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Cardiovascular Research 1999 44(2):303-314; doi:10.1016/S0008-6363(99)00232-1
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Effects of chronic treatment by amiodarone on transmural heterogeneity of canine ventricular repolarization in vivo: interactions with acute sotalol

Jocelyn Merota,b, Flavien Charpentiera, Jean-Marie Poirierd, Gérard Coutrisc and Jacques Weissenburgerd,*

aLaboratoire de Physiopathologie & Pharmacologie Cellulaires & Moléculaires, INSERM CJF 96-01, Nantes, France
bLaboratoire de Médecine, Ecole Vétérinaire de Nantes, Nantes, France
cService Central de Médecine Nucléaire et Biophysique, Hôpital Saint-Antoine, Paris, France
dService de Pharmacologie, Faculté de Médecine Saint-Antoine, Paris, France

* Corresponding author. Tel.: +33-140-01-1438; fax: +33-140-01-1404 weissenb{at}b3e.jussieu.fr

Received 23 March 1999; accepted 25 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The present study was designed to examine the effects of chronic amiodarone on the different ventricular cell subtypes in situ and to evaluate its interactions with sotalol. Methods: Three groups of dogs were studied. Group I (n=8) received no treatment. Group II (n=7) and group III (n=8) received, respectively, 100 and 200 mg amiodarone orally twice a day for 6 weeks to 8 months. In vivo studies were performed under halothane anesthesia 14 h after the last administration of amiodarone. Three leads ECG, femoral blood pressure and left ventricular intramural monophasic action potentials (MAP) were continuously recorded. Bradycardia was obtained by clamping the sinus node and β-blockade and the heart was driven by atrial pacing. Three weeks before the in vivo experiments, the cellular electrophysiologic properties of right ventricular tissues obtained by cardiac biopsy in six treated and six control dogs were studied with standard microelectrodes. Results: Amiodarone produced a dose-dependent decrease in plasma levels of triiodothyronine (T3; 5.9±0.4 pM in control dogs, 3.1±0.2 pM in group III, P<0.001) without affecting thyroxine (T4). Under anesthesia, the QT interval was 14% larger in group III compared to group I at a paced cycle length (PCL) of 1500 ms (P<0.05). This is consistent with the 10% increase in endocardial MAP duration in group III at the same PCL (P<0.05). There was no significant increase in transmural dispersion of MAP duration. In group I, sotalol induced a significant reverse use-dependent increase in MAP duration. This effect was reduced in group II and completely suppressed in group III. Amiodarone prevented the sotalol-induced increase in transmural dispersion of ventricular repolarization which was 69±12 ms in untreated dogs, 41±8 ms in group II (P<0.05) and 34±8 ms (P<0.05) in group III at PCL=1500 ms. Amiodarone also prevented the sotalol-induced ventricular tachyarrhythmias. In vitro, the action potential duration was longer in amiodarone-treated dogs that in control ones (208±5 ms versus 188±9 ms at PCL=1000 ms, P<0.05).The sotalol-induced prolongation of repolarization was reduced in amiodarone-treated dogs. Conclusion: Chronic treatment of dogs with amiodarone induced a moderate prolongation of the QT interval and MAP duration without affecting transmural dispersion of repolarization and inhibited the effects of acute sotalol, including the prolongation of repolarization, the increase in transmural dispersion of repolarization and the induction of arrhythmias.

KEYWORDS ECG, electrocardiogram; i.v., Intravenous; MAP, monophasic action potentials; NS, not significant; RR, Ventricular cycle length; VF, Ventricular fibrillation; VT, Ventricular tachycardia


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Amiodarone is one of the most potent antiarrhythmic drugs in the treatment of life-threatening arrhythmias. It was first referred to a class III antiarrhythmic agent [1] because early studies had shown that it prolongs action potential duration and effective refractory period of cardiac muscle [2,3]. However, more recent studies have shown that the pharmacological profile of amiodarone is far more complex. It possesses class I Na channel and class IV Ca channel blocking activity [4,5]. It has also non-competitive {alpha}- and β-adrenergic blocking effects and modulates thyroid function and phospholipid metabolism [6–8]. Moreover, its long-term effects differ from its acute ones. Its main acute effect is a depression of atrioventricular node conductivity with minimal effect on the QT interval and the effective refractory periods of atrial and ventricular muscle and of His-Purkinje tissue [9–11]. In contrast, long-term administration of amiodarone lengthens repolarization and refractoriness in most cardiac tissues (atria, ventricles and His-Purkinje system) as a function of time with little or no change in QRS duration (except at faster rates) and a modest increase in atrio-His bundle (AH) or His bundle-ventricular (HV) intervals [9–12]. Amiodarone also decreases sinus rate [10–12] and induces ultrastructural changes in myocardium [13].

