Modified method for the production of cardiac arrhythmias by ouabain in anesthetized cats

Modified Method for the Production of Cardiac Arrhythmias by Ouabain in Anesthetized cats

TELLAS. RAo, SHIVA D. SETH, USHA NAYAR,

AND

SUBHASH C. MANCHANDA

A modified method of producing ouabain-induced cardiac arrhythmias in chloralose-anesthetized cats has been developed, whereby prolonged arrh~hmias with low incidence of mortality (12%) are observed. Furthermore, the employment of a modified arrhythmia scale in the present model allowed a quantitative estimation of antiarrhythmic efficacy of test drugs and also made it possible to unravef the arrhythmogenic nature of these substances. This improved method could be conveniently used for the quantitative screening of both the antiarrhythmic or the arrhythmogenic nature of test drugs. Key Words: Modified thetized cats

method;

Ouabain

infusion;

Cardiac arrh~hm~as;

Anes-

INTRODUCTION

One of the most widely employed experimental models for the screening of antiarrhythmic substances is the digitalis-induced cardiac arrhythmias. This method has a great potential from a clinical-toxicological standpoint, in view of the occurrence of the relatively high incidence of potentially fatal cardiac arrh~hmias due to digitalis overdosage (Mason et al., 1973). The earlier methods for the evaluation of antiarrhythmic efficacy of drugs with the use of cardiac glycosides were based on two principles: I) the ability of the test drug to prevent the development of or 2) to abolish an existing arrhythmia. Due to the low margin of safety and the progressive nature of intoxication with cardiac glycosides, there has been a difficulty in standardization of this arrhythmia model (Szekeres, 1971). The standard experimental method of producing arrhythmias that has been employed for the screening of the potential of test drugs to abolish existing arrhythmias consists of injecting an initial large i.v. bolus dose followed by small bolus doses of cardiac glycosides at regular intervals of IO min until the arrhythmias are produced. However, it has been reported that about 75% of the total animals die of serious cardiac arrhythmias with this procedure (Szekeres, 1971; Forster et al., 1976) and is thus not a satisfactory model for the evaluation of test substances. Furthermore, the administration of a test substance during this progressive phase of ouabain-induced cardiotoxicity may cause a considerable problem in distinguishing between the drug-induced aggravation and the spontaneous aggravation of these

From the Departments

of Pharmacology (T.S.R., S.D.S.),

Physiology W.N.)

and Cardiology (S.C.M.),

All-India Institute of Medical Sciences, New Delhi, India. Address reprint requests to: Dr. S. D. Seth, Department Sciences, New Delhi-110 029, India.

]ournalof

Pharmacological

Methods

Q 1988 Elsevier Science Publishing

20,255-X3

of Pharmacology, All-India institute of Medical

(19W

Co., Inc., 655 Avenue of the Americas,

New York,

NY lWl0

256

T. S. Rao et al. arrhythmias. In light of these facts, the method has been modified to obtain prolonged arrh~hmias with relatively low incidence of mortality, which allowed a satisfactory quantitative evaluation of antiarrhythmic efficacy of test substances.

