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The effect of bretylium tosylate on the electrophysiologic properties of ventricular muscle and purkinje fibers
Experimental
Studies
The Effect of Bretylium Tosylate on the Electrophysiologic of Ventricular
J. THOMAS
BIGGER,
Muscle and Purkinje Fibers
Jr., MD*
CONRADE C. JAFFE. MD New York, New York
From the Departments of Pharmacology and Medicine, College of Physicians and Surgeons, Columbia University, New York, N. Y. This investigation was supported in part by U. S. Public Health Service Grant HE-12738 and in Dart bv a grant-in-aid from the New Yo’rk Heart issociation. Manuscript received December 4, 1969, accepted March 12, 1970. ::Senior Investigator of the New York Heart Association. Address for reprints: J. Thomas Bigger, Jr., MD, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W. 168 St., New York, N. Y. 10032.
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Properties
Preparations of papillary muscle and Purkinje fibers were obtained from dog hearts, perfused with oxygenated Tyrode solution in a tissue chamber, and observed both under control conditions and during exposure to bretylium tosylate in concentrations from 1 X 10 i M to 1 I’ 10 m( M. Transmembrane voltage was recorded from both ventricular muscle and Purkinje fibers in spontaneously beating and in electrically driven preparations. Bretylium did not suppress phase 4 depolarization or spontaneous firing in Purkinje fibers. After application of bretylium to spontaneously firing Purkinje fibers, the rate initially increased by 20 percent; in quiescent fibers, bretylium often induced spontaneous firing. Since neither effect was seen in preparations obtained from dogs treated with reserpine, each was attributed to bretylium-induced release of catecholamines from the adrenergic nerve terminals in the preparations. In concentrations of less than 1 :( lo-,& M, bretylium did not alter the resting transmembrane voltage or the action potential amplitude and overshoot; concentrations of 1 :< lo-m4 M caused a decrease in each of these variables. Bretylium, < 1 ?< lo---’ M, had no effect on the maximal rate of rise of phase z&o depolarization (peak \imax), membrane responsiveness or conduction velocity in Purkinje fibers. A concentration of 1 :+ 10-l M caused a decrease in peak \imax, shifted membrane responsiveness curves on their voltage axis, and decreased conduction velocity. Over a wide range of stimulation rates (30 to 150/min), bretylium caused a large increase in action potential duration and effective refractory period of both ventricular muscle and Purkinje fiber cells without lengthening the effective refractory period relative to action potential duration. Since bretylium lacked most of the electrophysiologic properties associated with antiarrhythmic drugs, its antiarrhythmic action against ventricular arrhythmias may, to a large extent, be due to its effect on adrenergic nerve terminals.
Rretylium tosylate, a quarternary benzylammonium salt, was reported in 1959 to be a relative of xylocholine which possessed sympathetic h1ockin.g activity and lacked undesirable muscarinic side effects.’ Rretylium is preferentially taken up by the adrenergic nerve terminals” m-here it prevents release of norepinephrine during direct or reflex sympat.hetic nerve stimulation.:k It interferes with norepinephrine release without significantly (1) altering the ultrastructural integrity of the adrenergic neural vesicles,’ (2) depressing preganglionic or postganglionic sympathetic nerve conduction.z (3) impairing sympathetic ganglionic
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Figure 1. Diagram of the typical experimental arrangement showing the free-running Purkinje fibers (false tendon) as a black strand between a segment of interventricular septum on the left and papillary muscle on the right. A pair of silver electrodes is placed on the surface of the papillary muscle (1) and used for stimulation. Transmembrane voltages from nearby ventricular muscle cells (2) and from an adjacent Purkinje fiber (3) were recorded using glass microelectrodes. The distance between the microelectrode sites at (2) and (3) ranged from 1 to 3 mm.
TOSYLATE
descending coronary artery’” or digitalis intoxicationl”; it has been found to increase the electrical ventricular fibrillation threshold in dogs subjected to thoracotomy alone’6 or to thoracotomy followed by acute coronary occlusion,17 and in closed chest dogs exposed to alternating current.lx This work has led to a limited therapeutic trial of bretylium as an antiarrhythmic agent in man. 14.1x-2’) The present study was undertaken to determine, in an isolated perfused preparation obtained from canine heart, the effects of bretylium on the electrophysiologic properties of the 2 cell types of the ventricle-ordinary ventricular muscle fibers and Purkinje fibers. These cell types were selected for study since alteration in their electrophysiologic behavior, particularly that of the Purkinje fiber, is thought to underlie the genesis of ventricular arrhythmias.“’ We also compared our findings with those obtained in previous studies of various antiarrhythmic drugs in the same preparation.22-25 We found significant differences between the electrophysiologic effects of bretylium on these 2 cell types and those of the previously studied antiarrhythmic drugs. We, therefore, propose that a major portion of bretylium’s antiarrhythmic action may result from its antiadrenergic effects rather than its direct effect on the cell membrane of ventricular muscle or Purkinje fibers.
Methods transmission,” (4) depleting the adrenergic nerve terminal of norepinephrine,7 or (5) diminishing the responsiveness of adrenergic receptors to adrenergic agonists.z,x Thus, the site of bretylium’s antiadrenergic action appears to be the adrenergic nerve terminal.!’ Bretylium has 2 other demonstrable actions on the adrenergic nerve terminal. Soon after injection into intact animals or application to various tissue preparations, bretylium causes release of norepinephrine from adrenergic nerve termina1s.8JnAlso, like cocaine, bretylium can impair the uptake of infused norepinephrine into the adrenergic nerve terminal,‘l so that it potentiates the effect of injected or infused norepinephrine on the adrenergic receptors.8-11 Recently, bretylium has been reported to possess significant antiarrhythmic activity. Levequel* reported in 1965 that high doses of bretylium prevented atria1 fibrillation induced by acetylcholine in dogs rendered hypokalemic by administration of glucose and insulin. More recently, investigators have shown the effectiveness of bretylium against experimental ventricular arrhythmias. Bretylium has been reported effective in preventing or abolishing ventricular arrhythmias induced by hypothermia,‘” hypothermia coupled with hemorrhagic shock,‘-’ acute ligation of the anterior
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Mongrel dogs weighing 10 to 14 kg were anesthetized with intravenously administered pentobarbital sodium (30 mg/kg). The heart was rapidly excised and dissected in oxygenated Tyrode solution at room temperature. Preparations containing Purkinje fibers and ventricular muscle were dissected from either ventricle and placed in a wax-lined tissue bath (Fig. 1). This bath was perfused with Tyrode solution, gassed with 95 percent oxygen and 5 percent carbon dioxide, at a constant rate of ‘7 ml/min ; the temperature of the perfusate was maintained at 36 -C 1C. The composition of the Tyrode solution and the techniques used for stimulating and recording have been described previously.*aJ6 The arrangement of preparations is shown in Figure 1. Surface electrodes at site 1 were used to stimulate the ventricular muscle. A microelectrode 5 4 mm away (site 2) was used to record transmembrane voltage of a ventricular muscle cell. Simultaneously, the transmembrane voltage of an adjacent Purkinje fiber and its first time derivative (Vmax) was recorded through a second microelectrode at site 3; the interelectrode distance was 1.8 -t 0.9 mm (mean & SEM). The effective refractory period of ventricular muscle and Purkinje fibers and the membrane responsiveness of the Purkinje fiber were measured while the preparation was stimulated at a constant rate of 75/min (cycle length 800 msec). Premature stimuli were introduced every seventh basic drive cycle. Premature stimuli were initially placed in electrical diastole and made progressively more premature until the most
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premature ventricular muscle action potential which propagated to and down the Purkinje fiber was identified (Purkinje fiber effective refractory period b. Slightly earlier test stimuli still produced a large amplitude ventricular muscle action potential which failed to excite the adjacent Purkinje fiber. Test stimuli were then made still more premature until the ventricular muscle cell isite 2) failed to respond (ventricular muscle effective refractory periotl I. Such
stimulation seqilences not only allowed rneasuYement of the effective refractory periotl in both ventriculal~ muscle and Purkinje fiber but also provided the opportunity to determine membrane responsivenessthe relationship between the maximal rate of rise of phase zero of the action potential (?mas I ant1 the transmembrane voltage at the time of excitation. \v;ts directl! estimated in Conduction velocit> Purkinje fibers by measuring the time required fol the propagating impulse to traverse the known lineal tlistance between two recording sites (sites A and E, Fig. 2 I. The instant in time at which peak iTmax of the action potential occurred at each recording site was determined by electronic differentiation of the 2 xand used as the indication tion potential upstrokes that the propagating impulse had arrived at the rccording site (Fig. 2). Eretylium tosylate was provided by Burroughs Wellcome & Co. as a powder which was initially dis-
lo-
I
263 I
;iOc?
.d
20 msec
msec
Further dilutions were in distilled water. out in Tyrode solution to obtain final conccnbetween 1 x 10 7 M ~‘0.04 /~g ml) and 1
solved carried trations X
tential 88 -t 3 mv. amplitude 114 t 4 mv and peak Vmax 430 2.- 70 volts ‘see). Action potential duration did not differ significantly from control values at concentrations of 1 ~.’ 10 i or 1 b 10 ” M hut was prolonged Rignificantly (P < 0.001) at concentrations of 2 1 .’ 10 A M (Fig.3 to 5). At concentrations of 1 X 10 ” M, x11increase in action potential duration \vas seen in all fibers (range in individual fibers
I
M (40 p’g ‘ml\.
