Electrophysiological effects of tiracizin and its metabolites in dog cardiac purkinje fibers

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0306-3623(94)00243-6

Gen. Pharmac.Vol. 26, No. 4, pp. 793-798, 1995 Copyright © 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/95$9.50+ 0.00

Electrophysiological Effects of Tiracizin and its Metabolites in Dog Cardiac Purkinje Fibers M I K L O S NI~METH, A N D R A S V A R R O and J UL I US GY. PAPP* Department of Pharmacology, Albert Szent-Gyfrgyi Medical University, Szeged, Hungary [Fax: 36 62 321 107] (Received 8 August 1994)

Abstract--1. Conventional microelectrode technique was used to study the effects of tiracizin and its two main metabolites on the action potential parameters in dog cardiac Purkinje fibers. 2. Tiracizin induced a use-dependent depression of the maximal rate of depolarization during the upstroke of the action potential (EC50= 0.323 ___0.059 #M, n = 5) and decreased the action potential duration (ECs0 = 0.230 + 0.024 #M, n = 5). 3. The two main metabolites exerted similar effects, but only at concentrations about one order of magnitude higher than tiracizin. 4. It was concluded that the electrophysiologicaleffects of tiracizin in dog Purkinje fibers correspond to those of Class I antiarrhythmic drugs also in cardiac Purkinje fibers. The main metabolites are less potent than the parent compound, therefore it is likely that their effects play a less important role in the beneficial effect of tiracizin in cardiac arrhythmias.

Key Words: Tiracizin, action potential, heart, Purkinje fibers

INTRODUCTION The most characteristic feature of a large group of antiarrhythmic drugs is the reduction of the maximal rate of depolarization during the upstroke of the action potential, which causes a decrease in the conduction velocity (Buchanan et aL, 1985). These drugs were classified by Vaughan Williams as Class I antiarrhythmics (Vaughan Williams, 1989). There are however distinct differences between the drugs belonging to the Class I group with regard to their effect on repolarization (Harrison, 1985) and also to their efficacy in cardiac ventricular muscle and Purkinje fibers (Varr6 et al., 1986), i.e. in both fiber types considered to be important in relation to the development of serious ventricular arrhythmias. Tiracizin (Fig. 1), a Class I antiarrhythmic drug has been found highly effective in abolishing experimental arrhythmias induced by aconitine, ouabain, CaCI2 or BaCI 2 in the rat, rabbit and dog (Femmer et al., 1985) and after occlusion of the coronary artery in conscious rats and dogs (Kaverina et al., 1985). Tiracizin has been reported to decrease the maximal rate of *To whom all correspondence should be addressed. ~P 26/4~J

depolarization during the upstroke of the action potential (f"max) in a use-dependent manner (Nilius et al., 1985a) and to lengthen repolarization in rabbit ventricular muscle (Nilius et al., 1985b). Pharmacokinetic studies revealed two main metabolites (Fig. 1) in the serum of tiracizin treated rats (Kiemm et al., 1985), but the possible antiarrhythmic and electrophysiological effects of these metabolites are unknown. Also, there are no data available about the effect of tiracizin on the action potential in cardiac Purkinje fibers. Therefore, in our present study the cellular electrophysiological effects of tiracizin and its two main metabolites were studied in dog cardiac Purkinje fibers. METHOD Adult mongrel dogs of either sex, weighing 5-8 kg were used. Following sodium pentobarbital (30 mg/kg i.v.) induced anaesthesia, their hearts were rapidly removed through right lateral thoracotomy. The hearts were immediately rinsed in oxygenated Tyrode's solution containing (in mM): Na ÷ 140, K ÷ 4, Ca 2+ 2.5, Mg 2+ 1, CI- 126, HCO3 25 and glucose 11. The pH of this solution was 7.35-7.45 when

