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Amiodarone, adrenoceptor responsiveness and iscaaemia- and reperfusion-induced arrhythmias
European Journal of Pharmacology,201 f 1991)103- 109 0 1991 Elsevier Science Publishers B.V. 0014-2QQ9/91/$03.50
103
ADONIS 0014299991005732
EJP 51971
Susan J. Coker and Russell Chess-Williams * Departmentof Pharmacologyand Therapeutics,Universityof Liuerpool,P.O. Box 147, Limpool L69 38x, U.K.
Received 24 January 1991, revised MS received 9 April 1991,accepted 14 May 1991
Experiments were performed in rats which had been pretreated with amiodarone 50 mg kg-’ day-’ p.o. for 4 weeks. In anaesthetized animals subject to 25 min of coronary artery occlusion, the rats which had received amiodarone had fewer ischaemia-induced ventricular premature beats than the controls (381 f. 106 compared with 315 f ~15, P = 0.070). The duration of a~hythmias induced by re~rfusion following 5 min of ischaemia was also less in the rats which had receivedami~arone than in the controls (43.8 + 6.8 and 16.0 f 3.1 s respectively). Pretreatment of rats with amiodarone reduced the maximum driving frequency of both isolated left atria and papillary muscles. There were no differences between the responses to (Y- or /3-adrenoceptor agonists or to calcium in papillary muscle preparations from amiodarone-pretreated and control rats. These results suggest that the antiarrhythmic activity of chronic abniodarone seen in the present study does not depend on changes in ventricular adrenoceptor responsiveness.
Amiodarone;
coronary
artery occlusion; Reperfusion;
1. In~duction
Although the antiarrhythmic drug amiodarone has been available for a number of years and many studies have been performed with it, its mechanisms of action are still not understood completely. Traditionally it is thought of as having Ciass III activity since the demonstration by Singh and Vaughan Williams (1970) of its ability to prolong action potential duration. However, a number of additional mechanisms have been postulated to contribute to its antiarrhythmic activity. These include blockade of inactivated sodium channels (Mason et al., 1984; Yabek et al., 1986), and an antis~pathetic effect (Charlier et al., 1968; Nokin et al., 1983). The Class III effects of amiodarone have also been shown to be very similar to those observed in hypothyroidism (Freedberg et al., 1970; Singh and Vaughan Williams, 1970) and it has been suggested that these effects depend upon the ability of ~iodarone to inter-
Correspondence to: S.J. Coker, Department of Pharmacology and Therapeutics, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. Tel. #.51.?94.55,5~ fax 44.51.794.55.40. * Present address: Department of Biomedical Sciences, University of Sheffield, Sheffield SlO 2TN, U.K.
Arrhythmias; Adrenoceptor
responsiveness
fere with th:: actions of thyroid hormones (Patterson et al., 1986). We have shown recently that P-adrenoceptor responsiveness is decreased whereas a-adrenoceptor responsiveness is increased in cardiac tissue from hypothyroid rats (Chess-Williams and Critchley, 1987; Chat-Williams and Coker, 1989). The latter study aIso demonstrated that the increase in ventricular cr-adrenoceptor responsiveness was accompanied by a marked reduction ;rll the incidence of ventricular fibrillation induced by either acute myocardial ischaemia or reperfusion. It has been reported that the antisympathetic effect of ami~ar~ne is not due to competitive antagonism at P-adrenoceptors (Charher, 1970) but may be due to a decrease in @adrenoceptor density (Nokin et al., 1983; Venkatesh et al., 1986). We are not aware of any studies on the effects of amiodarone on cr-adrenoceptor number. The findings described above led us to think that there may be some relationship between the ability of amiodarone to alter adrenoceptor responsiveness and its antiarrhythmic activity. The aim of the experiments performed in this study was to test this hypothesis. A pretimina~ account of the results has been presented to the Inte~ational Society for Heart Research Poker and Chess-Williams, 1989).
riments were performed using male Wistar rats bad been bred in the departmental animal unit. ats were housed in groups of 6 to 16, in a room rnal~t~~~ed at 20°C on a 12 h light/dark cycle. Food diet) and water were available ad libigroup of rats received amiodarone 50 mg 1 p.0. for 4 weeks and the controls were orahy dosed with equivalent volumes of distilled water each day. NI experiments were performed 18 to 24 h after administration of the last dose. In addition, all esperiments and data analysis were performed blind. 2.2. hmi-iw aperiments
After 4 weeks of pretreatment
with amiodarone
or
water. rats were anaesthetized with sodium pentobarbitone ( mg kg- ’ i-p.) and prepared for the induction
of arrhythmias due to ischaemia or reperfusion using the methods of Clark et al. (1980) and Kane et al. (19&Q as described in detail previously (Coker and Ellis, 1987). Carotid arterial blood pressure and a Lead 1 ECG were recorded continuously. A femoral vein was cannulated to permit the administration of further doses of anaesthetic, if required. After performing a left thoracotomy the pericardium was incised to expose the heart. The rats were ventilated with room air at a rate of 54 strokes mm-‘, a stroke volume of 1.0 to 1.5 and a positive end expiratory pressure of m H,O. The heart was then exteriorised briefly while a fine silk ligature was placed around the left coronary artery close to its origin. After a 10 min stabihsation period the coronary artery was occluded and the arrhythmias that occurred during the first 25 min of myocardial ischaemia were quantified in accordance with the Lambeth Conventions (Walker et al., 1988). In survivors the total number of ventricular premature beats (VPBs) was counted and divided into those occurring as singles, bigeminy or salvos (collectively termed ‘others’) and those occurring as ventricular tachycardia (VT, defined as four tir more consecutive VPBsI. The incidence and duration of VT and ventricular fibrillation (VF) were also recorded along with the mortality. Reperfusion-induced arrhythmias were studied in separate groups of rats. In these experiments both ends of the ligature around the coronary artery were passed through a small polythene button which was placed in contact with the heart. Coronary artery occlusion was achieved by applying tension and clamping the ligature against the button with a small pair of rubber-sheathed Dieffecbach forceps. After 5 min of myocardial ischaemia the forceps were removed, thus releasing ten-