Results Effect of Bretylium Tosylate Voltage-Time Course
on Transmembrane
The effect of bretylium tos;yCOUIW of transmen~branc~ voltage was studied in 15 Purkinje fibers. (‘oncentrations from 1 y 10 i to 5 ,: 10 ’ M exerted 110 significant effect on resting potential or overshoot. and thus no change in the total amplitude of the action potential occurred. At a 1 Y 10 ’ M concentration, the resting potential decreased from the COlltr01 ValUe of --91 I!- 2 mv (mean rt: SEM) to -87 i 2 mv (P < 0.05). At this concentration the overshoot was also slightly decreased so that. the total amplitude of the action potential decreased from the control value of 119 -I 4 mv t.o 110 -I5 mv (P < 0.01). In 5 other experiments, not reported in the previous results, the effects of bretylium were noted on fibers that were stretched during the pinning of the preparation in the tissue bath so that a decrease was seen in resting potential (84 i- 6 mv), amplitude (109 ?- 5 mv) and rate of rise of phase zero of the action potential (peak pmax 390 -t 50 volts ‘set). Under these conditions, application of bretylium tosylate. 1 X IO-” M or 5 x lo- z M, caused A significant increase in each of t.hese variables (resting poPurkinje
fibers:
late on the voltage-time
84
Figure 2. Method of measuring conduction velocity. The lower portion of the figure shows a diagram of the preparation used in studies of conduction velocity. A pair of silver electrodes was placed directly on the false tendon and used to stimulate the preparation. Microelectrodes were used to impale 2 sites in a single strand of the false tendon (sites A and B); the distance between sites A and B was measured with a calibrated ocular micrometer. The upper portion of the figure shows typical recordings obtained from such a preparation. The upper trace records time marks every 100 msec. Calibration for phase zero \imax is shown on the left (1,000 volts/set) and voltage calibration for the action potentials on the right (100 mv). The time calibration for the slow sweep speed is shown at the bottom left (200 msec) and for the rapid sweep speed at the bottom right (20 msec). Recordings obtained from site A are labeled A; those recorded from site B are labeled B. Action potentials A, and B, are recorded at slow sweep speeds from sites A and B. A, and B, are the upstrokes of action potentials recorded at sites A and B recorded at sweep speed 10 times faster. A, and B, are \imax of potentials A and B obtained by electronic differentiation and recorded at the rapid sweep speed. The conduction velocity of the propagating impulse was estimated by accurate measurement of the time between the 2 action potential upstrokes (phase zero) and the distance between the sites at which the action potentials were recorded and was expressed in meters per second.
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10 to 20 percent change, Fig. 3 to 5). Further prolongation ranging from 20 to 35 percent occurred at a concentration of 5 x lo-” M (Fig. 4 and 5). At a bretylium concentration of 1 X 1O-4 M some fibers showed slightly further prolongation, but the mean change in action potential duration was not significantly different from that at 5 x’ 10 mGM. The increase in action potential duration produced by bretylium was due largely to prolongation of the action potential plateau (phase 2) ; in some fibers, phase 3 was also slightly prolonged (Fig. 3 and 6). The effect of bretylium on action potential duration was examined in 10 fibers at drive cycle lengths from 400 to 2,000 msec-drive rates of 30 to 150 /min (Fig. 4). At all these cycle lengths and at a concentration of 1 X lo-” M, Purkinje fiber action potential duration was prolonged by about the same percent change from control (16 * 5 percent). At 5 ?: 10mGand 1 >< lo-.’ M, action
CL 1000
CL 500
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280
msec
I4 5 msec
150
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249 VM
‘0 i =
___\-
-1,
,.
-__1
potential duration was further prolonged (28 I 7 percent change). Study of ventricular Ventricular muscle: muscle transmembrane voltage was simultaneously performed with that of Purkinje fibers in 12 preparations (Fig. 3 to 5). Bretylium 5 1 x 10~’ M (40 &ml) produced no significant change in resting potent,ial or action potential amplitude in ventricular muscle. The general configuration of the action potential after bretylium tosylate was unaltered except for prolongation of duration (Fig. 3 and 6). Prolongation of the ventricular muscle action potential duration became significant at a concentration of 1 x 10~-z M (P < 0.025) and paralleled the change seen in Purkinje fibers (Fig. 5A). The percent change in action potential duration in ventricular muscle was slightly smaller at any given drug concentration than in Purkinje fiber (10 t 5 percent at 1 X lo-; M, 18 t 5 percent at both 5 x 10-j and 1 X 1O-4
CL 2000
290 153
-
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_&
PF
2 91 msec
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VM
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PF
‘0 &) -T;
PF VM
\A. 285
y
=1 msec
163 msec
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173md
,
;:;t;;,
mV msac
Figure 3. Effect of bretylium tosylate on action potential duration of canine Purkinje and ventricular muscle fibers. In the top trace of each panel, time marks are repeated every 100 msec. The other 2 traces record transmembrane action potentials from ventricular muscle (shorter action potential) and a Purkinje fiber (longer action potential). The 3 vertical columns show recordings made at cycle lengths (CL) of 500 msec (left column), 1,000 msec (center column), and 2,000 msec (right column). The horizontal rows show action potentials recorded under control conditions (top row) and during exposure to bretylium tosylate in concentrations of 1 x 10.” M (second row) and 1 x 10.’ M (third row). The action potential duration (measured to 95 percent repolarization) of the Purkinje fiber and ventricular muscle fiber is given under each frame. At 1 x 10.’ M, action potential duration was not significantly affected in either cell type (not shown). At 1 y lo-” M and 1 x 10-l M, Purkinje fiber action potential lengthened by 17 percent and that of the ventricular muscle cell by 12 percent at each drive rate.
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I loo 167
200
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Figure 4. Effect of bretylium tosylate on the action potential duration of Purkinje fibers (PF) and ventricular muscle (VM) fibers which were driven over a wide range of cycle lengths (400 to 2,000 msec). Action potential duration in milliseconds is plotted on the ordinate and the driven cycle length in msec on the abscissa. Each point represents the mean value of 10 experiments in Purkinje fibers (closed symbols, above) and 7 experiments in ventricular muscle (open symbols, below); vertical bars represent the standard error of the mean. After a 30 minute exposure to a concentration of 1 x lo-’ M (not shown), the action potential duration of PF and VM did not differ significantly from control values (P < 0.4). Exposure to bretylium in concentration of 1 x 10.” M or 1 x lo-” M significantly prolonged the action potential duration of both Purkinje fibers and ventricular muscle fibers (P < 0.001).
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PF
l
VM
o
A
M ) .The change in ventricular muscle action potential duration as R function of drive cycle length at any given concentration! of \,retylium ‘ivas studied in 7 preparations and was found to l,e similar to Purkinje tiller in that the percent change for ventricular muscle action potential duration ~vas al)out the same at all drive cycle lengths between 400 and 2,000 msec ( Fig. 4). Effect of Bretylium Refractory Period
PF
l
VM
1,
Figure 5. Action potential duration and effective refractory period of Purkinje and ventricular muscle fibers as a function of bretylium tosylate concentration in the perfusate. Mean observations made in 15 Purkinje fibers (PF, closed circles) and 12 ventricular muscle fibers (VM, open circles) are shown; the vertical bars represent the standard error of the mean. All preparations were driven at a cycle length of 800 msec (75/min). Action potential duration (A) and effective refractory period (B) are plotted on the ordinate as a function of the negative common logarithm of the molar bretylium concentration (that is, 6 represents 1 x lo-” M). Control observations (designated C on the abscissa) are plotted to the left of the vertical hatched bar. The apogee of increase in both action potential duration and effective refractory period as a function of bretylium concentration was found at a concentration of 5 x 10.” M.
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on Duration
of Effective
The effective refractory period I’urkinje fiber: \~a:: measured in 15 Purkinje fibers; a typical esperiment result is shown in Figure G. Vent ricular muscle was driven at a constant rate aiid premature actiolt potentials elicited iii ventricular muscle (VM:!) every tenth drive cvcle. The interval I,et\veen the last of a series of diiven responses and the earliest premature response in ventricular muscle which propagated to the adjacent Purkinje filler (shortest interval between T’M, and ‘I’M,) was measured. Under control toll(litions, the effective refractory period ill Purkinje fibers was 228 1 15 msec (mean 1: SEM). No sigllificullt change cmunwl at concentrations < 1 Y 10 Ii M, Ijut at ;I I,retglium tosylnte co*iceiitYation of1 IO r, M the Purkinje fiber effective refractor\- period was prolonged to 2.59 ri+ 19 msec, a significant change from the mean control value (P Y.’ 0.005) . When the concentration was in-
Figure 6. Effect of bretylium tosylate on the effective refractory period (ERP) of canine ventricular muscle (VM) and Purkinje fibers (PF). The preparation from which these recordings were made was arranged as in Figure 1. Purkinje fibers: The upper row of panels shows the earliest propagated PF response that could be elicited by a premature VM action potential (the PF ERP). Under control conditions (top left panel), the PF ERP was 214 msec; after a bretylium concentration of 1 Y lo-’ M. PF ERP was prolonged to 259 msec. Exposure to a concentration of 1 x lo-’ M (40 pg/ml) resulted in an increase in PF ERP to 274 msec and prolonged the propagation of impulses from ventricular muscle to the The 3 panels in the lower Purkinje fiber. Ventricular muscle: row depict the shortest Interval at which a premature electrical stimulus directly applied to VM (site 1 of Figure 1) evoked a propagated response in VM (VM ERP). At these short intervals, the impulse that propagates through the muscle does not cross the Purkinje fiber-ventricular muscle junction to activate the Purkinje fiber.