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gassed with 95% 02 and 5% CO2 at 37°C. Purkinje strands obtained from either ventricle were individually mounted in a tissue chamber (volume: 50 ml). Each preparation was initially stimulated (HSE Stimulator Type 215/I1) at a basic cycle length of 500 msec (frequency = 1 Hz), using 2 msec long rectangular constant current pulses isolated from ground across bipolar platinum electrodes in contact with the preparation. At least 1 hr was allowed for each preparation to equilibrate while they were continuously superfused with Tyrode's solution. The temperature was kept constant at 37°C. Transmembrahe potentials were recorded by conventional microelectrode technique. Microelectrodes filled with 3 M KCI having tip resistances of 5-20 Mf~ were connected to the input of a high impedance electrometer (HSE Microelectrode Amplifier Type 309) which was referenced to the ground. The first derivative of transmembrane voltage with respect to time (~'max) was electronically derived by a HSE Differential:or (Type 309) which had linear response over the range of 20-1000V/sec. The voltage outputs from all amplifiers were displayed on a dual beam memory oscilloscope (Tektronix 2230, 100MHz Digital Storage Oscilloscope). The maximum diastolic potential (MDP), action potential amplitude (APA), action potential durations measured at 50 and 90% repolarization (ADPs0_90) were evaluated by a software (developed in our Department by Dr Ott6 Hfila) with IBM 386

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computer connected to the digital output of the oscilloscope. When different steady state stimulation cycle lengths were applied, action potential parameters were measured at each cycle length following adaptation to the new pacing cycle length (usually after 2 min). The preparations had been superfused for 30 min with the compounds before measurements were started. The concentration dependent effect of tiracizin was established in a cumulative manner with measurements of the effects of new concentrations added to the nutritient solution at 30 min intervals. Whenever it was possible, the same impalement was maintained throughout the experiment. When impalement was lost during measurement, readjustment was attempted. If the readjusted parameters were not more than 10% deviant from the previous ones the experiments were continued, otherwise they were terminated. Tiracizin and its two metabolites were obtained from AWD G m b H (Dresden, Germany). Student's t test for paired data were used to compare the results statistically. Results were considered significant when p was found to be less than 0.05. RESULTS As Fig. 2 and Table 1 indicate, tiracizin decreased APDs0, APD90 and ~'m~x in a concentration

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H3C Tiracizin

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Metabolite 1 Metabolite 2 Fig. 1. The chemical structure of tiracizin (3-Carbethoxyamino-5-dimethylaminoacetyl-10 , ll-dihydro5H-dibenzazepin) and its two rnetabolites, metabolite 1 (3-amino-5-dimethylarninoacetyl-10, l l-dihydro5H-dibenzazepin) and metabolite 2 (3-arnino-5-methylamino-acetyl-10, 1l-dihydro-5H-dibenzazepin).

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Effects of tiracizin in Purkinje fibers

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Control

@ Tiracizin

O

I

~IL

0.03 I.tM

~1<

0.1 p.M

[]

0.3 p.M

@

i ~M

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Fig. 2. Original recordings from a representative experiment showing the concentration dependent effect o f tiracizin on the action potential duration and lYm~x in a dog cardiac Purkinje fiber. The stimulation frequency was 2 Hz.

dependent

manner

in d o g c a r d i a c P u r k i n j e fibers

spectively. T h e A P A w a s d e c r e a s e d s i g n i f i c a n t l y b y

at the stimulation frequency of 2 Hz. The corres-

tiracizin at c o n c e n t r a t i o n s h i g h e r t h a n 0 . 3 / z M . T h e

ponding

~'m~x were

effects o f t h e t w o m a i n m e t a b o l i t e s o f tiracizin re-

0.230 + 0.024 # M a n d 0.323 + 0 . 0 5 9 / z M (n = 5), re-

f e r r e d in t h e text a s m e t a b o l i t e 1 a n d m e t a b o l i t e 2

ECs0 v a l u e s f o r

APDg0 a n d

Table 1. The effects of tiracizin and its metabolites on the action potential parameters in dog cardiac Purkinje fibers at 2 Hz stimulation frequency Drug

MDP (mY)

APA (mV)

APDs0 (mscc)

APDg0 (mscc)

lYma~ (V/sec) 448.0 + 17.4 432.0_+17.4

Control Tiracizin 0.03/~M

-87.2 + 1.7 -89.2+0.6

119.0 + 2.9 120.0+1.9

180.4 + 15.8 177.2+20.7

0.1/zM

-87.8_+0.5

116.4_+2.1

0.3#M

-87.4+0.9

112.0_+3.4

I#M (n = 5)

-85.6+1.6

104.2-+2.9 p <0.05

153.6+_16.7 p < 0.01 115.2+_7.3 p <0.01 90.2-+8.9 p <0.01

241.8_+ 17.0 232.4 + 18.6 p < 0.05 211.6-+ 16.4 p < 0.01 180.6+_10.7 p <0.01 156.8_+5.9 p <0.01

