (Axon Instruments) controlled by pClamp 6.0.3 software (Axon Instruments) was used to generate voltage-clamp command protocols and acquire data. Currents were filtered at 2 kHz with a four-pole Bessel filter, digitally sampled at 4 kHz and stored on the hard disk of an Epson 486Dx/33 computer. I_K currents were recorded at a sampling frequency of 2 KHz and filtered at 1 KHz. Micropipettes were pulled from borosilicate glass capillary tubes (TW 150-6; World Precision Instruments, Inc., Sarasota, FL) on a programmable horizontal puller (Sutter Instruments, Novato, CA). When filled with the intracellular solution, the pipette tip resistance was 1 to 2 M. Series resistance compensation was set to 80%, and whole-cell capacitance compensation was optimized to minimize capacitive currents and reduce voltage errors. We only analyzed experiments in which access resistance was 1.2 M after compensation. The experiments to determine the effect of chloroquine on I_Na were performed at a temperature of 15°C. Recordings of calcium and potassium currents were performed at 35°C.

Solutions. Tyrode's solution had the following composition: 125 mM NaCl, 24 mM NaHCO₃, 0.42 mM NaH₂PO₄, 5.4 mM KCl, 1.8 mM CaCl₂, 1.05 mM MgCl₂, 11 mM glucose, and 10 mM taurine. The solution was equilibrated with 95% O₂,5% CO₂, pH 7.4. Nominally, calcium-free solution was prepared by omitting CaCl₂ from the Tyrode's solution. The high-potassium, low-chloride solution (KB medium) had the following composition: 80 mM potassium glutamate, 50 mM KCl, 20 mM taurine, 3 mM KH₂PO₄, 10 mM glucose, 10 mM HEPES, and 0.2 mM EGTA. The pH was adjusted to 7.4 with KOH.

Statistics. Data are expressed as means ± S.E.M. Statistical significance was evaluated by ANOVA and Dunnett's t test. Differences were considered significant at P < 0.05.

	Results

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Effects of Chloroquine on Action Potentials. Chloroquine increased action potential duration and decreased V_max) in a concentration-dependent (0.3-10 µM) manner in cat Purkinje fibers stimulated at a basic cycle length (BCL) of 1000 ms (Fig. 1A; Table 1). In addition, at concentrations of 3 and 10 µM, chloroquine decreased the maximum diastolic potential. Action potentials in ventricular myocytes were elicited by 5-ms pulses applied at a basic cycle length of 1000 ms, using the whole-cell perforated patch-clamp technique. Chloroquine at concentrations 1 to 10 µM increased action potential duration (APD) in a concentration-dependent manner (Fig. 1B; Table 1). At concentrations 3 and 10 µM, it decreased resting membrane potential, action potential peak, and plateau amplitudes (Fig. 1B; Table 1).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Effect of chloroquine on action potential. A, action potentials recorded from a Purkinje fiber during stimulation at 1 Hz, using standard microelectrode techniques, under control conditions and in the presence of chloroquine 1 and 10 µM. B, effect of chloroquine 1 and 10 µM on a ventricular myocyte action potential recorded during stimulation at 1 Hz, using the perforated patch technique.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of chloroquine on automaticity in cat Purkinje fibers. Spontaneous action potential firing under control conditions and in the presence of chloroquine (1-10 µM).

Effects of Chloroquine on I_Na. To improve voltage control, the experiments were performed at 15°C using a low-sodium external solution (under Materials and Methods). In patch-clamp experiments, the voltage dependence of I_Na activation and inactivation slowly drifts toward more negative potentials during the first 10 min after rupture of the membrane patch (Kimitsuki et al., 1990). In the present study, control I_Na recordings were initiated at least 15 min after the rupture of the membrane patch. To test the effects of chloroquine (0.3-10 µM) on I_Na, 40-ms depolarizing pulses were applied from a holding potential of 120 mV to membrane potentials ranging from 80 to +10 mV at a frequency of 0.1 Hz. Only a single concentration of chloroquine was tested in each myocyte. Chloroquine decreased peak current amplitude at all potentials studied (Fig. 3A). The drug did not change the threshold potential, the potential at which peak I_Na was maximum or the apparent reversal potential (Fig. 3B). The percentage block of I_Na peak amplitude was concentration-dependent. The concentration-dependent effect of the drug on peak current, measured at 40 mV, is shown in Table 2. The effects of chloroquine on I_Na at concentrations of 0.3 to 3 µM were completely reversible after washout, whereas at 10 µM the reversibility was about 80% (data not shown). A possible additional effect of chloroquine on I_Na at a stimulation frequency (1 Hz) closer to the physiological range was studied by applying trains of 20-ms pulses from a holding potential of 120 mV to a test potential of 20 mV. No significant use-dependent effects were observed (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of chloroquine on INa in cat ventricular myocytes. From a holding potential of 120 mV, test pulses to different membrane potentials were applied at a frequency of 0.1 Hz. A, current traces induced by test pulses to membrane potentials ranging from 70 to +10 mV, obtained under control conditions. B, current traces obtained in the presence of chloroquine 10 µM. C, I-V relationships for the peak current amplitude recorded under control conditions, and in the presence of chloroquine, mean ± S.E. of n = 5 cells are shown.

