THE EFFECT OF DIPHENYLHYDANTOIN ON CONDUCTION IN ISOLATED, BLOOD-PERFUSED DOG HEARTS. Accepted for publication February 15, 1968

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1 Tns Jouiuiu or PHRMCOLOGY ND EXPHRMENTL THZRPEUTC$ Copyright by The Williams & Wilkins Co. Vol. 161 No. 2 Printed in &.S.. THE EFFECT OF DPHENYLHYDNTON ON CONDUCTON N SOLTED, BLOOD-PERFUSED DOG HERTS BETTY. SSYNUK id PETER E. DRESEL Department of Pharmacology and Therapeutics, University of Manitoba Faculty of Medicine, Winnipeg, Canada ccepted for publication February 15, 1968 BSTRCT SSYNUX, Bz rry. m Pnrza E. DRESEL: The effect of diphenyihydantoin on conduction in isolated, blood-perfused dog hearts. J. Pharmacol. Exp. Therap. 161: , The effects of diphenylhydantom on conduction times through the atrium and the specialized conducting tiseue to the ventricular myocardium have been determined from records obtained with a bipolar surface electrode placed over the bundle of His of isolated, bloodperfused dog hearts. Diphenyihydantoin slowed conduction in the atrioventricular node to a greater extent than in any other part of the heart at all concentrations tested. Clinically effective concentrations slowed conduction only through the atrioventricular node. The conduction of atrial extrasystoles interpolated after every 10th driving stimulus was preferentially affected by diphenyihydantoin. Concentrations of diphenylhydantoin which had only minimal effects on atrioventricular nodal conduction time of the normal beat greatly increased the atrioventricular nodal conduction delay of an extrasystole. Diphenylhydantoin (DPH) is an effective antiarrhythniic agent (Conn, 1965; Bernstein et at., 1965; Harris et at., 1966). There is little agreement concerning the mechanism of its effects on cardiac conduction. trioventricular (V) conduction time is shortened both in anesthetized (Helfant et at., 1967; Bigger et at., 1967) and in unanesthetized (Rosati et al., 1967) dogs. Bigger et at. reported that the effect of DPH on conduction was not affected by cholinergic and adrenergic blockade. Rosati et at., on the other hand, found the effect to be abolished or reversed by cardiac denervation. The present study was undertaken to determine the direct effect of diphenyihydantoin on cardiac conduction. ntraatrial, atrium-his bundle and His-ventricular conduction can be measured simultaneously in the isolated, bloodperfused dog heart. Extracardiac drug actions which contribute to changes in conduction in intact animals do not affect the isolated preparation, yet conduction times do not differ appreciably from those seen in vivo. METHODS. The technique for perfusion of dog hearts, developed originally by lanis et at. (1958), has been described in detail by Kirk and Received for publication January, Supported by a grant from the Medical Research Council of Canada. Dresel (1965). The hearts were removed from young dogs (5.-92 kg) anesthetized i.v. with pentobarbital sodium (0 mg/kg) and were transferred immediately to cold, oxygenated Krebs- Henseleit solution. The pericardium and all adjacent tissues were removed. The hearts were perfused through the aorta with arterial blood from a heparin-treated donor dog. The donor dog ( kg) was anesthetized with i.v. pentobarbital sodium (0 mg/kg) and was ventilated by a Palmer deal respiration pump with either air or 100% oxygen. The perfusion pressure of the isolated heart was maintained at 100 mm Hg by adjusting the flow of blood through one head of a calibrated double-headed DeBakey roller pump interposed between the femoral artery of the donor dog and the recipient heart. Changes in perfusion pressure due to drug injections were minimal and of short duration. No attempt was made to adjust the flow to compensate for the pressure changes. The temperature of the perfusing blood was kept at 7 ± 0.5#{176}Cby a water-jacketed coil condenser interposed between the pump and the heart. Coronary venous outflow was collected in a funnel and returned to the femoral vein of the donor dog through the second head of the roller pump. The right atrium of the isolated heart was opened and the cut edges were retracted with stainless-steel hooks. Simultaneous recordings of atrial, His bundle and ventricular potentials were obtained with a bipolar silver contact sur- 191

2 192 SSYNUK ND DRESEL Vol. 161 face electrode placed over the bundle of His. The contact points, 02 mm in diameter, were mounted 2 mm apart with epoxy cement in the tip of a curved flexible polyethylene plastic probe. The hearts were driven at rates 20 to 0 beats/mm faster than the sinus rate (0-180 beats/mm) with 5-msec rectangular pulses of twice threshold voltage applied to bipolar steel clip electrodes attached to the right atrium near the sinus node. Tektronix (type ) stimulator was used to drive the heart at the basal frequency. n most experiments, an atrial extrasystole was interpolated at various intervals after every 10th driving stimulus. Output pulses from the driving stimulator triggered a scale-of-lo counter which in turn delivered a trigger impulse to a second Tektronix stimulator which could stimulate the heart at an adjustable interval after every 10th driving stimulus. The electrogram from the bundle of His was displayed on a Tektronix model 502 dual beam oscilloscope. The stimulator used to drive the heart at the basic frequency also triggered the sweep of the oscilloscope. For technical reasons B S VP VEX Fic. 1. Recordings (two regular beats, one extrasystole) obtained with a bipolar surface electrode placed over the bundle of His. S, stimulus artifact of driving pulse;, atrial potentials HN and action potentials from bundle of lls of normal beat and extrasystole, respectively; V and V, ventricular potentials of normal beat and extrasystole, respectively. Recordings shown in were obtained during the control period before drug; those