Tsalikakis_european Journal Of Pharmacology

European Journal of Pharmacology 564 (2007) 150 – 157 www.elsevier.com/locate/ejphar

Comparative antiarrhythmic efficacy of amiodarone and dronedarone during acute myocardial infarction in rats Maria G. Agelaki a , Constantinos Pantos b,⁎, Panagiotis Korantzopoulos a , Dimitrios G. Tsalikakis c , Giannis G. Baltogiannis a , Andreas Fotopoulos d , Theofilos M. Kolettis a b

a Department of Cardiology, University of Ioannina Medical School, 45110 Ioannina, Greece Department of Pharmacology, University of Athens School of Medicine, 75 Mikras Asias Avenue, 11527 Goudi, Athens, Greece c School of Computer Sciences, University of Ioannina, 45110 Ioannina, Greece d Department of Nuclear Medicine, University of Ioannina Medical School, 45110 Ioannina, Greece

Received 10 November 2006; received in revised form 20 February 2007; accepted 26 February 2007 Available online 7 March 2007

Abstract The effects of dronedarone, a non-iodinated derivative of amiodarone, on ventricular tachycardia and ventricular fibrillation post-myocardial infarction are not well established. Fifty-five Wistar rats were randomly allocated to a 2-week oral treatment with either vehicle (n = 18), amiodarone (30 mg/kg, n = 20), or dronedarone (30 mg/kg, n = 17). After acute coronary artery ligation, a single-lead electrocardiogram was continuously recorded for 24 h and episodes of ventricular tachycardia/fibrillation as well as mortality rates were analysed. Monophasic action potential recordings were obtained from the left ventricular epicardium at baseline and 24 h post-myocardial infarction. Thyroid hormones and catecholamines were measured using radioimmunoassay. Thyroid function was similar in the 3 groups. Compared to controls, amiodarone and dronedarone equally decreased the number of ventricular tachycardia/fibrillation episodes by approximately 75%. Both agents prevented the increase in monophasic action potential duration and in beat-to-beat variation. Norepinephrine levels were lower only after amiodarone treatment. Despite the observed antiarrhythmic effect, total mortality did not differ between groups (38.8% in controls, 30.0% in the amiodarone group and 58.8% in the dronedarone group), because of excess bradyarrhythmic mortality in both drug groups that reached significance in the dronedarone group. Dronedarone and amiodarone display similar antiarrhythmic efficacy post-myocardial infarction, partly by preventing repolarization inhomogeneity. However, dronedarone increases bradyarrhythmic mortality possibly secondary to its negative inotropic effects. © 2007 Elsevier B.V. All rights reserved. Keywords: Acute myocardial infarction; Ventricular arrhythmias; Amiodarone; Dronedarone; Mortality

1. Introduction Amiodarone is one of the most effective antiarrhythmic compounds currently available for the treatment of lifethreatening arrhythmias (Connolly, 1999). Amiodarone primarily inhibits K+ channels, but it also exhibits all other known antiarrhythmic mechanisms, including antiadrenergic activity and inhibition of Na+ and L-type Ca++ channels (Connolly, 1999). In clinical practice, the role of amiodarone is entrenched, but its side-effect profile during chronic administration remains of ⁎ Corresponding author. Tel.: +30 210 7462560; fax: +30 210 779 0841. E-mail address: [email protected] (C. Pantos). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.02.052

concern. The development of thyrotoxicity has been attributed to the iodinated nature of the molecule (Singh, 2004). Dronedarone, (SR 33589; N,N-dibutyl-3-[4-([2-butyl-5-methylsulphonamido] benzofuran-3-yl-carbonyl) phenoxypropylamine) a structurally related non-iodinated benzofuran-derivative of amiodarone, was developed to overcome these toxic effects. However, the issue of amiodarone-induced thyrotoxicity is not fully understood, as an additional iodine-independent mechanism has been suggested (Di Matola et al., 2000). Amiodarone is effective in the treatment of ventricular arrhythmias during acute myocardial infarction (Kodama et al., 1997), but the efficacy of dronedarone in this setting is not well established. Previous studies in anaesthetised animals indicated

