J. Physiol. (I959) I45,

Transcription

1 266 J. Physiol. (I959) I45, THE EFFECTS OF VAGAL STIMULATION ON THE ISOLATED PERFUSED RAT HEART BY J. M. BENFORADO From the Department of Pharmacology, University of Oxford (Received 3 July 1958) The isolated mammalian heart with intact vagus nerves has been employed previously for a study of the cardiac effects of vagal stimulation (Perry & Reinert, 1954; McEwen, 1956; Lynch & Essex, 1956). The use of an isolated preparation has the advantage of decreasing the number of variables present in such a study in the intact animal. However, previous investigators have recorded only mechanically, usually with a kymograph lever, and have reported effects on heart rate in a qualitative or semi-quantitative manner. In the present report the use of electrocardiographic recording has enabled the effects of vagal stimulation on sino-atrial rate, ventricular rate, heart block and on the production of ectopic beats and extrasystoles to be observed. In addition, the effects of physostigmine, acetylcholine, hexamethonium, atropine, cocaine and ouabain on the response to vagal stimulation have been examined. METHODS White male rats ( g) were anaesthetized with urethane (1.25 g/kg) administered intraperitoneally. The operation for isolation of the heart with intact vagus nerves was carried out under artificial respiration following the technique described for the rabbit heart by McEwen (1956). Temperature control ( C) of the Langendorff perfusion arrangement was as described by Saxby (1956). The apparatus which allowed the vagus nerves to be stimulated individually by shielded platinum hook electrodes is shown in Fig. 1. The nerves were kept moist by the constant dripping on to them of a warmed bathing solution. This solution, which was also employed for perfusion of the heart, was that described by McEwen (1956). It is a phosphatebicarbonate Ringer's solution with added sucrose and is gassed with 95% oxygen and 5% CO2. The use of separate reservoirs and heating coils prevented drugs in the fluid perfusing the heart from reaching the nerves. Two shielded platinum fish-hook electrodes, one in the right atrium and one in the left ventricle (see Fig. 1) permitted recording of an e.c.g. in which atrial and ventricular complexes were easily discernible. However, at the paper speed employed, no accurate evaluation of changes in PR interval could be made. E.c.g. records read from right to left. Stimulation of the nerves was effected from a square-wave stimulator and stimulus isolation unit. In preliminary experiments a study of the effect of frequency of stimulation over the range c/s showed that maximum effect was reached at 20 c/s and this frequency was subsequently employed. The duration of the stimulus was 0-3 msec and stimulus intensity (voltage delivered at the electrodes)

2 VAGAL EFFECTS ON RAT HEART 267 was varied from threshold to submaximal and supramaximal. Threshold voltage varied from experiment to experiment, being usually about 2 V. Drugs were either added to the perfusion fluid at the desired concentrations, or given directly into the cannula in a single dose; concentrations are expressed as g/l. of the base; doses are expressed as micrograms of the base. In all experiments a 30 min control period elapsed following the onset of perfusion before the commencement of nerve stimulation. Unless otherwise stated, the nerve was stimulated for a period of 10 sec. At least 5 min was allowed for recovery before repeating stimulation. B A B Fig. 1. Apparatus for Langendorff perfusion of the isolated rat heart with provision for e.c.g. recording and independent stimulation of each vagus nerve. Ball and socket joints with tension springs allow for appropriate positioning of glass electrode carriers and bathing fluid tubes. Perfusion and bathing fluids from separate warming coils and reservoirs. Filter-paper seal keeps the air in the Perspex cage saturated with water vapour. A, cannula for heart perfusion; B, tube for nerve bathing fluid; C, e.c.g. electrodes; D, stimulating electrode carrier; E, vagus nerve; F, filter paper. RESULTS Nerve viability In seventeen experiments, the right vagus alone was excitable in five, the left vagus alone excitable in three, and both vagi were excitable in nine experiments. Development of inexcitability with time, in nerves excitable at the start, did not occur. Most preparations were used for periods of 3-5 h, and seven for periods greater than 6 h. The factor limiting the duration of an experiment was the coronary flow, which decreased with time. Effects of stimulation The onset of vagal effect was usually abrupt, occurring immediately following stimulation or one to two beats later. In some experiments effects F

