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Cardiovascular Research 2002 55(2):290-299; doi:10.1016/S0008-6363(02)00438-8
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Effects of sildenafil on cardiac repolarization

Chern-En Chianga,*, Hsiang-Ning Lukb, Tsui-Min Wangc and Philip Yu-An Dinga

aDivision of Cardiology, Taipei Veterans General Hospital and National Yang-Ming University, 201, Sec 2, Shih-Pai Road, Taipei 112, Taiwan
bDepartment of Anesthesiology, Chang-Gung Memorial Hospital, Taipei, Taiwan
cGraduate Institute of Medical Science, Taipei Medical University, Taipei, Taiwan

cechiang{at}vghtpe.gov.tw

* Corresponding author. Tel.: +886-2-2875-7602; fax: +886-2-2874-5422

Received 17 December 2001; accepted 4 April 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Sudden death has occasionally been reported in patients taking sildenafil. The objective of this study was to investigate the effect of sildenafil on cardiac repolarization. Methods: We used conventional microelectrode recording technique in isolated guinea pig papillary muscles and canine Purkinje fibers, whole-cell patch clamp techniques in guinea pig ventricular myocytes, and in vivo ECG measurements in guinea pigs. Results: Action potential duration at 90% repolarization (APD90) was not affected by sildenafil in the therapeutic ranges (≤1 µM), but shortened by higher concentration (≥10 µM) in both guinea pig papillary muscles and canine Purkinje fibers. D-Sotalol prolonged APD90 in the same preparations with concentrations ≥1 µM in a reverse frequency-dependent manner. Co-administration of sildenafil (10 and 30 µM) abolished the APD-prolonging effects of D-sotalol (30 µM) and amiodarone (100 µM). Sildenafil, with concentrations up to 30 µM, had no significant effect on both the rapid (IKr) and the slow (IKs) components of the delayed rectifier potassium currents in guinea pig ventricular myocytes. Sildenafil dose-dependently blocked L-type Ca2+ current (ICa,L), but had no effect on persistent Na+ current in guinea pig ventricular myocytes. ECG recordings in intact guinea pigs revealed significant shortening of QTc interval by sildenafil (10 and 30 mg/kg orally). The QT-prolonging effects by D,L-sotalol (50 mg/kg) and amiodarone (100 mg/kg) were abolished by sildenafil (30 mg/kg). Conclusions: Sildenafil does not prolong cardiac repolarization. Instead, in supra-therapeutic concentrations, it accelerates cardiac repolarization, presumably through its blocking effect on ICa,L.

KEYWORDS K-Channel; Long QT syndrome; Membrane potential; Myocytes; Purkinje fiber; Repolarization


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Sildenafil (Viagra) is a highly selective inhibitor of cGMP-specific phosphodiesterase type 5 (PDE5) and has been widely used for the treatment of erectile dysfunction [1]. Post-marketing surveillance data after approval of sildenafil by the Food and Drug Administration (FDA) showed significant cardiovascular events [2,3]. As of February 1999, the number of spontaneous reports of death to FDA among men who had taken sildenafil was 401 [4]. In these patients, a total of 219 were presumed to be due to cardiac origin, including myocardial infarction and sudden cardiac death [4]. It is difficult to determine if these events were directly related to the use of sildenafil, although the early study by Morales et al. [5], a study in men with ischemic heart disease [6], the recent database comparing sildenafil to placebo [4], and the most recent observational cohort study [7] do not show an increase in either myocardial infarction or other serious cardiovascular events with this agent.

Recently, one study showed that sildenafil exerted direct cardiac electrophysiological effects similar to class III antiarrhythmic agents at concentrations that may be found in conditions of impaired drug elimination, during co-administration of CYP3A substrate/inhibitor, or after drug overdose, and suggested a potential explanation for sudden death during sildenafil treatment [8]. If this is true, there will be a great concern about the risks of long QT syndrome and torsade de pointes in patients taking sildenafil, especially in those patients who already took antiarrhythmic drugs [9], erythromycin [10], second generation antihistamine [11], and other QT-prolonging agents [12]. Patients with hypokalemia may also have increased risk of developing life-threatening arrhythmia during sildenafil treatment. New suggestions might have to be included in the expert consensus from American College of Cardiology/American Heart Association [13] to remind the sildenafil users of this potentially malignant drug effect.