MATERIALS AND METHODS Cats of either sex weighing between 2 to 5.25 kg were anesthetiied with alphachloralose (70-80 mg/kg i.v.) after initial induction with anesthetic ether and were allowed to breathe spontaneously. The left femoral arterial blood pressure (BP) and lead II electrocardiogram (ECG) were recorded on a Grass Model 7 Polygraph. The heart rate (FIR) was calculated from the ECG tracing. The rectal temperature of the animals was maintained at 38 * 1°C. Ouabain octahydrate (Sigma) dissolved in normal saline was infused through the femoral vein at a rate of 1 or 2 bg/kg/min (0.1 ml/min). The infusion of ouabain was terminated when two or more consecutive ectopic beats were first observed in the ECG. The cardiac arrhythmias were then monitored until the sinus rhythm was spontaneously restored. The time of occurrence of peak arrhythmias was also noted. In some animals after the arrhythmias had reached the peak severity, the effect of different doses of i.v. administered prostaglandins (PCs) I2 and F2n and their vehicles and also of lidocaine on these arrhythmias were tested. Lidocaine was administered as repeated bolus injections of 1 mglkg at S-min intervals until the arrhythmias were completely abolished or a total dose of 4 mg/kg was achieved. The reduction in the severity or complete suppression of cardiac arrhythmias by the drugs in those experiments in which this change was followed by recovery to initial (pre-drug) arrh~hmia was considered as antiarrhythmic effect. The time interval between the beginning of injection and the occurrence of maximal antiarrhythmic effect was designated as the onset of action and the time interval between the occurrence of maximal antiarrhythmic effect and the recovery to the starting arrhythmias was termed as the duration of effect. The restoration of sinus rhythm for more than 0.5 min indicated an antiarrhythmic effect, whereas the normalization of the cardiac rhythm disturbances for less than 0.5 min and the reduction in the severity of arrhythmias were recorded as improvement of arrhythmia. Similarly, the onset of arrhythmogenic effect was calculated as the time interval between the beginning of injection and the maximal arrhythmogenic effect, whiie the duration was determined as the time interval between the maximal arrhythmogenic effect and the recovery to the starting arrhythmias, respectively. The statistical analysis of arrhythmias was performed using the arrhythmia scale of Mest and Forster (1982) with some modifications. The modified scale used is as follows: Score 0: Sinus rhythm Score 20: Sporadic ventricular ectopics, supraventricular tachycardia with a ventricular rate of 150-200. Score 40: Bigeminy, more frequent occurrence of sporadic ventricular ectopics, supraventricular tachycardia with a ventricular rate of 200-250.

Ouabain-induced

Arrhythmia

Model

Score 60: Volleylike ventricular ectopics up to 4 inch succession, supraventricular tachycardia with a ventricular rate of 250-275. Score 80: Volleylike ventricular ectopics more than 4 inch succession, ventricular tachycardia and supraventricular tachycardia with a ventricular rate of more than 27.5. Score 100: Ventricular flutter, ventricular fibrillation and death. The differences in pre- and post-drug arrhythmia scores were calculated. These differences of 0 (no change in arrhythmia), 20, 40, 60, 80, and 100 were ranked as 1, 2, 3,4, 5, and 6 respectively. The statistical significance of these rankings in drugtreated groups was determined using Wilcoxon’s Two-Sample Rank Test for unpaired samples with correction for ties by comparing with appropriate vehicle control groups. The changes in HR (beats/min) and BP (mm Hg) produced by drugs were evaluated for statistical significance using Student’s t-test for paired values. salt. PC12 sodium salt (The Upjohn The drugs used were PGF2G tromethamine Company, MI, USA), ouabain octahydrate, alpha-chloralose (Sigma Chemical Co., St. Louis, MO USA), lidocaine ~Suhrid Geigy Ltd., India), anesthetic ether (Allemb~c Chemical Works Co. Ltd., India). Stock solution of 10 mglml of PGF2, was made in absolute ethanol, and further dilutions were made in normal saline just before use. PC& was made up as 10 mg/ml solution in 0.5% W/V solution of sodium hydroxide (pH 13) and further diluted with 0.0005% W/V sodium hydroxide solution for use. All other drugs used in the study were dissolved in normal saline. RESULTS Ouabain-induced