3 268 J. M. BENFORADO on sino-atrial rate alone were noted. In other experiments, in addition to these effects, the development of heart block was observed. This ranged from occasional dropped beats to varying degrees of partial block and to complete block (Figs. 2 and 3). The magnitude of both types of effect was related to intensity and duration of stimulation. Table 1 illustrates the effect of changing the stimulus intensity. At threshold voltage only slowing of sino-atrial rate was noted. As stimulus intensity was increased this effect was augmented, and in addition progressively increasing heart block occurred. Finally, at supramaximal voltage, complete heart block was seen immediately, although effects on sino-atrial rate developed more Isec Fig. 2. E.c.g. records from isolated rat heart perfused with a salt solution containing physo stigmine Initial rate was 426/mi. Left vagus was stimulated (supramaximal stimulus, 16 V) for 10 sec; during stimulation there was complete arrest. (a) shows varying block 40 sec after cessation of stimulation when sino-atrial rate was 408/min. (b) shows 3: 1 block 50 sec after cessation of stimulation. Complete recovery in 60 sec. slowly. In most experiments supramaximal stimulation caused sino-atrial arrest. Occasionally only a marked bradycardia was noted. The effect of submaximal and supramaximal stimulation on sino-atrial and ventricular rate's is illustrated in the graphs of Fig. 4A (upper and lower). With submaximal stimulation the sino-atrial rate dropped from 252 to 156 beats/mm and 2:1 heart block occurred. Supramaximal stimulation decreased the sino-atrial rate from 245 to 90 beats/mmi and complete block was present. Recovery was rapid in both instances. The effect of changing the duration of stimulation is shown in Table 2. The maximum effect on sino-atrial rate and heart block was seen with a 10 sec stimulus. As the duration of stimulation was increased two- and threefold, the sino-atrial rate began to recover while the block remained complete. However,

4 VAGAL EFFECTS ON. RAT HEART 269 A 1. I -L i i t i i i t i i i 0 i 1 F 1 i i f f f f 0 O i f i i f f i f f f i 1 t 1 1 i 0 i H9q 'j,11h I j- I. Z I.AK-... P, HH4 Ffii-lifil "LE U-No I &"4-& Li Li Li A"I psti 1t Inital rate was 1atu complete arrest (a) RecoIT rd45 sec after* 1: stimution opped. shows si-atria rt 9 tcl-i. ~~I. I 8g Idr-I - - * II{;PI J se s-{ tif zxsx1r LE'Ir L l Fig.~~~~~~R 3. E..g recod from isolted ;j ra her duin perfu\-tsio w+ith a sat L\ solto cotat)ining Fig.~~~~~ 3. E_-g reod from isolated Trat hie'rt.during p*erfimion with iasat souto physos~ colntaining. TI 11. o sini o-tii (Ia a v i ( Irats (beat o IsIng t systoles. timn 10-6I InitIa rate wa 19.mn n. stimulatiotnfo 30 amstimulus Icaused (c) 30 sec later,sino-atrial rate 17/ ; 21blc. Fulrcvrn13 sec. intensity during right vagus stimulation for 10 sec in an isolated perfused rat heart: stimulus, 20 c/is, 0-3 msec Conttrol rates* Changes in rate during 10 sec Stimulus intensity (beats/min) stimulation (beats/min) Threshold 3 V 270 s v Submaximal, 4V 240 sa v Occasional dropped beats Submaximal, 5V 264 sa Zero v 80t 63t 63t Zero Supramaximal, 17V 294 sa Zero v Zero Zero Zero Zero * Sino-atrial and ventricular rates the same; t 3: 1 heart block; t 2:1 heart block; complete heart block.

5 A J. M. BENFORADO 8 B ~~~~~/ IE w -o 4) < 300 r- A I L / X0, -S 0 I I Time (sec) Fig. 4. Sino-atrial and ventricular rates of isolated perfused rat heart. Effect of physostigmine on submaximal stimulation with 8 V (upper graphs) and supramaximal stimulation with 17 V (lower graphs) of the right vagus for 10 sec (20 c/s; 0 3 msec). A, without physostigmine; B, with physostigmine Ordinates, rate (beats/min). Abscissae, time (sec). Continuous lines, sino-atrial rate; dashes, ventricular rate during heart block, solid bar, period of stimulation. TABLE 2. Effect on sino-atrial (8a) and ventricular (v) rates (beats/min) of changing the duration of stimulation during right vagus stimulation in an isolated perfused rat heart; stimulus, 20 c/s, 0*3 msec, and supramaximal voltage, 17 V Rates at various times during stimulation Duration of (beats/min) stimulation Control rates*, A (sec) (beats/min) 10 sec 20 sec 30 sec 40 sec 60 sec sa 90 v Zerot sa v Zerot Zerot sa v Zerot Zerot Zerot sa v Zerot Zerot Zerot 63t 144 * Sino-atrial and ventricular rates the same; t complete heart block; t 2: 1 heart block.