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creased to 5 x IO-” M, the Purkinje fiber effective refractory period increased to 272 et 20 msec, which again was significantly longer than the control value (P < 0.001). Increasing the drug concentration to 1 X 10W4 M did not significantly alter the Purkinje fiber effective refractory period from the values found at 5 X 1O-S M. Ventricular muscle : The effective refractory period of ventricular muscle was determined in 12 preparations. In the case of ventricular muscle, the earliest propagating response in the muscle which could be elicited by an electrical stimulus at 4 to 5 times threshold was defined as the ventricular muscle effective refractory period (the shortest obtainable VM,-VM, interval) ; recordings from a typical experiment are shown in Figure 6. The control ventricular muscle effective refractory period was 179 * 8 msec (mean + SEM) ; again, concentrations 5 1 X lo-‘; M had no significant effect (Fig. 5). At a bretylium concentration of 1 X lo-” M a 5 I 2 percent prolongation in ventricular muscle effective refractory period occurred (P < 0.025) and at 5 x 10WS and 1 X lo-” M a 19 i 4 percent change was produced (P < 0.005) (Fig. 5B). Effect of Bretylium on Relation Between Action Potential Duration and Effective Refractory Period Most antiarrhythmic drugs cause the effective refractory period to lengthen in relation to action potential duration. The relation between these variables as a function of the bretylium concentration in the perfusate was examined in 15 Purkinje and 12 ventricular muscle fibers; the mean changes, in milliseconds, are plotted in Figure 7. Ventricular muscle : The mean change in action potential duration and effective refractory period in ventricular muscle was roughly equal at all concentrations of bretylium studied (1 X lo--’ to 1 x 10~~ M). Even at the higher concentrations there was no tendency for the points representing the relation between effective refractory period and action potential duration to fall significantly below a line of identity between these variables (Fig. 7). Purkinje fiber: In concentrations of 1 X 10W7 to 1 X lo-; M, bretylium did not alter the relation between effective refractory period and action potential duration in Purkinje fibers. At a concentration of 5 x 10-m-,the mean change in action potential duration was somewhat greater than that in effective refractory period so that the points representing these variables tend to fall above a line of identity. Certainly, below toxic concentrations experimental points show no tendency to fall below a line of identity for effective refractory period and action potential duration during ex-
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posure to bretylium, a finding which is significantly different from previously studied antiarrhythmic drugs.22v23*25 Effect of Bretylium on Velocity of Phase Zero Depolarization and Membrane Responsiveness in Purkinje Fibers The maximal rate of phase zero depolarization was measured in 12 Purkinje fibers during exposure to a range of bretylium tosylate concentrations from 1 x 1O-7 to 1 x 1O-4 M. Under control conditions the 12 fibers studied had a
ti+60 m I a
B
t 30 A ERP +90-
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+60 MSEC 0
n
a
a f30
~
-/
A ERP
I x10-5
0
5x10-5
m
I x10-4
cl
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Figure 7. Effect of bretylium tosylate on the relation between action potential duration (APD) and effective refractory period (ERP) in ventricular muscle (VM, 12 experiments) and Purkinje fiber (PF, 15 experiments). In both A and B, mean change in APD (A APD) in milliseconds is plottec! on the ordinate and mean change in ERP (I ERP) in msec on the abscissa. The symbols representing drug concentrations are shown at lower right. If no significant change in either APD or ERP occurs, the symbols cluster at or near the origin. The dark diagonal line beginning at the origin is the line of identity; if 1 APD and A ERP were equal at all drug concentrations, all symbols would fall on or near this line. A, ventricular muscle: At each drug concentration (from 1 x 10” M to 1 x lo-( M), the points fall near the line of identity. Thkse observations suggest that bretylium does not substantially alter the relation between APD and ERP in ventricular muscle. B,Purkinje fiber: At low concentrations (1 x 10-O M, closed circle), bretylium did not alter the relation between APD and ERP. At higher concentrations, the change in APD has become long relative to the change in ERP. See text for discussion.
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resting potential of -91 i 2 mv (mean t SEM) and a peak Qmax of 538 +- 12 volts/‘sec (mean t SEM). No change in either variable was seen at concentrations between 1 >: 1O-7 to 1 X lo-’ M. At a concentration of, 5 x lo-‘, M, bretylium did not change resting potential but did cause a minimal decrease in peak vmax to 495 2 25 volts see, a decrease of 8 percent from the control value (P < 0.1). At this concentration, there was considerable variation between individual experiments, some fibers showing negligible change, others changes as great as 15 percent from control values. During exposure to a bretylium concentration of 1 x lo- ’ M, most fibers showed a decrease in resting potential, and all showed a decrease in peak vmax; the mean resting potential at this concentration was 88 -i- 3 mv and peak ?max 382 5 14 volts set, a decrease of 29 percent from the control value (P < 0.001). The decrease in peak qmax (and overshoot) in each fiber seemed greater than could be accounted for by the change in resting potential. To examine further the effects of bretylium on qmax in Purkinje fibers, the i;max was determined over a wide range of transmembrane voltage (membrane responsiveness) by stimulating 10 fibers at selected voltage levels during the time course of action potential repolarization. vmax was then plotted as a function of transmembrane voltage (Fig. 8, membrane responsiveness). In each fiber studied, the observations made under control conditions fit well on the S-shaped curve computed using the Weidmann formula” (Fig. 8). In concentrations from 1 x 10~’ to 1 X lo-” M bretylium had no effect on this relation. Thus, at these concentrations, the transmembrane voltage at which vmax is 50 percent of the peak value (Vh) was unchanged from control values (71 5 1.6 mv) , and vmax at any given transmembrane voltage was the same as under control conditions. At 5 x lo-;’ M, bretylium shifted the relation to the right on the voltage axis by 1.6 I 0.5 mv, and peak vimax was reduced by 41 i 10 volts’sec (Fig. 8) ; these changes are of questionable significance. At a concentration of 1 )< lo- I M, the changes produced by bretylium were striking (Fig. 8). The relation between membrane voltage and vmax was shifted rightward on the voltage axis; VI, increased (became more negative) by 8.4 -t 1.3 mv. Peak vimax decreased by 159 ?X 32 volts/set. It can be concluded that the pronounced decrease in peak vimax seen during exposure to a bretylium concentration of 1 x 10 ’ M is not primarily due to the small decrease in resting potential but rather due to the marked decrease in membrane responsiveness ; the Vmax at any given level of
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Figure 8. A typical example of the effect of bretylium tosylate on the membrane responsiveness of a canine Purkinje fiber. Transmembrane voltage at the time of activation (in millivolts) is plotted on the abscissa; the corresponding phase zero \jmax (in volts per second) is plotted on the ordinate. Each control experimental point (filled circles) repiesents the mean of at least 3 determinations. Control peak Vmax was 560 volts/set and transmembrane voltage at 50 percent peak \jmax (V,, was 72 mv. Using these 2 measured variables (peak \imax and V,,), the S-shaped curve was computed by the Weidmann modification of the HodgkinHuxley formula.” Thirty minutes after a bretylium concentration of 1 v lo-” M measurements were identical to the control values (the points obtained at 1 x lo-” M are not shown). After a concentration of 5 x 10 i M (open triangles), v,, shifted 1 to 2 mv rightward on the voltage axis and peak Vmax decreased by 50 volts/set (at 90 mv transmembrane voltage). Thirty minutes after a bretylium concentration of 1 y: 10-l M (closed triangles), V,, shifted 9 mv rightward on the voltage axis and peak Vmax decreased by 240 volts/set; this change represents a pronounced decrease in responsiveness.