sion on the ligature and allowing reperfusion. The incidence of VT, VF and mortality was noted. In all animals an arterial blood sample (0.1 ml) was taken prior tk -0ronary artery occlusion and PO,, PCOl and pr were measured with a Corning 158 blood gas analyzer. If necessary the stroke volume of r;ic ventilation pump was adjusted to maintain blood gases within appropriate limits. The anaesthetized rats were maintained at a temperature of 37 to 38°C by means of a heated table. On each day that arrhythmia experiments were performed 6 rats (3 control, 3 amiodarone pretreated) were used in a randomised order. For these studies 24 rats were treated with amiodarone and 24 with water. One rat receiving amiodarone died during the 3rd week of treatment. Of the remaining 47 rats subject to either ischaemia-induced or reperfusion-induced arrhythmias 9 were excluded from the final data analysis for the following reasons: Arrhythmias prior to coronary artery occlusion (7); Arrhythmias prior to thoracotomy (1); Cardiac failure within the first 5 min after coronary artery occlusion (1). Thus the final group sizes were: ischaemia-induced arrhythmias, control (101, amiodarone (8); reperfusion-induced arrhythmias, control (91, amiodarone (11). 2.3. In citro experiments A further 20 rats were pretreated (10 amiodarone, 10 controls) for 4 weeks before being used for isolated tissue experiments. Rats were killed by a blow to the head and exsanguinated. Left atria and left ventricular papillary muscles were removed rapidly and set up in a Krebs-bicarbonate solution of the following composition (mMk NaCl 118.4, KC1 4.7, NaHCO, 25.0, MgSO, 1.2, KH rP0, 1.2, glucose 11.7, CaCl, 1.9, gassed with 95% 02-5% CO2 at 37°C. Each tissue was suspended under 0.8 g tension and paced at 1 Hz by square wave pulses of 5 ms duration at twice threshold voltage delivered via bipolar electrodes, from Grass S48 stimulators. Isometric developed tension was recorded on a Lectromed MX2 polygraph via Lectromed UFl force transducers (57 g sensitivity range). After an equilibration period of 30 min, during which the bathing fluid was changes several times, the maximum driving frequency of each tissue was measured. This was determined, at twice threshold voltage, by gradually increasing the driving frequency until the tissue failed to respond to each stimulus. After washing, a cumulative concentration-response curve to isoprenaline was obtained, followed after washout, by one to phenylephrine and then one to calcium. Desipramine (1 PM) and metanephrine (10 PM) were added to the tissue bath, 15 min before obtaining agonist responses, to block neuronal and extraneuronal uptake respectively. The concentration-response curves
105 TABLE
1
Baseline values for weight, heart rate (HR). systolic and dias!olic blood pressures (SBP, DBP), arterial blood pH, PCO, and PO2 before coronary artery occlusion in anaecthetized rats. n
Weight
(‘3)
HR (beats min - ‘1
SBP (mmHg)
DBP (mmHg)
PH (units)
PCO, (mmkg)
PO, (m;Hg)
7.4Sf0.01 7.4s f 0.01 7.46 f 0.02 7.48 f 0.02
30+1 30+1 27+1 26+1
s7+7 S3k6 79*7 82*4
Control-ischaemia Amiodarone-ischaemia
IO 8
338-1: 7 340+ 7
419* 13 423k 1.5
122+7 134*9
95+7 104+7
Control-reperfusion Amiodarone-reperfusion
9 11
37ti+ 8 342k 10
437*13 451* 9
123+7 135+4
96&7 109*4
to phenylephrine were obtained propranolol (1 PM).
in the presence
of
tained in tissues from amiodarone-pretreated trol animals.
and con-
2.4. Drugs 3. Results Amiodarone hydrochloride was a gift from Sanofi, Wythenshawe. A solution of 25 mg ml-’ was prepared by weighing out 2.5 g of amiodarone hydrochloride, adding 95 ml of distilled water, stirring carefully to wet the powder and heating the white suspension until it began to clear (at approximately 70°C). The solution was removed from the heat aird when it had cooled to room temperature it was made up to 100 ml with distilled water. This solution was stored at room temperature protected from light. Each rat received 2 ml kg-’ day-’ p.o. of this solution. Sodium pentobarbitone was purchased from May and Baker, Dagenham. All drugs for the in vitro experiments were purchased from Sigma, Poole, and were made up fresh in Krebs solution. The reagents for Krebs solution were of AnalaR grade bought from BDH, Poole or Fisons, Loughborough. 2.5. Statistical analysis Where appropriate values are expressed as means 2 S.E. of n experiments. In the in vivo experiments the differences in baseline parameters (e.g. haemodynamits) between grocps were compared with an independent t-test. A Mann-Whitney U-test was used to compare ischaemia-induced VPBs since these data may not be distributed normally. The incidence of events was compared with I-uher’s exact test. In the in vitro experiments increases in developed tension were plotted as a percentage of the maximum increase for each drug. Individual EC5,, values were determined and geometric mean EC,, values with 95% confidence limits calculated. An independent t-test was performed on logarithmic EC,, values to test for differences between control and amiodarone-treated rats. Mean absolute maximum developed tensions and mean increases in developed tension induced by each drug were calculated and independent t-tests applied to individual values to compare maximum responses ob-
3.1. In Lko experiments No significant differences were observed in baseline haemodynamics or blood gases prior to coronary artery occlusion between the control rats and those which had received amiodarone (table 1). In both amiodaronetreated groups (those used for ischaemia-induced arrhythmias and those used for reperfusion-induced arrhythmias) arterial blood pressure appeared to be higher than in controls but this was not statistically significant even when the values from both groups were pooled. Pretreatment of rats with amiodarone 50 mg kg-’ day-’ for 4 weeks did not significantly alter the incidence of VT, VF or mortality during the first 25 min of acute myocardial ischaemia (fig. 1). There was also no significant difference in the duration of VT during ischaemia; 57 + 21 s in the controls and 20 f 5 s (P = 0.110, independent t-test) in the rats which had received amiodarone. The total number of VPBs that occurred in the amiodarone-pretreated rats appeared to be lower than those that occurred in the controls (fig. 21, but this difference just failed to reach statistical VT
VF
Mortality
100
80
%
‘30 40 20
:
1
0I Fig. 1. The incidence of ventricular tachycardia (VT). ventricular fibrillation (VF) and the mortality resulting from 2.5 min of permanent coronary artery occlusion in anaesthetized rats which were pretreated with amiodarone (shaded columns, n = 8) or water (open columns, n = IO).
so.