Because of its complex electropharmacology, its mode of action is not well understood; nor is the reason for its low proarrhythmic profile compared to more classical class III agents that can induce ‘torsades de pointes’ and ventricular fibrillation [14] despite the very long QT intervals observed in some patients. Moreover, numerous patients who exhibited ‘torsades de pointes’ during sotalol treatment could be safely switched from sotalol to amiodarone [15]. The effects of amiodarone on INa and ICa could limit its class III potency the same way as mexiletine has been found to inhibit sotalol effects on repolarization and proarrhythmia [17,18].

Another interesting and attractive mode of action has been recently proposed. Sicouri et al. [19] have shown that chronic treatment with amiodarone in dogs abolishes transmural heterogeneity of ventricular repolarization, mainly by decreasing M cells action potential duration and preventing the occurrence of EADs. Some of us found similar results in human ventricle [20]. M cells, by developing disproportionate increases in action potential duration at slow rates, have been shown to be responsible for the genesis of EAD-induced triggered activity after exposure to antiarrhythmic agents such as quinidine and D,L-sotalol, but also other QT-prolonging drugs, such as erythromycin [21]. However, these experiments have been performed on isolated ventricular slabs and no information is available on the effects of chronic amiodarone on the electrophysiologic properties of the different ventricular cell types in situ.

The present study was therefore designed to examine the effects of chronic amiodarone on the different ventricular cell subtypes in situ and to examine the interactions between amiodarone and sotalol on transmural repolarization and proarrhythmic potency.


   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).

2.1 In vivo studies
2.1.1 Animals
Three independent groups of dogs were included in the study. Group I (n=8) received no treatment before the experiment. Group II (n=7) and group III (n=8) received, respectively 100 and 200 mg amiodarone orally twice a day (Cordarone®, a gift from Sanofi, Montpellier, France) after a first loading week at twice that dose. The duration of amiodarone treatment was 6 weeks (35±1 days, from 31 to 38) for the first six dogs (three dogs from group II and three from group III). Due to the fact that amiodarone did not produce large enough effects on QT interval in these dogs, the treatment was extended to 8 months (253±9 days, from 220 to 296) for the remaining animals. This prolongation did not produce more effects on baseline ECG. Therefore, the data obtained at 6 weeks were pooled with those obtained at 8 months. Three weeks before the scheduled in vivo experiments, six of these remaining dogs were submitted to a short anesthesia allowing right ventricular biopsy for in vitro experiments.

2.1.2 Instrumentation
In vivo acute studies were performed 14 h after the last administration of amiodarone. Before anesthesia, ECG was recorded in animals kept as quiet as possible and 10 ml of blood were sampled for determination of amiodarone, n-desethylamiodarone, T3, T4 and TSH concentrations.

The twenty-three adult beagles (12.5±1.0 kg; thirteen males and ten females) were anesthetized with i.v. thiopental (30 mg/kg; Nesdonal®, Rhône-Mérieux, France). After tracheal cannulation, anesthesia was maintained by halothane inhalation (0.9 to 1.2%; Halothane Belamont®, Laboratoires Belamont, France), while expiratory CO2 (Beckman, LB2) was adjusted to physiological values by control of the mechanical ventilation (Harvard pump). Three leads ECG (I, II and III) and femoral blood pressure were recorded continuously (Statham P23 XL).

The heart was exposed through a median sternotomy and suspended in a pericardial cradle. Two electrodes were sutured to the epicardial surface of the right atria for atrial pacing. Bradycardia (40 bpm) was obtained by a clamp of the sinus node area associated to a drug-induced β-blockade (propranolol i.v. injected as a bolus of 0.5 mg/kg followed by an infusion of 0.5 mg/kg/h).

Monophasic action potentials (MAPs) were recorded according to the unipolar method described by Nesterenko and co-workers [22,23] and recently validated by Horner et al. [24]. Unipolar MAPs were obtained by differential amplification of the signal obtained between an active electrode and a reference potassium electrode located within the first 2 mm under the epicardial surface at distance from any coronary vessel. Four MAPs were recorded using a 4F multipolar electrode (ATSM, Castelnaudary, France) located in the anterior wall of the left ventricle just after the first main branch of the LAD, close to the septum. The four levels were located at 2 mm intervals along the same axis. Care was taken to locate the most distal pole inside the left ventricular cavity, in immediate proximity to the endocardium. Then, the chest was closed by a plastic sheet and intrathoracic heat was maintained by adjusting the distance of an infra-red heating lamp. MAP signals were filtered (DC-500 Hz), amplified by Gould Universal amplifiers and recorded on paper (at 100 mm/s) and on DAT tapes.

2.1.3 Experimental protocol
The first ECG, blood pressure and MAP recordings were started 30 min after the end of the instrumentation. The cycle length dependency of ventricular repolarization was studied at steady-state by decreasing or increasing the atrial cycle length at 3-min intervals (1500, 1200, 1000, 800, 600 and 400 ms or reverse). The order of variation in stimulation rates was changed every experiment in order to limit the interactions between time effects (up to 15 min for a complete pacing sequence) and cycle length effects.