transmembrane voltage is markedly reduced from the value obtained at the identical level under control conditions. The decrease in membrane responsiveness was not seen until high concentrations of bretylium were used, i.e., those which also reduced the transmembrane resting potential (presumably a toxic effect). There was also no tendency for bretylium to produce a rightward shift of the steep portion of the S-shaped curve on the voltage axis before the decrease in peak Vmax, in contrast to quinidine and procainamide. Effect of Bretylium on Conduction in Purkinje Fibers The velocity with which an action potential propagated down a linear strand of Purkinje fiber was measured in 5 preparations which were arranged in the manner shown in Figure 2 and stimulated at a constant rate of 75 ‘min. The distance between microelectrodes A and B was 0.8 -i 0.24 cm. The average conduction velocity
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BRETYLIUM
EFFECT
OF BAETYLIUV
AND QUINIDINE
BRETYLIUM
CONTROL
ON AUTOMATIC
IxIO-~
PURKINJE
QUINIDINE
FIBER
1~10~~ 5 set
Figure 9. The effect of bretylium tosylate and quinidine sulfate on spontaneous firing of Purkinje fibers. In both A and B recordings were made from undriven preparations with a microelectrode located in site 3 of Figure 1. A, effect on a fiber which was firing spontaneously under control conditions. Each frame was recorded from the same Purkinje fiber site 30 minutes after exposure to the drug concentration listed below the frame. The control (upper row, left frame) spontaneous firing rate was 44/min. Note the typical diastolic (phase 4) depolarization. The sharp junction between action potential phase 4 and phase zero suggests that the pacemaker site is distant and that the impaled cell is depolarized by the propagating impulse. Exposure to a bretylium concentration of 5 x 10.” M produced an increased rate of firing during the initial 10 minute period of firing (not shown), but, after 30 minutes, the rate decreased to 3l/min (upper row, center frame). At a concentration of 1 x 10-l M (upper row, right frame), the firing rate increased to 40/min, the action potential prolonged in duration, maximal diastolic transmembrane voltage decreased (shifted toward zero), and terminal phase 4 depolarization became more concave upward. This change in the configuration of phase 4 suggests that (1) the pacemaker moved closer to the recording site or (2) more of the Purkinje fiber surface membrane participated in pacemaker activity. After exposure to quinidine, 1 x lo-” M, action potential configuration altered noticeably, but the firing rate remained unaltered at 45/min (lower row, left frame). Exposure to quinidine, 1 x lo-‘M, (lower row, right frame) caused a decrease in maximal diastolic voltage and action potential amplitude, decreased the slope of phase 4 depolarization and reduced the spontaneous firing rate to 25/min. B, effect on a fiber that was quiescent (not firing spontaneously) under control conditions. The fiber showed no evidence of spontaneous activity under control conditions (left frame). Spontaneous firing began 4 minutes after bretylium (1 x 10-l M) perfusion was begun. After 35 minutes, the spontaneous firing rate was 57/min (center frame) and remained stable during a subsequent 30 minute period of observation. Bretylium perfusion was then stopped, and spontaneous firing stabilized at 42/min in normal Tyrode solution. When quinidine (1 x lo-” M) was added, slowing began within 5 minutes and spontaneous activity ceased after 12 minutes (right frame).
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under control conditions was 3.1 -+ 0.51 meters/ sec. During exposure to a bretylium concentration of 1 x lo-” M, the conduction velocity increased to 3.4 f 0.62 meters;/sec, a change which was not statistically significant (P < 0.1) ; no change in resting potential or peak vmax was seen at concentration in these experiments. When these preparations were exposed to a bretylium concentration of 1 x 10-I M, the conduction velocity fell to 2.4 +- 0.89 meters ‘see, a 22 percent decrease (P < 0.05) ; resting potential decreased by 3.0 + 0.7 mv and peak vimax by 48 + 19 volts/see in these experiments. Effect of Bretylium on Purkinje Fiber Automaticity Automaticity is a property of Purkinje fibers which is responsible for in vivo escape rhythms and which may also be involved in the genesis of cardiac arrhythmias. All previously studied antiarrhythmic drugs have shown a significant ability to suppress automaticity in Purkinje fibers. The effects of bretylium tosylate on Purkinje fiber automaticity were studied in 14 preparations. Seven preparations showed stable spontaneous firing during a 1 to 2 hour period of control observation. The average control spontaneous firing rate was 39 i: 2.0 clepolarizat.ions ‘min. Preparations were then perfused with Tyrode solution containing bretylium. The initial concentrations used were 5 X 10msM in 4 experiments ; preparations that were initially exposed to a concent.ration of 5 x 10~~ M were all subsequently exposed to a concentration of 1 >( 10-l M. Two preparations were finally perfused with solution containing a bretylium concentration of 5 >( lOed M. All fibers studied showed an initial increase in rate when exposed to bretylium. This increase in rate peaked 10 to 15 minutes after adding bretylium, at which time the average rate was 47 -+ 2.4 depolarizations ‘min, an increase of 20 percent (P < 0.05). The rat.e then declined to 36 -i- 6.7 minutes and stabilized by 30 to 40 minutes after bretylium was begun; at this time, the spontaneous firing rate was not significantly different from the rate found under control conditions (Fig. 9A). This was even true in the 2 preparations exposed to a bretylium concentration of 5 S 10-j M. Bretylium, in all the concentrations employed in this series of experiments, did prolong the action potential duration in these unstimulated preparations and at concentrations of > 1 X lo--’ M caused a decrease in the maximal cliastolic potential. In 3 preparations, bretylium changed the voltage-time course of the terminal portion of phase 4 so that the transmembrane voltage-time course became more concave upward (Fig. 9A). This change suggests that (1) the site of the pacemaker in the preparation shifted
89
BIGGER AND JAFFE
closer to the microelectrode recording site or (2) more of the Purkinje fiber surface membrane area became automatic. Bretylium compared with quinidine: Kretylium’s inability to suppress automaticity in Purkinje fibers was unexpected. We, therefore, compared the response of 5 of the preparations to quinidine sulfate (Fig. 9A). After the response to bretylitim had been observed, these 5 preparations were perfused for an hour with drug-free Tyrode solution. Then Tyrode solution containing a quinidine concentration of either 5 c: 10 Z or 1 v 10 ’ M was used to perfuse the preuasation. The average spontaneous firing rate of the 5 preparations at the end of the drug-free perfusion period was 41 +- 2.2 depolarizations./min, a value not significantly different from the original control observations. After quinidine, the mean firing rate fell to 14 f~ 4/min, a 66 percent decrease (P < 0.01) ; in 1 preparation, spontaneous firing ceased altogether. It is thus evident that in comparable concentrations quinidine had a more powerful antiautomatic effect than bretylium and that the lack of significant antiautomatic eff’ect was not due to unusual resistance to suppressive drugs in the Purkinje fibers studied. Effects of pretreatment with reserpine: A known early effect of bretylium is release of norepinephrine from the adrenergic nerve terminals. It seems reasonable to propose that the initial increase in rate seen during perfusion with bretylium in our experiments was due to release of catecholamines, To provide further information on this point, experiments were performed on obtained from dogs previously preparations treated with reserpine. The protocol directed that 5 dogs be given 0.5 mgfkg of reserpine on 3 consecutive days and sacrificed on the fourth. Only 2 dogs survived to the time of sacrifice. Four preparations were obtained from these 2 animals, but only 2 showed spontaneous firing; these had rates of 14 and 20 depolarizations ‘min. Each preparation was first perfused with Tyrode solution containing a bretylium concentration of 5 Y 10Ws M and then with a solution containing a concentration of 1 x lO- I M. The significant increase in rate seen in unreserpinized preparations was absent in the 2 preparations pretreated with reserpine. After a 30 minute exposure to a bretylium concentration of 1 >c; IO--' M, the rate of 1 preparation was unaltered, and spontaneous activity ceased in the other. In addition, bretylium, in concentrations of 5 .Y 1O-S and 1 x 10-q M, did not initiate firing in the 2 quiescent reserpinized preparations ; application of norepinephrine did.