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that dronedarone and amiodarone have comparable antiarrhythmic efficacy (Finance et al., 1995; Manning et al., 1995). However, the variety in animal species and anaesthesia protocols utilized introduce limitations in the interpretation of antiarrhythmic effects. More importantly, in these studies, the time window for the observation of ventricular arrhythmias was confined to the immediate post-infarction period. The aim of the present study was twofold: first, to examine the effects of amiodarone and dronedarone on thyroid function; and, second and foremost, to examine the antiarrhythmic efficacy of amiodarone and dronedarone during acute myocardial infarction in the rat model. The rat not only exhibits a high frequency of ischaemic ventricular tachyarrhythmias, but also their occurrence corresponds to the time course observed in humans after acute myocardial infarction (Opitz et al., 1995). We used the conscious rat model that permits the study of ventricular tachyarrhythmias for extended periods of time, without the confounding effects of anaesthesia. To shed light into possible antiarrhythmic mechanisms of the two agents, monophasic action potential was recorded from the lateral left ventricular epicardium under general anaesthesia and plasma catecholamine levels were measured 24 h post-infarction. 2. Materials and methods The study was conducted in female Wistar rats, 20 ± 1 weeks old. The animals received humane care, the investigation complies with international guidelines and the protocol was approved by the local national authority. All rats were housed in individual cages, in a temperature controlled environment (21 ± 1 °C), with a 12:12-h light–dark cycle and were given water and standard rat chow ad libitum. 2.1. Drug administration The rats were randomized to control, amiodarone, or dronedarone groups. Both drugs were kindly provided by Sanofi-Aventis, Montpellier, France and were administered by gavage, once daily for 2 weeks, at a dose of 30 mg/kg. Before each administration, a fresh solution was prepared in 0.6% methylcellulose to obtain the necessary drug concentration in 3 ml. In the control group, 0.6% methylcellulose alone was given for the same time period. The rats were weighed at the beginning and at the end of the drug administration period. 2.2. Implantation of telemetry transmitter On the fourteenth day of drug administration, a continuous electrocardiogram telemetry transmitter (Dataquest, Data Sciences International, Transoma Medical, Arden Hills, MN, USA) was implanted in the abdominal cavity, using a previously described method (Opitz et al., 1995). The animals were intubated and mechanically ventilated (ventilator model 7025, Ugo Basile, Comerio, VA, Italy) and anaesthetised with isoflurane. The transmitter was secured in the abdominal cavity; the leads were tunnelled under the skin and attached to the underlying tissue. The positive electrode was sutured in a V4–V5 position and the

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negative electrode under the right axilla. The rats were housed in individual cages, placed on a receiver that continuously captured the signal, independently of animal activity. The signal was displayed in real-time with the use of a computer program (A.R.T. 2.2, Dataquest, Data Sciences International, Transoma Medical, Arden Hills, MN, USA) and stored for analysis. 2.3. Monophasic action potential recordings Twenty-four hours after telemetry transmitter implantation, the rats were re-anaesthetised and a left thoracotomy was performed, allowing dissection of the pectoral muscles. The heart was exposed and the pericardium was carefully removed. The method used in our laboratory for monophasic action potential recordings has been described previously (Baltogiannis et al., 2005). A probe (model 200, EP Technologies, Sunnyvale, CA, USA) was placed on the lateral left ventricular wall. The signal was amplified with the use of a preamplifier (model 300, EP Technologies, Sunnyvale, CA, USA) and filtered at 50 Hz using a digital notch filter (for elimination of power line interference). The signal was further filtered using a band pass filter, allowing a signal range between 0.05 Hz and 500 Hz. Two-minute recordings were stored into a personal computer, equipped with an analog-todigital converter (BNC 2110, National Instruments Corporation, Dallas, TX, USA). This recording duration is optimal, since it provides sufficient number of analysable beats, whilst causing minimal interference with the experimental procedure. The software utilized in this study, developed and validated in our Institution (Tsalikakis et al., 2003), permits recording and off-line analysis. For purposes of this study, the action potential duration at 90% of repolarization was measured at baseline and 24 h after acute coronary artery ligation. During analysis, non-sinus beats were excluded and 50 consecutive sinus beats per recording were analysed. The standard deviation of action potential duration at 90% of repolarization was calculated for each recording, as a measure of beat-to-beat variation, indicating electrical alternans (Franz, 1999). 2.4. Generation of acute myocardial infarction and blood sample collection Coronary artery ligation was performed, as described previously (Pfeffer et al., 1979), by an operator blinded to treatment assignment. The left coronary artery was encircled and ligated using a 6-0 suture, placed between the pulmonary artery cone and the left atrial appendage; following these anatomical landmarks ensures generation of similar infarct size in all experiments. The incision was sutured in two layers and the remaining air was aspirated from the thorax, allowing the resumption of spontaneous respiration. A six-lead electrocardiogram was obtained and ST-segment elevation was considered a proof of induced infarction. The animals were returned to their cage and recording was continued for 24 h, or until spontaneous death. Bradyarrhythmic death occurring during the first 5 min post-ligation was attributed to the surgical procedure and these animals were excluded from the study. No resuscitation attempts were allowed at any time during the study.