View this table: [in this window] [in a new window]   TABLE 2 Concentration-dependent effects of chloroquine on different ionic currents Numbers refer to percentage of block. INa was measured at 40 mV, holding potential (HP) = 120 mV; ICa-L was measured at +10 mV, HP = 70 mV; IK1 was measured at the HP of 60 mV; Ito was measured at +50 mV, HP = 60 mV; IK(tail) was measured at 40 mV following an activating pulse to +50 mV; IKs(tail) was measured at 40 mV following an activating pulse to +50 mV.

Effects of Chloroquine on I_Ca-L The experiments on calcium and potassium currents were performed at 35°C. The effect of chloroquine on I_Ca-L was studied by applying depolarizing pulses to membrane potentials ranging from 40 to +40 mV from a holding potential of 70 mV. Pulses were applied at a frequency of 0.1 Hz. Chloroquine at 10 µM decreased the peak amplitude of I_Ca-L measured at +10 mV by 32 ± 11% (n = 5) (Fig. 4). The drug did not alter the shape of the I_Ca-L-V relationship (Fig. 4). The effect of chloroquine on I_Ca-L at a test potential of +10 mV was concentration-dependent (Table 2). Each myocyte was treated with a single concentration of chloroquine. The effects of chloroquine at all concentrations used were partially reversible on washout, about 60% after 1 and 3 µM and about 40% after 10 µM (data not shown). Possible use-dependent block of I_Ca-L by chloroquine was studied using a 1-Hz train of 200-ms pulses to +10 mV, applied from a holding potential of 70 mV. No additional significant use-dependent effects were observed (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of chloroquine on ICa-L in cat ventricular myocytes. From a holding potential of 70 mV, test pulses to membrane potential ranging from 30 to +50 mV were applied at a frequency of 0.1 Hz. A, current traces elicited by pulses to 20, 10, 0, and +10 mV, obtained under control conditions and in the presence of chloroquine 10 µM. B, I-V relationships for the peak current amplitude, under control conditions and in the presence of chloroquine, mean ± S.E. of n = 5 cells are shown.

