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CARDIOVASCULAR

Multiple Cellular Electrophysiological Effects of a Novel Antiarrhythmic Furoquinoline Derivative HA-7 [N-Benzyl-7-methoxy-2,3,4,9-tetrahydrofuro[2,3-b]quinoline-3,4-dione] in Guinea Pig Cardiac Preparations

Graduate Institute of Clinical Medicinal Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (G.-J.C., Y.-S.L.); Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan (M.-J.S.); Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan (S.-C.K., T.-P.L.); and the First Cardiovascular Division of Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan (Y.-S.L.)

Received for publication July 6, 2005
Accepted September 15, 2005.

	Abstract

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We studied the electrophysiological and antiarrhythmic actions of HA-7 [N-benzyl-7-methoxy-2,3,4,9-tetrahydrofuro[2,3-b]quinoline-3,4-dione], a furoquinoline alkaloid derivative, in guinea pig heart preparations. In the perfused whole heart model, HA-7 caused a prolongation in the basic cycle length, ventricular repolarization time, and the atrioventricular (AV) nodal Wenckebach cycle length and prolonged the refractory period of the atrium, AV node, and His-Purkinje system. The atrioventricular conduction interval was also prolonged in a frequency-dependent manner. In isolated hearts, HA-7 significantly raised the threshold for experimental atrial fibrillation and reduced the occurrence of reperfusion-induced ventricular fibrillation. Conventional microelectrode-recording study shows that HA-7, but not d-sotalol, prolonged the action potential duration (APD) and decreased the maximum rate of depolarization in isolated atrial strips. In ventricular papillary muscles, higher concentrations of HA-7 caused a prolongation of APD₉₀ in a frequency-independent manner, whereas d-sotalol exerted a reverse frequency-dependent action on this parameter. Whole-cell patch clamp results on ventricular myocytes indicate that HA-7 decreased both the slow (I_Ks) (IC₅₀ = 4.8 µM) and fast component (I_Kr) (IC₅₀ = 1.1 µM) of the delayed rectifier K⁺ currents. Similar results could also be observed in atrial myocytes. The inward rectifier K⁺ current (I_K1) was also reduced somewhat by HA-7. HA-7 also suppressed the Na⁺ inward current (I_Na) (IC₅₀ = 2.9 µM) and inhibited the L-type Ca²⁺ current (I_Ca) (IC₅₀ = 4.0 µM, maximal inhibition = 69%) to a lesser extent. We conclude that HA-7 blocks multiple ionic currents and that these changes affect the electrophysiological properties of the conduction system as well as the myocardial tissues and may contribute to its antiarrhythmic efficacy.

HA-7 is a novel tetrahydrofuroquinoline alkaloid derivative that has been synthesized recently. We previously demonstrated that HA-7 could reverse the cardiac arrhythmias induced by postischemic reperfusion in isolated rat hearts and produce a moderate positive inotropic effect in rat cardiac tissues (Su et al., 1997). In the same study, it was shown that this antiarrhythmic activity may be related to the predominant blockade of the transient outward K⁺ current (I_to), a major repolarizing current in rat heart (Josephson et al., 1984), steady-state outward K⁺ current (I_SS), and Na⁺ channels. Thus, HA-7 may exert mixed class I and stronger class III antiarrhythmic properties (Su et al., 1997). In fact, most of the class III antiarrhythmic agents, such as sotalol, dofetilide, or amiodarone, are known to prolong cardiac APD and suppress re-entrant arrhythmia primarily via the blockade of the I_K, which is absent in rat cardiomyocytes but exists prominently as the main repolarizing current in guinea pig (Hume and Uehara, 1985) and human myocardium (Li et al., 1996). Therefore, the present study was conducted to examine whether HA-7 could also exert antiarrhythmic, electromechanical, and ion channel-modifying actions in guinea pig heart preparations and to compare its effects with those of a typical class III agent d-sotalol (Hohnloser and Woosley, 1994). In this study, the antiarrhythmic effect of HA-7 was evaluated on both the electrical stimulation-induced atrial fibrillation and the reperfusion-induced ventricular tachyarrhythmias in Langendorff perfused guinea pig hearts. The electrophysiological activity of HA-7 on the conduction system was assessed in perfused isolated hearts, whereas the mechanical and electrophysiological effects of HA-7 were assessed in guinea pig atrial strips and ventricular papillary muscles. Further investigations on ionic currents were also performed using the patch-clamp technique in isolated guinea pig cardiomyocytes.

	Materials and Methods

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All experiments were performed in accordance with the internationally accepted guidelines. Studies were approved by the Institute Committee of the Animal Care of the University of Chang Gung College of Medicine. Guinea pigs were housed in the animal care facility at the Chang Gung Memorial Hospital (Tao-Yuan, Taiwan). All animals were housed with a 12-h light/dark cycle. Food and water were available ad libitum.

Intracardiac Electrocardiogram-Recording Experiment
Animal Preparation. Adult male Hartly guinea pigs (250-300 g body weight, purchased from the Laboratory Animal Center of National Taiwan University Hospital) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and given heparin (300 units/kg, i.p.). Following anesthesia, the heart was excised via thoracotomy and the ascending aorta was retrogradely perfused at a rate of 4 ml/min/g cardiac tissue with normal Tyrode's solution (Chang et al., 2002). The solution was continuously gassed with 95% O₂ and 5% CO₂ to give a pH of 7.4 and was maintained at 37°C. To pace the atria, a high right atrial electrode was placed near the junction of the superior vena cava and right atrium. A bipolar electrode consisting of a tungsten spring-soldered silver wire was placed on the endocardium near the apex of the triangle of Koch to record the His bundle electrograms. In the next stage, the tips of the ventricular recording electrode were placed on opposite sides of the epicardium near the ventricular apex, thereby providing a recognizable T wave. In addition, a ventricular pacing electrode was placed on the pericardium near the right ventricular apex. Pacing studies were performed by utilizing a programmable stimulator (DTU 215; Bloom Electrophysiology, Fischer Imaging, Denver, CO). A pacing stimulus of 1 ms in duration and twice the diastolic threshold voltage was applied to the preparation through the bipolar atrial or ventricular electrodes. The electrograms were continuously monitored on an oscilloscope (TDS520D; Tektronix, Beaverton, OR), and pertinent data were recorded on a chart recorder (WindowGraf; Gould Instrument Systems Inc., Cleveland, OH).