Sildenafil has been associated with atrial fibrillation in susceptible patients [14–16]. In patients receiving sildenafil and concomitant administration of CYP3A4 inhibitors that resulted in several-fold increase in the plasma concentration of sildenafil, their QT internal was not prolonged [17]. Interestingly, in the single paper describing that sildenafil prolonged cardiac repolarization [8], conventional microelectrode recording techniques in mammalian cardiac papillary muscles [18,19] or Purkinje fibers [20,21] for the testing of the drug effect were not used. Instead, monophasic action potential (MAP) was recorded [22]. Furthermore, drug effect on the rapid (IKr) and the slow (IKs) components of the delayed rectifier potassium current has not been tested on mammalian cardiac myocytes, and the effect on the ECG (electrocardiography) parameters were unknown. Consequently, we undertook the present study to determine the effects of sildenafil on the ionic channels involved in action potential duration, and on cardiac repolarization in the mammalian hearts. We found that sildenafil did not prolong repolarization in guinea pig papillary muscles and canine Purkinje fibers. In guinea pig ventricular myocytes, sildenafil had no effect on either the IKr or the IKs of the delayed rectifier potassium currents. Neither did it block persistent Na+ current (IpNa). Nevertheless, sildenafil dose-dependently blocked L-type Ca2+ current (ICa,L). In vivo ECG recordings suggested that sildenafil did not prolong corrected QT (QTc) interval in guinea pigs. Moreover, in conditions of prolonged repolarization in the presence of antiarrhythmic agents, sildenafil did not have additive effect. Instead, with a concentration far above the therapeutic one, sildenafil shortened cardiac repolarization and QTc interval, and abolished the APD- and the QT-prolonging effects of antiarrhythmic agents, presumably through its blocking effect on ICa,L.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult Hartley guinea pigs (weighing 400–500 g) and mongrel dogs (weighing 15–20 kg) of either sex were used in this study.

2.1 Recording of action potentials
Action potentials were recorded with conventional intracellular recording technique [18,23]. The perfusing Tyrode solution was oxygenated with a gas mixture of 3% CO2 and 97% O2 and kept at 37 °C with a temperature-controlled circulator. The perfusion speed was set at 5 ml/min. The tissue preparation was driven by electrical pulses (duration=2 ms, frequencies of 0.1 Hz, 1 Hz and 2 Hz). The signals of action potentials were amplified with Axoclamp-2A (Axon Instruments, Foster City, USA) and recorded by a digital data recorder (model VR-10B, Instrutech, Great Neck, USA) for off-line analysis. The resting membrane potential (RMP), Vmax of the phase 0 depolarization, the action potential duration at 50% (APD50) and 90% (APD90) repolarization were measured when they were stable at different stimulation frequencies. To test the drug effect, we allowed 20 min perfusion of drug and another 20–40 min for washout. We also examined if co-administration of sildenafil with other antiarrhythmic agents known to prolong repolarization (D-sotalol and amiodarone) would have additive effects on cardiac repolarization.

2.2 Voltage-clamp experiments
Single ventricular myocytes were isolated from guinea pig left ventricle using enzymatic digestion method as described previously [24,25]. The single-pipette, whole-cell, voltage-clamp technique was used [26,27]. Membrane currents were recorded using a patch clamp amplifier (Axopatch 1-D, Axon) and experiments were performed at a temperature of 35 °C for K+ currents, or at room temperature (20–22 °C) for other currents. Voltage-clamp command pulses were generated by a digital-to-analog converter (DigiData 1200, Axon) controlled by the pCLAMP software (6.03, Axon). Junction potentials were zeroed before formation of the membrane–pipette seal. Several minutes after seal formation, the membrane was ruptured by gentle suction to establish the whole-cell configuration for voltage clamping. Cell capacitance was measured by integrating the capacitive transient evoked by applying a 5-mV hyperpolarizing step from a holding potential of –40 mV. The cell capacitance and series resistance were electrically compensated by 60–90%.

The protocols for the recording IKr and IKs were adapted from previous studies [28,29]. The membrane potential of myocytes was held at –40 mV to inactivate sodium current and subsequently depolarized by pulses lasting either 250 ms (IK250) or 5000 ms (IK5000). Test potentials of depolarization varied between –20 and +50 mV for IK250 but between 0 and +50 mV for IK5000. The activation currents and the tail currents were recorded. To verify the recorded currents, chromanol 293B (an IKs specific blocker) [30] and D-sotalol (an IKr specific blocker) [28] were used.