Qf SPBS
1200 -
Total
VT Others
VF
Mortality
as VT
80
Fit. 2. The number of i~haemia-induced ventricular premature beats (VPBs> that occurred in survivors during the first 25 min of myocardial ischaemia. The total number of VPBs has also been &WI after subdivision into those occurring as ventricular tachycardia (VT’) or singles, bigeminy or salvos, collectively termed ‘Others’. Each value is the mean f SE. Open co!mnns: controls, n = 6: shaded columns: amiodarone. n = 4. * P = 0.070, Mann-Whitney U-test.
s~i~c~ce fP = 0.070, Mann-jitney U-test) at the commonly accepted 5% level. When reperfusion-induced arrhythmias were examined amiodarone failed to si~i~cantly reduce the incidence of VT, VF or the mortality (fig. 3). However, closer examination of the data revealed a reduction in the duration of repe~sion-induced arrhythmias in survivors which Irad received amiodarone. Although there was no difference in the time to onset of arrhythmias after revision the time at which they ceased was significantly reduced (fig. 4).
Fig. 3. The incidence of ventricular tachycardia (VT), ventricular fibrillation (VF) and the mortality resulting from reperfusion after 5 min of myocardial ischaemia in anaesthetized rats. Open columns: controls, n = 9; shaded columns: amiodarone, n = 1I.
The resting tension developed by isolated left atria from ~iodarone-pretreated animals (0.61 + 0.10 g) was significantly (P < 0.05) lower than that developed by atria from controls (0.81 + 0.07 g). The maximum tensions developed to isoprenaline, phenyleph~ne and calcium were also reduced by amiodarone pretreatment, although this was only significant for responses to isoprenaline (table 2). When m~mum responses were calculated as the increase in developed tension induced by the drugs, maximum responses were again reduced but in this case the changes were only statistically significant for responses to phenylephrine and
TABLE 2 Geometric mean EC& values (with 95% confidence limits) and mean m~mum cardiac tissues from amiodarone-treated and control rats. n
EC,
responses
f S.E. for isoprenaline, phenylephrine MDT (PI
MTDT (gf
lsoprenaline Left atria-control Left a~a-~~a~ne Papillary-control Papillary-amiodarone
8 9 10 9
(nM) 10.8 f4.7- 25.2) 56.5 (22.4-142.53 b 137.4 (93.2-202.7) 233.9 ( 126.0-434.0)
1.74f0.10 1.37k8.13 = 0.93*0.10 A.01zto.07
0.94 f 0.08 0.77*0.07 0.35 f 0.05 0.47 f 0.05
Phe~leph~ne Left atria-control Left atria-amiodarone Papi!l~-~n~ol Papillary-amiodarone
8 9 10 9
(&MI 5.0 (3.1- 8.1) 4.4 (2.8- 6.8) 33.7 124.7- 46.1) 33.1 (19.3- 56.7)
1.05 f 0.09 0.81 f0.13 0.53~0.11 0.52 * 0.02
0.54f0.04 0.37 f 0.05 a 0.14*0.02 0.18kO.01
Calcium Left atria-control Left atria-amiodarone Papillary-control Papillary-amiodarone
5 6 5 6
(MM) 8.9 7.1 6.8 7.2
1.59*0.08 1.26f0.17 1.07rtO.16 1.03~0.11
1.09f0.07 0.78kO.11 a 0.73*0.1t 0.69 f 0.08
(7.0- 11.4) (4.9- 10.4) (4.8- 9.8) (Xl- 10.1)
and calcium in
Maximum responses are expressed as the maximum developed tension (MDT) and the maximum increase in developed tension (M t DT) induced by the drugs. a P < 0.85, b P < 0.01 compared with corresponding controls, independent t-test on absolute values of MDT and M f DT and on log EC, values.
107
Time (s) 60 1
Time to onset
Time to cease
Duration of VT
Fig. 4. The time to onset and the time to cease for reperfusion-induced arrhythmias along with the duration of reperfusion-induced ventricular tachycardia (VT). Each value is the meanfS.E. Open columns: controls, n = 9; shaded columns: amiodarone, n = 11. * P < 0.05compared with control, independent t-test.
In contrast to that observed with left atria, the resting tensions developed by isolated papillary musCks were similar in control (0.58 + 0.06 g) and ho_ darone-pretreated tissues (0.55 + 0.03 g). The hotropic responses of papillary muscles to isoprenaline, phenylephrine and calcium were not affected by pretreating animals with amiodarone. Neither the EC, values nor the maximum responses to these agents were altered (table 2). Amiodarone pretreatment did, however, result in significant (P < 0.05) reductions in maximum driving frequency in both left atria and papillary muscles. The values in control left atria were 14.6 + 0.8 Hz corn-pared with 11.9 + 0.5 Hz in left atria from rats which had received amiodarone. The corresponding values in papillary muscles were 12.1 + 0.5 Hz (control) and 10.1 f 0.5 Hz (amiodarone).
4. Discussion calcium (table 2). The sensitivity of left atria to isoprenaline, as indicated by EC,, values, was reduced in tissues from rats which had received amiodarone, but the sensitivity of these tissues to phenylephrine and calcium was not altered (fig. 5; table 2).
Developed
Tension
(g)
Left
Papillary lo-9
1o-6
,o-7 lsoprenaline
atria
muscle
,6-6
,6-5
,n-4
(M)
Fig. 5. Mean concentration-response curves to isoprenaline in left atria and papillary muscles from control (01 and amiodarone (0) pretreated rats. Each value is the mean f LE.. n = 8-10.