Discussion Electrophysiologic properties of antiarrhythmic agents : The electrophysiologic properties of quinidine,22,2U procainamide,“” diphenylhydan-
90
toin,Z’: pr0pt~10101” attd litlocaincYJ all have lien studied in canine Purkinje fiber and ventricular muscle; each of these agents possesses ;I significant antiarrhythmic effect against ventricular arrhythmias in man. Many difl’ercnces harTe 1~11 identified amon% these antiarrhythmic agents. Quinidine, procainamide ant1 p~op~-uJJ0101 decreiise diastolic escit,:Lbility, whereas comparable concentraiions of’ diphenylhgdantoin and lidocnine have minimal effects.“” Therapeutic ~on~cnli.~~iions of quiniditlc, procainamide and prop~*anolol also decrease peak <7mW nX3?-tbJ~arK’ I-espoltSjveMW :lJld coJldll~~ioJ1 velocity in Purkinje fibers; comparable conccntrations of diphenylhydantoin and lidocaine either do not alter these properties, or under some circumstances may increase each of these variablesL.X1.“-D.:NI Quinidine and procainamide cause small but definite increases in action potential duration and effective refractory period in ventricular muscle and Purkinje fibers”‘; diphenylpropranolol” and lidocaine”” cause hydantoin,‘:’ a decrease in both action potential duration and effective refractory period in each cell t.ype. There are, of course, other electrophysiologic differences among these agents. Quinicline’” and procainamidti” prolong atria1 action potential tluration. These agents and propranolol:‘” decrease peak vmax and slow conduction in rabbit atria1 fibers ; diphenylhydantoin”” and lidocaine:‘:: do not significantly affect atria1 action potential duration, either increase or do not change peak Qmax and do not slow conduction in atrium. Quinidine, procainamide and propranolol prolong atrioventricular refractory periods and conduction time in a variety of animals including man ; diphenylhydantoin and lidocaine do not prolong these variables and often even shorten the atrioventricular refractory period and conduction time, particularly when digitalis has been previously administered.:‘-’ Quinidine and procainamide produce unreliable results when used to combat arrhythmias caused by digitalis excess, while propranolol, diphenylhydantoin and lidocaine are highly effective.:‘f Two properties were uniformly concordant in studies of these 5 drugs in ventricuIar muscle-Purkinje fiber preparations. First, all 5 agents had a significant ability to depress automaticity in Purkinje fibers. Second, all 5 caused t.he effective refractory period of Purkinje fibers (and when studied, ventricular muscle fibers, as well) to become long relative to action potential duration; this was true although some of the drugs (quinidine and procainamide) prolong both action potential duration and effective refractory period and others (propranolol, diphenylhydantoin and lidocaine) shorten both. Because these two electrophysiologic changes were produced by all 5 antiarrhythmic drugs in concentrations comparable to in viva therapeutic concentration, they have
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BRETYLIUM
been considered of great importance. Indeed, it has been proposed that changes in these 2 properties can largely explain the antiarrhythmic properties of these agents.29 Comparison of antiarrhythmic effects of bretylium and other agents: Bretylium stands out in bold relief against the background of the 5 previously studied agents. Although bretylium resembled quinidine and procainamide in prolonging action potential duration and effective refractory period, bretylium primarily affects phase 2 of action potential duration, whereas quinidine and procainamide primarily lengthen phase 3. Furthermore, action potential lengthening caused by bretylium was not accompanied by significant change in Purkinje fiber peak iimax, membrane responsiveness or conduction velocity in concentrations thought to be comparable to therapeutic doses in man. The sequence of alterations in membrane responsiveness during perfusion with high (toxic) concentrations of bretylium was also different from that seen with quinidine or procainamide. With bretylium, peak iimax decreased concomitantly with a rightward shift of the S-shaped responsiveness relation on its voltage axis whereas quinidine and procainamide shifted the S-shaped curve rightward on the voltage axis and increased V,, (that is, made V,, more negative) before peak Vmax was altered. Most disturbing was the failure of bretylium to exert a significant effect on either of the 2 electrophysiologic properties that are so strikingly modified by previously studied antiarrhythmic drugsautomaticity and the relation between effective refractory period and action potential duration. First, bretylium failed to show powerful effects, if any, in suppressing automaticity in Purkinje fibers. In fact, initially this drug caused a significant increase in the spontaneous firing rate of in vitro Purkinje fibers (Fig. 9). Second, bretylium did not significantly lengthen the effective refractory period relative to action potential duration in either Purkinje or ventricular muscle fibers. High (toxic) concentrations of bretylium did not alter the relation or actually made action potential duration slightly long relative to effective refractory period. seen in this study Only 2 actions of bretylium might. reasonably be considered to have antiarrhythmic effects by acting directly on ventricular myocardial cells. First, bretylium prolongs the action potential ancl lengthens refractory period without slowing conduction. This would alter the wave length in a reentrant pathway and might, thus. abolish reentrant arrhythmias. Second, in partially depolarized fibers, bretylium caused a prompt, if mild, increase in resting transmembrane voltage and, thus, in phase zero amplitude and jimas. This effect may be due to the early
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JANUARY
1971
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catecholamine releasing action of bretylium, since catecholamines hyperpolarize partially depolarized Purkinje fibers and thereby increase phase zero amplitude and vmax.:s8 This effect could enhance conduction and abolish reentrant rhythms.s0 Although it remains to be demonstrated conclusively that bretylium is an effective antiarrhythmic drug in man, an increasing number of observations in intact animals and man lend credence to the hypothesis that its action is antiarrhythmic, particularly in cases of ventricular arrhythmia.34~1”~“” The findings of our study suggest that the effects of brktylium on the electrophysiologic properties of the cell membrane of Purkinje and ventricular muscle fibers do not entirely explain its antiarrhythmic effects in terms of present concepts of antiarrhythmic drug action. Indeed, the predominant mechanism through which bretylium acts as a cardiac antiarrhythmic agent may not involve, to a major degree, its effects on cell membranes of cardiac muscle. Adrenergic neural blocking action of bretylium : The role of the autonomic nerves and nerve terminals in the genesis of cardiac arrhythmias has been stressed recently in the case of arrhythmias occurring after cardiac coo1ing,3’i hypoxiaXG and excessive doses of digitalis.:‘i It has been suggested that drugs that depress neural function exert antiarrhythmic effects, whereas those that do not depress neural function lack antiarrhythmic activity.:” This hypothesis receives its strongest support in the instance of beta adrenergic agents used to treat digitalis-induced arrhythmias : at essentially equipotent blocking doses, the agents that are neural depressants are antiarrhythmic; those without this effect are not.“’ Han et aPR have shown that stimulation of cardiac sympathetic nerves, as well as hypothermia, myocardial ischemia and ouabain intoxication, increase the temporal dispersion of the refractory period in the canine ventricle and suggested that this would promote reentrant rhythms and fibrillation. Yanowitz et al. showed that unilateral increases or decreases in cardiac sympathetic nerve activity can lead to marked alterations in the duration of electrical systole and markedly change the sequence of repolarization in the canine ventricle.:“’ Roberts et al.:“’ have suggested that in so far as each cardiac axon or nerve terminal has the capacity for independent activity, the cardiac neural system may become discoordinated. The discordant cardiac neural activity could fractionate the conductivity, automat,icity and excitability of the ventricles and lead to arrhythmias.ZG It is reasonable to suppose that when such discordant cardiac neural activity plays a role in the genesis of cardiac arrhythmias, bretylium might have a beneficial effect through its adrenergic neural blocking action.
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References 1. Boura ALA, Copp FC, Green AF: New antiadrenergic compounds. Nature (London) 184:70-71, 1959. 2. Boura ALA, Copp FC, Duncombe WG, et al: The selective accumulation of bretylium in sympathetic ganglia and their post ganglionic nerves. Brit J Pharmacol 15:265270, 1960 3. Boura ALA, Green AF: The action of bretylium: adrenergic neurone blocking and other effects. Brit J Pharmacol 14536-548. 1959 ultrastructurelles provoquCes 4 Clementi F: Modifications tnedicaments par quelques sur les terminaisons nerveuses adrknergiques et sur la medullaire surrCnale. Experientia 21:171-176, 1965 of adrenergic nerve conduc5. Exley KA: The persistence tion after TM,,, or bretylium in the cat. In. Adrenergic Mechanisms (Vane JR. Wolstenholme GEW. O’Conner M, ed). London, J.A. Churchill, 1960, p 158-161 Role of norepinephrine in autos 6. Brodie BB, Costa E: noniic ganglia in regulation of blood pressure. In, Hypertension, Recent Advances (Brest AN, Moyer JH. ed). Philadelphia, Lea & Febiger. 1961. p 354-360 Tissue amine levels and sympa7. Cass R, Spriggs TLB: thetic blockade after guanethidine and bretylium. Brit J Pharmacol 17:442-450, 1961 SM, Furchgott RF: The sympathomimetic ac8. Kirpekar tion of bretylium on Isolated atria and aortic smooth muscle. J Pharmacol Exp Ther 143:64-76, 1964 Adrenergic neurone blocking 9. Boura ALA, Green AF: agents. Ann Rev Pharmacol 5:183-212, 1965 of Initial 10. Gokhale SD, Gulati OD, Kelkar VV: Mechanism adrenergic effects of bretylium. Brit J Pharmacol 20: 362-377, 3 963 G, Axelrod J, Patrick RW: Actions of bretylium 11. Hertting and guanethidine on the uptake and release of [“HI-noradrenaline. Brit .I Pharmacol 1.8:161-166. 1962 Anti-arrhythmic action of bretylium. Na12. Leveque PE: ture (London) 207:203-204, 1965 13. Nielsen KC, Owman C: Control of ventricular fibrillation during induced hypothermia in cats after blocking the adrenergic neurons with bretylium. Life Sci 7:159-168, 1968 M: Prevention and treatment of ventricular 14. Bacaner arrhythmias and ventricular fibrillation with bretylium tosylate. Univ Minn Med Bull 38:317--319, 1967 actions of 15. Ellis CH, Barnes M, Cozzi M: Antiarrhythmic bretylium (abstr). Fed Proc 27:406, 1968 Bretylium tosylate for suppression of in16. Bacaner M: duced ventricular fibrillation. Amer J Cardiol 17:528534, 1966 D: Bretylium tosyiate for 17. Bacaner M, Schrienemachers the suppression of ventricular fibrillation after experimental myocardial infarction. Nature (London) 220:494496, 1968 18. Torresani J, Heuillet G, Djourno J, et al: Essai de prevention de la fibrillation ventriculaire par le Bretylium. Arch Mal Coeur 61:982-992, 1968 19. Bacaner M: Treatment of ventricular fibrillation and other acute arrhythmias with bretylium tosylate. Amer J Cardiol 21:530-543, 1968 20. Redleaf PD, Lerner IJ: Thiazide-induced hypokalemia with associated maior ventricular arrhythmias. JAMA
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22.
23.
24.
25.
26.
27.
28.
29.
30.
31. 32.
33.
34. 35.
36.
37.
38. 39.