Experimental Protocol. An average of four stable cycle lengths of spontaneous heartbeats was taken as the basic cycle length. The right atrium was then paced at a constant rate that was slightly faster than the spontaneous heart rate. At this constant pacing rate, the intra-atrial (sinoatrial conduction interval), AV nodal (AH), and His-Purkinje (His-ventricular conduction interval) conduction times as well as the ventricular repolarization time (VRT) were measured. Incremental right atrial pacing was used to determine the Wenckebach cycle length at which the 1:1 AV nodal conduction pattern was lost. Atrial premature extrastimulation (S₂) was then delivered to the high right atrium after a train of constant rate atrial pacing (S₁S₁) for eight beats to obtain the refractory periods of the atria, AV node, and His-Purkinje system. Ventricular effective refractory period was similarly determined using a ventricular extrastimulation study protocol.

Evaluation of Antiarrhythmic Properties
Global Ischemia/Reperfusion-Induced Arrhythmias. The isolated guinea pig heart was retrogradely perfused through the aorta with normal Tyrode's solution at a constant perfusion pressure. A PE60 cannula attached to a side arm of the constant-pressure Langendorff perfusion apparatus was placed in the right atrium. Following a 10-min equilibration period, hearts were administered drug or vehicle for 10 min. After this preischemic period, the aortic cannula was clamped to terminate aortic flow and to institute global no-flow ischemia. Simultaneously with ischemia onset, the right atrial cannula was opened to enable intracavitary superfusion. The rate of atrial superfusion flow was controlled at 6 ml/min to maintain a heart rate of 200 beats/min. The electrograms were recorded from a low atrial and a ventricular recording electrode and were continuously monitored on an oscilloscope, and pertinent data were recorded on a chart recorder. After 30 min, the aortic cannula was unclamped to permit reperfusion, whereas the right atrial superfusion cannula was clamped and the duration and incidence of arrhythmias was recorded. Vehicle or drug was included in the perfusion solution throughout the time after the preischemic period.

Measurement of Atrial Fibrillation Threshold. Previous studies have shown that atrial vulnerability is enhanced by adenosine, which causes shortening of APD and refractoriness (Kabell et al., 1994; for a review, see van der Hooft et al., 2004). We induced AF with a combination of adenosine and electrical stimulation in Langendorff perfused guinea pig hearts. The fibrillating current was generated from a programmable stimulator (DTU 215) and consisted of a train of 50-square wave pulses of 2-ms duration at a frequency of 50 Hz for 1 s. The pulse train was delivered to the right atrium after every eighth basic paced beat. The current was increased gradually from an intensity twice the diastolic threshold. Atrial fibrillation threshold (AFT) was defined as the minimum amount of current required to induce AF, which was sustained for at least 30 s. To avoid damaging the atrial tissue, we did not deliver a current greater than 14 mA. If AF could not be induced when the current was increased to 14 mA, the AFT was provisionally determined to be 14.1 mA.

Electromechanical Recordings. Action potentials were recorded from guinea pig cardiac muscle preparations with a conventional intracellular recording technique (Chang et al., 2002). In brief, the left atrial strip or ventricular papillary muscle was placed in a tissue chamber and perfused at a rate of 20 ml/min with normal Tyrode's solution. The perfusing solution was oxygenated (95% O₂ and 5% CO₂) and kept at 37°C with a temperature controller. External stimuli (1-ms duration, 1.5x the diastolic threshold voltage) were delivered at a basic rate of 1 Hz by an electronic stimulator (S88; Grass Instrument Division, Astro-Med, Inc., West Warwick, RI) via bipolar platinum electrodes. Each preparation was stretched to a length at which maximum-developed force was evoked and allowed to equilibrate for at least 1.5 to 2 h before the commencement of the experiments. Transmembrane potentials were amplified with Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA), and the mechanical response was measured with a bridge amplifier (Quad Bridge amplifier; ADInstruments Pty Ltd., Castle Hill, Australia). Action potentials and contractions were recorded by a digital recorder (PowerLab/4sp; ADInstruments) via Chart software (version 4.0.2; ADInstruments) for off-line analysis.

Single-Cell Isolation. Single atrial and ventricular cells from adult guinea pigs were enzymatically dissociated as described previously (Isenberg and Klöckner, 1982). In brief, the excised heart was mounted on a modified Langendorff perfusion system and retrogradely perfused through the aorta with a normal HEPES-buffered Tyrode's solution (pH 7.4) gassed with 100% O₂ and maintained at 37 ± 0.2°C. The perfusion medium was then changed to a nominally Ca²⁺-free HEPES-buffered Tyrode's solution. After 5 min, the perfusate was switched to the same solution containing 0.3 mg/ml collagenase (Type II; Sigma-Aldrich, St. Louis, MO) and 0.1 mg/ml protease (Type XIV; Sigma-Aldrich). After a 7 to 15-min digestion, the residual enzyme solution was removed by more than 5-min perfusion with "modified KB medium" (10 mM taurine, 25 mM KCl, 10 mM KH₂PO₄, 70 mM glutamate, 0.5 mM EGTA, and 22 mM dextrose titrated to pH 7.4 with KOH) (Isenberg and Klöckner, 1982). Thereafter, the atrium was separated from the ventricle, and myocytes from both tissues were dispersed and then stored in KB medium for later use. Only quiescent and Ca²⁺-tolerant cells with clear striations were used for experiments.

Voltage-Clamp Recording. Whole-cell recordings were performed following the technique described by Hamill et al. (1981). Atrial or ventricular cells were bathed at room temperature (25-27°C) in a normal Tyrode's solution. Patch electrodes were fabricated from glass capillaries (o.d., 1.5 mm; i.d., 1.0 mm; A-M Systems, Inc., Carlsborg, WA) using a two-stage vertical puller (model PP-830; Narishige, Tokyo, Japan) and Microforge (model MF-830; Narishige). The electrode was 2 to 5 M when filled with the pipette solution. Membrane currents were recorded using an integrating patch-clamp amplifier (Axopatch 200B; Molecular Devices). Command pulses were generated by a 16-bit D/A converter (Digidata 1320A; Molecular Devices) controlled by the pCLAMP software (version 8.0.2; Molecular Devices). Electrode junction potentials (5-10 mV) were measured and compensated before formation of the pipette-membrane seal. A high-resistance seal (5-10 G) was obtained before the disruption of the membrane patch. Usually, more than 5 min was allowed for adequate cell dialysis after disruption of the membrane patch.