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Twenty-four hours post-ligation, the survivors were reanaesthetised, blood was collected by internal jugular venous puncture, centrifuged immediately and the serum was stored at − 20 °C. The site of previous left thoracotomy was re-opened and monophasic action potential recordings were repeated at the same epicardial sites. The rats were subsequently sacrificed using a lethal dose of potassium chloride and the heart was harvested for measurement of infarct size. 2.5. Infarct size The method used for measurement of infarct size has been described previously (Ytrehus et al., 1994). The heart was excised, frozen (in −20 °C for 1 h), hand-cut in five 2 mm slices, incubated (in triphenyltetrazolium chloride for 15 min at 37 °C) and fixed (in 10% formalin for 20 min). The slices were scanned and the areas of infarcted and non-infarcted myocardium were measured from both slice sides and averaged. Planimetry was performed using a previously validated software program (Image Tool, University of Texas, USA). The measured areas were multiplied by slice thickness to determine the volumes of infarcted and non-infarcted myocardium for each slice. These values were summed and infarct size (expressed as a percentage) was defined as the ratio between infarcted and total left ventricular volume. 2.6. Measurement of thyroid hormone and catecholamine serum levels Serum levels of thyroxine, triiodothyronine, thyroid stimulating hormone, epinephrine and norepinephrine were measured using radioimmunoassay kits, obtained from BioSource Europe S.A., Nivelles, Belgium. Antibodies to thyroperoxidase and thyroglobulin were measured using radioimmunoassay kits, obtained from BRAHMS Aktiengesellschaft, Henningsdorf, Germany. 2.7. Sinus heart rate We analysed continuous 5-min electrocardiogram recordings, from which non-sinus beats were excluded. Sinus heart rate was calculated from the mean value of these RR intervals at baseline, at the 5th, 30th and 60th minute post-ligation and hourly thereafter. 2.8. Arrhythmia analysis The acquired electrocardiogram tracings were displayed and analysed off-line independently by two of the authors, blinded to treatment assignment. We report the number of ventricular tachycardia and ventricular fibrillation episodes, according to the guidelines provided by the Lambeth Conventions for determination of experimental arrhythmias (Walker et al., 1988). The duration of each ventricular tachycardia or fibrillation episode was measured using the time-scale provided by the recording software. Ventricular tachycardia was defined as 4 or more consecutive ventricular ectopic beats, and ventricular fibrillation as a signal in which individual QRS deflections could not easily be distinguished from one another. Even with these guidelines,

separating ventricular tachycardia from fibrillation was often difficult, and this has been the experience of others (Opitz et al., 1995). Therefore, in this study, we report ventricular tachycardia and fibrillation collectively, as ventricular tachyarrhythmias. For each rat, ventricular tachyarrhythmias were divided by the actual survival time (i.e. the time at risk for experiencing a tachyarrhythmia) and are reported as number of episodes per h alive. Since different mechanisms have been suggested to account for ventricular tachyarrhythmias occurring during the first and the following hours after coronary occlusion, the number of ventricular tachyarrhythmia episodes (per h alive) is also reported separately for phase I (defined as the first hour post-ligation) and for phase II (defined as the time interval from the 61st minute to the end of the recording or to spontaneous death) (ClementsJewery et al., 2005). Tachyarrhythmic death was defined as ventricular asystole, preceded by a sustained ventricular tachyarrhythmia episode and bradyarrhythmic death as ventricular asystole, preceded by bradycardia (b 200 bpm) associated with complete heart block. 2.9. Statistical analysis All values are given as mean ± S.E.M. Student's t-test was used for the comparison of thyroid hormones between animals with different weight responses in the amiodarone group. Differences in continuous variables between the 3 groups were compared using one-way analysis of variance, followed by posthoc Tukey's multiple comparisons test. To assess changes of continuous variables over time, we used analysis of variance for repeated measures and we report F-values, degrees of freedom, and P-values of main effects. The continuous variables describing the arrhythmia frequencies were not normally distributed and were compared using the Kruskal–Wallis analysis of variance. This test was used also for assessment of variance in mortality rates; if present, differences between groups were compared with chi square (after Yates' correction). Statistical significance was defined at an alpha level of 0.05. 3. Results We studied 60 female Wistar rats, weighing 213± 2 g. Of these, 2 rats died during telemetry transmitter implantation and 3 during ligation. Thus, the final study population consisted of 55 animals, of which 18 (218 ± 3 g) were allocated vehicle, 20 (214± 6 g) amiodarone and 17 (205 ± 4 g) dronedarone. After the 14-day treatment period, the body weight increased by 10.0± 3.6 g in controls, by 1.3 ± 2.8 g in the amiodarone group and by 11.4± 5.7 g in the dronedarone group. Differences between groups failed to reach statistical significance (F = 2.31, degrees of freedom = 2, P = 0.11). The lower mean increase in the amiodarone group (albeit statistically insignificant) was due to weight loss, observed in 3 rats during treatment. 3.1. Sinus heart rate There was a statistical variance (F = 11.6, degrees of freedom = 2, P b 0.001) in baseline heart rate, being higher in

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Fig. 1. Heart rate post-myocardial infarction in the three groups. Note the differences between dronedarone and control after 15th hour and between amiodarone and control after the 18th hour.