Other plateau currents, such as ICa,L and IpNa were also examined. ICa,L was induced by a single 300-ms voltage pulse to +10 mV from the holding potential of –40 mV once every 30 s. The amplitude of ICa,L was the difference between the peak inward current and the current at the end of the test pulse. The I–V relationship of ICa,L was obtained by plotting the peak current amplitude in response to voltage pulses to potentials between –40 and +70 mV from the holding potential in 10-mV increments at 0.2 Hz. The IpNa was recorded according to the method of Sakmann et al. [31].

2.3 Electrocardiographic recording
Adult guinea pigs were anesthetized with intraperitoneal urethane (1.2 g/kg). Heart rate, PR interval, QRS duration, and QT interval were recorded at baseline condition just before the administration of various agents or water (sham group) via nasogastric tube, and every 30 min until 2 h. QTc was calculated with Bazett's formula [32]: QTc=QT/(RR)1/2. ECG parameters were measured in Lead II [33].

2.4 Drugs and solutions
The composition of Tyrode solution for the recording of action potential was (in mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 1, NaHCO3 12, glucose 5.0. The external solution for recording of IKr and IKs contained (in mM): NaCl 145, KCl 4, MgCl2 1, Hepes 10, glucose 5, and nisoldipine 0.2 µM (pH 7.4, titrated with NaOH), while the pipette solution contained (in mM): KCl 140, Hepes 5, creatine 5, K2ATP 5, glucose 5.5 (pH 7.2, titrated with KOH) [21]. The external solution for recording of ICa,L was composed of (in mM) choline Cl 137, CsCl 4, CaCl2 1.8, MgCl2 0.5, 4-aminopyridine (4-AP) 2, Hepes 10, glucose 5.5 (pH adjusted to 7.4 with CsOH), while the pipette solution contained (in mM) CsCl 110, tetraethylammonium (TEA) 20, Hepes 5, phosphocreatine 5, MgATP 5, EGTA 20 (pH 7.2, titrated with CsOH). The compositions of the external solution and pipette solution for recording of IpNa were the same as those described by Sakmann et al. [31].

Sildenafil was kindly provided by Pfizer. Chromanol 293B was kindly provided by Aventis. D,L-Sotalol and D-sotalol were gifts from Bristol–Myers–Squibb. Amiodarone and all the chemicals of the Tyrode solution were purchased from RBI-Sigma (St. Louis, MO, USA). Nisoldipine was provided by Miles Pharmaceuticals (New Haven, USA). Sildenafil, D-sotalol, chromanol 293B, amiodarone, and nisoldipine were prepared in DMSO. Possible vehicle effect was excluded by using a maximal vehicle concentration of less than 0.1%.

2.5 Data analysis and statistics
The data are expressed as mean±S.E.M. unless otherwise specified. Only single papillary muscle and single Purkinje fiber were obtained from each guinea pig and each dog, respectively. We used a computer-based statistical package (SPSS version 9.0, SPSS, Chicago, IL, USA). Two-tailed Student's t-test was used for statistical analyses. Two-way ANOVA was also used, when indicated, to analyze the experimental data, with one way being between groups and the other way being within group. If significance was found, a post hoc analysis (Newman–Keuls) was used. The difference was considered statistically significant when P value was less than 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effects of sildenafil on action potential
Fig. 1A shows that sildenafil, with concentrations up to 1 µM, had no effects on the APD50 and the APD90 of guinea pig papillary muscles when stimulated with frequencies of 1 Hz and 0.1 Hz. Higher concentrations (10 and 30 µM) of sildenafil actually shortened the APD50 and the APD90 dose-dependently. In contrast, D-sotalol (≥1 µM) prolonged the APD50 and the APD90 at both stimulation frequencies, showing dose-dependent effects as well (Fig. 1B). Table 1 demonstrates that the percent prolongation of APD90 with D-sotalol was apparently more at 0.1 Hz than at 1 Hz for both 10 and 30 µM, suggesting a reverse frequency-dependent effect. On the other hand, sildenafil dose-dependently shortened APD90 at both stimulation frequencies. Fig. 2 shows representative illustrations of the effect of sildenafil on guinea pig papillary muscles. Sildenafil 10 and 30 µM significantly shortened APD and depressed the plateau phase. The effects of sildenafil and D-sotalol of each concentration except 30 µM could be completely washed out within 20–40 min. Sildenafil had no effects on the RMP and Vmax of phase 0 depolarization with concentrations up to 30 µM.


