The Different Effects of Phenothiazines on Cardiac Action Potential Duration

Cardiac side effects such as QT-interval prolongation and the appearance of torsade de pointes by non-cardiac drugs have been of main concern (De ponti et al. 2000; Redfern et al. 2003). Antipsychotic drugs may cause QTc prolongation, ventricular tachycardia and sudden death in patients (Bigger & Weld 1982; Glassman 1984; Liberatore & Robinson 1984; Buckley & Sandres 2000; Glassman & Bigger 2001). Especially thioridazine and chlorpromazine, both of the phenothiazine type, have been suggested to be associated with prolongation of the QT interval by blocking the human ether-a-go-go-related gene (hERG) and K+ channels (Drolet et al. 1999; Tie et al. 2001; Thomas et al. 2003). Many antipsychotic drugs can also affect Na+ and/or Ca2+ channels (Studenik et al. 1998; Buckley & Sandres 2000; Welch & Chue 2000). However, the effects of the phenothiazines on action potential generated by various cardiac ion channels with the exception of delayed rectifier potassium channels (e.g. hERG) are poorly understood. Therefore, in the present study we investigated the effects of three phenothiazines (thioridazine, chlorpromazine, and trifluoperazine) on the cardiac action potential duration recorded in rabbit Purkinje fibers. 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). Female New Zealand white rabbits (1.8–2 kg) were anaesthetized with pentobartital sodium (45 mg/kg intravenously). Their hearts were rapidly removed and placed in Normal Tyrode solution (mM): 143 NaCl; 5.4 KCl; 5.0 HEPES; 0.33 NaH2PO4; 0.5 MgCl2; 16.6 Glucose; 1.8 CaCl2; pH 7.4 aerated with O2 gas. Purkinje fibers were excised from the left ventricle and stored in a chamber superfused with Normal Tyrode solution at 37±0.5 °. The preparations were stimulated at a basal rate (frequency=1 Hz, duration=2 msec., voltage=1.5–2 V). Two hr were allowed for each preparation to equilibrate while continuously superfused with Normal Tyrode solution. Action potentials were recorded with a glass microelectrode filled with 3M KCl and connected to a Geneclamp 500B (Axon Instruments, CA, USA). Action potential duration at 50% and 90% repolarization (APD50 and APD90) was automatically measured using Notocord HEM program (NOTOCORD, France) at a sampling rate of 50 kHz. Before drug treatment, action potential parameters were measured for 1 hr to establish stable control value recording. To test the drug effects, we allowed 20 min. perfusion of drug. Fibers were considered suitable for study if the following criteria: a) the resting membrane potential was more negative than −80 mV, b) the APD90 ranged between 200 and 300 msec. Statistical analysis was performed by comparing the differences between the drug treated groups and the control group using GraphPad InStat (version 3.05, GraphPad Software Inc., CA, USA). Dunnett's multiple comparison test was conducted and data was considered to be significant when P<0.05 or P<0.01. All drugs (thioridazine, chlorpromazine, trifluoperazine) used in this study were obtained from Sigma-Aldrich Co. (MO, USA) and were dissolved in NT solution to achieve a stock solution for the required concentrations. All drugs shortened the APD50 in a dose-dependent manner (fig. 1A). The effects were statistically significant at concentrations of 3 and 10 μM (mean reduction=16.9±3.4%; 32.6±6.1% P<0.01) for chlorpromazine and 10 μM for thioridazine and trifluoperazine (mean reduction=24.4±9.7%; 40.3±10% P<0.01). Fig. 1B shows that thioridazine prolonged the APD90 at concentrations of 1 μM and above (mean increase=9.0±2.9%; 9.2±2.6%; 9.7±1.9% P<0.05). By contrast, chlorpromazine and trifluoperazine had no significant effect on the APD90 at all concentrations. Effects of phenothiazines on (A) action potential duration at 50% repolarization (APD50) and (B) action potential duration at 90% repolarization (APD90) by thioridazine (•), trifluoperazine (○) and chlorpromazine (▾). Values represent the means±S.E.M. of 4 animals per group. *P<0.05, **P<0.01, versus control. The adverse effects of the antipsychotics on QT-prolongation, arrhythmias and sudden death are well-known. Most studies on QT-prolongation have been limited to the hERG-Ikr channel investigation. However, various ion channels are able to alter the action potential configuration and drugs may interact with more than one channel target to produce either offsetting or synergistic effects on the action potential. Therefore, assessing changes in action potential duration in each phase can be a comprehensive approach to identifying electrophysiological effects of drugs on the heart. In previous reports, thioridazine and chlorpromazine were classified as low-potency phenothiazines, whereas trifluoperazine belonged to high-potency phenothiazines (table 1) (Glassman & Bigger 2001). High-potency antipsychotic drugs were commonly considered safer than low-potency antipsychotic drugs. Phenothiazines previously evaluated for their effects on hERG currents have been listed (table 1) (Fayer 1986; Warner et al. 1996). In the present study, we compared the effects of high and low potency phenothiazines on the action potential duration. Thioridazine inhibited hERG currents with an IC50 value of 1.07 μM (Tie et al. 2000) and prolonged APD90 at 1 μM. However, at higher concentrations, the APD90 value remained unchanged; furthermore, chlorpromazine and trifluoperazine produced a slight trend in APD90 reduction in a dose-dependant manner (fig. 1B). In previous reports, the shortened APD50 and/or APD90 values were associated with the depression of the plateau potential in phase 2, which may be attributed to the inhibition of L-type Ca2+ channels (Pacher et al. 2000; Chiang et al. 2002). Some phenothiazines were demonstrated to block Ca2+ channels (Scamps et al. 1989; Lee et al. 1999). Consistent with the previous reports, our data suggested that the effect of hERG channel blockade was reduced by additional drug effects on non-hERG channels, especially L-type Ca2+ channels. We observed that APD90 was exclusively prolonged by thioridazine in rabbit Purkinje fibers, and that chlorpromazine and trifluoperazine had no effect on APD90 prolongation. These observations raise the possibility that thioridazine may have greater effects on hERG channel blockade and/or lesser effects on Ca2+ channel blockade in comparison with chlorpromazine and trifluoperazine. Thioridazine was most potent achieving a 9–10% increase in APD90 at concentration up to 1 μM (fig. 1B) and associated with numerous cases of torsade de pointes when compared with other phenothiazine drugs (Mehtonen et al. 1991). Moreover, therapeutic plasma concentration of thioridazine required high concentration and far exceed IC50 value of hERG assay (table 1). Therefore, we could prove that thioridazine have more proarrhythmic potential when compared with other phenothiazine drugs such as chlorpromazine and trifluoperazine. Even if a drug blocks hERG channel, it is not fully predictive of proarrhythmic risk since ventricular repolarization, determined by the duration of the cardiac action potential, depends upon the characteristics of drug block on individual currents (Martin et al. 2004). Moreover, our unpublished observations indicate that phenothiazines had no effects on the resting membrane potential and total amplitude with all concentration but, at high concentrations decreased the maximum velocity (Vmax) and disturbed action potential generation. These effects can be attributed to the inhibition of fast Na+ channels, which are involved in the upstroke of the action potential at phase 0. Therefore, further multi-channel (at least Ca2+ and Na+ channels) studies integrating hERG and action potential duration assay will be necessary to elucidate the electrophysiological effects of drugs on the heart. This work was supported by National research laboratory grant from the Ministry of Science and Technology (M1-0302-00-0003-03-J00-00-003-10).