QT intervals were measured between the beginning of the QRS and the end of the T wave. The lead exhibiting the highest T wave amplitude throughout the experiment was selected a posteriori for all QT measurements (usually the lead II). MAP duration was measured between the activation time (at a position, 50% of the MAP amplitude backward from the MAP activation spike) and the end of the repolarization. This later point was obtained by the intersection between the steepest tangent of phase 3 and the isoelectric line. Transmural dispersion of repolarization was defined as the maximal difference in MAP duration among the four transmural electrodes.

After these baseline measurements, D,L-sotalol (sotalol chlorhydrate, a gift from Bristol, Paris La Défense, France), dissolved and diluted in sterile isotonic saline solution, was administered as a 4.5 mg/kg i.v. injection in 10 min followed by an infusion of 1.5 mg/kg/h maintained for 90 min. The dose of sotalol was chosen in order to obtain plasma levels at the highest third of the therapeutic range in humans. This dose is known in our laboratory to produce severe QT prolongation and arrhythmias in conscious [17,25] as well as halothane-anesthetized dogs [26]. Because the dogs were already β-blocked, the only expected effects of sotalol were limited to its class III effects. Electrophysiological measurements were performed 30 min after starting the perfusion of sotalol. Between the measurements, the heart rate was maintained at a cycle length of 1000 ms.

In addition to this predefined protocol, we continuously monitored ECG and MAP for any unusual events including arrhythmias. After completion of sotalol infusion, the dog was killed with potassium chloride injected in the right ventricle and the heart excised. A 1x1 cm (1 cm3) transmural segment was cut from the left ventricle, close to the area of MAP recordings, and divided in three relatively equal layers parallel to the epicardial surface. The samples were immediately frozen (–80°C or liquid nitrogen). The weights of these samples were: 235±28 mg (epicardium), 284±29 mg (midmyocardium) and 259±32 mg (endocardium).

2.1.4 Amiodarone and hormone determinations
Plasma levels of T3 and T4 were determined using chemiluminescence immunoassay (ACS180, Chiron Diagnostic).

For amiodarone and n-desethylamiodarone (n-DEA) determinations, plasma samples (250 µl) were extracted at pH 5.4 with 5 ml hexane. After a 10-min stirring and centrifugation, the organic layer was decanted and evaporated at 40°C under N2. The residue was reconstituted in mobile phase (hexane—ethanol, 75:25 v/v, containing 0.2% 1.1 M perchloric acid). Amiodarone and n-DEA were assayed with a normal-phase HPLC method using a Lichrosolv Si 100 column (Merck, Nogent-sur-Marne, France). The limit of quantification of the assay was 10 ng/ml.

Tissue levels of amiodarone and n-DEA were measured by the Laboratoire de Biochimie et de Biologie Moléculaire of the University Hospital of Lille (France). Tissue samples were homogenized with an equal volume of distilled water. Amiodarone and n-DEA were extracted with two volumes of acetonitrile (per volume H2O). After a short vortex-mix and centrifugation, the supernatant was evaporated at 70°C under N2. After reconstitution, the residue was analyzed by a reversed-phase HPLC method, according to Jandreski and Vanderslice [27]. This method allowed a limit of quantification of 0.2 µg/g.

2.2 In vitro studies
For this study, six beagle dogs (three males and three females) treated with amiodarone for 8 months and seven control dogs (two beagles and five mongrel dogs; three males and four females) were included.

2.2.1 Endomyocardial biopsies
Dogs were anesthetized with an i.v. bolus of ketamine (0.7 mg/kg; Imalgène®, Rhône-Mérieux, France) and xylazine (0.7 mg/kg; Rompun®, Bayer, Germany). The biotome (Cordis 7F/2.2 mm; Cordis, Viry-Châtillon, France) was advanced to the heart under X-ray control by way of the right jugular vein through a catheter. Cardiac tissue was excised from the right ventricular endomyocardium on each biopsy. Each specimen was immersed quickly in a cold modified Tyrode's solution containing (in mM): NaCl, 120; NaH2PO4, 0.33; MgCl2, 1.1; CaCl2, 2; KCl, 5; glucose, 5; HEPES (N-[2-Hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid]), 10; pH adjusted to 7.4 with NaOH. The number of specimens ranged from four to five in each dog.

2.2.2 Experimental protocols
The tissues were mounted in a Lucite tissue bath perfused with an oxygenated (95% O2–5% CO2) Tyrode's solution warmed to 37±0.5°C and containing (in mM): NaCl, 124; NaHCO3, 27; NaH2PO4, 1.8; KCl, 4; MgCl2, 0.5; CaCl2, 2.7; glucose, 5 (pH 7.4). The flow rate in the tissue chamber was 12 ml/min. Transmembrane recordings were obtained as previously described [28].