206:1032-1034, 1968 Hoffman BF: The electrophysiology of heart muscle and the genesis of arrhythmias. In. Mechanisms and Therapy of Caidiac Arrhvthmias IDreifus LS. Likoff W. ed1. , New York, Grune & Stratton, 1‘966, p 27-34 Hoffman BF: Action of quinidine and procaine amide on single fibers of dog ventricle and specialized conducting system. Anais Acad Brasil Ci&c 29:365-368, 1958 Bigger JT Jr, Bassett AL, Hoffman BF: Electrophysiological effects of diphenylhydantoin on canine Purkinje fibers. Circ Res 22:221-236. 1968 Davis LD, Temte JV: Effects of propranolol on the transmembrane potentials of ventricular muscle and Purkinje fibers of the dog. Circ Res 2:661-677. 1968 Bigger JT Jr, Mandel WJ: Effect of lidocaine on the electrophysiological properties of ventricular muscle and Purkinje fibers. J Clin Invest 49:63-77, 1970 Strauss HC, Bigger JT Jr, Bassett AL, et al: Actions of diphenylhydantoin on the electrical properties of isolated rabbit and canine atria. Circ Res 23:463-477, 1968 Weidmann S: Effect of the cardiac membrane potential on the rap d availability of the sodium-carrying system. J Physiol (London) 127:213-224, 1955 Weidmann S: Effects of calcium ions and local anesthetics on electrical properties of Purkinje fibers. J Physiol (London) 129:568-582. 1955 Hoffman BF, Bigger JT Jr: Antiarrhythmic drugs. In, Drill’s Pharmacology in Medicine, fourth edition (DiPalma JR, ed). New York, McGraw-Hill, 1971, chap 40 Bigger JT Jr, Mandel WJ: Effect of lidocaine on conduction in canine Purkinje fibers and at the ventricular muscle-Purkinje fiber junction. J Pharmacol Exp Ther 172:239-254, 1970 Szekeres L, Vaughan Williams EM: Antifibrillatory action. J Physiol (London) 160:470-482, 1962 Morales-Aguilera A, Vaughan Williams EM: The effects on cardiac muscle of p-receptor antagonists in relation to their activity as local anesthetics. Brit J Pharmacol 24: 332-338, 1965 Mandel WJ, Bigger JT Jr: Effects of lidocaine on the sinoatrial node and atrial fibers (abstr). Amer J Cardiol 25:113, 1970 Bigger JT Jr, Heissenbuttel RH: Clinical use of antiarrhythmic drugs. Postgrad Med 47:119-125, 1970 Hoffman BF, Singer DH: Appraisal of the effects of catecholamines on cardiac electrical activity. Ann N Y Acad Sci 139:914-939, 1967 Szekeres L: Principles of the pharmacotherapy of the disorders of cardiac rhythm. Hungarian Med J 15:39-45, 1967 Roberts J, Levitt B, Standaert FG: Autonomic nervous system and control of cardiac rhythm. Nature (London) 214:912-913, 1967 Han J, Moe GK: Nonuniform recovery of excitability in ventricular muscle. Circ Res 14:44-60, 1964 Yanowitz F, Preston JB, Abildskov JA: Functional distribution of the right and left stellate innervation to the ventricles: production of neurogenic electrocardiographic changes by unilateral alteration of sympathetic tone. Circ Res 18:416-428, 1966
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Studies
The Effect of Bretylium Tosylate on the Electrophysiologic of Ventricular
J. THOMAS
BIGGER,
Muscle and Purkinje Fibers
Jr., MD*
CONRADE C. JAFFE. MD New York, New York
From the Departments of Pharmacology and Medicine, College of Physicians and Surgeons, Columbia University, New York, N. Y. This investigation was supported in part by U. S. Public Health Service Grant HE-12738 and in Dart bv a grant-in-aid from the New Yo’rk Heart issociation. Manuscript received December 4, 1969, accepted March 12, 1970. ::Senior Investigator of the New York Heart Association. Address for reprints: J. Thomas Bigger, Jr., MD, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W. 168 St., New York, N. Y. 10032.
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Properties
Preparations of papillary muscle and Purkinje fibers were obtained from dog hearts, perfused with oxygenated Tyrode solution in a tissue chamber, and observed both under control conditions and during exposure to bretylium tosylate in concentrations from 1 X 10 i M to 1 I’ 10 m( M. Transmembrane voltage was recorded from both ventricular muscle and Purkinje fibers in spontaneously beating and in electrically driven preparations. Bretylium did not suppress phase 4 depolarization or spontaneous firing in Purkinje fibers. After application of bretylium to spontaneously firing Purkinje fibers, the rate initially increased by 20 percent; in quiescent fibers, bretylium often induced spontaneous firing. Since neither effect was seen in preparations obtained from dogs treated with reserpine, each was attributed to bretylium-induced release of catecholamines from the adrenergic nerve terminals in the preparations. In concentrations of less than 1 :( lo-,& M, bretylium did not alter the resting transmembrane voltage or the action potential amplitude and overshoot; concentrations of 1 :< lo-m4 M caused a decrease in each of these variables. Bretylium, < 1 ?< lo---’ M, had no effect on the maximal rate of rise of phase z&o depolarization (peak \imax), membrane responsiveness or conduction velocity in Purkinje fibers. A concentration of 1 :+ 10-l M caused a decrease in peak \imax, shifted membrane responsiveness curves on their voltage axis, and decreased conduction velocity. Over a wide range of stimulation rates (30 to 150/min), bretylium caused a large increase in action potential duration and effective refractory period of both ventricular muscle and Purkinje fiber cells without lengthening the effective refractory period relative to action potential duration. Since bretylium lacked most of the electrophysiologic properties associated with antiarrhythmic drugs, its antiarrhythmic action against ventricular arrhythmias may, to a large extent, be due to its effect on adrenergic nerve terminals.
Rretylium tosylate, a quarternary benzylammonium salt, was reported in 1959 to be a relative of xylocholine which possessed sympathetic h1ockin.g activity and lacked undesirable muscarinic side effects.’ Rretylium is preferentially taken up by the adrenergic nerve terminals” m-here it prevents release of norepinephrine during direct or reflex sympat.hetic nerve stimulation.:k It interferes with norepinephrine release without significantly (1) altering the ultrastructural integrity of the adrenergic neural vesicles,’ (2) depressing preganglionic or postganglionic sympathetic nerve conduction.z (3) impairing sympathetic ganglionic
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Figure 1. Diagram of the typical experimental arrangement showing the free-running Purkinje fibers (false tendon) as a black strand between a segment of interventricular septum on the left and papillary muscle on the right. A pair of silver electrodes is placed on the surface of the papillary muscle (1) and used for stimulation. Transmembrane voltages from nearby ventricular muscle cells (2) and from an adjacent Purkinje fiber (3) were recorded using glass microelectrodes. The distance between the microelectrode sites at (2) and (3) ranged from 1 to 3 mm.
TOSYLATE
descending coronary artery’” or digitalis intoxicationl”; it has been found to increase the electrical ventricular fibrillation threshold in dogs subjected to thoracotomy alone’6 or to thoracotomy followed by acute coronary occlusion,17 and in closed chest dogs exposed to alternating current.lx This work has led to a limited therapeutic trial of bretylium as an antiarrhythmic agent in man. 14.1x-2’) The present study was undertaken to determine, in an isolated perfused preparation obtained from canine heart, the effects of bretylium on the electrophysiologic properties of the 2 cell types of the ventricle-ordinary ventricular muscle fibers and Purkinje fibers. These cell types were selected for study since alteration in their electrophysiologic behavior, particularly that of the Purkinje fiber, is thought to underlie the genesis of ventricular arrhythmias.“’ We also compared our findings with those obtained in previous studies of various antiarrhythmic drugs in the same preparation.22-25 We found significant differences between the electrophysiologic effects of bretylium on these 2 cell types and those of the previously studied antiarrhythmic drugs. We, therefore, propose that a major portion of bretylium’s antiarrhythmic action may result from its antiadrenergic effects rather than its direct effect on the cell membrane of ventricular muscle or Purkinje fibers.
Methods transmission,” (4) depleting the adrenergic nerve terminal of norepinephrine,7 or (5) diminishing the responsiveness of adrenergic receptors to adrenergic agonists.z,x Thus, the site of bretylium’s antiadrenergic action appears to be the adrenergic nerve terminal.!’ Bretylium has 2 other demonstrable actions on the adrenergic nerve terminal. Soon after injection into intact animals or application to various tissue preparations, bretylium causes release of norepinephrine from adrenergic nerve termina1s.8JnAlso, like cocaine, bretylium can impair the uptake of infused norepinephrine into the adrenergic nerve terminal,‘l so that it potentiates the effect of injected or infused norepinephrine on the adrenergic receptors.8-11 Recently, bretylium has been reported to possess significant antiarrhythmic activity. Levequel* reported in 1965 that high doses of bretylium prevented atria1 fibrillation induced by acetylcholine in dogs rendered hypokalemic by administration of glucose and insulin. More recently, investigators have shown the effectiveness of bretylium against experimental ventricular arrhythmias. Bretylium has been reported effective in preventing or abolishing ventricular arrhythmias induced by hypothermia,‘” hypothermia coupled with hemorrhagic shock,‘-’ acute ligation of the anterior
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Mongrel dogs weighing 10 to 14 kg were anesthetized with intravenously administered pentobarbital sodium (30 mg/kg). The heart was rapidly excised and dissected in oxygenated Tyrode solution at room temperature. Preparations containing Purkinje fibers and ventricular muscle were dissected from either ventricle and placed in a wax-lined tissue bath (Fig. 1). This bath was perfused with Tyrode solution, gassed with 95 percent oxygen and 5 percent carbon dioxide, at a constant rate of ‘7 ml/min ; the temperature of the perfusate was maintained at 36 -C 1C. The composition of the Tyrode solution and the techniques used for stimulating and recording have been described previously.*aJ6 The arrangement of preparations is shown in Figure 1. Surface electrodes at site 1 were used to stimulate the ventricular muscle. A microelectrode 5 4 mm away (site 2) was used to record transmembrane voltage of a ventricular muscle cell. Simultaneously, the transmembrane voltage of an adjacent Purkinje fiber and its first time derivative (Vmax) was recorded through a second microelectrode at site 3; the interelectrode distance was 1.8 -t 0.9 mm (mean & SEM). The effective refractory period of ventricular muscle and Purkinje fibers and the membrane responsiveness of the Purkinje fiber were measured while the preparation was stimulated at a constant rate of 75/min (cycle length 800 msec). Premature stimuli were introduced every seventh basic drive cycle. Premature stimuli were initially placed in electrical diastole and made progressively more premature until the most
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premature ventricular muscle action potential which propagated to and down the Purkinje fiber was identified (Purkinje fiber effective refractory period b. Slightly earlier test stimuli still produced a large amplitude ventricular muscle action potential which failed to excite the adjacent Purkinje fiber. Test stimuli were then made still more premature until the ventricular muscle cell isite 2) failed to respond (ventricular muscle effective refractory periotl I. Such
stimulation seqilences not only allowed rneasuYement of the effective refractory periotl in both ventriculal~ muscle and Purkinje fiber but also provided the opportunity to determine membrane responsivenessthe relationship between the maximal rate of rise of phase zero of the action potential (?mas I ant1 the transmembrane voltage at the time of excitation. \v;ts directl! estimated in Conduction velocit> Purkinje fibers by measuring the time required fol the propagating impulse to traverse the known lineal tlistance between two recording sites (sites A and E, Fig. 2 I. The instant in time at which peak iTmax of the action potential occurred at each recording site was determined by electronic differentiation of the 2 xand used as the indication tion potential upstrokes that the propagating impulse had arrived at the rccording site (Fig. 2). Eretylium tosylate was provided by Burroughs Wellcome & Co. as a powder which was initially dis-
lo-
I
263 I
;iOc?