During measurement of K⁺ currents, the L-type Ca²⁺ currents (I_Ca) were blocked by the addition of 5 µM nifedipine to the bathing solution, and Na⁺ and T-type Ca²⁺ channels were voltage-inactivated by maintaining the holding potential at -40 mV. To study the effect of HA-7 on I_Ks,5 µM E4031 (Sanguinetti and Jurkiewicz, 1990) was used to block I_Kr. When the effect on I_Kr was measured, 30 µM (-)-[3R,4S]-chromanol 293B (Bosch et al., 1998) was used to block I_Ks. For measurement of Ca²⁺ and Na⁺ inward currents, the K⁺ currents were blocked by adding CsCl (2 mM) to the bathing solution and internal dialysis of the cells with Cs⁺ and tetraethylammonium-containing pipette solution. I_Na was studied in nifedipine (5 µM)-containing low Na⁺ Tyrode's solution ([Na⁺] = 54 mM, with NaCl replaced by N-methyl-D-glucamine) and dialysis of the cell with Na⁺-containing (10 mM) Cs⁺ pipette solution.

Time-dependent I_K amplitude was measured as the difference from the initial instantaneous current after settling of the capacity transient to the final current level during a given voltage step to various test potentials. I_K tail current amplitude was measured as the difference from the steady-state holding current level to the peak tail current amplitude. Inward rectifier K⁺ current (I_K1) was measured as the difference from the zero current level at the end of 200-ms voltage steps. The amplitude of I_Na was measured as the peak inward current with respect to the zero current level. The amplitude of I_Ca was the difference between the peak inward current and the current at the end of the test pulse. Reduction of both I_Ca and I_K with time ("rundown") was observed initially after rupture of the membrane patch. The rundown phenomenon was more prominent during the initial 3- to 8-min access of the patch pipette to the interior of the cell with no significant change over 30 min thereafter. When I_Ca (evoked at 0 mV) was normalized to the value at 1 min after disruption of membrane patch, it decreased to 83 ± 3, 73 ± 4, 68 ± 4, 69 ± 5, and 67 ± 5% (n = 11) during the subsequent 3, 6, 9, 12, and 18 min, respectively. Similarly, I_K tail current (evoked at +60 mV) decreased to 84 ± 4, 72 ± 4, 69 ± 3, 68 ± 5, and 69 ± 6% (n = 13) during the subsequent 3, 6, 9, 12, and 18 min, respectively. Therefore, experiments were performed only on those cells with stable I_Ca or I_K 10 min after cell rupture. Current recordings were filtered at 10 kHz bandwidth and then sampled at 100 kHz and stored on the hard disk of an IBM-AT-compatible computer using an on-line data acquisition program (Clampex, pCLAMP). The data were analyzed using a pCLAMP software (Clampfit).

Solutions and Drugs. The normal Tyrode's solution contained 137 mM NaCl, 5.4 mM KCl, 1.1 mM MgCl₂, 11.9 mM NaHCO₃, 0.33 mM NaH₂PO₄, 1.8 mM CaCl₂, and 11 mM dextrose. The HEPES-buffered Tyrode's solution contained 137 mM NaCl, 5.4 mM KCl, 1.2 mM KH₂PO₄, 1.22 mM MgSO₄, 1.8 mM CaCl₂, 22 mM dextrose, and 6 mM HEPES titrated to pH 7.4 with NaOH. The internal pipette filling solution contained 120 mM aspartic acid, 20 mM KCl, 1 mM MgCl₂, 5 mM K₂ATP, 5 mM sodium creatine phosphate, 0.2 mM NaGTP, 5 mM EGTA, and 10 mM HEPES adjusted to pH 7.2 with KOH. The Cs⁺-containing pipette solution contained 130 mM CsCl, 5 mM EGTA, 15 mM tetraethylammonium chloride, 5 mM dextrose, and 10 mM HEPES adjusted to pH 7.2 with CsOH. HA-7 was synthesized by our co-investigators Professors S.-C. Kuo and T.-P. Lin. Adenosine, nifedipine, E4031 dihydrochloride, and all of the chemicals of the physiological solution were purchased from Sigma-Aldrich. (-)-[3R,4S]-Chromanol 293B was purchased from Tocris Cookson Inc. (Ellisville, MO). d-Sotalol hydrochloride was a gift from Bristol-Myers Squibb Co. (Stamford, CT). HA-7, nifedipine, d-sotalol, and (-)-[3R,4S]-chromanol 293B were dissolved in dimethyl sulfoxide (DMSO). Other drugs were dissolved in physiological saline before the start of the experiment. In control experiments, the final concentration of DMSO up to 0.1% produced no significant effect on muscle contractions and electrophysiological parameters of the cells. HA-7 was dissolved in 100% DMSO as a stock solution of the highest concentration of 0.2 M. Final concentrations (0.1, 0.3, 1, 3, 10, 30, and 100 µM) of HA-7 were obtained by cumulative adding aliquots of corresponding stock solutions (1, 2, 10, 20, 50, 200, and 200 mM) to the physiological or external solutions to limit the final concentration of DMSO to approximately 0.096%. All of the stock solutions of d-sotalol, an agent with better solubility, were dissolved in 50% DMSO with distilled water. When stock solutions (the highest concentration was 0.2 M) of d-sotalol were given in a cumulative manner to obtain each final concentration from 3 to 300 µM, the total amount of DMSO approximated to 0.087%. During the antiarrhythmic experiments, we used stock solutions with different concentrations to obtain an identical final vehicle (DMSO) concentration of 0.06%.