Cardiac Arrh~hmias

The 2 ~g/kg/min i,v. infusion of ouabain resulted in ventricular arrhythmias in all the five animals tested. The mean arrhythmogenic dose of ouabain to produce two or more consecutive ectopics was 83.4 + 7.6 r&kg. In four of these five animals, the severity of arrhythmias progressively increased and ultimately death occurred due to ventricular fibrillation. In one animal, ventricular tachycardia lasted for 205 min. The cardiac arrhythmias produced by 1 pg/kg/min i.v. ouabain included ventricular ectopics, bigeminy, ventricular tachycardia, supraventricular tachycardia, and AV conduction disturbances. AV conduction defect preceded the ventricular arrhythmia in one out of eight control animals studied. The mean arrhythmogenic dose of ouabain to produce two or more consecutive ectopics was 79.5 rt 5.8 Fg/ kg. In one control animal, ventricular arrhythmias progressively increased in severity and resulted in death due to ventricular fibrillation 5 min after the termination of ouabain infusion. In the rest of the seven control animals, the ventricular arrhythmias reached the peak severity within IO-15 min after the termination of ouabain infusion and lasted for a minimum of 23 min to maximum of 160 min with a mean of 68.7 2 16.5 min. The ouabain-induced hemodynamic changes included a gradual reduction in HR associated with a simultaneous progressive increase in BP during its infusion. The

257

258

T. S. Rao et al. TABLE 1 DOSE (@K&~IN) 2.0

1.0

Time-course of Mean Cardiovascular Responses to i.v. Ouabain in Anesthetized VALUEDURING OUA n

PARAMETER

INITIAL VALUE

15

30

HR

190.6 f 10.5

179.6 2 7.6'

176.4 k 9.4

BP HR BP

115.2 2 9.4 185.4 f 11.4 111.3 * 7.9

126.0 2 10.2' 181.0 2 ll.od 120.6 ” 7.6d

127.4 ? 10.9' 174.8 t 9.Sd 128.4 t- 6.1d

45

5

8

Values are expressed as mean 2 SE; n = number of experiments. a The numerals in parentheses indicate the number of experiments was being continued. b The numerals indicate the number of surviving cats. c p < 0.05. d p < 0.01,as compared to initial values.

171.1 * 9.9d 134.9 2 6.7

in which the infusion of oubain

bradycardia of this phase was maintained till the appearance of arrhythmia in six animals. In the remaining two animals, this bradycardia was followed by a rapid increase in HR at 4 and 20 min before the occurrence of arrhythmias and the time of peak increase coincided with the occurrence of arrhythmias. In one of these two animals there was a simultaneous steep increase in BP. In contrast to HR, the progressive increase in BP seen during the above slow phase did not sustain, and it returned to preinfusion level or below this level at the time of occurrence of arrhythmias in five out of eight animals. In two animals the phase of initial increase in BP maintained until the arrhythmias occurred. After the arrhythmias had reached peak severity, the HR maintained at a level higher than the preouabain values, whereas the BP remained at a lower level. The time course of changes in HR and BP produced by ouabain is shown in Table 1. Similar changes were seen in cats used for screening the effects of drugs on ouabain-induced arrhythmias. The incidence of mortality due to severe arrhythmias caused by ouabain was 12%. Effect of Drugs

on Ouabain-induced

Arrhythmias

PC& (2-16 Fg/kg i.v.), when administered immediately after the arrhythmias had reached the peak severity, effectively suppressed the ouabain-induced ventricular and supraventricular arrhythmias. However, in few cats, PGFZa, although statistically insignificant, aggravated these arrhythmias. The vehicle of PGFza had no effect on arrhythmias (Figure 1). The antiarrhythmic and arrhythmogenic effects of PGFZuwere of short duration (Table 2). PG12 (4-16 pg/kg i.v.) markedly suppressed the ouabain-induced ventricular and supraventricular arrhythmias and, unlike PGFZa, failed to aggravate the ouabaininduced arrhythmias. The vehicle of PG12 failed to alter the ouabain-induced arrhythmias (Figure 2). The 4, 8, and 16 kg/kg doses of PG12 restored the sinus rhythm in 1,2, and 3 and improved the arrhythmias in two, five, and three out of five, eight, and seven animals tested. The antiarrhythmic effects of PG12were of short duration