in B were obtained immediately alter a 1-mm infusion of diphenylhydantoin (16 g/ml). Basic cycle length, 500 msec; interval between extrasystoles, 162 msec. The rapid deflection of atrial and His bundle potentials have been retouched. (see DscussoN), the stimulator triggered by the scale-of-lo counter did not. Thus, the response to the first driving stimulus alter the interpolated extrasystole was recorded, whereas the interpolated extrasystole was not. The first response alter the interpolated beat may itself be regarded as a second extrasystole and will be referred to as the extrasystole. The oscilloscope tracings were recorded on 5-mm film by a Shackman camera. Single sweeps were recorded except in those experiments in which an extrasystole was interpolated after every 10th driving stimulus. n these, two normal beats and the immediately following (second) extrasystole were recorded on each frame (fig. 1). Exposures were taken every 20 sec for 2 to min before drug administration, every 15 to 20 sec for 5 to 10 min alter drug administration was begun, and then every 0 to 60 sec until the conduction intervals had returned to the preadministration state. Diphenyihydantoin was injected over 1 mm into the aortic cannula of the isolated heart. The concentration of the drug in micrograms per milliliter of perfusing blood was calculated from the rate of injection and the volume of blood delivered by the pump. No more than three injections were made in any one experiment. The intervals between doses of drug were 5 to 90 mi Diphenyihydantoin was also infused i.v. into the donor dog. The diphenylhydantoin used was the sodium salt, supplied by Sigma Chemical Company, or Dilantin, supplied by Parke, Davis and Co. stock solution of the drug was prepared before each experiment. One hundred milligrams of the drug were dissolved in 2 ml of 0.1 N sodium hydroxide. Distilled water was then added to a total volume of 10 ml. n appropriate volume of the stock solution was then diluted to.0 ml with 0.9% sodium chloride for intraarterial administration. The commercial diluent was not used because it was found to prolong the -H conduction time of the isolated heart. We observed no effects with our diluent in the volumes used for administering drug (2 experiments). RESULTS. The effects of intraarterial injections of diphenyihydantoin. typical record obtained with a bipolar surface electrode placed over the bundle of His is shown in figure 1. S is the stimulus artifact of the atrial drive. is due to depolarization of atrial tissue immediately adjacent to the bundle of His. S and for the normal beats and for the atrial extrasystole are superimposed in the record. HN and VN represent the local potentials of

3 1968 DPH ON CRDC CONDUCTON 19 the bundle of His and ventricular tissue adjacent to the His electrode during conduction of the normal beat. H and V1 represent the local potentials of the bundle of His and ventricular tissue during conduction of the atrial extrasystole. The S- interval is a measure of the intraatrial conduction time; the -H interval represents primarily conduction through the -V node; the H-V interval represents conduction time from the bundle of His to right ventricular muscle. Figure 1 demonstrates that the S- and H-V intervals of the extrasystole are the same as those of the normal beat whereas the -H interval is lengthened. ll intervals remained constant for several hours if the electrode remained in place. The H potential was identified in all experiments by the intraarterial injection of acetyicholine (10-50 g) or by increasing the rate of atrial stimulation, procedures which have been shown previously (lanis et at., 1958; Kirk and Dresel, 1965) to prolong only the -H interval. Diphenyihydantoin (0.5-0 /Lg/ml) was given by injection into the arterial inflow in 10 experiments. n of these experiments no extrasystole was interpolated. The effect of an intermediate concentration of diphenyihydantom (16 g/ml) is seen in figure lb. This concentration caused a small but significant increase in the -H, interval and a considerably greater increase in the -H interval. Neither the S- nor the H-V intervals of the normal beat or of the extrasystole were changed by the drug. Figure 2 shows that a concentration of diphenyihydantoin (5,Lg/ml) which had no effect on the conduction times of the normal beat caused a significant increase in the -H conduction time of the extrasystole. The minimal concentration which was found to cause an increase in -H conduction time of an extrasystole was 1.5 g/ml. Concentrations greater than jg/mi were necessary to cause significant increases in -H conduction times of the regular driven beats. n effect on atrial conduction (S- interval) was seen only after concentrations greater than 50 jg/mi. minimal increase in the H-V interval of the normal beat was seen only at the highest concentration tested (0 jg/mi). Conduction of extrasystoles was not studied at concentrations above 60 jg/mi. The absolute amplitudes of the potentials and the relationship between the amplitudes of the normal and extrasystolic potentials varied from preparation to preparation and from one injection to another within a given preparation. The decrease in the amplitudes of the, H, and potentials after administration 10 no n 80 M #{19} O HV S - -2.: $ lime - MNUTES Fia. 2. Time course of the effects of diphenylhydantoin (16,sg/ml) on the S-, -H and H-V intervals of cardiac conduction and the differential effects of diphenyihydantoin (16 and 5 zg/ml) on the -H interval of the regular driven beat (-Hzq) and the extrasystole (-H). bscissa: time in minutes after start of infusion (stippled bar) of DPH. Ordinate: intervals in milliseconds. Curves are drawn from determinations made at 20- to 60-sec intervals. The S- and H-V intervals are plotted for only the regular beat and only at the higher concentration used.