Figure 1
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  		Fig. 1 Effects of sildenafil on action potential duration (APD) of guinea pig papillary muscles. (A) Lower concentrations (≤ 1 µM) of sildenafil had no effect. Higher concentrations (≥ 10 µM) significantly shortened both the APD50 and APD90. n = 18, 10, 10, 14, 12, 10 for baseline, 0.01, 0.1, 1, 10, 30 µM, respectively. *P<0.05 versus baseline; #P<0.01 versus baseline. APD50 and APD90: APD at 50 and 90% repolarization. (B) Lower concentrations (≤ 0.1 µM) of D-sotalol had no effect. Higher concentrations (≥ 1 µM) significantly prolonged both the APD50 and APD90. n = 15, 8, 8, 12, 11, 10 for baseline, 0.01, 0.1, 1, 10, 30 µM, respectively. Other labelings and abbreviations are the same as those of (A).

 

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  		Table 1 Percent changes in APD90 of guinea pig papillary muscles by sildenafil and D-sotalol

 

Figure 2
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  		Fig. 2 Representative illustrations of the effect of sildenafil on the action potential of guinea pig papillary muscles. (A) 1 Hz; (B) 0.1 Hz. Sildenafil (10 and 30 µM) shortened the APD in both stimulation frequencies.

 
For canine Purkinje fibers, sildenafil had no effects on the APD90 at concentrations up to 1 µM, but shortened it at higher concentrations (10 and 30 µM) at both stimulation frequencies (Fig. 3). The APD50 was shortened by sildenafil (1, 10 and 30 µM) at both stimulation frequencies. In the same preparations, D-sotalol (1, 10 and 30 µM) significantly prolonged the APD90 at both stimulation frequencies. The percent changes of APD90 were shown in Table 2. Reverse frequency-dependent prolongation by D-sotalol was observed. Fig. 4 represents an example of the effects of sildenafil on canine Purkinje fibers. The plateau phase was depressed by higher concentrations of sildenafil (10 and 30 µM). Sildenafil had no effects on the RMP and Vmax of phase 0 depolarization of canine Purkinje fibers with concentrations up to 30 µM.


Figure 3
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  		Fig. 3 Effects of sildenafil on action potential duration (APD) of canine Purkinje fibers. n = 10, 4, 4, 8, 7, 6 for baseline, 0.01, 0.1, 1, 10, 30 µM. Other labelings and abbreviations are the same as those in Fig. 1.

 

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  		Table 2 Percent changes in APD90 of canine Purkinje fibers by sildenafil and D-sotalol

 

Figure 4
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  		Fig. 4 Representative illustrations of the effect of sildenafil on the action potential of canine Purkinje fibers. (A) 1 Hz; (B) 0.1 Hz. Sildenafil (10 and 30 µM) shortened the APD in both stimulation frequencies.

 
3.2 Sildenafil reversed the APD-prolonging effect of antiarrhythmic agents
Fig. 5 shows that instead of prolonging repolarization sildenafil in fact antagonized the effect of QT-prolonging agents. D-Sotalol 30 µM prolonged APD90 and revealed a reverse frequency-dependent effect (Fig. 5A, n = 6). In the presence of D-sotalol, sildenafil dose-dependently shortened APD90. Sildenafil 30 µM abolished the APD-prolonging effect of D-sotalol at 0.1 and 1 Hz, and further shortened the APD90 to be less than the baseline when stimulated with 2 Hz. Fig. 5B (n = 6) shows that amiodarone 100 µM prolonged APD90 without a reverse frequency-dependent effect and sildenafil dose-dependently shortened the APD90. Sildenafil 30 µM completely blunted the APD-prolonging effect of amiodarone. Likewise, when stimulated with 2 Hz, sildenafil 30 µM decreased the APD90 to an even lower level compared with baseline.


Figure 5
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  		Fig. 5 Sildenafil reversed the APD-prolonging effects of antiarrhythmic agents. *P<0.05 when compared with baseline; #P<0.05 when compared with D-sotalol alone (A) or amiodarone alone (B).