Ouabain-induced

Arrhythmia

Model

Cats PEAKSVALUE

BAlN lNFusi0~~ (MIN) 75

60

1 MIN AFTER

DURING

ARRHYTHMIA

ARRHYTHMIA

ARRHYTHMIA

183.6 2 12.5

181.4 2 12.0

205 (1)

124.8 2 9.9’ 180.8 2 13.0 124.1 2 10.9

121.8 ? 13.5 197.1 * 10.8 114.6 * 12.5

85 (1) 214.4 2 9.4’ (7) 90.0 2 6.3’ (7)

90

190.7 ? 26.6 (3) 117.3 * 14.1 (3)

166.3 ‘- 11.5 (6) 125.7 2 13.0 (6)

161.6 2 8.3d (7) 132.0 ? 7.1’ (7)

1 MIN BEFORE

(Table.2). Lidocaine effectively suppressed the ouabain-induced ventricular and supraventricular arrhythmias in five out of six animals tested (Figure 2). The mean dose of lidocaine required to produce its maximal antiarrhythmic effect was 1.6 4 0.2 mg/kg. The antiarrhythmic effect of lidocaine was of short duration (Table 2).

DISCUSSION

The data presented indicate that the slow i.v. infusion of ouabain at a rate of 1 pg/kg/min and its subsequent termination immediately after the first appearance of a minimum of two consecutive ectopic beats causes prolonged cardiac arrhythmias of ventricular and supraventricular origin. With this mode of administration of ouabain, the arrhythmias reached the peak severity within 15 min after the termination of ouabain infusion. The incidence of mortality in the present method is 12%, and, in contrast,

TABLE 2 Cats

75% mortality

was commonly

observed

with the use of original

The Effects of PCs Fzu, I2 and Lidocaine on i.v. Ouabain-induced

method

Arrhythmias in

ANTIARRHMHMIC EFFECT DOSE (PC/KC I.V. DRUG

BOLUS)

2.0 4.0 8.0

PGFz,

PC Fz, PG Fzu PGFz, PC12 PGl* PC12 Lidocaine

1.6+

16.0 4.0 8.0 16.0 0.2mg/kg

N

n

5 5 8

2 2 5

23 5 8 7 6

14 3 7 6 5

ONSET

DURATION

(MlN)

(MIN)

0.5, 0.75 1.0, 1.5 1.1 t 0.5 0.9 0.44 0.54 0.32

2 -+ t ? -

0.2 0.1 0.1 0.06

ONSET n

4.5, 4.0 8.0, 5.0 3.721.1

1 1 1

4.9 2.3 2.6 3.6 4.7

4

t ? t t ?

0.8 0.6 0.7 0.8 1.7

(SEC)

60 20 20 28.8 i- 1.3

DURATION bd

8.0 1 .o 2.5 4.0, 1.75 (2)

The data are given as either individual absolute values or mean 2 SE. N indicates the total number of animals in each group whereas n represents the number of animals that responded to drugs with the particular effect under which it is mentioned. The numerals in parentheses indicate the number of surviving cats.

259

260

T. S. Rao et al. a

BEFORE AFTER

Iv--

DRUG DRUG

PGIF, 2cc ANTIARRHYTHMIC

l-

EFFECT

ARRHYTHMOGENIC

EFFECT

lEH1Cl.E OF paF2

a:

1 1II! I

I-

I-

l

/-

l-

+*

1 ~_

2

2

5

14 ~

~

4

8

p!il/kg

16

1

1 ~

2

1

4

4

8

16

0.5

ml

FIGURE 1. The antiarrhythmic and arrhythmogenic effects of 2 (n = 5), 4 (n = 5), 8 (n = 8) and 16 (n = 23) &kg i.v. doses of PGFzu on i.v. ouabain-induced cardiac arrhythmias in chloralose-anesthetized cats. The numerals just below the horizontal axis represent the doses of PGFzo,, whereas the numerals inside the histograms indicate the number of animals exhibiting the corresponding effect at the respective dose levels. The arrh~hmia scores were calculated as described in the text. The effects of PC& on cardiac arrh~hmias were statistically compared with its vehicle control groups using Wilcoxon’s two-sample rank test for unpaired samples with correction for ties. *P < 0.05; **P < 0.01.