Effects of Chloroquine on Potassium Currents. I_K1 was elicited with hyperpolarizing and depolarizing pulses of 500-ms duration to membrane potentials ranging from 100 to 40 mV from a holding potential of 60 mV. Figure 5A shows current traces obtained under control conditions and in the presence of 3 µM chloroquine. The effect of chloroquine on I_K1 measured at the end of the pulses is shown in Fig. 5B. Chloroquine at 3 µM significantly decreased current amplitude in a voltage-dependent manner. I_K1 measured at 40 mV was decreased by 94%, at 60 mV by 86%, and at 100 mV by 67%. In addition, during hyperpolarizing pulses to membrane potentials of 90 and 100 mV, a time-dependent unblock was observed (Fig. 5A, bottom). Table 2 shows the concentration-dependent block by chloroquine. The effects of chloroquine on I_K1 at all concentrations used were completely reversible on washout (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of chloroquine on IK1 in cat ventricular myocytes. From a holding potential of 60 mV, test pulses to membrane potentials ranging from 100 to 40 mV were applied at a frequency of 0.1 Hz were applied. A, current traces elicited by test pulses to 100, 90, 80, 60, and 40 mV, obtained under control conditions and in the presence of chloroquine 3 µM. B, I-V relationships for the current measured at the end of the pulses, under control conditions and in the presence of chloroquine, mean ± S.E. of n = 7 cells are shown.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of chloroquine on Ito in cat ventricular myocytes. From a holding potential of 60 mV, test pulses to membrane potentials ranging from 30 to +50 mV were applied at a frequency of 0.1 Hz. A, current traces elicited by pulses to 30, 10, +10, +30, and +50 mV, obtained under control conditions and in the presence of chloroquine 10 µM. B, I-V relationships for the peak current amplitude, under control conditions and in the presence of chloroquine, mean ± S.E. of n = 5 cells are shown.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of chloroquine on IK of cat ventricular myocytes. From a holding potential of 40 mV, test pulses to membrane potentials ranging from 30 to +50 mV were applied at a frequency of 0.05 Hz, tail current was measured upon repolarization to the holding potential of 40 mV. A, current traces elicited by test pulses to 30, 10, +10, +30, and +50 mV, obtained under control conditions and in the presence of chloroquine 3 µM. B, I-V relationships for the time-dependent current activated during the depolarizing pulses, under control conditions and in the presence of chloroquine, mean ± S.E. of n = 7 cells are shown. C, I-V relationships for the tail current amplitude, under control conditions and in the presence of chloroquine, mean ± S.E. of n = 7 cells are shown.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Chloroquine (3 µM)-sensitive time-dependent current. A, current traces of the chloroquine-sensitive current (control current minus current recorded in the presence of chloroquine, digitally subtracted), obtained at test potentials of 30, 10, +10, and +30 mV. B, I-V relationship of the chloroquine-sensitive, time-dependent current amplitude. C, I-V relationship of the chloroquine-sensitive, tail current amplitude. Chloroquine-sensitive current was obtained by digital subtraction of currents recorded in the presence of chloroquine, from control records. Mean ± S.E. of n = 7 cells are shown.

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Effects of chloroquine on IKs of cat ventricular myocytes. From a holding potential of 40 mV, test pulses to membrane potentials ranging from 30 to +50 mV were applied at a frequency of 0.05 Hz, tail current were recorded upon repolarization to the holding potential of 40 mV. A, current traces elicited by test pulses to 30, 10, +10, +30, and +50 mV, in the presence of MK-489 3 µM and in the presence of MK-499 3 µM + chloroquine 10 µM. B, I-V relationships for the time-dependent current activated during the test depolarizing pulses, in the presence of MK-489 alone and in the presence of MK-489 plus chloroquine, mean ± S.E. of n = 4 cells are shown. C, I-V relationships for the tail current amplitude, in the presence of MK-489 alone and in the presence of MK-489 plus chloroquine, mean ± S.E. of n = 4 cells are shown.

	Discussion

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We found that chloroquine (0.3-10 µM) induced a decrease of V_max, prolonged APD, and decreased maximum diastolic potential in cat isolated Purkinje fibers and ventricular myocytes. Chloroquine also increased the firing frequency of spontaneous activity in cat Purkinje fibers. In addition, after 60 min of superfusion with a concentration 10 µM, in four of five cat Purkinje fibers spontaneous firing was abolished. In experiments under voltage-clamp conditions, chloroquine inhibited I_K1 > I_Kr > I_Na > I_Ca-L. The transient outward potassium current, I_to, and the slow component of I_K were not modified by chloroquine. The blocking effects of chloroquine on I_K1 were significantly voltage-dependent, the block increased with depolarization and decreased with hyperpolarization. This profile of voltage dependence is consistent with a positively charged molecule blocking the channel from the intracellular side and entering the pore to such an extent as to be subjected to the transmembrane electrical field (Snyders et al., 1992; Benavides-Haro and Sanchez-Chapula, 2000).

Chloroquine administered at therapeutic concentrations reaches plasma concentrations of 0.29 to 0.48 µM (Webster, 1992). In a retrospective study of patients with acute chloroquine overdose, the mean amount ingested by 167 patients was 4.5 ± 2.8 g. The mean blood chloroquine concentration on admission was 20.5 ± 13.4 µM (Clemessy et al., 1996). Riou et al. (1988) found in severe chloroquine poisoning, blood levels ranging from 40 to 80 µM. Therefore, the concentrations used in the present study are clinically relevant. Chloroquine, administered at therapeutic concentrations, has been found to produce different cardiovascular effects such as fall in blood pressure, slowing of ventricular conduction, and electrocardiographic changes such as lengthening of QRS and QT intervals (Bustos et al., 1994). Acute poisoning with chloroquine has been reported to produce cardiac failure, rhythm disturbances, atrioventricular block (Guedira et al., 1994), impaired intraventricular conduction, and cardiac arrest (Taboulet and Bismuth, 1994).