controls (345 ± 9 bpm), compared to either the amiodarone group (300 ± 6 bpm, P b 0.001), or to the dronedarone group (300 ± 5 bpm, P b 0.001). A statistical variance (F = 43.03, degrees of freedom = 17, P b 0.0001) was found in heart rate until the 15th hour post-ligation, without an effect between groups (F = 0.17, degrees of freedom = 2, P = 0.84). Subsequently, and until the end of the observation period, heart rate in the dronedarone group was higher (P b 0.030), compared to the control group. Higher (P b 0.035) heart rate was found for the amiodarone group compared to controls at the 18th hour postligation until the end of the observation period. No differences were found between amiodarone and dronedarone at any time during the entire observational period (Fig. 1). 3.2. Infarct size Mean infarct size was 38.3 ± 1.4% in the control group, 37.8 ± 1.1% in the amiodarone group and 39.9 ± 1.8% in the dronedarone group, without significant differences between groups (F = 0.48, degrees of freedom = 2, P = 0.62). 3.3. Ventricular tachyarrhythmias A statistical variance was present between groups (H = 11.88, degrees of freedom = 2, P = 0.0026), due to significantly fewer ventricular tachyarrhythmia episodes in the amiodarone group (4.0 ± 2.0 episodes/h, P = 0.022) and in the dronedarone group (3.6 ± 2.4 episodes/h, P = 0.023), compared to controls (16.2 ± 4.5 episodes/h). However, no statistical variance was found in the mean episode duration (6.9 ± 1.7 s in controls, 4.7 ± 1.1 s in the amiodarone group and 12.4 ± 8.6 s in the dronedarone group, H = 0.51, degrees of freedom = 2, P = 0.77). The decrease in the number of episodes (per h alive) was evident during both post-ligation phases (phases I and II, as defined above). There was a significant variance in the number of ventricular tachyarrhythmia episodes (per h alive) during phase I (H = 9.81, degrees of freedom = 2, P = 0.0074). During this phase, there were 4.7 ± 2.2 episodes in controls, 0.4 ± 0.4 in amiodaroneand 0.1 ± 0.1 in dronedarone-treated rats. A variance was also present during phase II (H = 6.52, degrees of freedom = 2, P = 0.038), the number of episodes being 278.0 ± 117.6 in

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Fig. 2. Ventricular tachycardia (VT) and ventricular fibrillation (VF) episodes in the three groups.

controls, 86.1 ± 45.4 in the amiodarone group and 62.1 ± 50.5 in the dronedarone group. Fig. 2 depicts the hourly distribution of ventricular tachyarrhythmia episodes during both phases. 3.4. Monophasic action potential duration There was a statistical variance in left ventricular action potential duration at 90% of repolarization (F = 25.62, degrees of freedom = 2, P b 0.0001), that was due to an increase (P b 0.001) in controls 24 h post-ligation, compared to baseline. In contrast, no significant changes were observed over time in any drug group (Table 1). Furthermore, a statistical variance was found in beat-to-beat variability of left ventricular action potential duration (F = 7.46, degrees of freedom = 2, P = 0.0035), due to an increase (P = 0.0030) 24 h post-ligation (compared to baseline) in controls. No significant changes were observed over time in any drug group (Table 1). 3.5. Thyroid hormones and thyroid antibodies No differences were found in triiodothyronine (F = 0.19, degrees of freedom = 2, P = 0.82), thyroxine (F = 0.59, degrees of freedom = 2, P = 0.55), or thyroid stimulating hormone (F = 0.77, degrees of freedom = 2, P = 0.46) levels 24 h postligation between groups. In addition, there was no indication of thyroiditis, as evidenced by comparable levels of anti-thyroperoxidase (F = 0.93, degrees of freedom = 2, P = 0.40) and antithyroglobulin (F = 0.88, degrees of freedom = 2, P = 0.42) between groups (Table 2). However, there was a strong trend (P = 0.051) towards a higher thyroxine/triiodothyronine ratio in the 3 rats that

Table 1 Action potential duration at 90% of repolarization (APD90) and beat-to-beat variation in APD90 in the three groups Control APD90 baseline (ms) 93.1 ± 4.7 APD90 24 h (ms) 120.2 ± 5.2a Beat-to-beat variation baseline (ms) 4.14 ± 0.45 Beat-to-beat variation 24 h (ms) 11.85 ± 2.92a P b 0.01 compared to baseline.

a

Amiodarone Dronedarone 88.2 ± 2.4 85.4 ± 2.1 3.30 ± 0.21 2.70 ± 0.21

87.5 ± 2.5 88.5 ± 3.0 3.14 ± 0.26 3.28 ± 0.28

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Table 2 Serum levels of triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), and thyroperoxidase (Anti-TPO), and thyroglobulin (Anti-Tgn) antibodies in the 3 groups

T3 (nmols/l) T4 (nmols/l) TSH (μIU/ml) Anti-TPO (μIU/ml) Anti-Tgn (μIU/ml)

Control

Amiodarone

Dronedarone

3.11 ± 0.27 99.50 ± 4.97 0.41 ± 0.03 62.33 ± 2.55 Not detectable

3.20 ± 0.31 91.42 ± 5.56 0.47 ± 0.03 57.18 ± 3.18 0.03 ± 0.02

3.41 ± 0.25 94.64 ± 6.10 0.46 ± 0.04 57.84 ± 2.19 Not detectable

No significant differences were found.

lost weight (33.48 ± 2.38) during amiodarone treatment, compared to the remaining animals in the same group (26.99 ± 1.44).

4. Discussion The strategies for the treatment of life-threatening ventricular tachyarrhythmias have changed markedly during the past years. The use of class I antiarrhythmic agents has declined, because of increased proarrhythmia rates, and class III agents have become the mainstay of pharmacologic therapy (Wellens, 2004). During the past decade, there has been an active pharmacological research towards the development of class III agents that will combine the efficacy and low proarrhythmia rates of the prototype drug amiodarone, but without its untoward effects (Khan, 2004). In the present study, we investigated the effects of amiodarone and dronedarone on thyroid function and on ventricular tachyarrhythmias during acute coronary ligation in rats.