Data Analysis. Statistical analysis was made using an analysis of variance (ANOVA) for repeated measures with Dunnett's t test for multiple comparisons. Incidences of arrhythmias were compared by a ² procedure followed by pairwise comparison by use of a Fisher's exact test. With the exception of incidences of arrhythmias, all of the data are expressed as mean ± S.E.M. Differences at a P < 0.05 level were considered statistically significant. Concentration-response curves were fitted by an equation of the form,

(1)

where E is the effect at concentration C, E_max is maximal effect, IC₅₀ is the concentration for half-maximal block, and n_H is the Hill coefficient.

	Results

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Modification of the Electrophysiological Properties of the Cardiac Conduction System. Intracardiac recording detected the atrial activity, His potential, and ventricular activity (Fig. 1). Changes in the electrophysiological parameters of the cardiac conduction system in guinea pig hearts after application of HA-7 and d-sotalol are summarized in Table 1. The basic cycle length was significantly lengthened by HA-7 at a low concentration (10 µM), and this effect was concentration-dependent. At higher concentrations (30 µM), the conduction through the AV node (AH interval) and the AV nodal Wenckebach cycle length and refractory period were prolonged. Additionally, HA-7 prolonged the atrial refractory period at a concentration of 30 µM or higher. At higher concentrations (100 µM), the VRT was also prolonged. The conduction interval through the atrial tissue (sinoatrial conduction interval) and the His-Purkinje system (His-ventricular conduction interval) was not significantly affected. In the present experimental protocol, the AV node usually became refractory to premature extrastimulation before the His-Purkinje system became refractory. Therefore, only the functional refractory period of the His-Purkinje system (shortest conducted V₁V₂ interval) was measured. This was significantly prolonged by HA-7 at a concentration of 30 µM or higher. As compared with HA-7, d-sotalol caused less significant effects on the atrial, AV nodal, and His-Purkinje system parameters but caused comparable or greater effects on basic cycle length and VRT. Both HA-7 and d-sotalol tended to prolong the refractory period of the ventricle, although this change did not reach statistical significance. The effect of d-sotalol on some of the parameters only partially reversed during the 60-min washout.

View larger version (74K): [in this window] [in a new window]   Fig. 1. Representative ventricular electrograms recorded at spontaneous sinus rhythm (left) and His bundle electrograms recorded during atrial pacing at 300 ms after HA-7 of the guinea pig heart. A, atrial depolarization; H, His bundle depolarization; S, stimulation artifact; T, ventricular repolarization; V, ventricular depolarization.

The atrial cycle length-response relations for the negative dromotropic effects (figure not shown) indicated that prolongation of the AH conduction interval caused by HA-7 was greater at faster atrial pacing rates than at slower rates. Such frequency-dependent effects on the AV node could also be observed in the d-sotalol treatment group. HA-7 at 100 µM prolonged the AH interval from 64 ± 2 to 80 ± 4 ms (P < 0.01, n = 9) and from 66 ± 2 to 92 ± 5 ms (P < 0.001, n = 9) at atrial cycle lengths of 300 and 260 ms, respectively. Equimolar d-sotalol prolonged this parameter from 56 ± 2 to 68 ± 4 ms (P < 0.05, n = 8) and from 58 ± 2 to 85 ± 10 ms (P < 0.01, n = 8) at atrial cycle lengths of 300 and 260 ms, respectively.

Antiarrhythmic Testing in Isolated Hearts. In the reperfusion-induced VF model, one group of hearts was used as a vehicle control (0.06% DMSO) group, whereas the other two groups were exposed to either HA-7 or d-sotalol (30 and 100 µM). Arrhythmia induced by reperfusion of the globally ischemic heart appeared within 10 to 30 s. There was no significant difference in both the incidence and duration of VF for either compound at 30 µM as compared with vehicle controls (Table 2). When the drug concentration was increased to 100 µM, the incidence of VF was significantly reduced in HA-7 but not in d-sotalol group, whereas the VF duration was significantly reduced in both groups. Also, HA-7 caused a more prominent shortening of mean duration of VF than that of d-sotalol.

In the control condition, AF could not be induced by a train of stimuli at an intensity up to 14 mA in Langendorff perfused hearts. After the application of 20 µM adenosine, AFT was decreased to approximately 1.5 mA, which was associated with a marked decrease in AERP (Table 3). The addition of 100 µM HA-7 significantly increased AFT and reversed the adenosine-induced decrease in ERP. Our unpublished data showed that this later effect might not be mediated by the blockade of the adenosine receptor. In contrast, the vehicle or d-sotalol had no apparent effect on AFT (Table 3).

Effects of HA-7 on Action Potentials and Contractile Force in Atrial Strips. The effects of HA-7 (30 and 100 µM) superfusion on the action potential in a left atrial strip paced at 1 Hz are shown in Fig. 2a (left panel). HA-7 at 100 µM significantly lengthened the action potential durations measured both at 50 and 90% repolarization (APD₅₀ and APD₉₀) from 36 ± 2 to 48 ± 3 ms (P < 0.05, ANOVA, n = 8) and from 82 ± 5 to 102 ± 5 ms (P < 0.05, n = 8), respectively, without causing considerable change in the resting membrane potential (RMP) (control mean value = -79 ± 2 mV) and the action potential amplitude (APA) (control = 108 ± 1 mV). At the same time, HA-7 (100 µM) caused a significant decrease in the maximal rate of depolarization (V_max) from 163 ± 7 to 134 ± 7 V/s (P < 0.05, n = 8). Concurrent with a prolongation of APD, HA-7 (30 and 100 µM) produced an increase in contractile force (CF) (figure not shown) to 160 ± 6 (P < 0.001, n = 8) and 246 ± 18% (P < 0.01, n = 8) of the control, respectively. In contrast, d-sotalol had no apparent effect on the action potential parameters and CF in atrial strips at any of the concentrations (10-100 µM) studied. Typical action potential tracings before and after treatment with 100 µM d-sotalol were shown in the right panel of Fig. 2a. The APD₅₀, APD₉₀, and V_max values before treatment were 27 ± 2 ms, 67 ± 3 ms, and 136 ± 8 V/s (n = 10), and these values were changed to 31 ± 4 ms, 76 ± 4 ms, and 135 ± 11 V/s, respectively (all P > 0.05, n = 10) after treatment with 100 µM d-sotalol. The same concentration of d-sotalol only caused a small increase in CF to 107 ± 6% (P > 0.05, n = 10) of the control.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Original superimposed tracings showing the effects of HA-7 and d-sotalol on the action potentials in guinea pig left atrial strips driven at 1 Hz (a) and ventricular papillary muscles driven at 1 and 3 Hz (b and c), respectively. The records were taken under control (CTRL) conditions and after exposure to the corresponding drug concentrations. Drugs were applied in a cumulative manner. For clarity, only the data obtained at 100 µM were shown in a (right tracing), b, and c. Data at different frequencies were obtained from the same impalement.