-82.0 +_ 1.2 -81.5_+0.8

119.3 _+ 1.8 115.7-+1.8 p < 0.05

138.7 _+21.0 97.7+_13.4 p < 0.01

227.3_+ 11.4 176.5+_6.0 p < 0.01

510.0_+ 15.1 393.3+_14.1 p < 0.01

-84.8-+ 1.5 -82.8-+1.0

118.0+__1.2 109.8_+4.3 p < 0.05

159.6+ 13.6 100.0_+14.2 p < 0.01

229.2+ 12.1 178.6_+7.8 p < 0.01

386.0-+21.1 268.0_+25.0 p < 0.01

Control Metabolite 1 5/tM (n = 6) Control Metabolite 2 5/JM (n = 5)

384.0-+20.4 p < 0.01 336.0+22.9 p <0.01 264.0_+10.3 p <0.01

MDP ~ maximal diastolic potential; APA = action potential amplitude; APDs0 = 50% rcpolarization; APD~ ~ 90% rcpolarization; lYm.x~ maximal rate of depolarization.

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C) Control A •

5 lttMMetabolite 1

0 mV

B

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C)

Control

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5 I.tM Melabolite 2

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Fig. 3. Original recordings from representative experiments showing the effects of 5/~M metabolite 1 (A) and 5/~M metabolite 2 (B) on the action potential and Vma~in dog cardiac Purkinje fiber. The stimulation frequency was 2 Hz. were investigated at a single concentration of 5/~ M. This concentration was chosen after preliminary dose finding experiments to match approximately the effects of tiracizin at the ECs0 concentrations. As Fig. 3 and Table 1 show both metabolite 1 and

metabolite 2 exerted similar effects on the action potential parameters at 5/~M concentration as was observed with tiracizin at lower concentrations. The frequency-dependent effects of tiracizin (0.1 pM) and its metabolites (5/~M) on the APD and

797

Effects of tiracizin in Purkinje fibers Table 2. The frequency-dependenteffectsof tiracizinand its metaboliteson APDg0 and l;'r~x in dog cardiac Purkinjefibers APD~ Drug

V~

1 Hz

3 Hz

I Hz

3 Hz

Tiracizin 0A/aM (n = 5)

81.8 _+ 3.2% p <0.01

85.4 + 2.9% p <0.01

89.2 _+ 2.4% p <0.01

75.4 _+ 7.7% p <0.01

Metabolite 1 5gM (n = 6 )

79.1+1.8% p <0.01

81.1+__1.5% p <0.01

86.7___1.4% p <0.01

74.5_2.2% p <0.01

Metabolite 2 5 #M (n = 5)

74.2 ___4.6% p < 0.01

83.7 + 3.0% p < 0.01

77.5 + 2.6% p < 0.01

67.8 + 3.3% p < 0.01

APDgo = 90% of repolarization; 17m~x = maximal rate of depolarization; Data are expressed as % of drug-free values.

[ ' ~ were studied by varying the stimulation frequency between 1 and 3 Hz. The results obtained are shown in Table 2. The APD shortening effects of tiracizin and metabolite 1 did not depend on the stimulation frequency, while with metabolite 2 somewhat more APD shortening occurred at the low (1 Hz) than at the high (3 Hz) stimulation rate. Tiracizin and its two metabolites depressed ~'maxmore at the high (3 Hz) than at the low (1 Hz) stimulation frequency, suggesting use-dependent ~'m~xblock by the compounds. DISCUSSION The main finding of the present experiments is that tiracizin and its two main metabolites are capable of shortening repolarization and depressing ~'ma~, the latter in a frequency-dependent manner. The electrophysiological changes evoked by tiracizin and its two metabolites were similar to those of Class I antiarrhythmic drugs, i.e. they shortened repolarization in Purkinje fiber and within a relatively wide range of stimulation frequencies they depressed ~'max

•

The observed shortening of APD could be related to the decrease of the TTX-sensitive inward current carried by sodium during the plateau phase of the action potential described as "window current" (Attwell et al., 1979) or by depressing a slowly inactivating component of the fast inward sodium current (Carmeliet, 1987). Tiracizin was reported earlier to depress both ~'max(Nilius et al., 1985a) and inward sodium current (Nilius et al., 1985a) in a frequency-dependent manner in rabbit ventricular and in frog atrial trabecular muscles, respectively. Our results agree with these previous observations. In rabbit ventricular muscle at high (5-8 mg/l) concentrations tiracizin lengthened APD (Nilius et al., 1985b). There are no voltage-clamp data available so far about the effect of tiracizin on the repolarizing potassium currents, but it is possible that the drug