4 19 SSYNJUK ND DRESEL. Vol g_.. 20.g < a a a U $0 Conc. DPH (jig/mi) U 110 U 150 Fia.. Maximum increases in the -H interval of both the regular beat and the extrasystole plotted against the concentrations of diphenyihydantoin used., maximum increases in the -H interval during conduction of the regular beat. i.i, maximum increases in the -H interval during conduction of the atrial extrasystole. The values plotted were obtained from 10 experiments. The fine horizontal line has been drawn to show the limit of reliability of the measurements. of the drug, seen in figure 1, was not a consistent finding. No changes in the duration of any of the potentials were observed after injection of DPH. The time course of the effect of diphenylhydantoin is illustrated in figure 2. The maximal effect was seen within 1.5 mm of the start of the injection of the drug at the concentrations used. Return to control values occurred within to 6 mm. The maximal effect was seen consistently within 1 to mm at all concentrations studied in the 10 experiments. The duration of the responses varied among preparations and was not clearly related to concentration. The minimum duration of a significant effect was mm; the maximum observed was 1 mm. The absolute maximum increases in the -H interval of both the normal and the extrasystolic beats produced by DPH in all 10 experiments are plotted in figure. The fine horizontal line is drawn to show the limit of reliability of the measurements. The drug effect on the -H, interval was always greater than the effect on the -H, interval. The time between the recorded extrasystole and the immediately preceding beat was between 5 and 55% of the basic cycle length in 6 of the 7 experiments. The magnitude of the drug effects did not vary systematically with this interval. n fact, the drug effect shown in figures 1 and 2, obtained in the single other experiment in which this time interval was significantly less than one-half the basic cycle length (162 msec at 500-msec cycle length), also did not differ from those obtained in the other experiments. t seemed possible that the effect of DPH on conduction of the extrasystole only appeared to be greater because the base-line -H interval was also longer than -H,. We therefore expressed the maximal drug effect as percentage of the base-line intervals. Table 1 shows that the percentage increase in -H was greater than the percentage increase in -H, in response to all but one drug injection. The percentage increases were equal in this one case. t is apparent, therefore, that the drug effect on the extrasystoles can be considered a differential one. The effects of diphenylhydantoin injected into the donor dog. Diphenylhydantoin was given i.v. to the donor dog in experiments. njection of 1, and 5 mg/kg was completed in 1 to 2 mm. slight bradycardia and a small decrease in blood pressure was produced in two

5 1968 DPH ON CRDC CONDUCTON 195 donor animals given doses of and 5 mg/kg of diphenythydantoin. The recipient hearts showed a marked increase in -H conduction time and a slight increase in S- conduction time. H-V conduction time was unchanged. No extra-.ystoles were interpolated in these experiments. The smallest dose (1 mg/kg) had no effect on the electrocardiogram or the blood pressure of the donor animal but produced a slight but significant prolongation of the -H interval of the recipient heart. The time course of the effects produced by diphenyihydantoin on the recipient hearts was similar to that seen when the drug was injected into the aortic cannula. DscussoN. The use of the isolated, bloodperfused heart has two major advantages in the study of cardiac conduction. First, it permits the study of drug effects in the absence of nervous factors operating in the intact dog. Second, we have now shown that it is feasible to study drug effects on the conduction of extrasystoles. -H conduction of extrasystoles, slow even in the absence of the drug, is preferentially affected by diphenyihydantoin. t would be of interest to determine whether other antiarrhythmic agents have a similar action. Surprisingly, we found no correlation between the maximum drug effect and the interval between the recorded extrasystole and the immediately preceding beat. This may have been due primarily to our inability to record the true interpolated extrasystole; the interpolated beat cannot trigger the sweep on the oscilloscope because at the sweep speeds necessary