The results of this study indicate that chronic pretreatment of rats with amiodarone had some antiarrhythmic effects in vivo, prolonged the effective refractory period of cardiac muscle measured in vitro but had no effect on ventricular a- or &adrenocep:ptor responsiveness. Previous reports have indicated that the same treatment regime in rats (Nokin et al., 1983) and similar dose ranges in rabbits (Venkatesh et al., 1986) decreased myocardial @-adrenoceptor density although corresponding functional studies were not performed. Although in left atria we found an increase in the isoprenaline EC,,, suggesting decreased P-adrenoceptor sensitivity, and a reduction in the m-urn increase in developed tension with phenylephrine the latter effect was also observed for responses to calcium. This suggests that these apparent changes in (Yand /3-adrenoceptor responsiveness may just be the result of a non-specific depressant effect of amiodarone on atria1 muscle. Further support for this suggestion comes from the evidence that the resting developed tension in left atria from amiodarone-pretreated rats was lower than that in controls whereas no such differences were seen in papillary muscle preparations. It is possible that we failed to observe any changes in ventricular adrenoceptor responsiveness because the plasma or myocardial concentrations of amiodarone were below the threshold required to produce such effects. The experiments which first showed the antisympathetic actions of amiodarone, were performed in vivo with high doses of amiodarone Kharlier et & 1968; Charlier, 1970). Similarly, the effects Of ami@ darone in vitro, on p-adrenoceptor mediated changes in heart rate in rabbit isolated right atria and on a-&eDoceptor mediated contractions of isolated rat aorta were fairly small, non-competitive and only OC-
ular arrhythmias induced chaemia or reperfusion.
by acute
myocardial
is-
ever, set:
s~~~i~~ca~treductions in the and ventricular ~r~~arati~~s. This suggests that we did achieve ~~~~~~~~~t~~~s s~f~cig~t to alter the effective refracriod of cardiac muscle a~tho~gb no changes in tory ve~t~~~~~a~adrenoeeptor responsiveness occurred. In our ~~~~~~~s study in rats made hypothyroid by chronic ~ret~eatm~~t with 6.pr~pyItbi~uraci1 ~~hess-Williams and coker, 1989). we observed reductions in maximum d~~~~~ frequency ~vhicb were of similar magnitude to those seen here. but a marked increase in ventricular a-adrenoceptor responsiveness also occurred. This indicates that amiodarone does not cause the same changes in adrenoce$ors as seen in propylthiouracilinduced h~ot~yroidism. The relative&+ poor antia~bythmic activity of amiodarone observed in this study was disappointing. The dose of 50 mg kg-’ day-’ p-o. for 4 weeks was chosen because it had been shown by Nokin et al. (19831 to reduce cardiac &adrenoceptor density in rats. In addition. it was more than doubie the dose of 20 mg kg-’ day- ’ p.a for 3 weeks which had been reported by Winslow et al. (1987) to have minor activity against is~haem~a-induced arrb~hmias in rats. Despite using a sigher dose we were not able to see any greater antiarrbyt~rni~ effect although it should be noted that all our embedments were performed 18 to 24 h after administration of the last dose of amiodarone, since we were interested in the effects of chronic treatment rather than acme effects. In contrast to the results seen follo~v~~gchronic dosing, Riva and Hearse (1989) have demonstrated marked reductions in ischaemia-induced VT and re~e~~sion-induced VF after a single i.v. dose of 5 mg kg-’ of amiodarone. This suggests that in rats, chronic dosing at daily intervals may be too infrequent to achieve drug levels which produce good antiarrh~hmic activity. Although the antiarrhythmic effects we measured in the present study were not as dramatic as some rewted by others, significant effects were observed. The maximum driving frequency of atrial and ventricular muscle was reduced, the number of ischaemia-induced VPBs was less and the duration of reperfusion-induced arrhythmias was shorter in amiodarone-pretreated rats. In addition, left atria1 ~-adr~no~eptor sensitivity was reduced. However, no alterations in ventricular adrenoceptor responsiveness could be found. Although we cannot exclude the possibility that alterations in adrenOCe@Or responsiveness may contribute to the antiarrhythmic activity of higher concentrations of amiodarone, the present results do not support the hypothesis that amiodarone-induced changes in adrenoceptor responsiveness contribute to its activity against ventric~~~~~~~
dying
~~~¶u~~c~ of both atria1
Financial support for this study was provided by Sanofi UK Ltd.
Charlier. R., 1970, Cardiac actions in the dog of a new antagonist of adrenergic excitation which does not produce competitive bkrckade of adrenoceptors, Br. J. Pharmacol. 39, 668. Charlier. R., G. Deltour, A. Baudine and F. Chaillet, 1968. Pharmacology of amiodarone, an anti-angina1 drug with a new biological profile, Anneim. Forsch. 18, 1408. Chess-Williams, R. and S.J. Coker, 1989, Ventricular fibrillation is reduced in hypothyroid rats with enhanced myocardial cr-adrenoceptor responsiveness, Br. J. Pharmacol. 98,95. Chess-Williams, R. and D.J.P. Critchley, 1987, Cardiac admnoceptor responsiveness in hypothyroid rats, Br. J. Pharmacol. 90, 289P. Clark. C., M.I. Foreman, K.A. Kane, EM. McDonald and J.R. Parratt, 1980, Coronary artery ligation in anaesthetised rats as a method for the production of experimental dysrhythmias and for the determination of infarct size, J. Pharmacol. Meth. 3, 357. Coker, S.J. and R. Chess-WiIliams, 1989, Effects of amiodarone on ischaemia and reperfusion induced arrhythmias and on adrenoceptor responsiveness, J. Mol. Cell. Cardiol. 21 (Suppl. II), 13. Coker, S.J. and A.M. Ellis, 1987, Ketanserin and ritanserin can reduce re~rfusion-induced but not ischaemia-induced arrhythmias in anaesthetised rats, J Cardiovasc. Pharmacol. IO, 479. Freedberg, A.S., J.G. Papp and E.M. Vaughan Williams, 1970, The effect of altered thyroid state on atria1 intracellular potentials, 1. Physioi. 