(Forster et al., 1976; Mest et al., 1974; Szekeres, 1971). The original method of producing arrhythmias consisted of injecting ouabain initially as a large bolus dose of 60 Pg/kg i.v. followed by small bolus doses of IO kg/kg every IO min until the arrhythmias are produced. However, with the use of this procedure, arrhythmias progressively increase in severity in majority of the animals (75%) and consequently lead to death (Forster et al., 1976; Mest et al., 1974; Szekeres, 1971). Therefore, the subsequent administration of any test drug during this progressive phase of ouabain-induced cardiotoxicity may pose considerable problems in distinguishing between the drug-induced aggravation and the spontaneous aggravation of these arrhythmias. Thus, it may be difficult to determine the arrhythmogenic potential of the test substances and may also underestimate the true antiarrhythmic efficacy of the drugs under these experimental conditions. One of these two possibilities has been evidenced by the results of the present study, which indicate that PGFza may cause either antiarrhythmic or arrhythmogenic effect in any given animal-the former being predominant. This is contrary to the earlier reports revealing only its antiarrhythmic efficacy (Forster, 1976; Forster et al., 1973; Mest et al., 1974). The failure to observe the arrhythmogenic potential of PCFza in the earlier investigations

Ouabain-induced

Arrhythmia

Model

may be due to the above-mentioned difficulty in distinguishing between the PGFZcrinduced aggravation and the spontaneous progressive aggravation of ouabain-induced arrhythmias. Since in the present study PCF,, was tested after the arrhythmias had reached the peak severity, the aggravation of arrhythmias observed after its administration can be ascribed to its inherent arrhythmogenic property and is not due to spontaneous aggravation of these arrhythmias. It is important to mention that there is a possibility of the spontaneous recovery of ouabain-induced arrhythmias being interpreted as the antiarrhythmic effect of test drugs. The occurrence of such interpretations is particularly likely when the test drugs are administered in the later part of the duration of ouabain-induced arrhythmias. However, this problem has been avoided by administering the drugs immediately after the arrhythmias had reached the peak severity and by observing for the reappearance of initial arrhythmias after the drug effect is subsided.

0

BEFORE

m

AFTER

DRUG DRUG

PGI,

VEHJCLE OF PGI,

LIDOCAINE

SALINE

!

4

8

pg/b

16

0.5 ml

1.620.2

0.5

mg/kg

ml

FIGURE 2. The antiarrh~hmi~ effects of 4 (n = 5), 8 fn = 81, and 16 (n = 7) &g/kg i.v. doses of PClz and lidocaine fn = 6) on iv. ouabain-induced cardiac arrhythmias in chloraloseanesthetized cats. The numerals just below the horizontal axis represent the doses of PGi2 or the mean dose of lidocaine, whereas the numerals inside the histograms indicate the number of animals exhibiting the corresponding effect at the respective dose levels. The arrhythmia scores were calculated as described in the text. The statistical significance of the effects of PC12 as compared to its vehicle (n = 5), and the lidocaine versus saline (n = 5) were calculated by employing Wilcoxon’s two-samples rank test for unpaired samples with correction for ites. *P < 0.05; **P < 0.01; ***P < 0.001.