To investigate the possible cellular electrophysiological mechanisms responsible of the slowing in ventricular conduction, electrocardiographic changes, and rhythm disturbances, the effects of chloroquine on action potential and the main ionic currents underlying the ventricular action potential were studied. Using microelectrode techniques in multicellular preparations, it was reported that chloroquine depresses the action potential V_max, suggesting that the drug inhibits I_Na (Harris et al., 1988). In the present work, we confirmed this assumption by showing that chloroquine at concentrations of 1 µM and higher inhibited I_Na. Class I antiarrhythmic drugs can be proarrhythmic due to facilitation of re-entrant arrhythmias caused by excessive slowing of conduction (Winkle et al., 1981; Rinkenberger et al., 1982; Morganroth and Horowitz, 1984). These proarrhythmic effects are more frequent in association with coronary artery disease (Morganroth, 1987). The excessive slowing of conduction induced by chloroquine due to inhibition of I_Na can be worsened by the decrease of the diastolic membrane potential, which can increase the fraction of inactivated sodium channels.

Chloroquine has also been found to produce hypotension, an effect attributed to depression of cardiac contractility on the basis of the observation that systolic blood pressure fell before diastolic pressure (Olatunde, 1970). Chloroquine in dogs produces significant reductions in cardiac contractility and vascular resistance (Sofola, 1980). In the present work, we found that chloroquine at concentrations of 1 µM and higher inhibited I_Ca-L. This effect can at least partially explain the depression in cardiac contractility induced by the drug. In addition, during acute chloroquine poisoning, third degree atrioventricular block (Verny et al., 1992; Guedira et al., 1998) and cardiac arrest (Clemessy et al., 1996) have been reported. These effects may also be explained by the inhibition of I_Ca-L and I_Na.

In addition to the impaired intraventricular conduction and the excessive lengthening in QT interval as a possible mechanism responsible for the proarrhythmic effects of chloroquine, the increase in automaticity induced by the drug can also be a potential cause of arrhythmias. The decrease in diastolic membrane potential and increase in automaticity induced by chloroquine can be explained by its blocking effects on I_K1. During phase 4 of the Purkinje fibers action potential, slow depolarization results from activation of the pacemaker current (I_f) (DiFrancesco and Noble, 1985). Because the maximum diastolic potential is near 90 mV, the decay of the delayed rectifying outward current contributes only a very small current during phase 4. On the other hand, I_K1 carries a very significant outward current during the pacemaker depolarization, which largely balances the inward current carried by I_f channels. For this reason, factors that change I_K1 have a large effect on Purkinje fiber rhythm and the maximum diastolic potential (Noble, 1995). However, a direct effect of chloroquine on I_f or electrogenic exchangers and pumps such as Na⁺/K⁺ pump cannot be discounted.

The most frequent cardiovascular manifestations during chloroquine treatment are the electrocardiographic changes, diminution of the T wave, and prolongation of the QTc interval (Bustos et al., 1994; Bouree, 1997). These effects can be explained by the action potential duration increase induced by the drug. Clinical and animal data support the hypothesis that acquired forms of long QT syndrome result from prolonged repolarization that leads to early afterdepolarizations and triggered arrhythmias (Surawicz, 1989). Early afterdepolarizations can be induced by block of potassium currents such as I_K1 or I_Kr (Kaseda et al., 1989), by activation of L-type Ca²⁺ current (January and Riddle, 1989), or inhibition of Na⁺ current inactivation (Boutjdir and El-Sherif, 1991). The most common mechanism of drug-induced torsades de pointes is I_Kr inhibition. In our experiments, chloroquine did not induce early afterdepolarizations; however, under voltage-clamp conditions, the most prominent effect of chloroquine on cardiac membrane currents was a marked reduction of I_K1 and I_Kr. The marked reduction of both I_K1 and I_Kr can explain the prolongation of action potential duration induced by the drug (Harris et al., 1988). On the other hand, the reduction of I_Na and I_Ca-L induced by chloroquine may limit the prolongation of action potential duration and the appearance of afterdepolarizations.

In conclusion, we have found that the antimalarial drug chloroquine at clinically relevant concentrations inhibited several currents in cat ventricular myocytes. These effects can explain most of the electrophysiological modifications and proarrhythmic effects reported for chloroquine in mammalian cardiac preparations.