Effects of HA-7 on Action Potentials and Contractile Force in Papillary Muscles. In papillary muscles paced at 1 Hz, HA-7 at 100 µM caused a moderate but insignificant increase in contractile force (155 ± 17% of the control) (figure not shown) and prolongation of APD₉₀ (from 186 ± 5 to 201 ± 6 ms, n = 9) (Fig. 2b, left panel). The higher concentrations of HA-7 (150 µM) caused a further increase in CF to 183 ± 29% of the control value (P < 0.01, n = 9) accompanied by a significant prolongation of APD₉₀ from 186 ± 5 to 205 ± 5 ms (P < 0.05, n = 9). HA-7 had a tendency to decrease V_max, although this effect did not reach statistical significance. At higher concentrations up to 150 µM, HA-7 decreased the V_max from 189 ± 9 to 163 ± 14 V/s (P > 0.05, n = 9). HA-7 produced little change in the RMP (control = -87 ± 1 mV), APA (control = 122 ± 1 mV), APD₂₅, or APD₅₀ (control = 157 ± 4 ms), although there was a trend for APD₂₅ to be shortened slightly (from 103 ± 4 to 95 ± 4 ms at 150 µM). Despite the small effect on left atrial action potentials, d-sotalol had a marked effect on APD of papillary muscles at a stimulation frequency of 1 Hz (Fig. 2c, left panel). d-Sotalol at 100 µM significantly lengthened the APD₅₀ and APD₉₀ from 149 ± 4 to 181 ± 5 ms (P < 0.001, n = 8) and from 175 ± 4 to 213 ± 4 ms (P < 0.001, n = 8), respectively. d-Sotalol did not affect the RMP, APA, V_max, or APD₂₅ (data not shown). Unlike the positive inotropy produced by HA-7, d-sotalol produced little change in CF as previously reported by Yang et al. (1992). d-Sotalol at 100 µM caused a slight decrease in CF to 81 ± 4% (P > 0.05, n = 8) of the control.

Frequency-Dependent Effects of HA-7. To investigate whether HA-7 can exert any use- or frequency-dependent action, we also studied its effects at different stimulation frequencies in papillary muscles. Stimulation frequency was increased sequentially to 0.1, 0.5, 1, 2, and 3 Hz (3 min each), and the steady-state action potentials were recorded before and during the application of HA-7 (30-150 µM) and d-sotalol (10-100 µM). Typical action potential tracings obtained at driven rates of 1 and 3 Hz before and after treatment with 100 µM HA-7 and d-sotalol are shown in Fig. 2, b and c, respectively. The results for the changes of APD₉₀ are shown in Fig. 3. In untreated control groups, when the stimulation frequency was changed from 0.1 to 3 Hz, the APD initially slightly increased and then decreased (Fig. 3, a and b), whereas the other parameters, RMP, APA, and V_max, were not changed significantly (data not shown). The prolonging effect of HA-7 on APD₉₀ was similar throughout the pacing frequency range (Fig. 3, a and c); i.e., it did not show true frequency dependence. At a stimulation frequency of 3 Hz, HA-7 (100 and 150 µM) significantly lengthened the APD₉₀ from 140 ± 5 to 158 ± 5 (P < 0.05, n = 9) and 161 ± 5 ms (P < 0.05, n = 9), respectively. In contrast, d-sotalol (30 µM) caused greater prolongation of APD₉₀ at lower pacing frequencies (from 0.1 to 1 Hz) than at higher frequencies (Fig. 3, b and d), indicating that the reverse frequency-dependent effect on APD is similar to previous reports (Schmitt et al., 1991). At a 3-Hz pacing rate, 100 µM d-sotalol only slightly prolonged the APD₉₀ from 115 ± 7 to 132 ± 9 ms (P > 0.05, n = 8).

View larger version (44K): [in this window] [in a new window]   Fig. 3. Concentration- and frequency-dependent effects of HA-7 and d-sotalol on APD90 recorded from papillary muscles. APD90 before and during application of HA-7 (a, n = 9) or d-sotalol (b, n = 8) was plotted as a function of stimulation frequency. Frequency dependence of APD90 was measured at the end of the predrug control period and again after exposure to each drug concentration tested for the cumulative concentration-response curve. c and d, effects of cumulative increases in HA-7 (n = 9) and d-sotalol (n = 8) on percentage change of APD90 at 0.1-, 0.5-, 1-, 2-, and 3-Hz pacing conditions. Each value represents mean ± S.E.M.

Figure 4a shows superimposed current traces in response to various depolarizing voltage steps under control conditions and during exposure to and after washout of 3 µM HA-7. HA-7 decreased the amplitude of activation currents and the tail currents of I_Ks. Average current-voltage (I-V) relationships for the activation currents and the tail currents recorded before and after additions of 1, 3, and 10 µM HA-7 are shown in Fig. 4, b and c, respectively. Figure 5a shows sample current traces, corresponding to the labeled measurements depicted in Fig. 5b, obtained before, during, and after cumulative application of 3, 10, and 30 µM HA-7. Figure 5b is a plot that indicates the time course of the changes in the magnitude of I_Ks tail current; this protocol was imposed on the cell every 60 s. From experiments similar to those shown in Fig. 5a, we derived the concentration-response relation for the inhibition of tail current (Fig. 5d) in which the data are expressed as percentage inhibition of predrug tail amplitude after step depolarizations to +60 mV. HA-7 decreased I_Ks,tail in a concentration-dependent manner. Under control conditions (in the presence of E4031), the mean I_Ks,tail density was 3.2 ± 0.8 pA/pF (n = 8). The overall data were well described by the Hill equation with a mean IC₅₀ of 4.8 ± 1.7 µM, a coefficient of 0.85 ± 0.10, and a maximal inhibition of 103 ± 5% (n = 8). In comparison with HA-7, d-sotalol produced less effect on I_Ks (Fig. 5, c and d). HA-7 also inhibited the I_Ks currents of atrial myocytes (Fig. 7, a and c). The mean amplitude of I_Ks,tail in atrial myocytes elicited at the test potential of +60 mV was 2.7 ± 0.6 pA/pF (n = 8). The averaged IC₅₀ for the effect of HA-7 on I_Ks,tail was 3.0 ± 1.1 µM with a maximal inhibition of 99 ± 3% (n_H = 0.84 ± 0.09, n = 8).