.d
20 msec
msec
Further dilutions were in distilled water. out in Tyrode solution to obtain final conccnbetween 1 x 10 7 M ~‘0.04 /~g ml) and 1
solved carried trations X
tential 88 -t 3 mv. amplitude 114 t 4 mv and peak Vmax 430 2.- 70 volts ‘see). Action potential duration did not differ significantly from control values at concentrations of 1 ~.’ 10 i or 1 b 10 ” M hut was prolonged Rignificantly (P < 0.001) at concentrations of 2 1 .’ 10 A M (Fig.3 to 5). At concentrations of 1 X 10 ” M, x11increase in action potential duration \vas seen in all fibers (range in individual fibers
I
M (40 p’g ‘ml\.
Results Effect of Bretylium Tosylate Voltage-Time Course
on Transmembrane
The effect of bretylium tos;yCOUIW of transmen~branc~ voltage was studied in 15 Purkinje fibers. (‘oncentrations from 1 y 10 i to 5 ,: 10 ’ M exerted 110 significant effect on resting potential or overshoot. and thus no change in the total amplitude of the action potential occurred. At a 1 Y 10 ’ M concentration, the resting potential decreased from the COlltr01 ValUe of --91 I!- 2 mv (mean rt: SEM) to -87 i 2 mv (P < 0.05). At this concentration the overshoot was also slightly decreased so that. the total amplitude of the action potential decreased from the control value of 119 -I 4 mv t.o 110 -I5 mv (P < 0.01). In 5 other experiments, not reported in the previous results, the effects of bretylium were noted on fibers that were stretched during the pinning of the preparation in the tissue bath so that a decrease was seen in resting potential (84 i- 6 mv), amplitude (109 ?- 5 mv) and rate of rise of phase zero of the action potential (peak pmax 390 -t 50 volts ‘set). Under these conditions, application of bretylium tosylate. 1 X IO-” M or 5 x lo- z M, caused A significant increase in each of t.hese variables (resting poPurkinje
fibers:
late on the voltage-time
84
Figure 2. Method of measuring conduction velocity. The lower portion of the figure shows a diagram of the preparation used in studies of conduction velocity. A pair of silver electrodes was placed directly on the false tendon and used to stimulate the preparation. Microelectrodes were used to impale 2 sites in a single strand of the false tendon (sites A and B); the distance between sites A and B was measured with a calibrated ocular micrometer. The upper portion of the figure shows typical recordings obtained from such a preparation. The upper trace records time marks every 100 msec. Calibration for phase zero \imax is shown on the left (1,000 volts/set) and voltage calibration for the action potentials on the right (100 mv). The time calibration for the slow sweep speed is shown at the bottom left (200 msec) and for the rapid sweep speed at the bottom right (20 msec). Recordings obtained from site A are labeled A; those recorded from site B are labeled B. Action potentials A, and B, are recorded at slow sweep speeds from sites A and B. A, and B, are the upstrokes of action potentials recorded at sites A and B recorded at sweep speed 10 times faster. A, and B, are \imax of potentials A and B obtained by electronic differentiation and recorded at the rapid sweep speed. The conduction velocity of the propagating impulse was estimated by accurate measurement of the time between the 2 action potential upstrokes (phase zero) and the distance between the sites at which the action potentials were recorded and was expressed in meters per second.
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10 to 20 percent change, Fig. 3 to 5). Further prolongation ranging from 20 to 35 percent occurred at a concentration of 5 x lo-” M (Fig. 4 and 5). At a bretylium concentration of 1 X 1O-4 M some fibers showed slightly further prolongation, but the mean change in action potential duration was not significantly different from that at 5 x’ 10 mGM. The increase in action potential duration produced by bretylium was due largely to prolongation of the action potential plateau (phase 2) ; in some fibers, phase 3 was also slightly prolonged (Fig. 3 and 6). The effect of bretylium on action potential duration was examined in 10 fibers at drive cycle lengths from 400 to 2,000 msec-drive rates of 30 to 150 /min (Fig. 4). At all these cycle lengths and at a concentration of 1 X lo-” M, Purkinje fiber action potential duration was prolonged by about the same percent change from control (16 * 5 percent). At 5 ?: 10mGand 1 >< lo-.’ M, action
CL 1000
CL 500
msec
280
msec
I4 5 msec
150
msec
249 VM
‘0 i =
___\-
-1,
,.
-__1
potential duration was further prolonged (28 I 7 percent change). Study of ventricular Ventricular muscle: muscle transmembrane voltage was simultaneously performed with that of Purkinje fibers in 12 preparations (Fig. 3 to 5). Bretylium 5 1 x 10~’ M (40 &ml) produced no significant change in resting potent,ial or action potential amplitude in ventricular muscle. The general configuration of the action potential after bretylium tosylate was unaltered except for prolongation of duration (Fig. 3 and 6). Prolongation of the ventricular muscle action potential duration became significant at a concentration of 1 x 10~-z M (P < 0.025) and paralleled the change seen in Purkinje fibers (Fig. 5A). The percent change in action potential duration in ventricular muscle was slightly smaller at any given drug concentration than in Purkinje fiber (10 t 5 percent at 1 X lo-; M, 18 t 5 percent at both 5 x 10-j and 1 X 1O-4
CL 2000
290 153
-
msec msec
_&
PF
2 91 msec
325
msec
346
VM
160 msec
169
msec
174 msec
msec
PF
‘0 &) -T;
PF VM
\A. 285
y
=1 msec
163 msec
msec
173md
,
;:;t;;,
mV msac
Figure 3. Effect of bretylium tosylate on action potential duration of canine Purkinje and ventricular muscle fibers. In the top trace of each panel, time marks are repeated every 100 msec. The other 2 traces record transmembrane action potentials from ventricular muscle (shorter action potential) and a Purkinje fiber (longer action potential). The 3 vertical columns show recordings made at cycle lengths (CL) of 500 msec (left column), 1,000 msec (center column), and 2,000 msec (right column). The horizontal rows show action potentials recorded under control conditions (top row) and during exposure to bretylium tosylate in concentrations of 1 x 10.” M (second row) and 1 x 10.’ M (third row). The action potential duration (measured to 95 percent repolarization) of the Purkinje fiber and ventricular muscle fiber is given under each frame. At 1 x 10.’ M, action potential duration was not significantly affected in either cell type (not shown). At 1 y lo-” M and 1 x 10-l M, Purkinje fiber action potential lengthened by 17 percent and that of the ventricular muscle cell by 12 percent at each drive rate.
1971
,
VM
I loo 167
200
VOLUME 27, JANUARY
TOSYLATE
400
CYCLE
600
8OC1
LENGTH
1000
2000
- MSEC
Figure 4. Effect of bretylium tosylate on the action potential duration of Purkinje fibers (PF) and ventricular muscle (VM) fibers which were driven over a wide range of cycle lengths (400 to 2,000 msec). Action potential duration in milliseconds is plotted on the ordinate and the driven cycle length in msec on the abscissa. Each point represents the mean value of 10 experiments in Purkinje fibers (closed symbols, above) and 7 experiments in ventricular muscle (open symbols, below); vertical bars represent the standard error of the mean. After a 30 minute exposure to a concentration of 1 x lo-’ M (not shown), the action potential duration of PF and VM did not differ significantly from control values (P < 0.4). Exposure to bretylium in concentration of 1 x 10.” M or 1 x lo-” M significantly prolonged the action potential duration of both Purkinje fibers and ventricular muscle fibers (P < 0.001).