decreases potassium conductances at high concentrations which may result in a prolongation of APD in ventricular muscle. The reported APD lengthening by tiracizin in ventricular muscle differed from our results, We have found a pronounced shortening of APD in dog Purkinje fiber. It is known that TTX shortens repolarization markedly in Purkinje, but only moderately in ventricular muscle fibers (Coraboeuf et al., 1979). If the APD shortening effect of tiracizin is due to depression of the sodium window current, as we speculated previously, this effect on the window current may overcome any possible repolarization lengthening effect in Purkinje, but not in ventricular muscle. It is important to note that the effective concentration of the metabolites is with one order of magnitude higher than that of the parent compound, tiracizin. This may suggest that these metabolites play a less important role in the antiarrhythmic effect of tiracizin. However, without available data about plasma and tissue concentrations of tiracizin and its metabolites, contribution of the latter compounds to the well documented antiarrhythmic effect after tiracizin administration (Femmer et al., 1985; Kaverina et al., 1985) cannot be ruled out. Acknowledgements--This work was supported by the Arzneimittelwerk Dresden GmbH and by grant (OTKA 676) from the Hungarian National Scientific Research Foundation.

REFERENCES Attwell D., Cohen I., Eisner D., Ohba M. and Ojeda C. (1979) The steady state TTX-sensitive ("window") sodium current in cardiac Purkinje fibers. Pfliigers Arch. 379, 137-142. Buchanan J. W., Saito T. and Gettes L. S. (1985) The effects of antiarrhythmic drugs, stimulation frequency, and potassium-induced resting membrane potential changes on conduction velocity and dV/dtm,~ in guineapig myocardium. Circulation Res. 56, 696-703. Carmeliet E. (1987) Slow inactivation of the sodium current in rabbit cardiac Purkinje fibers. Pfliigers Arch. 408, 18-26.

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Coraboeuf E., Deroubaix E. and Coulombe A. (1979) Effect of tetrodotoxin on action potentials of the conducting system in the dog heart. Am. J. Physiol. 236, 561-567. Femmer K., Poppe H., Heer S. and Bartsch R. (1985) Zur anti-arrhythmischen Wirkung yon 3-Carbethoxyamino-5-dimethylaminoacetyl-iminodiben-zyl-hydrochlorid (BonnecorR, AWD 19-166, GS 015) an der experimentellen Arrhythmie durch Aconitin, Ouabain, Calciumchlorid und Bariumchlorid. Pharmazie 40, 836-840. Harrison D. C. (1985) Antiarrhythmic drug classification: New science and practical applications. Am. J. Cardiol. 56, 185-187. Kaverina N. V., Liskovzer V. V., Grigoreva J. K., Senova Z. P., Sulakvelidze M. T. and Grizenko A. N. (1985) Antifibrillatorische und antiarrhythmische Wirksamkeit von 3-Carl~thoxyamino-5-dimethyl-amino-acetyl-iminodibenzyl-hydrochlorid (Bonn~or a, AWD 19-166, GS 015) an Modellen der Myocardisch/imie. Pharmazie 40, 845-847. Klemm W., Morgenroth U., Joram U. and Rodionov A. P.

al.

(1985) Pharmakokinetic und Metabolisierung von 3Carbet hoxiamino-5-dimethylamin-acetyl-imino-dibenzylhydrochlorid (Bonnecor~, AWD 19-166, GS 015) an der Ratta. Pharmazie 40, 864-867. Nilius B., Benndorf K. Schfittler K. and Boldt W. (1985a) Use-dependent depression of fast sodium current in heart muscle by the new antiarrhythmic substance BonnecorR (AWD 19-166, GS 015). Pharmazie 40, 852-854. Nilius B., Schfittler K., Benndorf K. and Boldt W. (1985b) Electrophysiological effects of a new antiarrhythmic substance Bonnecor (AWD 19-166, GS 015) on mammalian myocardium. Pharmazie 40, 847-851. Varro A., Nakaya Y., Eiharrar V. and Surawicz B. (1986) Effect of antiarrhythmic drugs on the cycle lengthdependent action potential duration in dog Purkinje and ventricular muscle fibers. J. Cardiovasc. Pharmac. 8, 178-185. Vaughan Williams E. M. (1989) Classification of antiarrhythmic actions. In Antiarrhythmic Drugs. (Edited by Vaughan Williams E. M. and Campell T. J.), pp. 45-67. Springer-Verlag, Berlin.