 
3.3 Effect of sildenafil on IK250 and IK5000
Fig. 6A shows that sildenafil 30 µM had no significant effect on the activation currents and the tail currents of IK250 in guinea pig ventricular myocytes (mean reduction=0.3±2.6%, P>0.05 for the former; 0.8±2.1%, P>0.05 for the latter). In the same cell, D-sotalol 30 µM inhibited both the activation currents (mean reduction 24.5±3.2%, P<0.001 compared with control) and the tail currents (mean reduction 31.2±4.3%, P<0.001 compared with control) of IK250, and the inhibitory effect could be partially washed out. This finding suggests that sildenafil had no effects on D-sotalol-sensitive current, presumably IKr. The finding was reproducibly observed in a total of 25 cells (from a total of 10 different guinea pigs). Fig. 6B demonstrates that D-sotalol 30 µM inhibited both the activation currents (mean reduction 23.3±3.8%, P<0.001 compared with control) and the tail currents (mean reduction 32.4±3.5%, P<0.001 compared with control) of IK250. On top of D-sotalol, sildenafil 30 µM had no additional effect, suggesting that sildenafil had no effect on D-sotalol-resistant currents. Similar findings were observed in a total of 17 cells (from 10 different guinea pigs). In all the cells tested, we did not find any evidence that sildenafil blocked IK250.


Figure 6
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  		Fig. 6 The effects of sildenafil on IK250 and IK5000. (A) Sildenafil 30 µM had no effect on both the activating currents and the tail currents of IK250, but D-sotalol 30 µM had inhibitory effect on both currents in the same preparations. (B) D-Sotalol 30 µM inhibited both the activating currents and the tail currents of IK250. The addition of sildenafil 30 µM had no additional effect. (C) Sildenafil 30 µM had no effect on both the activating currents and the tail currents of IK5000, but chromanol 293B 10 µM blocked both currents in the same preparation. (D) Chromanol 293B 10 µM blocked both the activating currents and the tail currents of IK5000. The addition of sildenafil 30 µM had no discernible effect on IK5000.

 
Fig. 6C shows that sildenafil 30 µM had no effect on both the activation currents and the tail currents of IK5000 in guinea pig ventricular myocytes (mean reduction=0.2±1.6%, P>0.05 for the former; 0.6±1.9%, P>0.05 for the latter). In the same cell, chromanol 293B 10 µM blocked both the activation currents (mean reduction 60.2±4.2%, P<0.001 compared with control) and the tail currents (mean reduction 69.3±3.1%, P<0.001 compared with control) of IK5000, and the blocking effect could be partially washed out. This finding suggests that sildenafil had no effects on chromanol 293B-sensitive current, presumably IKs. The finding was reproducibly observed in a total of 18 cells (from a total of six different guinea pigs). Fig. 6D demonstrates that chromanol 293B 10 µM inhibited both the activation currents (mean reduction 62.7±3.5%, P<0.001 compared with control) and the tail currents (mean reduction 67.6±2.9%, P<0.001 compared with control) of IK5000. On top of chromanol 293B, sildenafil 30 µM had no additional effect, suggesting that sildenafil had no effect on chromanol 293B-resistant currents. Similar findings were observed in a total of 15 cells (from six different guinea pigs).

3.4 Effects of sildenafil on ICa,L and IpNa
Fig. 7 shows the inhibitory effect of sildenafil on ICa,L. Fig. 7A shows a representative tracing of the blocking effect of sildenafil on ICa,L. Fig. 7B was the time course of the blocking effect of Fig. 7A. Similar effects have been demonstrated in seven experiments. Fig. 7C demonstrated the current–voltage relationship (I–V curve) of ICa,L in the absence and presence of sildenafil. Sildenafil exerted a concentration-dependent inhibition of ICa,L, but there was no shift in the reversal potential, nor any change in the voltage dependence of peak ICa,L (0 mV). The dose–response curve was shown in Fig. 7D. The half-maximum inhibition concentration (IC50) was 27.2±6.3 µM.


Figure 7
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  		Fig. 7 Effects of sildenafil on ICa,L. (A) Typical example of the blocking effect of sildenafil on ICa,L. (B) Time course of blocking effect of panel (A). (C) Effect of sildenafil on the I–V curve of ICa,L. n = 16, 4, 7, 5 for control, 1 µM, 30 µM and 100 µM, respectively. (D) The dose–response curve of the inhibition of ICa,L by sildenafil. The curve could be fitted by the Hill equation: (1–Imin)/(1+[IC50/x]n)+Imin, where Imin represents the relative current in the maximally effective concentration and x represents concentration of sildenafil. It was constructed from peak ICa,L measured in response to a voltage pulse to +10 mV in each concentration of sildenafil.