6 w_ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~15v1IL rl L r A \l1.... VAGAL EFFECTS ON RAT HEART 271 with a stimulus duration of 60 sec recovery from block was complete while sino-atrial rate recovered to only 62% of the control value. Either ectopic beats or extrasystoles or both were noted in most experiments. These occasionally occurred during stimulation but were most often seen in the period following cessation of stimulation. The ectopic beats (Fig. 5) were observed as bizarre e.c.g. complexes. These were occasionally prefibrillatory. The extrasystoles (Fig. 3) were coupled to sino-atrial or nodal beats. Fig. 6 shows extrasystoles with fixed coupling to nodal ectopic beats. -. f~~~~~~~~~~~~~~~~~~1.-=it Tow''1atftlrrvwsrsannXztttr Isec Fig. 5. E.c.g. record from isolated perfused rat heart. One burst of ectopic beats during a 10 sec period of supramaximal stimulation (16 V) of right vagus. Onset of stimulation at arrow. Isec Fig. 6. E.c.g. record from isolated rat heart perfused with salt solution containing physostigmine 10r. Extrasystoles with fixed coupling to nodal ectopic beats 5 sec after cessation of a 10 wse period of supramaximal stimulation (16 V) of left vagus. During recovery after cessation of stimulation, tran,sient increases in rate above the control value were noted in only a few experiments. Even in these experiments, such increases were constant and evident only during the first win. A comparison of the results of independent right and left vagal stimulation in preparations in which both nerves were viable showed no differenceis in the effect on sino-atrial rate, heart block, ectopic beats, extrasystoles or increaws in rate following cessation of stimulation. Effects of drugs Acetylckoline. As expected, constant perfusion with acetylcholine (l0-7), in the absence, of vagal stimulation, caused a decrease in sino-atrial rate. The,

7 272 J. M. BBNFORADO decrease in two experiments was 75 and 76 %. However, partial recovery from this effect was noted during continued perfusion. The presence of physostigmine (10-7) markedly augmented the effects of 0 5-1O,ug, as well as of a 10-7 concentration of acetylcholine. In each of two experiments complete cardiac arrest without the establishment of a lower idioventricular pacemaker was observed with 10-7concentrations of both acetylcholine and physostigmine. During this period, rare volleys of extrasystoles were noted. Coupled extrasystoles, occurring during washing out with acetylcholine, resembled those seen in the same experiment during recovery from vagal stimulation. Physostigmine. In each of seven experiments physostigmine (10-) was observed to augment the magnitude of the effects of vagal stimulation on the sino-atrial rate and on heart block (when present), and further to delay markedly the recovery from these effects when stimulation ceased. This is illustrated in Fig. 4, which shows the augmentation by physostigmine of the effects of submaximal and supramaximal stimulation on sino-atrial rate and on heart block. The relatively more rapid recovery of the sino-atrial rate as compared with the block is also evident. In the seven experiments the mean decrease in the sino-atrial rate produced by physostigmine (106) in the absence of vagal stimulation was 22 % (range 10-35). Hexamethonium and atropine. In five experiments, two of which were done with physostigmine (106), hexamethonium (1.5 x 104) abolished the effects of vagal stimulation. Fifteen to twenty minutes were required for this concentration of hexamethonium to exert its full blocking effect and the antagonism proved reversible with washing. This concentration had no effect on the change in rate caused by a large (10OO,g) injection of acetylcholine. However, in two out of three experiments the effect of a IO,ug dose of acetylcholine was reduced by 29-39%. In four experiments, three of which were done with physostigmine (106), atropine (10-7) abolished the effects of vagal stimulation within 5 min and the antagonism proved reversible with washing. This concentration of atropine antagonized the effects of small doses of acetylcholine ( g) and reduced the effect of a large dose (10OOg). Increases in rate, ectopic beats or extrasystoles were never observed during vagal stimulation in the presence of hexamethonium or atropine. Ouabain. In two experiments, one in the presence of physostigmine (104), ouabain (10-) augmented the effects of vagal stimulation as well as those of a small dose (0.5 jig) of acetylcholine. The augmentation was reversed by washing. This concentration of ouabain itself decreased the sino-atrial rate by 10-23% in the two experiments. These results, obtained in the experiment without physostigmine, are illustrated in Table 3. Cocaine. In experiments on the perfused rabbit heart isolated together with the vagus nerves, McEwen (1956) observed that there was an increase in the