3.6. Catecholamines Epinephrine levels 24 h post-ligation were 5.81 ± 0.72 μg/l in controls, 7.38 ± 2.05 μg/l in the amiodarone group and 11.16 ± 4.16 μg/l in the dronedarone group, without significant differences between groups (F = 1.44, degrees of freedom = 2, P = 0.25). In contrast, norepinephrine levels 24 h post-ligation displayed a significant variance (F = 7.83, degrees of freedom = 2, P = 0.0022), that was due to lower (P = 0.0017) levels in the amiodarone group (23.13 ± 8.80 μg/l), compared to controls (74.60 ± 2.30 μg/l). Norepinephrine levels in the dronedarone group (59.55 ± 19.43 μg/l) were comparable (P = 0.54) to controls. 3.7. Mortality 3.7.1. Total mortality During the 24-h observational period, total mortality was 38.8% (7/18 rats) in the control group, 30% (6/20 rats) in the amiodarone group and 58.8% (10/17 rats) in the dronedarone group. These differences did not reach statistical significance (H = 3.17, degrees of freedom = 2, P = 0.20). However, there were significant differences in the mode of death. 3.7.2. Tachyarrhythmic mortality No rat (0%) in the amiodarone group died of ventricular tachyarrhythmia and this percentage was lower (P = 0.017) compared to controls (6 rats, 33.3%, 2 during phase I and 4 during phase II). In the dronedarone group, 2 (11.7%) rats (both during phase II) died of ventricular tachyarrhythmia, this rate being comparable (P = 0.26) to controls. 3.7.3. Bradyarrhythmic mortality There was excess bradyarrhythmic mortality after treatment with either amiodarone or dronedarone that reached statistical significance only in the latter group. All bradyarrhythmic deaths occurred during phase II and were observed in 1 (5.5%) control rat (5.5 h post-ligation) and in 6 (30.0%) rats (17.9 ± 1.1 h postligation) from the amiodarone group (P = 0.11). Compared to controls, the incidence of bradyarrhythmic deaths was higher (P = 0.015) in the dronedarone group (8 rats or 47.0%, 12.5 ± 1.2 h post-ligation, earlier (P = 0.0079) compared to bradyarrhythmic mortality in the amiodarone group).

4.1. Effects of dronedarone and amiodarone on thyroid function We confirm the previously reported (Pantos et al., 2002) lack of a significant effect on thyroid hormone levels after chronic dronedarone administration. Previous studies on chronic amiodarone administration in rats found a modest decrease in plasma levels of triiodothyronine, accompanied by an increase in serum thyroxine (Aanderud et al., 1984; Kodama et al., 1997; Sogol et al., 1983). In accordance with these studies, we report the development of hyperthyroidism in 3 animals, evidenced by a higher thyroxine/triiodothyronine ratio. However, the overall changes in body weight and in thyroid function indices failed to reach statistical significance between groups. The explanation for the less pronounced effect of amiodarone on thyroid function in our animal population may be twofold: First, a lower dose, shorter treatment duration and/or shorter follow-up period was present in our study. Second, we measured thyroid hormones 24 h post-ligation; a considerable amount of evidence (Klein and Ojamaa, 2001; Pantos et al., 2004) indicates that thyroid hormone levels decline during acute infarction, as an adaptive response to preserve energy homeostasis. Thus, any potential differences induced by treatment with amiodarone might have been eliminated in the post-ligation period. 4.2. Antiarrhythmic efficacy In our study, a comparable antiarrhythmic effect was found for amiodarone and dronedarone. Both drugs decreased the number of ventricular tachyarrhythmia episodes, without affecting the mean episode duration. Interestingly, their antiarrhythmic action appears to be prominent during both post-infarction phases. Although both agents are thought to act mainly by prolonging repolarization, no increase in the action potential duration was observed in our study, prior to infarct generation. Species differences may account for this finding, since ventricular repolarization in rats depends primarily on a large transient outward K+ current (Cheng and Kodama, 2004). Thus, blockade of the delayed rectifier K+ current by amiodarone may not prolong repolarization (Rees and Curtis, 1993). Indeed, variable results have been reported after amiodarone (Kadoya et al., 1985; Kodama et al., 1992) or dronedarone administration (van Opstal

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Fig. 3. Example of bradyarrhythmic death. Note the abrupt onset of bradycardia, following sinus tachycardia.