View larger version (31K): [in this window] [in a new window]   Fig. 4. Effects of HA-7 on IKs in guinea pig ventricular myocytes. a, superimposed current traces obtained during 3-s depolarizing pulses to potentials ranging from 0 to +60 mV in 10-mV steps applied from a holding potential of -40 mV at 0.1 Hz before (control) and during 5-min exposure to 3 µM HA-7 and after washout of the drug. b and c, average current density-voltage relations for time-dependent activation currents (b) and tail currents (c) of IKs recorded under control conditions and during exposure to increasing concentrations of HA-7. Data are expressed as mean ± S.E.M. (n = 8).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Concentration-related block of IKs in ventricular myocytes by HA-7 and d-sotalol. a, superimposed current traces recorded from a cell before and after cumulative HA-7 treatment. IKs was activated using depolarizing voltage steps of 3-s duration clamped from the holding potential of -40 mV to the test potential of +60 mV (protocol inset in top panel). b, the tail current amplitude is plotted as a function of time during sequential application of HA-7. Letters on the curve correspond to traces in a: a, at the beginning of the experiment (control); b, c, and d, in the presence of 3, 10, and 30 µM HA-7, respectively. c, superimposed IKs current traces recorded before and after cumulative d-sotalol treatment. d, concentration-response curve for the inhibition of HA-7 (n = 8) and d-sotalol (n = 8) on IKs tail current. Percentage inhibition of tail current amplitude corresponding to the control value was plotted against drug concentration. Symbols represent mean ± S.E.M. The solid line was drawn by fitting to the Hill equation.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Concentration dependence of inhibition on IKs and IKr in atrial myocytes by HA-7. a and b, representative superimposed IKs and IKr current traces, respectively, generated by the protocol as shown in the top panels in the absence or presence of HA-7. c and d, concentration-response curve for the blocking effect of HA-7 on IKs (n = 8) and IKr (n = 6) tail currents, respectively. Symbols represent mean ± S.E.M.

Figure 6 depicts that HA-7 could also suppress the I_Kr currents in guinea pig ventricular myocytes. The superimposed I_Kr current traces before and during exposure to and following washout of 1 µM HA-7 are shown in Fig. 6a. I_Kr tail current density-voltage relationships are plotted in Fig. 6b for control and after application of 0.3, 1, and 3 µM HA-7. Concentration-dependent inhibition of HA-7 on I_Kr evoked at a test potential of +30 mV was plotted in Fig. 6d. Under control conditions (in the presence of (-)-[3R,4S]-chromanol 293B), the mean I_Kr density was 0.15 ± 0.02 pA/pF (n = 6). The overall data are well described by the Hill equation with a mean IC₅₀ of 1.1 ± 0.3 µM, a coefficient of 1.02 ± 0.14, and a maximal inhibition of 104 ± 3% (n = 6). d-Sotalol also showed a comparable inhibitory effect on the I_Kr current (Fig. 6, c and d). The calculated IC₅₀ for the effect of d-sotalol on I_Kr,tail was 1.7 ± 0.4 µM with a maximal inhibition of 107 ± 2% (n_H = 1.01 ± 0.12, n = 7). In addition, HA-7 also significantly inhibited I_Kr in atrial myocytes (Fig. 7, b and d). The mean I_Kr,tail density in atrial myocytes was 0.44 ± 0.05 pA/pF (n = 6). The calculated IC₅₀ for the effect of HA-7 on I_Kr,tail was 2.1 ± 0.8 µM with a maximal inhibition of 114 ± 6% (n_H = 0.77 ± 0.17, n = 6).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effects of HA-7 and d-sotalol on IKr in ventricular myocytes. a, representative recordings of IKr obtained under control conditions (left), in the presence of 1 µM HA-7 (right), and upon 8 min of washout (bottom left). The current was activated by 250-ms-long depolarizing voltage pulses from holding potential of -40 mV to various test potentials ranging from -20 to +50 mV in 10-mV increments. b, current-voltage relationship of IKr tail current density under control conditions and in the presence of 0.3, 1, and 3 µM HA-7. Data are given as mean ± S.E.M. (n = 6). c, superimposed IKr current traces evoked at +30 mV recorded before and after cumulative d-sotalol treatment. d, concentration-response curve for the blocking effect of HA-7 (n = 6) and d-sotalol (n = 7) on IKr tail current. Symbols represent mean ± S.E.M.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Effects of HA-7 on IK1 in guinea pig ventricular myocytes. a, sample traces used to construct I-V relationships in the absence (control) and after 5-min exposure to 30 µM HA-7. Currents were serially elicited (from -20 to -120 mV for 200 ms in 10-mV steps) after a prepulse of -40 mV. Dashed lines indicate zero current level. b, average current density-voltage relationships of steady-state component of IK1 in control and after 5-min exposure to 1, 3, 10, and 30 µM HA-7 (n = 6).