The tissues were allowed to recover for at least 1 h before the experiments were started. During this period, they were paced at a cycle length of 1000 ms by field or bipolar stimulation trough teflon-coated silver wire electrodes. Stimulus pulse width was 0.5 to 1.5 ms and the amplitude was twice the diastolic threshold.

To study the effects of pacing on repolarization, the preparations were paced at cycle lengths decreasing from 8000 to 300 ms. Action potential characteristics were measured at steady state for each pacing cycle length. We measured the resting potential (RP), the action potential amplitude (AMP), the maximum upstroke velocity of phase 0 of the action potential (Vmax), the phase 1 and phase 2 amplitudes (differences between RP and the potential at the end of phase 1 or the top of phase 2, respectively) and the action potential duration at 30% (APD30), 50% (APD50), 70% (APD70) and 90% (APD90) of full repolarization.

The same pacing protocol was performed in presence of D,L-sotalol (10–5 and 10–4 M, Sotalex i.v., Laboratoires Allard, Bristol-Myers-Squibbs, France) after 15 min of superfusion. Between the pacing protocols, the tissues were paced at a cycle length of 1000 ms.

2.3 Statistical analysis
Data are expressed as mean±SEM. For in vivo studies, data were analyzed using SPSS version 6.1/98. For each group of dogs, the effect of cycle length and sotalol were analyzed using a multi-factorial analysis of variance (ANOVA). Between-group comparisons were performed using ANOVA, on baseline values and on variations from baseline values. These ANOVA were followed by Student's t-tests and Bonferroni adjustments when necessary to compare pairs of results.

For in vitro studies, statistical analysis was performed with Student's t-test and two-way analysis of variance completed by a t-test for multiple comparisons (Bonferroni procedure) when adequate. Statistical significance was set at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Dogs receiving 100 mg of amiodarone twice a day (group II) had trough plasma levels of amiodarone ranging from 0.11 to 0.59 µg/ml. At twice this dose (group III), they ranged from 0.47 to 1.83 µg/ml. These levels were relatively low with respect to the high doses administered. However, the average level of amiodarone observed with the highest dose was around 1 µg/ml, a value close to that observed in Latini's study [29]. The amiodarone/n-DEA ratio was similar in the two groups, 2.80±0.25 in group II and 2.19±0.35 in group III (NS).

Amiodarone concentrations were 50 times higher in tissues than in plasma (Table 1). A non-uniform distribution of amiodarone within the anterior left ventricular wall was observed at the lower dose. At that dose, amiodarone was 50% more concentrated in the epicardium (20±4 µg/g) than in any other myocardial layers (P<0.05). At the higher dose, epicardial amiodarone level reached 85±12 µg/g, i.e. 90 times more than the corresponding plasma concentration, but the epicardium exhibited only a 12% larger (NS) concentration than the other layers. n-DEA exhibited a much higher tissue/plasma ratio than amiodarone, especially in group III (171±49 vs. 91±23, P<0.05), but the concentrations of n-DEA remained relatively low in both groups (30 to 50% of the parent drug) comparatively to clinical studies, despite the long duration of treatment.


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  		Table 1 Cardiac tissue levels (in ng/mg) of amiodarone and n-desethylamiodaronea

 
Amiodarone treatment produced a dose-dependent decrease in triiodothyronine (T3) without affecting thyroxine (T4). The plasma concentrations of T3 were 5.9±0.4 pM in control animals (group I), 4.3±0.4 pM in dogs from group II (P<0.01 vs. group I) and 3.1±0.2 pM in dogs from group III (P<0.001 vs. both group I and group II). These findings are in agreement with clinical data [30].

3.1 In vivo studies
In all dogs, the arterial blood pressure remained constant throughout the whole experiments with no significant differences between the three groups.

3.1.1 Effects of amiodarone on baseline electrophysiological properties
Before anesthesia, the data collected on surface ECG were similar in all three groups except for sinus rate and QT interval duration (Table 2). Treatment with the highest dose of amiodarone (group III) induced a 47% increase in RR interval. QT interval duration was also significantly increased by amiodarone. However, this last effect can be related to the increase in sinus cycle length since no significant differences could be detected between the three groups on corrected QT intervals (274±8 ms, 277±11 ms and 254±10 ms in groups I, II and III, respectively, Bazett's correction).


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  		Table 2 Effects of anesthesia on QT interval durationa

 
Halothane anesthesia induced a prolongation of the QT interval. At matching RR intervals, QT intervals were larger during anesthesia than in the conscious state (Table 2) in all three groups. At distance from anesthesia induction, QT intervals were significantly longer in group III than in groups I and II (381±15 ms vs. 334±11 ms and 335±10 ms, respectively, at a cycle length of 1500 ms, P<0.05). This 14% difference matched with the 10% longer endocardial MAP duration observed in group III and suggests that the lack of significance of the measurements made before anesthesia may be due to interacting variables like autonomic stress-induced modulations of repolarization.