et al., 2001; Varro et al., 2001) and may be dose-dependent (Le Bouter et al., 2004). The most important novelty of our study is the extension of the post-infarction observation period, to include the assessment of phase II ventricular tachyarrhythmias. Their onset occurs after a quiescent period and coincides with the gradual transition of reversible into irreversible myocardial injury (Clements-Jewery et al., 2005). Thus, the reduction in phase II tachyarrhythmias after either amiodarone or dronedarone treatment, may be secondary (a) to the ‘pure’ antiarrhythmic action and (b) to cardioprotective effects of these agents, leading to a reduction of border zone myocardial ischaemia. Most phase II tachyarrhythmias originate from this zone (i.e., between infarcted and normal myocardium), due to changes in the electrophysiological milieu, favouring re-entry and abnormal automaticity (Clements-Jewery et al., 2005). The possible anti-ischaemic properties of amiodarone include coronary vasodilation (Guiraudou et al., 2004) and scavenging of oxygen free radicals (Ide et al., 1999). Dronedarone is also a coronary vasodilator (Guiraudou et al., 2004), but this agent may exert unique cardioprotective actions, by inhibiting 3,5,3′-triiodothyronine binding to thyroid receptor-alpha-1 (Pantos et al., 2005a,b). These actions may decrease intracellular Ca2+ content (Pantos et al., 2005a,b), an important regulator of ischaemia-induced ventricular tachyarrhythmias (Priori and Napolitano, 2005). Our study provides evidence in support of the cardioprotective effects of both, amiodarone and dronedarone. In controls, we found a prolongation of left ventricular repolarization and electrical alternans 24 h post-ligation. Both these characteristics, indicative of ischaemia and increased arrhythmogenesis (Franz, 1999; Horacek et al., 1984; Mohabir et al., 1991), were absent in both drug groups. Similar anti-ischaemic action, evidenced by action potential preservation, was reported for amiodarone and dronedarone in the ischaemic isolated rat heart (Rochetaing et al., 2001). Thus, the decrease in temporal dispersion of ventricular repolarization after infarction, either as a direct electrophysiologic action, or indirectly, as an anti-ischaemic effect, is an important antiarrhythmic mechanism, common for both agents.

Another interesting finding of the present study was the decreased norepinephrine plasma concentrations in amiodarone-, but not in dronedarone-treated rats. This finding is in agreement with the previously reported partial norepinephrine depletion in the heart after amiodarone administration (Du et al., 1995). Amiodarone enhances intraneuronal norepinephrine metabolism, leading to an impaired release during sympathetic activation. (Du et al., 1995). This represents an additional antiarrhythmic mechanism, unique for amiodarone, and deserves further study. 4.3. Bradyarrhythmic mortality We report a trend towards an increase in bradyarrhythmic deaths after chronic amiodarone treatment. In the rat model, bradyarrhythmic mortality is generally considered representative of death due to heart failure (Opitz et al., 1995). Indeed, in our experiments, all these deaths were preceded by sinus tachycardia, followed by an abrupt onset of complete atrioventricular block and ventricular asystole (Fig. 3). These observations may be secondary to the negative inotropic effects of amiodarone (Drvota et al., 1999) and are compatible with findings in clinical studies, where increased mortality was reported after chronic amiodarone treatment in patients with heart failure (Bardy et al., 2005). A prominent finding of our study was the significant increase in bradyarrhythmic mortality after chronic dronedarone administration, displaying identical electrocardiographic features to those observed in the amiodarone group. Dronedarone, in addition to its (common with amiodarone) antiadrenergic and negative inotropic effects (Djandjighian et al., 2000), antagonises thyroid hormone binding to receptor-alpha-1 and downregulates the expression of alpha myocin heavy chain. These effects may further deteriorate left ventricular function, especially in the acute infarction setting, where thyroid hormone signalling is altered (Pantos et al., 2005a,b). Our results are consistent with findings in the clinical ANDROMEDA trial that was prematurely terminated, due to a possible increased risk of death due to heart failure.

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4.4. Limitations We feel that our study advances current thinking on the pharmacologic management of ventricular tachyarrhythmias during acute myocardial infarction. However, some potential limitations should be acknowledged. First, plasma and/or tissue levels of the drugs were not assessed. Second, no evaluation of left ventricular function was performed. Lastly, a synergistic effect of amiodarone and anaesthesia on left ventricular dysfunction has been described (Rooney et al., 1995), that might have interfered with monophasic action potential measurements. However, these effects are very short-lived and unlikely to have confounded our overall results. 4.5. Conclusions Chronic oral amiodarone or dronedarone administration decreases ventricular tachyarrhythmias in the rat model of acute myocardial infarction. However, dronedarone increases bradyarrhythmic mortality, most likely attributable to heart failure. Future studies should evaluate whether lower pharmacological dosages of these agents would maintain the antiarrhythmic effect, but without the untoward bradyarrhythmic effects. Acknowledgements We wish to thank Sanofi-Aventis, Montpellier, France, for providing amiodarone and dronedarone and Boston Scientific/ Iatriki Efzin, Athens, Greece, for offering the MAP probe and preamplifier. The contribution of the Cardiovascular Research Institute, Ioannina and Athens, Greece, is acknowledged. Anastasia Alevizatou, RN, provided valuable help during the experiments and Tzihad Albouharali, MD, performed all radioimmunoassay measurements. Eleni Goga, MSc, was an excellent research coordinator. References Aanderud, S., Sundsfjord, J., Aarbakke, J., 1984. Amiodarone inhibits the conversion of thyroxine to triiodothyronine in isolated rat hepatocytes. Endocrinology 115, 1605–1608. Baltogiannis, G.G., Tsalikakis, D.G., Mitsi, A.C., Hatzistergos, K.E., Elaiopoulos, D., Fotiadis, D.I., Kyriakides, Z.S., Kolettis, T.M., 2005. Endothelin receptorA blockade decreases ventricular arrhythmias after myocardial infarction in rats. Cardiovasc. Res. 67, 647–654. Bardy, G.H., Lee, K.L., Mark, D.B., Poole, J.E., Packer, D.L., Boineau, R., Domanski, M., Troutman, C., Anderson, J., Johnson, G., McNulty, S.E., ClappChanning, N., Davidson-Ray, L.D., Fraulo, E.S., Fishbein, D.P., Luceri, R.M., Ip, J.H., 2005. Amiodarone or an implantable cardioverter-defibrillator for congestive heart failure. N. Engl. J. Med. 352, 225–237. Cheng, J.H., Kodama, I., 2004. Two components of delayed rectifier K+ current in heart: molecular basis, functional diversity, and contribution to repolarization. Acta Pharmacol. Sin. 25, 137–145. Clements-Jewery, H., Hearse, D.J., Curtis, M.J., 2005. Phase 2 ventricular arrhythmias in acute myocardial infarction: a neglected target for therapeutic antiarrhythmic drug development and for safety pharmacology evaluation. Br. J. Pharmacol. 145, 551–564. Connolly, S.J., 1999. Evidence-based analysis of amiodarone efficacy and safety. Circulation 100, 2025–2034. Di Matola, T., D'Ascoli, F., Fenzi, G., Rossi, G., Martino, E., Bogazzi, F., Vitale, M., 2000. Amiodarone induces cytochrome c release and apoptosis through