Effects of HA-7 on Inward Na⁺ and L-Type Ca²⁺ Currents. I_Na was elicited every 30 s with a step depolarization from -80 to -20 mV. Figure 9ai shows the superimposed current traces from typical experiments with 1 and 3 µM HA-7. Concentration-dependent inhibition of HA-7 on I_Na was shown in Fig. 9aii. Under control conditions, the mean I_Na density was 52.1 ± 5.1 pA/pF (n = 9). The calculated IC₅₀ was 2.9 ± 0.6 µM with a maximal inhibition of 108 ± 3% (n_H = 1.44 ± 0.23, n = 9). Compared with HA-7, peak I_Na was decreased by 2 ± 5, 17 ± 7, 30 ± 8, 35 ± 9, and 45 ± 12% (n = 9) after 3, 10, 30, 100, and 300 µM d-sotalol, respectively. HA-7 also inhibited the I_Na currents of atrial myocytes. Peak I_Na was decreased by 26 ± 5, 69 ± 4, and 92 ± 3% (n = 5) after 1, 3, and 10 µM HA-7, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 9. a, effects of HA-7 on INa in guinea pig ventricular myocyte. ai, superimposed current traces of INa recorded in the control conditions and after exposure to 1 and 3 µM HA-7. INa was elicited by a 30-ms depolarizing pulse from a holding potential of -80 to -20 mV. Dashed lines indicate zero current level. aii, concentration-response curve for the inhibition of INa by HA-7. Symbols represent mean ± S.E.M. (n = 9). b, effects of HA-7 on ICa in guinea pig ventricular myocyte. bi, I-V relationships for ICa recorded under control conditions and during exposure to 1 and 3 µM HA-7. Currents were elicited during 300-ms depolarizing pulses ranging from -40 to +50 mV in 10-mV steps from a holding potential of -40 mV. Symbols represent mean ± S.E.M. (n = 6). bii, concentration-response curve for the inhibition of ICa by HA-7. Symbols represent mean ± S.E.M. (n = 8). Inset shows original recordings of ICa evoked at 0 mV (holding potential = -40 mV) under control conditions (a), after a 5-min exposure to 10 µM HA-7 (b), and after 8-min washout (c). Stimulation frequency was 0.1 Hz. Dashed lines indicate zero current level.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The major findings from this study are as follows. 1) HA-7 prolonged conduction intervals and the refractoriness of the cardiac conduction system. 2) HA-7 lengthened the repolarization in both atrial and ventricular muscles. 3) HA-7 exhibited a lack of reverse frequency-dependent prolongation of APD. 4) HA-7 was more effective than d-sotalol in reducing experimental atrial and ventricular arrhythmias. 5) HA-7 exerted equipotent I_Kr-blocking activity as d-sotalol but was more potent on I_Ks, I_K1, I_Na, and I_Ca. Using a typical class III agent d-sotalol as a reference compound, the results of this study demonstrate that a nonselective ion channel blocker HA-7 was more effective in modifying electrophysiological parameters in atrial and ventricular tissue and, by virtue of these changes, HA-7 may possess a higher antiarrhythmic effectiveness than d-sotalol in guinea pig heart.

In this study, the experimental AF could be easily induced by a high-frequency atrial stimulation with low intensity during adenosine perfusion in isolated guinea pig hearts. The shortening of ERP resulting from the activation of adenosine-activated K⁺ current [I_K(Ado)] and the subsequent shortening of APD by adenosine (Watanabe et al., 1996) likely underlies the induction of AF in this study. The dominant mechanism of this atrial arrhythmia is considered to be re-entry (Nattel, 2002). In general, drugs that lengthen the cardiac APD and refractoriness (i.e., class III agents) should prevent premature excitation of cardiac cells and should suppress the incidence of re-entrant tachyarrhythmias, such as atrial and ventricular fibrillation (Singh and Nademanee, 1985). Our study has shown that higher concentrations of HA-7 reverted the shortened atrial ERP caused by adenosine toward control levels. The improved suppressive effect of HA-7 compared with d-sotalol on experimental AF in the present model is correlated with the more prominent increase of AERP and atrial APD by HA-7.

The mechanism(s) underlying the genesis of the ischemia/reperfusion-induced lethal arrhythmias (ventricular tachycardia and VF) are complex, but a number of factors have been implicated as potential culprits. These detrimental factors, which mostly occur during early reperfusion, may include the formation of oxygen free radicals, the heterogeneity of tissue injury and recovery, and the subsequent electrophysiological disturbances including the re-entry and enhanced automaticity, such as oscillatory after-potentials (Manning and Hearse, 1984; Pogwizd and Corr, 1987). In the reperfusion-induced arrhythmia model, HA-7 was applied from the preischemic through the reperfusion period and might cause some pharmacological effects over this period, so that could promptly prevent or ameliorate the arrhythmias that occur early at subsequent reperfusion. The efficacy of HA-7 to reduce both the incidence and mean duration of VF was greater than d-sotalol, perhaps through the prolonging effects on ventricular APD, VRT interval, or ventricular effective refractory period. This may suggests that HA-7 may act via a mechanism that includes the prolongation of repolarization and the subsequent suppression of re-entry-induced ventricular tachyarrhythmias. Additionally, through the blockade of I_Na and I_Ca, HA-7 may reduce the influx of these ions and, as such, help to suppress the occurrence of oscillatory after-potentials or extrasystoles provoked by Ca²⁺ overload during ischemia and reperfusion. Moreover, in contrast to the inherent reverse frequency-dependent effect of d-sotalol that may attenuate its action during conditions of high-frequency cardiac rhythm, such as ventricular tachycardia or VF, the lack of reverse frequency-dependent effect of HA-7 may maintain its antiarrhythmic action in the same conditions.

The balance between plateau inward and outward currents determines the APD. In this study, it is highly possible that the prolongation of APD in atrial and papillary muscles by HA-7 may be related to its inhibition on I_K (i.e., I_Kr and I_Ks), although a slight reduction of outward I_K1 current may also contribute to APD prolongation. Because HA-7 also inhibited I_Ca, this effect may counterbalance the inhibition of outward currents that could explain the slightly shortening of APD₂₅ of papillary muscle. Similar to previous observations (Carmeliet, 1985; Malécot and Argibay, 1999), d-sotalol was found to prolong APD via inhibition of K⁺ outward currents (mainly I_Kr) in this study. With respect to the effect on APD, d-sotalol exhibits greater potency on guinea pig ventricular fibers than on atrial fibers. Similar difference in tissue sensitivity was noted in an early study reported by Campbell (1987). Because I_K is largely responsible for repolarization in sinus node fibers (Schram et al., 2002), it is reasonable to speculate that both HA-7- and d-sotalol-induced bradycardiac effects are due to inhibition of I_K in the sinus node and that inhibition of I_Ca by HA-7 may also play a part. However, further studies are required to determine the direct effects of HA-7 on this preparation.