207, 357. Kane, K.A.. J.R. Parratt and F.M. Williams, 1984, An investigation into the characteristics of reperfusion-induced arrhythmias in the anaesthetized rat and their susceptibili~ to antiarrhythmic agents, Br. J. Pharmacol. 82, 349. Mason, J.W., L.M. Hondeghem and B.C. Katzung, 1984, Block of inactivated sodium channels and of de~la~zat~on-induced automaticity in guinea pig papillary muscle by amiodarone. Circ. Res. 55, 277. Nokin, P., M. Clinet and P. Schoenfeld, 1983, Cardiac P-adrenoceptor modulation by amiodarone, Biochem. Pharmacol. 32,2473. Patterson, E., K.M. Walden, M.B. Khazaeli, D.G. Montgomery and B.R. Lucchesi. 1986, Cardiac electrophysiologic effects of acute and chronic amioda~ne administration in isolated perfused rabbit heart: altered thyroid metabolism, J. Pharmacol. Exp. Ther. 239. 179. Polster, P. and J. Broekhuysen, 1976, The adrenergic antagonism of amiodarone, Biochem. Pharmacol. 25, 131. Riva, E. and D.J. Hearse, 1989. Anti-arrhythmic effects of amiodarone and desethylamiodarone on malignant ventricular arrh~hmias arising as a consequence of ischaemia and reperfusion in the :A, testhetised rat, Cardiovasc. Res. 23, 331. Singh, B.N. and E.M. Vaughan Wubams, 1970, The effect of amiodarone, a new anti-angina1 drug, on cardiac muscle. Br. J. Pharmacoi. 39, 657. Venkatesh, N., J.F. Padbury and B.N. Singh, 1986, Effects of amiodarone and desethylamiodarone on rabbit myocardial j3-adrenoceptors and serum thyroid hormones - absence of relationship to serum and myocardial drug concentrations, J. Cardiovasc. Pharmacol. 8, 989. Walker, M.J.A.. M.J. Curtis, D.J. Hearse, R.W.F. Campbell, MJ
109 Janse, D.M. Yellon, SM. Cobbe, S.J. Coker, J.B. Harness, D.W.G. Harron, A.J. Higgins, D.J. Julian, M.J. Lab, AS. Manning, B.J. Northover, J.R. Parratt, R.A. Riemersma, E. Riva, D.C. Russell, D.J. Sheridan, E. Winslow and B. Woodward, 1988, The Lambeth Conventions: Guidelines for the study of arrhythmias in ischaemia, infarction and reperfusion, Cardiovasc. Res. 22, 447. Winslow, E., R.J. Marshall and F.G. Hope, 1987, Effects of diet-induced hypokalaemia on the antiarrhythmic and electrophysiologi-
cal actions of prolonged oral treatment with either amiodarone or disopyramide in the anaesthetised rat. J. Cardiovasc. Pharmarol. 9, 267. Yabek. S.M., R. Kato and B.N. Singh. 1986, Effects of amiodarone and its metabolite, desethylamiodarone, on the electrophysiologic properties of isolated cardiac nmscle, J. Cardiovasc. Pharmacol. 8, 197.
103
ADONIS 0014299991005732
EJP 51971
Susan J. Coker and Russell Chess-Williams * Departmentof Pharmacologyand Therapeutics,Universityof Liuerpool,P.O. Box 147, Limpool L69 38x, U.K.
Received 24 January 1991, revised MS received 9 April 1991,accepted 14 May 1991
Experiments were performed in rats which had been pretreated with amiodarone 50 mg kg-’ day-’ p.o. for 4 weeks. In anaesthetized animals subject to 25 min of coronary artery occlusion, the rats which had received amiodarone had fewer ischaemia-induced ventricular premature beats than the controls (381 f. 106 compared with 315 f ~15, P = 0.070). The duration of a~hythmias induced by re~rfusion following 5 min of ischaemia was also less in the rats which had receivedami~arone than in the controls (43.8 + 6.8 and 16.0 f 3.1 s respectively). Pretreatment of rats with amiodarone reduced the maximum driving frequency of both isolated left atria and papillary muscles. There were no differences between the responses to (Y- or /3-adrenoceptor agonists or to calcium in papillary muscle preparations from amiodarone-pretreated and control rats. These results suggest that the antiarrhythmic activity of chronic abniodarone seen in the present study does not depend on changes in ventricular adrenoceptor responsiveness.
Amiodarone;
coronary
artery occlusion; Reperfusion;
1. In~duction
Although the antiarrhythmic drug amiodarone has been available for a number of years and many studies have been performed with it, its mechanisms of action are still not understood completely. Traditionally it is thought of as having Ciass III activity since the demonstration by Singh and Vaughan Williams (1970) of its ability to prolong action potential duration. However, a number of additional mechanisms have been postulated to contribute to its antiarrhythmic activity. These include blockade of inactivated sodium channels (Mason et al., 1984; Yabek et al., 1986), and an antis~pathetic effect (Charlier et al., 1968; Nokin et al., 1983). The Class III effects of amiodarone have also been shown to be very similar to those observed in hypothyroidism (Freedberg et al., 1970; Singh and Vaughan Williams, 1970) and it has been suggested that these effects depend upon the ability of ~iodarone to inter-
Correspondence to: S.J. Coker, Department of Pharmacology and Therapeutics, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. Tel. #.51.?94.55,5~ fax 44.51.794.55.40. * Present address: Department of Biomedical Sciences, University of Sheffield, Sheffield SlO 2TN, U.K.
Arrhythmias; Adrenoceptor
responsiveness
fere with th:: actions of thyroid hormones (Patterson et al., 1986). We have shown recently that P-adrenoceptor responsiveness is decreased whereas a-adrenoceptor responsiveness is increased in cardiac tissue from hypothyroid rats (Chess-Williams and Critchley, 1987; Chat-Williams and Coker, 1989). The latter study aIso demonstrated that the increase in ventricular cr-adrenoceptor responsiveness was accompanied by a marked reduction ;rll the incidence of ventricular fibrillation induced by either acute myocardial ischaemia or reperfusion. It has been reported that the antisympathetic effect of ami~ar~ne is not due to competitive antagonism at P-adrenoceptors (Charher, 1970) but may be due to a decrease in @adrenoceptor density (Nokin et al., 1983; Venkatesh et al., 1986). We are not aware of any studies on the effects of amiodarone on cr-adrenoceptor number. The findings described above led us to think that there may be some relationship between the ability of amiodarone to alter adrenoceptor responsiveness and its antiarrhythmic activity. The aim of the experiments performed in this study was to test this hypothesis. A pretimina~ account of the results has been presented to the Inte~ational Society for Heart Research Poker and Chess-Williams, 1989).