an iodine-independent mechanism. J. Clin. Endocrinol. Metab. 85, 4323–4330. Djandjighian, L., Planchenault, J., Finance, O., Pastor, G., Gautier, P., Nisato, D., 2000. Hemodynamic and antiadrenergic effects of dronedarone and amiodarone in animals with a healed myocardial infarction. J. Cardiovasc. Pharmacol. 36, 376–383. Drvota, V., Haggblad, J., Blange, I., Magnusson, Y., Sylven, S., 1999. The effect of amiodarone on the beta-adrenergic receptor is due to a downregulation of receptor protein and not to a receptor–ligand interaction. Biochem. Biophys. Res. Commun. 255, 515–520. Du, X.J., Esler, M.D., Dart, A.M., 1995. Sympatholytic action of intravenous amiodarone in the rat heart. Circulation 91, 462–470. Finance, O., Manning, A., Chatelain, P., 1995. Effects of a new amiodarone-like agent, SR 33589, in comparison to amiodarone, d,l-sotalol, and lignocaine, on ischemia-induced ventricular arrhythmias in anesthetized pigs. J. Cardiovasc. Pharmacol. 26, 570–576. Franz, M.R., 1999. Current status of monophasic action potential recording: theories, measurements and interpretations. Cardiovasc. Res. 41, 25–40. Guiraudou, P., Pucheu, S.C., Gayraud, R., Gautier, P., Roccon, A., Herbert, J.M., Nisato, D., 2004. Involvement of nitric oxide in amiodarone- and dronedaroneinduced coronary vasodilation in guinea pig heart. Eur. J. Pharmacol. 496, 119–127. Horacek, T., Neumann, M., von Mutius, S., Budden, M., Meesmann, W., 1984. Nonhomogeneous electrophysiological changes and the bimodal distribution of early ventricular arrhythmias during acute coronary artery occlusion. Basic Res. Cardiol. 79, 649–667. Ide, T., Tsutsui, H., Kinugawa, S., Utsumi, H., Takeshita, A., 1999. Amiodarone protects cardiac myocytes against oxidative injury by its free radical scavenging action. Circulation 100, 690–692. Kadoya, M., Konishi, T., Tamamura, T., Ikeguchi, S., Hashimoto, S., Kawai, C., 1985. Electrophysiological effects of amiodarone on isolated rabbit heart muscles. J. Cardiovasc. Pharmacol. 7, 643–648. Khan, M.H., 2004. Oral class III antiarrhythmics: what is new? Curr. Opin. Cardiol. 19, 47–51. Klein, I., Ojamaa, K., 2001. Thyroid hormone-targeting the heart. Endocrinology 142, 11–12. Kodama, I., Suzuki, R., Kamiya, K., Iwata, H., Toyama, J., 1992. Effects of longterm oral administration of amiodarone on the electromechanical performance of rabbit ventricular muscle. Br. J. Pharmacol. 107, 502–509. Kodama, I., Kamiya, K., Toyama, J., 1997. Cellular electropharmacology of amiodarone. Cardiovasc. Res. 35, 13–29. Le Bouter, S., El Harchi, A., Marionneau, C., Bellocq, C., Chambellan, A., van Veen, T., Boixel, C., Gavillet, B., Abriel, H., Le Quang, K., Chevalier, J.C., Lande, G., Leger, J.J., Charpentier, F., Escande, D., Demolombe, S., 2004. Long-term amiodarone administration remodels expression of ion channel transcripts in the mouse heart. Circulation 110, 3028–3035. Manning, A.S., Bruyninckx, C., Ramboux, J., Chatelain, P., 1995. SR 33589, a new amiodarone-like agent: effect on ischemia- and reperfusion-induced arrhythmias in anesthetized rats. J. Cardiovasc. Pharmacol. 26, 453–461. Mohabir, R., Franz, M.R., Clusin, W.T., 1991. In vivo electrophysiological detection of myocardial ischemia through monophasic action potential recording. Prog. Cardiovasc. Dis. 34, 15–28. Opitz, C.F., Mitchell, G.F., Pfeffer, M.A., Pfeffer, J.M., 1995. Arrhythmias and death after coronary artery occlusion in the rat. Continuous telemetric ECG monitoring in conscious, untethered rats. Circulation 92, 253–261. Pantos, C., Mourouzis, I., Delbruyere, M., Malliopoulou, V., Tzeis, S., Cokkinos, D.D., Nikitas, N., Carageorgiou, H., Varonos, D., Cokkinos, D., Nisato, D., 2002. Effects of dronedarone and amiodarone on plasma thyroid hormones and on the basal and postischemic performance of the isolated rat heart. Eur. J. Pharmacol. 444, 191–196. Pantos, C., Malliopoulou, V., Varonos, D.D., Cokkinos, D.V., 2004. Thyroid hormone and phenotypes of cardioprotection. Basic Res. Cardiol. 99, 101–120. Pantos, C., Mourouzis, I., Malliopoulou, V., Paizis, I., Tzeis, S., Moraitis, P., Sfakianoudis, K., Varonos, D.D., Cokkinos, D.V., 2005a. Dronedarone administration prevents body weight gain and increases tolerance of the heart to ischemic stress: a possible involvement of thyroid hormone receptor alpha1. Thyroid 15, 16–23.