for adequate display of all the complexes, the instrument is not ready to accept the triggering signal of the interpolated beat. We therefore had to be satisfied with recording the responses to the next regular beat, which was coupled to the interpolated beat by an interval of (basal driving interval-coupling interval of the interpolated beat). This beat therefore can be considered to be the second of two extrasystoles. We have shown that diphenylhydantoin in concentrations found to be effective clinically (Bigger et at., 1966) affects conduction only through the atrioventricular node. Higher concentrations had a small effect on intraatrial conduction; intraventricular conduction was not slowed even at the highest concentrations tested. Our results agree with those reported Dog No DPH pg/mi bsolute Change Change Normal TBLE 1 Maximum increases of the -H intervals of normal and extrasystolic beats after diphenyihydantoin msec Normal by Rosati et at. (1967), but differ from those of Bigger et a!. (1967) and Helfant et at. (1967). ll three groups found V-nodal conduction to be facilitated by the drug. Rosati et at., however, found that diphenyihydantoin slowed sinus rate and V conduction after cardiac denervation. This latter observation suggests that the direct effect of diphenyihydantoin is a depressant one. Rosati et at. suggested that the effect of diphenylhydantoin is due to an atropine-like action but Bigger et al. found that cholinergic and adrenergic blocking agents did not alter significantly the effect of this agent. Helfant et at. studied the drug in dogs with intact vagi. These investigators did not speculate on the possible nature of the V facilitation which they observed. Slowing of V conduction in man has been reported by Finkelman and rieff (192). No effect was seen in man by Gupta etal. (1967). The effects of diphenythydantoin on intracardiac conduction differ from those of two other antiarrhythmic drugs, quinidine and amodiaquin,

6 196 SSYNUK ND DRESEL Vol. 161 studied previously in the blood-perfused dog heart (Kirk and Dresel, 1965). Quinidine affects atrial, V-nodal and intraventricular conduction equally at low concentrations and has a predominant effect on atrial conduction at somewhat higher concentrations. modiaquin, like diphenylhydantoin, affects primarily V-nodal conduction. Unlike DPH, however, amodiaquin has significant effects on atrial and intraventricular conduction even at low concentrations. t appears possible therefore that drugs will be found to have differing spectra of activity on conduction which may in some cases be correlated with their antiarrhythmic effects. CKNOWLEDGMENTS. The technical assistance of Mr. Lothar Schluter and Mr. ndrew Bagnail in certain phases of the study is gratefully acknowledged. REFERENCES LNS, J., GoNzis H. ND LopEz, E.: The electrical activity of the bundle of His. J. Physiol. (London) 12: 7-10, BERNSTEN, H., Gou), H., LNG, T. W., PPPELBUM, S., BZK, V. ND CORDY, E.: Sodium diphenylhydantoin in the treatment of recurrent cardiac arrhythmias. J. mer. Med. ss. 191: , BGGER, J. T., JR., llmas, P. D. ND WENBERG, D..: Effects of diphenylhydantoin on cardiac conduction and repolarization (bstract). mer. J. Cardiol. 19: 119, BGGER, J. T., SCHMDT, D. H. ND Ku r r, H.: The relationship between the antiarrhythmic effect and the plasma level of diphenylhydantoin sodium (Dilantin). Bull. N.Y. cad. Med. 2: 109, CONN, R. D.: Diphenylhydantoin sodium in cardiac arrhythmias. New Engl. J. Med. 272: , FNKELMN,. ND REFF,. J.: Untoward effects on phenytoin sodium in epilepsy. J. mer. Med. ss. 118: 09-, 192. GUPT, WERS, D. N., UNL, M. W. R.: Effects 0., BSHOUR, F.. of diphenylhydantoin ND (Dilantin) on peripheral and coronary circulation and myocardial contractility in the experimental animal. Dis. Chest 51: , HRRS, P. D., BGGER, J. T., WENBERG, D. ND MLM, J. R.: Use of diphenylhydantoin in ventricular arrhythmias following open heart surgery. Surg. Forum 17: , HELFNT, R. H., SCHERLG, B. J. ND DMTO,. N.: The electrophysiological properties of diphenylhydantoin sodium as compared to procaine amide in the normal and digitalis-intoxicated heart. Circulation 6: , KRK, B. W. ND DRESEL, P. E.: Effects of amodiaquin and Qulnidlne on cardiac conduction. Can. J. Physiol. Pharmacol. : 29-8, ROST, R.., xxner, J.., ScHi&L, S. F. ND WLLCE,. G.: nfluence of diphenyihydantoin on electrophysiological properties of the canine heart. Circulation Res. 21: , 1967.