Fig. 1A shows the effects of amiodarone treatment on the duration of the endocardial MAP at different RR intervals. MAP duration in dogs from group II (100 mg twice daily) did not differ significantly from that in group I. In dogs from group III, we observed a slight increase in MAP duration compared to group I (significant at the two longest cycle lengths; Table 3, Figs. 1A and 3Go) and a small increase in transmural dispersion (NS, Fig. 1B).


Figure 1
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  		Fig. 1 Effects of amiodarone treatment (x-axis) on (A) endocardial monophasic action potential duration (MAPd; y-axis) and (B) transmural dispersion of MAP duration (y-axis), measured at different RR intervals. (*P<0.05 versus control).

 

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  		Table 3 Effects of D,L-sotalol on ventricular repolarization of untreated and amiodarone-treated dogs paced at a cycle length of 1500 msa

 

Figure 3
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  		Fig. 3 ECG (lead II) and transmural MAP recordings obtained in (A) a control dog and (B) an amiodarone-treated dog (group III) at a cycle length of 1500 ms in control conditions (thin lines) an after sotalol infusion (thick lines). EPI, epicardium; subEPI, subepicardium; subENDO, subendocardium; ENDO, endocardium. In the amiodarone-free dog (A), sotalol dramatic effect on MAP duration predominated at the deepest layers while sotalol exhibits only a slight residual effect similar in all layers in the amiodarone-treated animal (B). The hump observed at the end of the MAP recording obtained in the endocardium of a control dog (A thick) after sotalol infusion may be due to early after-depolarizations or to electrotonic interactions with the underlying layers with longer action potentials. The end of the MAP recordings obtained in the epicardium are shown by dotted lines, in control conditions (thin) an after sotalol infusion (thick). Horizontal bars, 500 ms; vertical bars, 1 mV for ECG and 20 mV for MAP.

 
Besides the effects of amiodarone on ventricular repolarization, amiodarone lengthened the atrio-ventricular conduction time. The duration between the atrial stimulation artifact and the ventricular complex was significantly longer in group II (133±9 ms at a cycle length of 1500 ms) and group III (141±10 ms) than in group I (112±4 ms). The difference between groups II and III was not significant. On the other hand, intraventricular conduction was not affected by amiodarone. Transmural conduction time (CT) and QRS duration in the three groups were almost identical (CT was 13±3 ms, 13±2 ms and 12±2 ms; QRS was 31±2 ms, 29±2 ms and 33±2 ms in groups I, II and III, respectively, at a cycle length of 1500 ms).

3.1.2 Electrophysiological interactions with D,L-sotalol
Fig. 2A shows the effects of sotalol on the endocardial MAP duration in the three groups of dogs. In dogs from group I, sotalol induced a significant increase in MAP duration. This effect was markedly reverse use dependent, i.e. it was much larger at longer RR intervals than at short ones. In dogs from group II, the reverse use dependence of sotalol was largely reduced: at 1500 ms, the MAP prolongation was three times less than in group I. At the highest dose, amiodarone almost completely suppressed the effects of sotalol, and this at all RR intervals. For example, during sotalol infusion, the mean endocardial MAP duration was only 374±16 ms in group III, but 473±37 ms in the control group at a cycle length of 1500 ms (P<0.001).


Figure 2
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  		Fig. 2 Sotalol-induced increase in (A) endocardial monophasic action potential duration (MAPd; y-axis) and (B) transmural dispersion of MAP duration (y-axis), as a function of the dose of amiodarone (x-axis) and for different RR intervals. (*P<0.05 versus control).

 
Amiodarone also prevented the sotalol-induced increase in transmural dispersion of ventricular repolarization. Indeed, in control dogs, sotalol increased dispersion of MAP duration more largely as RR intervals increased (Fig. 2B), by increasing mainly endocardial and subendocardial MAP durations (Fig. 3A). This effect of sotalol was considerably reduced in dogs from group II (especially at longer RR intervals) and almost suppressed in dogs from group III (Fig. 3B). During infusion, transmural dispersion was 69±12 ms in untreated dogs, 41±8 ms in group II (P<0.05) and 34±8 ms (P<0.05) in group III at a cycle length of 1500 ms (Table 3).

Following sotalol administration, three out of eight control dogs exhibited ventricular tachyarrhythmias of ‘torsades de pointes’ type (Fig. 4). Arrhythmias were observed in none of the dogs treated with amiodarone.