 
Sildenafil has no significant effect on IpNa. The IpNa measured at –20 mV was –0.32±0.04 pA/pF at baseline and was –0.31±0.05 pA/pF after sildenafil 30 µM infusion (n = 8, P>0.05).

3.5 Effect of sildenafil on ECGs
The ECG parameters of guinea pigs receiving sildenafil (10 mg/kg) are shown in Table 3. The HR, PR interval and the QRS duration did not change significantly. The QT interval shortened at 30 and 60 min. The most striking change was in the QTc interval that shortened significantly and was maximally abbreviated at 2 h.


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  		Table 3 Electrocardiographic changes by oral sildenafil

 
3.6 Sildenafil reversed the QT-prolonging effect of antiarrhythmic agents (Fig. 8)
QTc interval did not change significantly in the sham group (n = 6). Sildenafil decreased the QTc interval dose-dependently (P<0.05 for sildenafil 10 mg/kg versus baseline, n = 10; P<0.01 for sildenafil 30 mg/kg versus baseline, n = 11). D,L-Sotalol 50 mg/kg significantly prolonged the QTc interval (P<0.01 versus baseline, n = 12). Co-administration of sildenafil 10 mg/kg with D,L-sotalol 50 mg/kg did not significantly alter the QT-prolonging effect of the latter (P>0.05 versus D,L-sotalol alone, n = 10). However, co-administration of sildenafil 30 mg/kg with D,L-sotalol 50 mg/kg significantly attenuated the QT-prolonging effect of the latter (P<0.05 versus D,L-sotalol alone, n = 10). Similar findings were observed with amiodarone. Amiodarone 100 mg/kg lengthened the QTc interval significantly (P<0.01 versus baseline, n = 10). Co-administration of sildenafil 10 mg/kg (n = 8) did not induce any significant change, but sildenafil 30 mg/kg significantly abolished the effect of amiodarone (P<0.05 versus amiodarone alone, n = 10). Actually the QTc of the guinea pig receiving both amiodarone 100 mg/kg and sildenafil 30 mg/kg did not differ from that of baseline (P>0.05).


Figure 8
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  		Fig. 8 The QTc interval from different experimental groups. The changes in QTc were maximal at 2 h after administration of drugs, which were shown here. *P<0.05 versus baseline; #P<0.01 versus baseline; +P<0.05 versus sotalol alone or amiodarone alone.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This is the first comprehensive study of the effect of sildenafil on cardiac repolarization. The major findings are that sildenafil in a therapeutic concentration does not block IKr and IKs in guinea pig ventricular myocytes, nor does it prolong cardiac repolarization in guinea pig papillary muscles and canine Purkinje fibers. Sildenafil has no significant effect on IpNa, but dose-dependently blocks ICa,L. In a supra-therapeutic concentration that might possibly be encountered during drug overdose or impaired metabolism, sildenafil shortens cardiac APD90 and the QTc interval, and antagonizes the APD- and QT-prolonging effects of antiarrhythmic agents, presumably through its blocking effect on ICa,L.

Sildenafil is a specific PDE5 inhibitor with a selectivity of 4600-fold over its inhibitory effect on PDE3, a major PDE in the cardiac myocytes [13]. The IC50 of its inhibitory effect on PDE5 is 3.5 nM compared to 16.2 µM for PDE3 [34]. The total plasma concentration after an oral dose of 100 mg for an adult male is between 450 nM [17] and 1 µM (US Prescribing Information, Pfizer, NY, 1998), rising to about 4-fold when ritonavir, an anti-viral protease inhibitor with potent CYP3A4 inhibitory effect, was co-administered [17]. In the present study, we examined the effect of sildenafil in a wide range of concentrations from 10 nM to 30 µM, which, at the upper limit, is equivalent to about 750 times the therapeutic free plasma concentration (30 µM/40 nM). We found lower concentrations of sildenafil did not affect APD but higher concentrations shortened the APD. In all the specimens we had tested (n = 18 for guinea pig papillary muscles, and n = 10 for canine Purkinje fibers), we have not found any one in which sildenafil prolonged the APD90. It is not reasonable to claim that higher doses (≥30 µM) would have different effect since in concentrations higher than 1 µM sildenafil actually decreased APD dose-dependently. In fact, 100 µM sildenafil further decreased APD90 (not shown).