riments were performed using male Wistar rats bad been bred in the departmental animal unit. ats were housed in groups of 6 to 16, in a room rnal~t~~~ed at 20°C on a 12 h light/dark cycle. Food diet) and water were available ad libigroup of rats received amiodarone 50 mg 1 p.0. for 4 weeks and the controls were orahy dosed with equivalent volumes of distilled water each day. NI experiments were performed 18 to 24 h after administration of the last dose. In addition, all esperiments and data analysis were performed blind. 2.2. hmi-iw aperiments
After 4 weeks of pretreatment
with amiodarone
or
water. rats were anaesthetized with sodium pentobarbitone ( mg kg- ’ i-p.) and prepared for the induction
of arrhythmias due to ischaemia or reperfusion using the methods of Clark et al. (1980) and Kane et al. (19&Q as described in detail previously (Coker and Ellis, 1987). Carotid arterial blood pressure and a Lead 1 ECG were recorded continuously. A femoral vein was cannulated to permit the administration of further doses of anaesthetic, if required. After performing a left thoracotomy the pericardium was incised to expose the heart. The rats were ventilated with room air at a rate of 54 strokes mm-‘, a stroke volume of 1.0 to 1.5 and a positive end expiratory pressure of m H,O. The heart was then exteriorised briefly while a fine silk ligature was placed around the left coronary artery close to its origin. After a 10 min stabihsation period the coronary artery was occluded and the arrhythmias that occurred during the first 25 min of myocardial ischaemia were quantified in accordance with the Lambeth Conventions (Walker et al., 1988). In survivors the total number of ventricular premature beats (VPBs) was counted and divided into those occurring as singles, bigeminy or salvos (collectively termed ‘others’) and those occurring as ventricular tachycardia (VT, defined as four tir more consecutive VPBsI. The incidence and duration of VT and ventricular fibrillation (VF) were also recorded along with the mortality. Reperfusion-induced arrhythmias were studied in separate groups of rats. In these experiments both ends of the ligature around the coronary artery were passed through a small polythene button which was placed in contact with the heart. Coronary artery occlusion was achieved by applying tension and clamping the ligature against the button with a small pair of rubber-sheathed Dieffecbach forceps. After 5 min of myocardial ischaemia the forceps were removed, thus releasing ten-
sion on the ligature and allowing reperfusion. The incidence of VT, VF and mortality was noted. In all animals an arterial blood sample (0.1 ml) was taken prior tk -0ronary artery occlusion and PO,, PCOl and pr were measured with a Corning 158 blood gas analyzer. If necessary the stroke volume of r;ic ventilation pump was adjusted to maintain blood gases within appropriate limits. The anaesthetized rats were maintained at a temperature of 37 to 38°C by means of a heated table. On each day that arrhythmia experiments were performed 6 rats (3 control, 3 amiodarone pretreated) were used in a randomised order. For these studies 24 rats were treated with amiodarone and 24 with water. One rat receiving amiodarone died during the 3rd week of treatment. Of the remaining 47 rats subject to either ischaemia-induced or reperfusion-induced arrhythmias 9 were excluded from the final data analysis for the following reasons: Arrhythmias prior to coronary artery occlusion (7); Arrhythmias prior to thoracotomy (1); Cardiac failure within the first 5 min after coronary artery occlusion (1). Thus the final group sizes were: ischaemia-induced arrhythmias, control (101, amiodarone (8); reperfusion-induced arrhythmias, control (91, amiodarone (11). 2.3. In citro experiments A further 20 rats were pretreated (10 amiodarone, 10 controls) for 4 weeks before being used for isolated tissue experiments. Rats were killed by a blow to the head and exsanguinated. Left atria and left ventricular papillary muscles were removed rapidly and set up in a Krebs-bicarbonate solution of the following composition (mMk NaCl 118.4, KC1 4.7, NaHCO, 25.0, MgSO, 1.2, KH rP0, 1.2, glucose 11.7, CaCl, 1.9, gassed with 95% 02-5% CO2 at 37°C. Each tissue was suspended under 0.8 g tension and paced at 1 Hz by square wave pulses of 5 ms duration at twice threshold voltage delivered via bipolar electrodes, from Grass S48 stimulators. Isometric developed tension was recorded on a Lectromed MX2 polygraph via Lectromed UFl force transducers (57 g sensitivity range). After an equilibration period of 30 min, during which the bathing fluid was changes several times, the maximum driving frequency of each tissue was measured. This was determined, at twice threshold voltage, by gradually increasing the driving frequency until the tissue failed to respond to each stimulus. After washing, a cumulative concentration-response curve to isoprenaline was obtained, followed after washout, by one to phenylephrine and then one to calcium. Desipramine (1 PM) and metanephrine (10 PM) were added to the tissue bath, 15 min before obtaining agonist responses, to block neuronal and extraneuronal uptake respectively. The concentration-response curves
105 TABLE
1
Baseline values for weight, heart rate (HR). systolic and dias!olic blood pressures (SBP, DBP), arterial blood pH, PCO, and PO2 before coronary artery occlusion in anaecthetized rats. n
Weight
(‘3)
HR (beats min - ‘1
SBP (mmHg)
DBP (mmHg)
PH (units)
PCO, (mmkg)
PO, (m;Hg)
7.4Sf0.01 7.4s f 0.01 7.46 f 0.02 7.48 f 0.02
30+1 30+1 27+1 26+1
s7+7 S3k6 79*7 82*4
Control-ischaemia Amiodarone-ischaemia
IO 8
338-1: 7 340+ 7
419* 13 423k 1.5
122+7 134*9
95+7 104+7
Control-reperfusion Amiodarone-reperfusion
9 11
37ti+ 8 342k 10
437*13 451* 9
123+7 135+4
96&7 109*4
to phenylephrine were obtained propranolol (1 PM).
in the presence
of
tained in tissues from amiodarone-pretreated trol animals.