HA-7 moderately prolonged ventricular APD₉₀ at low and high heart rates, whereas the effect of d-sotalol was largely attenuated at fast rates. This property of d-sotalol is similar to earlier data on most class III antiarrhythmic agents (Katritsis and Camm, 1993). This reverse frequency-dependent effect (Hondeghem and Snyders, 1990) may lead to an increase in the dispersion of repolarization and favor the occurrence of cardiac arrhythmias. Jurkiewicz and Sanguinetti (1993) proposed that the reverse frequency-dependent effect on APD of typical class III agents is a consequence of selective blockade of I_Kr. It has been suggested that I_Ks accumulation at increased frequencies decreases the relative importance of I_Kr, reducing the impact of I_Kr blockade on APD prolongation (Jurkiewicz and Sanguinetti, 1993). They suggested that compounds that inhibit I_Ks might be devoid of reverse use-dependence. Actually, it has been shown that an agent that blocks both components of I_K might have a more consistent effect on action potentials at different rates and an improved safety profile over a specific I_Kr blocker (Sager et al., 1993). In this study, as compared with d-sotalol that blocked I_Kr selectively, HA-7 inhibited both I_Kr and I_Ks, so it is possible that the lack of reverse use-dependent effect of HA-7 may arise from its nonselective blockade of both I_K components. However, other mechanisms, as will be discussed later or yet to be identified, may also play some role.

Our study demonstrated that HA-7 decreased the V_max value of the atria and, to a lesser extent, the ventricular papillary muscles in a concentration-dependent manner. This effect is consistent with the inhibition of the I_Na by this agent, which likely contributes to the antiarrhythmic and AERP and His-Purkinje system functional refractory period prolongation actions by this agent. In rat ventricular myocytes, our previous report showed that HA-7 exerted use-dependent inhibition on I_Na and may bind preferentially to the inactivated state of Na⁺ channels (Su et al., 1997). In this context, the greater V_max-suppressing effect of HA-7 in atria than in papillary muscles may be explained by the less negative resting membrane potential and more prominent drug-induced APD prolongation of atrial tissues. Because of such properties, atrial tissues would have a higher ratio of inactivated Na⁺ channels that may enhance the channel blockade action of HA-7. In addition, the use-dependent inhibition on I_Na may also be expected to confer the greater suppressive actions on atrial or ventricular tachyarrhythmias of HA-7 than d-sotalol.

In keeping with the greater frequency-dependent depressant effects on the AV nodal conduction (i.e., A-H interval lengthening), HA-7 caused a greater prolongation of the Wenckebach cycle length and AV nodal effective refractory period than d-sotalol. These actions may be mainly related to its Ca²⁺ channel blockade activity. However, the inhibition of K⁺ channels may also contribute to the prolongation of AV nodal effective refractory period. The prolongation of A-H interval, AV nodal ERP, and Wenckebach cycle length by HA-7 at shorter atrial cycle lengths indicates that the negative dromotropic effect of this drug became greater as the atrial rate increased. Drugs that exacerbate the physiological frequency-dependent modulatory effects of atrial rate on AV nodal conduction delay (Meijler et al., 1996) provide additional protection against excessive ventricular rate during rapid atrial fibrillation or flutter (Ganz and Friedman, 1995). In this study, HA-7 exerted efficacy against experimental atrial arrhythmias. However, whether or not HA-7 could also filter the atrial impulse and control the ventricular rate during rapid atrial arrhythmias through AV nodal suppression remains to be established.

In conclusion, HA-7 possessed a multiple K⁺, Na⁺, and Ca²⁺ current-blocking profile without reverse frequency dependence in guinea pig heart preparations. The antiarrhythmic efficacy on either electrically induced AF or reperfusion-induced VF in guinea pig heart was greater than that of a typical class III agent d-sotalol. It is well known that patients with ischemic heart disease are particularly susceptible to events of atrial or ventricular tachyarrhythmias and that the latter may even culminate in sudden death. Our findings suggest that HA-7 may be useful for the prevention of such arrhythmias associated with ischemic heart disease. The modest positive inotropy by HA-7 may be viewed as a potential therapeutic advantage over d-sotalol and other similar cardiodepressant agents. Furthermore, it has been suggested that the block of I_Ca and/or I_Na, by reducing the risk of early after-depolarization development with excessive APD prolongation, attenuating excessive repolarization delay and temporal dispersion of repolarization may contribute to the reduced proarrhythmic potential of the multiple channel blockers (Abrahamsson et al., 1996; Amos et al., 2001). Consequently, because of its multifaceted actions, it can be reasonably expected that HA-7 may possess promising antiarrhythmic efficacy but lesser or negligible proarrhythmic risk.

	Acknowledgements

 
We thank Chin-Mei Kuo and Miao-Sui Lin for technical assistance and also gratefully acknowledge Bristol-Myers Squibb Co. for providing d-sotalol.

	Footnotes

 
This work was supported by grants from the National Science Council (NSC 91-0420-B-002-123) of Taiwan and from Chang Gung Medical Research Foundation (CMRP 1231).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.092106.

ABBREVIATIONS: AH, atrio-His bundle conduction interval; ERP, effective refractory period; AERP, atrial effective refractory period; AF, atrial fibrillation; AV, atrioventricular; CF, contractile force; APA, action potential amplitude; APD_{25, 50, 90}, action potential duration measured at 25, 50, and 90% repolarization; DMSO, dimethyl sulfoxide; E4031, N-(4-[(1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidyl)-carbonyl]phenyl)methanesulfonamide; (-)-[3R,4S]-chromanol 293B, N-[(3R,4S)-6-cyano-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl]-N-methylethanesulfonamide; HA-7, N-benzyl-7-methoxy-2,3,4,9-tetrahydrofuro[2,3-b]quinoline-3,4-dione; ANOVA, analysis of variance; RMP, resting membrane potential; VF, ventricular fibrillation; VRT, ventricular repolarization time; AFT, atrial fibrillation threshold.

Address correspondence to: Dr. Gwo-Jyh Chang, Graduate Institute of Clinical Medicinal Sciences, College of Medicine, Chang Gung University, 5 Fu-Shing St, Kwei-Shan, Tao-Yuan, Taiwan. E-mail: gjchang{at}adm.cgmh.org.tw

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