Our results are different from those of other investigators [8]. It is difficult to explain the discrepancy. The major differences were in the methodology. First, we used guinea pig papillary muscles and canine Purkinje fibers, and conventional methods for testing drug effect on APD. Other investigators used MAP recording technique, a popular method in arrhythmia research [22]. Another major difference lies in the pacing cycle lengths. Geelen et al. used cycle lengths of 150–250 ms [8]. One might argue that these pacing rates might be more physiological in guinea pigs. However, in most previous publications studying drug effects on mammalian papillary muscles [18,19,21,35–38], pacing cycle lengths of 300–5000 ms were used, comparable to what we used in the present study. In fact, longer pacing cycle lengths are more likely to induce APD prolongation and early afterdepolarization [12], since most IKr blockers demonstrated reverse frequency-dependent effect [12]. If sildenafil could induce APD prolongation with a pacing cycle length of 250 ms, it could be expected that more pronounced APD prolongation would be observed with a pacing cycle length of 1000 ms.

Based on our findings in the cardiac action potential of two different mammalian species, the whole cell voltage clamp study in guinea pig ventricular myocytes, and the in vivo ECG recordings of guinea pig receiving sildenafil, we suggest that sildenafil does not prolong cardiac repolarization. Actually, in the only two patients in whom ventricular tachyarrhythmias were documented after ingestion of sildenafil, monomorphic ventricular tachycardia, instead of torsade de pointes, was observed [39]. The recent data from an observational cohort study of 9748 patients also did not disclose any patient with torsade de pointes [7]. The cause of increased cardiovascular mortality, if any, remains to be determined.

Although the magnitude of IKr was small relative to fully activated IKs, the two currents were of similar magnitude when measured during a relatively short pulse protocol (250 ms) at membrane potentials (–20 to +20 mV), typical of the plateau phase of cardiac action potential [28,29]. In the present study, we did not observe any inhibitory effect of sildenafil on IK250. But in the same preparations, D-sotalol 30 µM exhibited a 20–30% inhibition that was consistent with previous study [28], suggesting that sildenafil does not block IKr. Our findings are different from those of a previous study [8]. It might be due to different experimental systems being used. IKs represents the major component of IK5000 activating and tail currents [28,29]. With the aid of chromanol 293B, we have verified that sildenafil had no effect on IKs.

The mechanism for the APD-shortening effect of higher concentrations of sildenafil needs to be addressed. The whole configuration of action potential depends on a fine-tuning of different outward and inward ionic currents. Any small decrease in the inward currents or increase in the outward currents would have significant effect on the conformation of action potential when they occur in the plateau phase. Thus, there are several possibilities for the APD-shortening effects of sildenafil. First, very high dose sildenafil cross-reacts with PDE3 and increases intracellular cAMP content that will enhance IK and cAMP-dependent chloride currents (both have APD-shortening effect). Second, sildenafil blocks ICa,L. Third, sildenafil blocks IpNa. In the present study, we did not observe any significant effect of sildenafil on IpNa. However, we found that sildenafil had blocking effect on ICa,L, with an estimated IC50 of 27 µM. We also observed that higher concentrations of sildenafil depressed the plateau of action potential. It is suggested that in a supra-therapeutic range sildenafil blocks ICa,L, shortens APD90, and antagonizes the APD-prolonging effects of antiarrhythmic agents.

In the present study, we did not observe significant changes in the HR, PR interval, and the QRS duration in the guinea pigs receiving sildenafil 10 mg/kg, a dose capable of raising plasma drug level to about three times the therapeutic one (Data on file. Pfizer). However, QTc interval was significantly shortened, a finding parallel to the APD-shortening effect by high dose sildenafil. Although we found an antagonistic effect of high dose sildenafil on the QT-prolonging effect of D,L-sotalol and amiodarone, its clinical significance needs to be determined.

Time for primary review 10 days


   Acknowledgements
 
This study was supported, in part, by Institutional Research Grants from Taipei Veterans General Hospital (VGH 90-067 and VGH 90-300) and by National Science Council (NSC 90-2314-B-075-041). The authors gratefully acknowledge Pfizer for kindly providing sildenafil, and Aventis for chromanol 293B.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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M. E. Werner, A. L. Meredith, R. W. Aldrich, and M. T. Nelson
Hypercontractility and impaired sildenafil relaxations in the BKCa channel deletion model of erectile dysfunction
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R181 - R188.
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