and con-
2.4. Drugs 3. Results Amiodarone hydrochloride was a gift from Sanofi, Wythenshawe. A solution of 25 mg ml-’ was prepared by weighing out 2.5 g of amiodarone hydrochloride, adding 95 ml of distilled water, stirring carefully to wet the powder and heating the white suspension until it began to clear (at approximately 70°C). The solution was removed from the heat aird when it had cooled to room temperature it was made up to 100 ml with distilled water. This solution was stored at room temperature protected from light. Each rat received 2 ml kg-’ day-’ p.o. of this solution. Sodium pentobarbitone was purchased from May and Baker, Dagenham. All drugs for the in vitro experiments were purchased from Sigma, Poole, and were made up fresh in Krebs solution. The reagents for Krebs solution were of AnalaR grade bought from BDH, Poole or Fisons, Loughborough. 2.5. Statistical analysis Where appropriate values are expressed as means 2 S.E. of n experiments. In the in vivo experiments the differences in baseline parameters (e.g. haemodynamits) between grocps were compared with an independent t-test. A Mann-Whitney U-test was used to compare ischaemia-induced VPBs since these data may not be distributed normally. The incidence of events was compared with I-uher’s exact test. In the in vitro experiments increases in developed tension were plotted as a percentage of the maximum increase for each drug. Individual EC5,, values were determined and geometric mean EC,, values with 95% confidence limits calculated. An independent t-test was performed on logarithmic EC,, values to test for differences between control and amiodarone-treated rats. Mean absolute maximum developed tensions and mean increases in developed tension induced by each drug were calculated and independent t-tests applied to individual values to compare maximum responses ob-
3.1. In Lko experiments No significant differences were observed in baseline haemodynamics or blood gases prior to coronary artery occlusion between the control rats and those which had received amiodarone (table 1). In both amiodaronetreated groups (those used for ischaemia-induced arrhythmias and those used for reperfusion-induced arrhythmias) arterial blood pressure appeared to be higher than in controls but this was not statistically significant even when the values from both groups were pooled. Pretreatment of rats with amiodarone 50 mg kg-’ day-’ for 4 weeks did not significantly alter the incidence of VT, VF or mortality during the first 25 min of acute myocardial ischaemia (fig. 1). There was also no significant difference in the duration of VT during ischaemia; 57 + 21 s in the controls and 20 f 5 s (P = 0.110, independent t-test) in the rats which had received amiodarone. The total number of VPBs that occurred in the amiodarone-pretreated rats appeared to be lower than those that occurred in the controls (fig. 21, but this difference just failed to reach statistical VT
VF
Mortality
100
80
%
‘30 40 20
:
1
0I Fig. 1. The incidence of ventricular tachycardia (VT). ventricular fibrillation (VF) and the mortality resulting from 2.5 min of permanent coronary artery occlusion in anaesthetized rats which were pretreated with amiodarone (shaded columns, n = 8) or water (open columns, n = IO).
so.
Qf SPBS
1200 -
Total
VT Others
VF
Mortality
as VT
80
Fit. 2. The number of i~haemia-induced ventricular premature beats (VPBs> that occurred in survivors during the first 25 min of myocardial ischaemia. The total number of VPBs has also been &WI after subdivision into those occurring as ventricular tachycardia (VT’) or singles, bigeminy or salvos, collectively termed ‘Others’. Each value is the mean f SE. Open co!mnns: controls, n = 6: shaded columns: amiodarone. n = 4. * P = 0.070, Mann-Whitney U-test.
s~i~c~ce fP = 0.070, Mann-jitney U-test) at the commonly accepted 5% level. When reperfusion-induced arrhythmias were examined amiodarone failed to si~i~cantly reduce the incidence of VT, VF or the mortality (fig. 3). However, closer examination of the data revealed a reduction in the duration of repe~sion-induced arrhythmias in survivors which Irad received amiodarone. Although there was no difference in the time to onset of arrhythmias after revision the time at which they ceased was significantly reduced (fig. 4).
Fig. 3. The incidence of ventricular tachycardia (VT), ventricular fibrillation (VF) and the mortality resulting from reperfusion after 5 min of myocardial ischaemia in anaesthetized rats. Open columns: controls, n = 9; shaded columns: amiodarone, n = 1I.
The resting tension developed by isolated left atria from ~iodarone-pretreated animals (0.61 + 0.10 g) was significantly (P < 0.05) lower than that developed by atria from controls (0.81 + 0.07 g). The maximum tensions developed to isoprenaline, phenyleph~ne and calcium were also reduced by amiodarone pretreatment, although this was only significant for responses to isoprenaline (table 2). When m~mum responses were calculated as the increase in developed tension induced by the drugs, maximum responses were again reduced but in this case the changes were only statistically significant for responses to phenylephrine and
TABLE 2 Geometric mean EC& values (with 95% confidence limits) and mean m~mum cardiac tissues from amiodarone-treated and control rats. n
EC,
responses
f S.E. for isoprenaline, phenylephrine MDT (PI
MTDT (gf
lsoprenaline Left atria-control Left a~a-~~a~ne Papillary-control Papillary-amiodarone
8 9 10 9
(nM) 10.8 f4.7- 25.2) 56.5 (22.4-142.53 b 137.4 (93.2-202.7) 233.9 ( 126.0-434.0)
1.74f0.10 1.37k8.13 = 0.93*0.10 A.01zto.07
0.94 f 0.08 0.77*0.07 0.35 f 0.05 0.47 f 0.05
Phe~leph~ne Left atria-control Left atria-amiodarone Papi!l~-~n~ol Papillary-amiodarone
8 9 10 9
(&MI 5.0 (3.1- 8.1) 4.4 (2.8- 6.8) 33.7 124.7- 46.1) 33.1 (19.3- 56.7)
1.05 f 0.09 0.81 f0.13 0.53~0.11 0.52 * 0.02
0.54f0.04 0.37 f 0.05 a 0.14*0.02 0.18kO.01
Calcium Left atria-control Left atria-amiodarone Papillary-control Papillary-amiodarone
5 6 5 6
(MM) 8.9 7.1 6.8 7.2
1.59*0.08 1.26f0.17 1.07rtO.16 1.03~0.11
1.09f0.07 0.78kO.11 a 0.73*0.1t 0.69 f 0.08
(7.0- 11.4) (4.9- 10.4) (4.8- 9.8) (Xl- 10.1)
and calcium in
Maximum responses are expressed as the maximum developed tension (MDT) and the maximum increase in developed tension (M t DT) induced by the drugs. a P < 0.85, b P < 0.01 compared with corresponding controls, independent t-test on absolute values of MDT and M f DT and on log EC, values.