Figure 4
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  		Fig. 4 Representative torsade de pointes recorded in a control dog after sotalol infusion. (A) ECG (lead II) and blood pressure (BP) recordings of the whole episode. (B and C) ECG and transmural MAP recordings, respectively, before and at the onset of the torsade de pointes. EPI, epicardium; subEPI, subepicardium; subENDO, subendocardium; ENDO, endocardium.

 
3.2 In vitro studies
3.2.1 Electrophysiological characteristics of endomyocardial preparation
Six dogs treated with amiodarone were used for this study. Three were given 100 mg of amiodarone twice daily and the three others were given 200 mg twice daily. Since we did not observe any differences in electrophysiological characteristics of endomyocardial preparations between the two groups, we decided to pool the preparations.

Table 4 summarizes the electrophysiological characteristics of endomyocardial preparations from control and amiodarone-treated dogs at a pacing cycle length of 1000 ms. In control conditions, resting potential, action potential amplitude and Vmax were similar in both groups, suggesting that amiodarone had little effects on the activation phase of the action potential. Conversely, the repolarization phase was modified. Action potential duration at all levels of repolarization was longer in treated dogs than in control ones (although statistical significance was reached only for APD90).


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  		Table 4 Effects of D,L-sotalol on cellular electrophysiological characteristics of canine endomyocardial preparations paced at a cycle length of 1000 msa

 
Fig. 5 shows the effects of pacing cycle length on the repolarization process in control and amiodarone-treated dogs. The amiodarone-induced prolongation of action potential duration was more pronounced at long cycle lengths and was quite absent at short ones. Graphs obtained from treated and control dogs are statistically different (P<0.05, two-way ANOVA).


Figure 5
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  		Fig. 5 Effects of amiodarone on canine endomyocardial repolarization. Effects of pacing cycle length (PCL) on action potential duration at 30, 50, 70 and 90% of full repolarization (APD30, APD50, APD70 and APD90, respectively) of endomyocardial biopsies obtained from control dogs (n=7 samples; circles) and amiodarone-treated dogs (n=8 samples; triangles) under control conditions. (*P<0.05 versus control).

 
3.2.2 Effects of D,L-sotalol
Sotalol (10–5 and 10–4 M) prolonged repolarization in both groups of dogs (Fig. 6A). However, this prolongation was larger in control than in treated dogs. Indeed, under sotalol 10–4 M, the increase in APD90 was 15% in control dogs and only 7% in treated dogs at a cycle length of 1000 ms. This was observed at all cycle lengths studied (Fig. 6B). Consequently, under 10–4 M of sotalol, there was no more difference in APD90 between the two groups of dogs. The other action potential parameters were not modified.


Figure 6
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  		Fig. 6 Effects of sotalol on cellular electrophysiological characteristics of canine ventricular endomyocardium. (A) Superimposed action potentials recorded on endomyocardial biopsies from a control dog (top) and an amiodarone-treated dog (bottom) under control conditions (CTRL) and in presence of sotalol 10–4 M (SOT) at two different pacing cycle lengths: 500 ms (left) and 2000 ms (right). Vertical bars, 50 mV; horizontal bars, 50 ms. (B) Effects of pacing cycle length (PCL; x-axis) on the increase in APD90 induced by sotalol 10–4 M (y-axis) on endomyocardial biopsies of control dogs (circles; n=7 samples) and amiodarone-treated dogs (triangles; n=8 samples). (*P<0.05 versus control).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Our study shows for the first time the effects of long-term amiodarone on repolarization of the different ventricular myocardial cell subtypes in situ. Chronic amiodarone had unexpectedly slight effects on canine ventricular repolarization in basal conditions, but decreased dramatically the prolongation of ventricular repolarization and the increase in transmural dispersion produced by sotalol.

4.1 Amiodarone concentrations
Despite the high dose (about 8 mg/kg twice a day) of amiodarone administered in group II, the plasma concentrations stayed largely below the so-called ‘therapeutic range’ of 1–2.5 mg/l [for review, see [31,32] In Group III, at twice this dose, plasma concentrations reached the lower third of that range. The fact that high doses of amiodarone are necessary in dogs to reach significant plasma levels is a common observation [19,33]. This is usually related to a poor absorption of amiodarone. Another difference with clinical studies is the low level of metabolite concentrations which never exceeded 50% of the parent drug in the plasma as well as in the myocardium. This is consistent with most other papers [29,34,35], except one canine study [36] and is probably related to breed differences in liver enzymes. The difference between clinical and canine studies are even more striking if tissue concentrations are considered. In human, the concentrations of n-DEA are usually 3 to 4 times those of amiodarone [37–39], in sharp contrast with the 50% observed in our dogs.