M.G. Agelaki et al. / European Journal of Pharmacology 564 (2007) 150–157 Pantos, C., Mourouzis, I., Saranteas, T., Paizis, I., Xinaris, C., Malliopoulou, V., Cokkinos, D.V., 2005b. Thyroid hormone receptors alpha1 and beta1 are downregulated in the post-infarcted rat heart: consequences on the response to ischaemia–reperfusion. Basic Res. Cardiol. 100, 422–432. Pfeffer, M.A., Pfeffer, J.M., Fishbein, M.C., Fletcher, P.J., Spadaro, J., Kloner, R.A., Braunwald, E., 1979. Myocardial infarct size and ventricular function in rats. Circ. Res. 44, 503–512. Priori, S.G., Napolitano, C., 2005. Intracellular calcium handling dysfunction and arrhythmogenesis: a new challenge for the electrophysiologist. Circ. Res. 97, 1077–1079. Rees, S.A., Curtis, M.J., 1993. Selective IK blockade as an antiarrhythmic mechanism: effects of UK66,914 on ischaemia and reperfusion arrhythmias in rat and rabbit hearts. Br. J. Pharmacol. 108, 139–145. Rochetaing, A., Barbe, C., Kreher, P., 2001. Beneficial effects of amiodarone and dronedarone (SR 33589b), when applied during low-flow ischemia, on arrhythmia and functional parameters assessed during reperfusion in isolated rat hearts. J. Cardiovasc. Pharmacol. 38, 500–511. Rooney, R.T., Marijic, J., Stommel, K.A., Bosnjak, Z.J., Aggarwal, A., Kampine, J.P., Stowe, D.F., 1995. Additive cardiac depression by volatile anesthetics in isolated hearts after chronic amiodarone treatment. Anesth. Analg. 80, 917–924. Singh, S., 2004. Trials of new antiarrhythmic drugs for maintenance of sinus rhythm in patients with atrial fibrillation. J. Interv. Card. Electrophysiol. 10 (Suppl 1), 71–76. Sogol, P.B., Hershman, J.M., Reed, A.W., Dillmann, W.H., 1983. The effects of amiodarone on serum thyroid hormones and hepatic thyroxine 5′-monodeiodination in rats. Endocrinology 113, 1464–1469.

157

Tsalikakis, D.G., Fotiadis, D.I., Kolettis, T., Michalis, L.K., 2003. Automated system for the analysis of heart monophasic action potentials. Comput. Cardiol. 30, 339–342. van Opstal, J.M., Schoenmakers, M., Verduyn, S.C., de Groot, S.H., Leunissen, J.D., van Der Hulst, F.F., Molenschot, M.M., Wellens, H.J., Vos, M.A., 2001. Chronic amiodarone evokes no torsade de pointes arrhythmias despite QT lengthening in an animal model of acquired long-QT syndrome. Circulation 104, 2722–2727. Varro, A., Takacs, J., Nemeth, M., Hala, O., Virag, L., Iost, N., Balati, B., Agoston, M., Vereckei, A., Pastor, G., Delbruyere, M., Gautier, P., Nisato, D., Papp, J.G., 2001. Electrophysiological effects of dronedarone (SR 33589), a noniodinated amiodarone derivative in the canine heart: comparison with amiodarone. Br. J. Pharmacol. 133, 625–634. Walker, M.J., Curtis, M.J., Hearse, D.J., Campbell, R.W., Janse, M.J., Yellon, D.M., Cobbe, S.M., Coker, S.J., Harness, J.B., Harron, D.W., 1988. The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion. Cardiovasc. Res. 22, 447–455. Wellens, H.J., 2004. Cardiac arrhythmias: the quest for a cure: a historical perspective. J. Am. Coll. Cardiol. 44, 1155–1163. Ytrehus, K., Liu, Y., Tsuchida, A., Miura, T., Liu, G.S., Yang, X.M., Herbert, D., Cohen, M.V., Downey, J.M., 1994. Rat and rabbit heart infarction: effects of anesthesia, perfusate, risk zone, and method of infarct sizing. Am. J. Physiol. 267, H2383–H2390.