107
Time (s) 60 1
Time to onset
Time to cease
Duration of VT
Fig. 4. The time to onset and the time to cease for reperfusion-induced arrhythmias along with the duration of reperfusion-induced ventricular tachycardia (VT). Each value is the meanfS.E. Open columns: controls, n = 9; shaded columns: amiodarone, n = 11. * P < 0.05compared with control, independent t-test.
In contrast to that observed with left atria, the resting tensions developed by isolated papillary musCks were similar in control (0.58 + 0.06 g) and ho_ darone-pretreated tissues (0.55 + 0.03 g). The hotropic responses of papillary muscles to isoprenaline, phenylephrine and calcium were not affected by pretreating animals with amiodarone. Neither the EC, values nor the maximum responses to these agents were altered (table 2). Amiodarone pretreatment did, however, result in significant (P < 0.05) reductions in maximum driving frequency in both left atria and papillary muscles. The values in control left atria were 14.6 + 0.8 Hz corn-pared with 11.9 + 0.5 Hz in left atria from rats which had received amiodarone. The corresponding values in papillary muscles were 12.1 + 0.5 Hz (control) and 10.1 f 0.5 Hz (amiodarone).
4. Discussion calcium (table 2). The sensitivity of left atria to isoprenaline, as indicated by EC,, values, was reduced in tissues from rats which had received amiodarone, but the sensitivity of these tissues to phenylephrine and calcium was not altered (fig. 5; table 2).
Developed
Tension
(g)
Left
Papillary lo-9
1o-6
,o-7 lsoprenaline
atria
muscle
,6-6
,6-5
,n-4
(M)
Fig. 5. Mean concentration-response curves to isoprenaline in left atria and papillary muscles from control (01 and amiodarone (0) pretreated rats. Each value is the mean f LE.. n = 8-10.
The results of this study indicate that chronic pretreatment of rats with amiodarone had some antiarrhythmic effects in vivo, prolonged the effective refractory period of cardiac muscle measured in vitro but had no effect on ventricular a- or &adrenocep:ptor responsiveness. Previous reports have indicated that the same treatment regime in rats (Nokin et al., 1983) and similar dose ranges in rabbits (Venkatesh et al., 1986) decreased myocardial @-adrenoceptor density although corresponding functional studies were not performed. Although in left atria we found an increase in the isoprenaline EC,,, suggesting decreased P-adrenoceptor sensitivity, and a reduction in the m-urn increase in developed tension with phenylephrine the latter effect was also observed for responses to calcium. This suggests that these apparent changes in (Yand /3-adrenoceptor responsiveness may just be the result of a non-specific depressant effect of amiodarone on atria1 muscle. Further support for this suggestion comes from the evidence that the resting developed tension in left atria from amiodarone-pretreated rats was lower than that in controls whereas no such differences were seen in papillary muscle preparations. It is possible that we failed to observe any changes in ventricular adrenoceptor responsiveness because the plasma or myocardial concentrations of amiodarone were below the threshold required to produce such effects. The experiments which first showed the antisympathetic actions of amiodarone, were performed in vivo with high doses of amiodarone Kharlier et & 1968; Charlier, 1970). Similarly, the effects Of ami@ darone in vitro, on p-adrenoceptor mediated changes in heart rate in rabbit isolated right atria and on a-&eDoceptor mediated contractions of isolated rat aorta were fairly small, non-competitive and only OC-
ular arrhythmias induced chaemia or reperfusion.
by acute
myocardial
is-
ever, set:
s~~~i~~ca~treductions in the and ventricular ~r~~arati~~s. This suggests that we did achieve ~~~~~~~~~t~~~s s~f~cig~t to alter the effective refracriod of cardiac muscle a~tho~gb no changes in tory ve~t~~~~~a~adrenoeeptor responsiveness occurred. In our ~~~~~~~s study in rats made hypothyroid by chronic ~ret~eatm~~t with 6.pr~pyItbi~uraci1 ~~hess-Williams and coker, 1989). we observed reductions in maximum d~~~~~ frequency ~vhicb were of similar magnitude to those seen here. but a marked increase in ventricular a-adrenoceptor responsiveness also occurred. This indicates that amiodarone does not cause the same changes in adrenoce$ors as seen in propylthiouracilinduced h~ot~yroidism. The relative&+ poor antia~bythmic activity of amiodarone observed in this study was disappointing. The dose of 50 mg kg-’ day-’ p-o. for 4 weeks was chosen because it had been shown by Nokin et al. (19831 to reduce cardiac &adrenoceptor density in rats. In addition. it was more than doubie the dose of 20 mg kg-’ day- ’ p.a for 3 weeks which had been reported by Winslow et al. (1987) to have minor activity against is~haem~a-induced arrb~hmias in rats. Despite using a sigher dose we were not able to see any greater antiarrbyt~rni~ effect although it should be noted that all our embedments were performed 18 to 24 h after administration of the last dose of amiodarone, since we were interested in the effects of chronic treatment rather than acme effects. In contrast to the results seen follo~v~~gchronic dosing, Riva and Hearse (1989) have demonstrated marked reductions in ischaemia-induced VT and re~e~~sion-induced VF after a single i.v. dose of 5 mg kg-’ of amiodarone. This suggests that in rats, chronic dosing at daily intervals may be too infrequent to achieve drug levels which produce good antiarrh~hmic activity. Although the antiarrhythmic effects we measured in the present study were not as dramatic as some rewted by others, significant effects were observed. The maximum driving frequency of atrial and ventricular muscle was reduced, the number of ischaemia-induced VPBs was less and the duration of reperfusion-induced arrhythmias was shorter in amiodarone-pretreated rats. In addition, left atria1 ~-adr~no~eptor sensitivity was reduced. However, no alterations in ventricular adrenoceptor responsiveness could be found. Although we cannot exclude the possibility that alterations in adrenOCe@Or responsiveness may contribute to the antiarrhythmic activity of higher concentrations of amiodarone, the present results do not support the hypothesis that amiodarone-induced changes in adrenoceptor responsiveness contribute to its activity against ventric~~~~~~~
dying
~~~¶u~~c~ of both atria1
Financial support for this study was provided by Sanofi UK Ltd.
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