8 VAGAL EFFECTS ON RAT HEART 273 rate and force of ventricular contractions in some preparations after the inhibition caused by vagal stimulation. When cocaine was added to the perfusion fluid this increase was seen in all preparations and was much greater. However, in the present experiments on the rat heart an increase in rate after cessation of stimulation was rarely seen, and when cocaine (4.5 x 106) was added to the perfusion fluid an augmentation of the increase was observed only in one out of three experiments. In two experiments cocaine (1-8 x 10-5) abolished the effects of vagal stimulation without altering the, effect of small doses (05-lO,ug) of acetylcholine. TABLE 3. Effect of ouabain (10-5) on changes in sino-atrial rate (beats/min) during right vagus stimulation at different intensities for 10 sec and following ACh in an isolated perfused rat heart. Stimulus, 20 c/s, 0-3msec After washing Control Ouabain out period (10-5) ouabain Before stimulation During threshold stimulation (0.8V) Before stimulation During submaximal stimulation (15V) Before stimulation During supramaximal stimulation (20 V) Zero Zero 324 Zero Before ACh During ACh 0*5 /&g 82 Zero 74 DISCUSSION Lynch & Essex (1956) reported an average of 108 min for the duration of vagal excitability in the isolated perfused guinea-pig heart. The success in the present experiments with the prolonged functioning of the vagus nerves parallels that noted for the rabbit heart by McEwen (1956). This can be ascribed to the use of the same perfusion fluid which, by minimizing oedema in the heart, may prolong survival of intracardiac vagal fibres. The failure to achieve excitability of both nerves in some experiments probably signifies damage done during the cannulation and tying of the aorta, since both nerves were usually excitable in vivo after dissection. In many experiments vagal stimulatioh produced a sinus bradyeardia and complete block, which would have appeared as cardiac arrest under conditions in which only ventricular contraction was recorded on a kymograph. The use of the e.c.g. made it possible to separate the effects of vagal stimulation on sino-atrial rate and on heart block. Differences between these effects, observed at various intensities of stimulus, may be the result of differences in excitability of those vagal fibres destined for the sinus node as compared with those leading to the atrio-ventricular bundle. During prolonged stimulation partial to complete recovery from vagal effects was observed, the recovery of the sino-atrial rate being more prolonged than that of heart block. Similar partial 18 PHYSIO. CXLV

9 274 J. M. BENFORADO recovery of the sino-atrial rate from the effects of acetylcholine was noted in experiments using constant perfusion. These observations support the idea of Obrink & Essex (1953) that vagal escape may be due to a progressive decrease in responsiveness of the heart to acetylcholine. Adaptation to constant concentrations of acetylcholine was described for sympathetic ganglia by Krivoy & Wills (1956). The use of physostigmine brought out differences in the time courses of recovery following cessation of stimulation, the heart block recovering more slowly than the sino-atrial rate. This was unexpected in view of the reversed time courses of recovery during periods of prolonged stimulation cited above, and could be the result of quantitative difference in cholinesterase inhibition at the two sites. More appealing, however, is the idea that diffusibility of acetylcholine from the region of the sinus node may be relatively more rapid. Obrink & Essex (1953) also reported that, in experiments on intact rabbits and guinea-pigs, vagal stimulation could produce cardiac arrest, but acetylcholine (even in large doses) did not. Intracellular liberation of acetylcholine at the sinus node during vagal stimulation was suggested as a reason for the difference. No such difference was found between vagal stimulation and acetylcholine in the present experiments on the isolated rat heart. Further, the absence of the appearance of an idioventricular pace-maker in the experiments in which continuous perfusion with acetylcholine and physostigmine produced cardiac arrest, suggest an inhibitory effect of acetylcholine on pacemakers below the atrioventricular node. Such an effect during idioventricular rhythm in the isolated perfused human foetal heart was described by Baker (1953). McEwen (1956) found that at the end of vagal stimulation of the isolated rabbit heart the rate and amplitude often increased. The increase'was greater in some hearts than in others and often disappeared after perfusion for a few hours. In the rat heart increases in rate after cessation of vagal stimulation were seen only occasionally, and in only one out of three experiments was the increase augmented by cocaine. Again, unlike the effect on the rabbit heart, no increases in rate were apparent during perfusion with hexametbonium. If the inconstant increases observed were related to the presence of adrenergic post-ganglionic fibres, atropine should certainly have enhanced such increases. In experiments in which these increases were absent atropine should have unmasked them. However, in the presence of atropine vagal stimulation produced no changes at all. It therefore seems unlikely that adrenergic postganglionic vagal fibres are present in the rat heart. This, and the fact that atropine prevented the appearance of ectopic beats and extrasystoles, usually seen during or after nerve stimulation, means that such arrhythmias are probably not related to an adrenergic neurohumoral mechanism. Scherf & Schott (1953) have documented the appearance of ectopic.beats and extra-