261

262

T. S. Rao et al.

The infusion of ouabain at a rate of 2 ~~kg/rnin caused m~rtaii~ in 80% animals due to severe arrhythmias as compared to 12% mortality observed with its 1 @g/kg/ min infusion. This suggests that the rate of infusion is a critical factor in eliciting prolonged arrhythmias with low incidence of mortality. As the duration of the arrhythmias was sufficiently prolonged with the use of the present method it was possible to determine the onset, duration, and dose-effect relationship of antiarrhythmic drugs and, in particular, the short-acting substances like PGs studied in the present work. The short duration of the effects of PGs on arrhythmias is in keeping with their fast metabolic degradation in vivo (Ferreira et al., 1967). With the employment of the present arrhythmia scale along with the proposed statistical tests, it is also possible to make a comparative and quantitative estimation of ant~arrhythmic efficacy of test drugs. The antiarrhythmic effect of PC& observed in the present study is in agreement with the earlier reports (Mest and Forster, 1981). The standard antiarrh~hmic drug lidocaine effectively suppressed the ouabain-induced arrhythmias. The slow increase in BP during the slow phase of the hemodynamic changes caused by ouabain infusion is consistent with its known vasoconstrictor action (Briggs and Shibata, 1966, Mason and Braunwafd, 1964; Ross et al., 19601. The simultaneous reduction in HR maay be due to the baroreceptor mediated reflex effect (Daggett and Weisfeldt, 1965). However, it was observed in some experiments that the bradycardia sustained even when the BP after the slow rise gradually declined to basal (pre-ouabain) values with the continuing infusion of ouabain. This may suggest additional mechanisms contributing to the bradycardia caused by ouabain. In this context, it may be noted that ouabain causes sensitisation of baroreceptors fGillis et al., 19751 and elicitation of Bezold-Jarisch reflex fhnellville, 1952; Sleight et al., 1969; Thames, 19791, which may in part mediate its bradycardiac effect. The phase of decline in the BP after its initial slow rise with continuing infusion may be due to the reflex effects of the above-mentioned buffering mechanisms and also the direct cardiotoxic action of high dose of ouabain. With regard to the rapid phase consisting of large and rapid increments in HR and BP, it has been convincingly demonstrated that the ouabain-induced cardiac arrhythmias are due in part to its sympathetic neuroexcitatory effects (Gillis and Quest, 1980). The rapid increase in HR and BP at the time of occurrence of arrh~hmias, therefore, may be due to this excessive sympathetic drive to the periphery. in conclusion the low incidence of mortality, the production of prolonged arrhythmias, the tack of progressive phase in which arrhythmias increased in severity resulting in death of the animals and the occurrence of peak arrhythmias within 15 min after the termination of ouabain infusion together make the present method more appropriate for the accurate quantitative estimation of antiarrhythmic and arrhythmogenic potentials of test drugs. Thanksare due to Dr. 1. E. Pike of The Upjohn Company, Michigan, U.S.A., for the generous gift of PC;F& , and PC~Z: Indian Council of Medical Research, New Delhi, tndia for financial support; and the Director, AU-India institute of Medical Sciences, New Delhi, India for awarding fellowship to T. S. Rae.

Ouabain-induced

Arrhythmia

Model

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Mellville KG (1952) On the mechanism of the cardiovascular action of digitalis: Observations on the influence of flaxedil, atropine or vagotomy. 1. Pharmacol Exp Ther 106:208-218. Mest HJ, Forster W (1981) Is PC& arrhythmogenic? Investigations on ouabain-induced arrhythmias in cats. Prostaglandins Med 7:411-419. Mest HJ, Forster W (19821 Influence of IS-ketometabolites and 13, ~4-dihydro-15-keto-metabelites of PC4 and PGFaa, as well as of 6 ketoPGF,,, as a metabofite of PC& on aconitine induced cardiac arrhythmia in rats. Acta Biol Med Germ 41:819-822. Mest HJ, Mentz P, Forster W (1974) Effects of prostaglandins on experimental arrhythmias. Pal / Pha~macoi Pharm 26:151-158.

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Thames MD (1979) Acetylstrophanthidin-induced reflex inhibition of canine renal sympathetic nerve activity mediated by cardiac receptors with vagal afferents. Circ Res 44:8-15

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