Tissue amiodarone and n-DEA both contribute to the drug efficacy. Myocardial concentrations of 10–20 µg/g amiodarone were shown to reduce mortality from 91 to 21% in dogs submitted to coronary re-occlusion [36]. In that unique study, n-DEA myocardial concentrations were around 50 µg/g. Because the metabolite shares roughly the same electrophysiological effects than amiodarone, both drugs must be considered for correlation between concentrations and effects. So, with respect to that later study and clinical observation, the tissue concentrations of amiodarone+metabolite that we found in group II were low. Nevertheless, those encountered in group III were clearly within the concentration range found in humans.

4.2 Amiodarone effects on ventricular repolarization
Our results show that chronic amiodarone produced a 10% increase in the QT interval in group III, a result consistent with previous clinical (see [31] for review, [40]) as well as experimental studies [19], and even with the unique study of Anderson et al. [33]. The authors have observed a 10–15% prolongation of ventricular repolarization after a 3-week treatment with amiodarone at doses five times higher than those given in our group III, leading to tissue levels of amiodarone and n-DEA 50% higher than in our study.

The effects of amiodarone on the QT interval are consistent with the 10% increase in MAP duration that we observed at the endocardial and subendocardial layers at the longer pacing cycle lengths Similarly, in our in vitro study, amiodarone induced a 10–15% increase in action potential duration (depending on pacing cycle length). These results are similar to those of Sicouri et al. [19] who found that chronic amiodarone (30–40 mg/kg) prolongs the action potential by 15–20% in endocardium and by 5–10% in epicardium. Similar values (15%) were also found by Papp et al. [41] with 50 mg/kg/day for 6 weeks. Despite the considerable doses of amiodarone (66 to 130 mg/kg/day) given by Anderson et al. [33], they did not obtain more than a 10% increase in epicardial repolarization of anesthetized dogs. Therefore, it seems that in vitro as well as in vivo, action potentials from canine myocardium are only moderately prolonged by chronic amiodarone while much higher sensitivity has been shown in rabbits (+34% with 20 mg/kg for 6 weeks; 2).

Another interesting finding of our study is that chronic amiodarone, in contrast to other class III agents, does not increase transmural heterogeneity. In vitro, chronic amiodarone has no effect or shortens (at longer cycle lengths) action potential duration in Purkinje fibers [41] and in M cells [19], therefore leading to a suppression of intramural heterogeneity of repolarization. Similar results were obtained in human ventricular tissues [20]. There is an apparent contradiction between these results and ours, since amiodarone increased MAP duration in all myocardial layers. A similar contrast between in vitro and in vivo measurements was recently observed with quinidine in dogs [42,43]. The reason for such in vivo/in vitro controversial results remains a matter of debate, but the action potential shortening effect of amiodarone observed in some experimental models is puzzling with respect to the clinical experience.

In human medicine, if amiodarone produces a 10–15% increase in QT intervals in most patients, huge but harmless prolongations are not uncommon after long-term treatments. The reason why these very long QT intervals are not specially alarming remains unknown. Our initial goal was to check the hypothesis of an ‘homogenizing’ effect of amiodarone on transmural dispersion. But as we did not observe such huge prolongations despite extremely long duration of treatment (up to 9 months), an extrapolation of our results to the clinical particularities of huge amiodarone-induced QT prolongations remains speculative.

4.3 Amiodarone prevents sotalol effects on ventricular repolarization
In control dogs, sotalol induced a marked prolongation of repolarization both in vivo and in vitro. In vivo, this prolongation varied among the myocardial layers, leading to a marked increase of the transmural dispersion of repolarization. At the sites of recording, the increase in MAP duration was very much larger in endocardial and subendocardial layers, as illustrated on Fig. 3A. These effects of sotalol were reverse use dependent, i.e. they were larger at long cycle lengths than at short ones (Fig. 2B). They provoked the occurrence of ‘torsades de pointes’ in three out of eight control dogs at longer RR intervals (Fig. 4). Amiodarone prevented the sotalol-induced prolongation of MAP in all myocardial layers and consequently the increase in transmural dispersion of repolarization (Fig. 2B). We observed a slight increase in MAP duration equivalent in all sites of recording (Fig. 3B). Interestingly, this effect was already observed at low doses (in group II), despite an apparent lack of direct effects of amiodarone on repolarization. Papp et al. [41] observed also that amiodarone (5 µM) applied directly to Purkinje fibers abolished totally early afterdepolarizations produced by dofetilide. In endomyocardial biopsies of dogs treated with amiodarone, sotalol increased the action potential duration, less than in the untreated dogs. These results are similar to those obtained by Sicouri et al. [19] using thin slices of ventricular tissue. They showed that amiodarone completely prevented the effects of sotalol in epicardium, and more importantly, in M cells. This latter effect may contribute to the antiarrhythmic action of amiodarone in our model, since M cells are supposed to contribute largely to the development of arrhythmias related to early afterdepolarizations [21].