10 VAGAL EFFECTS ON RAT HEART 275 systoles following vagal stimulation in the intact animal. They stressed the need for predisposing conditions (aconitine, digitalis, BaCl2) and the fact that changes in normal rate were not required for the occurrence of these arrhythmias. In the present experiments ectopic beats and extrasystoles were observed only concurrently with marked decreases in sino-atrial and ventricular rates, and were most usually seen during sino-atrial arrest and during recovery. It would seem that, in the isolated rat heart, suppression of higher pace-maker activity is a necessary antecedent to the arrhythmias produced by vagal stimulation in the absence of any of the predisposing factors cited by Scherf & Schott. The potentiation of the effects of acetylcholine and of vagal stimulation by ouabain, observed in these experiments, was described previously for the isolated perfused rat heart by Perry & Reinert (1954). In their experiments the ouabain was given by single injection rather than by continuous perfusion and the potentiation was recorded kymographically. SUMMARY 1. When isolated rat hearts with intact vagus nerves were perfused with an appropriate solution, the vagus nerves remained viable for periods greater than six hours. 2. The use of e.c.g. recording made it possible to observe the different effects of vagal stimulation on the sino-atrial rate, on heart-block and in causing arrhythmia. 3. Physostigmiine and ouabain augmented the effects of vagal stimulation, whereas hexamethonium and atropine antagonized them. The absence of any vagal effects after atropine made the presence of adrenergic post-ganglionic vagal fibres unlikely. 4. Experiments with ACh and physostigmine suggested an inhibitory effect of ACh on pace-makers below the atrio-ventricular node. This research was done during the tenure of a Life Insurance Medical Research Fellowship, New York, N.Y. I am indebted to Professor J. H. Burn for suggesting the problem and for the opportunity of working in his laboratory. Thanks are due to 0. B. Saxby and D. Groves for constructing the apparatus used. The electrocardiograph, Cossor model 1314, was given to the department by the Wellcome Trust. REFERENCES BAxEm, J. B. E. (1953). Some observations upon isolated perfused human foetal hearts. J. Physiol. 120, KiLIvoY, W. A. & Wuis, J. H. (1956). Adaptation to constant concentrations of acetylcholine. J. Pharmacol. 116, LYNCH, P. R. & ESSEx, H. E. (1956). Restoration of chronotropic effects of vagus stimulation on isolated perfused heart of the guinea-pig. Amer. J. Physiol. 186, McEwEN, L. M. (1956). The effects on the isolated rabbit heart of vagal stimulation and its modification by cocaine, hexamethonium and ouabain. J. Physiol. 131,

11 276 J. M. BENFORADO OBRNtK, K. J. & ESSEX, H. E. (1953). Chronotropic effects of vagal stimulation and acetylcholine on certain mammalian hearts with special reference to the mechanism of vagal escape. Amer. J. Phy8iol. 174, Pmy, W. L. M. & REINET, H. (1954). The action of cardiac glycosides on autonomic ganglia. Brit. J. Pharmacol. 9, SAXEIY, 0. B. (1956). Temperature control in the Langendorff heart perfusion. J. Physiol. 133, 4-5P. SCHBF, D. & ScHortr, A. (1953). Extrasystolea and Allied Arrhythmias, pp London: Heinemann.