Propranolol has been shown to reduce transmural heterogeneity and arrhythmogenesis related to β-adrenergic stimulation in an experimental LQT1 model produced by specific IKs-blockade [44]. In the present study, the elevated sympathetic tone produced by open-chest surgery may have been limited by the propranolol-induced β-adrenergic blockade. But in control dogs, propranolol did not avoid sotalol-induced arrhythmias, QT prolongation and MAP transmural dispersion.

The in vitro result of the present study did not totally reproduce the in vivo data. Sotalol produced a large increase in QT interval: 150 ms at BCL=1500 ms (Fig. 2), but only a slight increase in action potential duration on endocardial biopsies: 30 ms at PCL=1000 ms (Fig. 6). Sotalol is known to have dramatic effects on M cells and Purkinje fibers and only these effects were shown to be reversed by amiodarone [19,41]. Endocardial or epicardial cells are much less sensitive [19]. Therefore, the difference between in vivo and in vitro results suggests the participation of M cells to sotalol-effect in our in vivo model.

These antagonistic effects of amiodarone on sotalol are close to those of mexiletine, a class Ib antiarrhythmic drug. In a previous study, some of us have shown that mexiletine antagonizes the effects of sotalol on QT interval and its proarrhythmic effects in a canine model of ‘torsades de pointes’ [17]. Similarly shortening of an abnormally prolonged QT interval was observed in LQT3 patients [45] and could also be obtained in LQT3 models [18]. The same type of interaction has been observed in conscious dogs with quinidine, another sodium channel inhibitor [46]. These results raise the hypothesis that the effects of amiodarone on sodium channels could explain the sotalol-amiodarone interaction on ventricular repolarization [16].

4.4 Clinical and physiological implications
Our study contributes to the understanding of the low incidence of proarrhythmic effects of amiodarone in contrast to other Vaughan—Williams class III agents.

We have shown that chronic amiodarone makes the myocardium relatively insensitive to sotalol effects on ventricular repolarization and on transmural dispersion of repolarization. Dispersion of repolarization is a critical factor in the development of long QT associated repolarization abnormalities and arrhythmias such as ‘torsades de pointes’ [21,47]. M cells contribute largely in the development of these anomalies by generating early afterdepolarizations and providing the substrate for intramural reentry [21,48]. These two mechanisms are exacerbated by drugs having class III properties and bradycardia. A recent study performed in a model of anthopleurin-A-induced long QT syndrome in dogs has shown that a large dispersion of repolarization precipitated the induction of polymorphic ventricular tachycardia resembling ‘torsade de pointes’ [49]. Therefore, the reduction in transmural dispersion observed when sotalol is administered together with amiodarone should provide the substrate of an unexpected antiarrhythmic profile for that paradoxical association.

Of course, the clinical relevance of these results are questionable. So far, such association between two class III antiarrhythmic agents are and will remain prohibited because they share the same effect on ventricular repolarization and therefore should potentiate the QT-dependent proarrhythmic risk. The present study like other studies from our laboratory using different models provide arguments suggesting that such potentiation is far from obvious [17,50]. Amiodarone—quinidine or amiodarone—sotalol combinations have been tested in patients. Long-term amiodarone—quinidine combination offered no advantage over amiodarone alone [51] but potentiation of the proarrhythmic risk was not observed [52]. A single study involved sotalol—amiodarone combination and concluded to the advantage of the combination. The fact that no proarrhythmic effects occurred is meaningless in such small groups (n=13) [53]. These clinical data are too small to allow reasonable speculations except that amiodarone could at least remain a good second choice treatment in patients who exhibited ‘torsades de pointes’ when submitted to other class III agents [15]. Our results provide also some theoretical advantages to drugs with combined mechanisms upon drugs with specific ion current targets like the latest class III agents.

But is there still a need for new antiarrhythmic drugs? Despite the failure of amiodarone to reduce all-cause mortality rates in CAMIAT, EMIAT and CHS-STAT trials, despite the advantage of ICD (implantable cardiac defibrillator) over conventional antiarrhythmic drugs (AVID study) and despite the impressive list of severe side effects produced by amiodarone, that drug remains an acceptable compromise in number of clinical cases at least to prevent the recurrence of VT/VF as demonstrated by the CASCADE trial. Therefore, the need to continue anti-arrhythmic drug development to ameliorate the risk/benefit ratio of these treatments [54] is obvious to date and in the future even if their use could be restricted to ICD recipients to prevent the delivery of electrical shock therapy.

Time for primary review 21 days.


   Acknowledgements
 
This work was supported in part by the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Association de Recherche en Physiopathologie et Pharmacologie (ARPP). We express our gratitude to Eliane Fligiel for her help in performing the in vivo experiments and to Pascal Gervier for his care with the animals. We thank Pr. Jean-Claude Le Nihouannen (DVM) from the Laboratoire de Chirurgie (Ecole Vétérinaire de Nantes, France) for giving us the opportunity to use the equipment necessary to perform cardiac biopsies on dogs.


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

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