85
BIGGER
AND
JAFFE
PF
l
VM
o
A
M ) .The change in ventricular muscle action potential duration as R function of drive cycle length at any given concentration! of \,retylium ‘ivas studied in 7 preparations and was found to l,e similar to Purkinje tiller in that the percent change for ventricular muscle action potential duration ~vas al)out the same at all drive cycle lengths between 400 and 2,000 msec ( Fig. 4). Effect of Bretylium Refractory Period
PF
l
VM
1,
Figure 5. Action potential duration and effective refractory period of Purkinje and ventricular muscle fibers as a function of bretylium tosylate concentration in the perfusate. Mean observations made in 15 Purkinje fibers (PF, closed circles) and 12 ventricular muscle fibers (VM, open circles) are shown; the vertical bars represent the standard error of the mean. All preparations were driven at a cycle length of 800 msec (75/min). Action potential duration (A) and effective refractory period (B) are plotted on the ordinate as a function of the negative common logarithm of the molar bretylium concentration (that is, 6 represents 1 x lo-” M). Control observations (designated C on the abscissa) are plotted to the left of the vertical hatched bar. The apogee of increase in both action potential duration and effective refractory period as a function of bretylium concentration was found at a concentration of 5 x 10.” M.
86
on Duration
of Effective
The effective refractory period I’urkinje fiber: \~a:: measured in 15 Purkinje fibers; a typical esperiment result is shown in Figure G. Vent ricular muscle was driven at a constant rate aiid premature actiolt potentials elicited iii ventricular muscle (VM:!) every tenth drive cvcle. The interval I,et\veen the last of a series of diiven responses and the earliest premature response in ventricular muscle which propagated to the adjacent Purkinje filler (shortest interval between T’M, and ‘I’M,) was measured. Under control toll(litions, the effective refractory period ill Purkinje fibers was 228 1 15 msec (mean 1: SEM). No sigllificullt change cmunwl at concentrations < 1 Y 10 Ii M, Ijut at ;I I,retglium tosylnte co*iceiitYation of1 IO r, M the Purkinje fiber effective refractor\- period was prolonged to 2.59 ri+ 19 msec, a significant change from the mean control value (P Y.’ 0.005) . When the concentration was in-
Figure 6. Effect of bretylium tosylate on the effective refractory period (ERP) of canine ventricular muscle (VM) and Purkinje fibers (PF). The preparation from which these recordings were made was arranged as in Figure 1. Purkinje fibers: The upper row of panels shows the earliest propagated PF response that could be elicited by a premature VM action potential (the PF ERP). Under control conditions (top left panel), the PF ERP was 214 msec; after a bretylium concentration of 1 Y lo-’ M. PF ERP was prolonged to 259 msec. Exposure to a concentration of 1 x lo-’ M (40 pg/ml) resulted in an increase in PF ERP to 274 msec and prolonged the propagation of impulses from ventricular muscle to the The 3 panels in the lower Purkinje fiber. Ventricular muscle: row depict the shortest Interval at which a premature electrical stimulus directly applied to VM (site 1 of Figure 1) evoked a propagated response in VM (VM ERP). At these short intervals, the impulse that propagates through the muscle does not cross the Purkinje fiber-ventricular muscle junction to activate the Purkinje fiber.
The
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BRETYLIUM
creased to 5 x IO-” M, the Purkinje fiber effective refractory period increased to 272 et 20 msec, which again was significantly longer than the control value (P < 0.001). Increasing the drug concentration to 1 X 10W4 M did not significantly alter the Purkinje fiber effective refractory period from the values found at 5 X 1O-S M. Ventricular muscle : The effective refractory period of ventricular muscle was determined in 12 preparations. In the case of ventricular muscle, the earliest propagating response in the muscle which could be elicited by an electrical stimulus at 4 to 5 times threshold was defined as the ventricular muscle effective refractory period (the shortest obtainable VM,-VM, interval) ; recordings from a typical experiment are shown in Figure 6. The control ventricular muscle effective refractory period was 179 * 8 msec (mean + SEM) ; again, concentrations 5 1 X lo-‘; M had no significant effect (Fig. 5). At a bretylium concentration of 1 X lo-” M a 5 I 2 percent prolongation in ventricular muscle effective refractory period occurred (P < 0.025) and at 5 x 10WS and 1 X lo-” M a 19 i 4 percent change was produced (P < 0.005) (Fig. 5B). Effect of Bretylium on Relation Between Action Potential Duration and Effective Refractory Period Most antiarrhythmic drugs cause the effective refractory period to lengthen in relation to action potential duration. The relation between these variables as a function of the bretylium concentration in the perfusate was examined in 15 Purkinje and 12 ventricular muscle fibers; the mean changes, in milliseconds, are plotted in Figure 7. Ventricular muscle : The mean change in action potential duration and effective refractory period in ventricular muscle was roughly equal at all concentrations of bretylium studied (1 X lo--’ to 1 x 10~~ M). Even at the higher concentrations there was no tendency for the points representing the relation between effective refractory period and action potential duration to fall significantly below a line of identity between these variables (Fig. 7). Purkinje fiber: In concentrations of 1 X 10W7 to 1 X lo-; M, bretylium did not alter the relation between effective refractory period and action potential duration in Purkinje fibers. At a concentration of 5 x 10-m-,the mean change in action potential duration was somewhat greater than that in effective refractory period so that the points representing these variables tend to fall above a line of identity. Certainly, below toxic concentrations experimental points show no tendency to fall below a line of identity for effective refractory period and action potential duration during ex-
VOLUME
27,
JANUARY
1971
TOSYLATE
posure to bretylium, a finding which is significantly different from previously studied antiarrhythmic drugs.22v23*25 Effect of Bretylium on Velocity of Phase Zero Depolarization and Membrane Responsiveness in Purkinje Fibers The maximal rate of phase zero depolarization was measured in 12 Purkinje fibers during exposure to a range of bretylium tosylate concentrations from 1 x 1O-7 to 1 x 1O-4 M. Under control conditions the 12 fibers studied had a
ti+60 m I a
B
t 30 A ERP +90-
PF
+60 MSEC 0
n
a
a f30
~
-/
A ERP
I x10-5
0
5x10-5
m
I x10-4
cl
MSEC.
Figure 7. Effect of bretylium tosylate on the relation between action potential duration (APD) and effective refractory period (ERP) in ventricular muscle (VM, 12 experiments) and Purkinje fiber (PF, 15 experiments). In both A and B, mean change in APD (A APD) in milliseconds is plottec! on the ordinate and mean change in ERP (I ERP) in msec on the abscissa. The symbols representing drug concentrations are shown at lower right. If no significant change in either APD or ERP occurs, the symbols cluster at or near the origin. The dark diagonal line beginning at the origin is the line of identity; if 1 APD and A ERP were equal at all drug concentrations, all symbols would fall on or near this line. A, ventricular muscle: At each drug concentration (from 1 x 10” M to 1 x lo-( M), the points fall near the line of identity. Thkse observations suggest that bretylium does not substantially alter the relation between APD and ERP in ventricular muscle. B,Purkinje fiber: At low concentrations (1 x 10-O M, closed circle), bretylium did not alter the relation between APD and ERP. At higher concentrations, the change in APD has become long relative to the change in ERP. See text for discussion.
87
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resting potential of -91 i 2 mv (mean t SEM) and a peak Qmax of 538 +- 12 volts/‘sec (mean t SEM). No change in either variable was seen at concentrations between 1 >: 1O-7 to 1 X lo-’ M. At a concentration of, 5 x lo-‘, M, bretylium did not change resting potential but did cause a minimal decrease in peak vmax to 495 2 25 volts see, a decrease of 8 percent from the control value (P < 0.1). At this concentration, there was considerable variation between individual experiments, some fibers showing negligible change, others changes as great as 15 percent from control values. During exposure to a bretylium concentration of 1 x lo- ’ M, most fibers showed a decrease in resting potential, and all showed a decrease in peak vmax; the mean resting potential at this concentration was 88 -i- 3 mv and peak ?max 382 5 14 volts set, a decrease of 29 percent from the control value (P < 0.001). The decrease in peak qmax (and overshoot) in each fiber seemed greater than could be accounted for by the change in resting potential. To examine further the effects of bretylium on qmax in Purkinje fibers, the i;max was determined over a wide range of transmembrane voltage (membrane responsiveness) by stimulating 10 fibers at selected voltage levels during the time course of action potential repolarization. vmax was then plotted as a function of transmembrane voltage (Fig. 8, membrane responsiveness). In each fiber studied, the observations made under control conditions fit well on the S-shaped curve computed using the Weidmann formula” (Fig. 8). In concentrations from 1 x 10~’ to 1 X lo-” M bretylium had no effect on this relation. Thus, at these concentrations, the transmembrane voltage at which vmax is 50 percent of the peak value (Vh) was unchanged from control values (71 5 1.6 mv) , and vmax at any given transmembrane voltage was the same as under control conditions. At 5 x lo-;’ M, bretylium shifted the relation to the right on the voltage axis by 1.6 I 0.5 mv, and peak vimax was reduced by 41 i 10 volts’sec (Fig. 8) ; these changes are of questionable significance. At a concentration of 1 )< lo- I M, the changes produced by bretylium were striking (Fig. 8). The relation between membrane voltage and vmax was shifted rightward on the voltage axis; VI, increased (became more negative) by 8.4 -t 1.3 mv. Peak vimax decreased by 159 ?X 32 volts/set. It can be concluded that the pronounced decrease in peak vimax seen during exposure to a bretylium concentration of 1 x 10 ’ M is not primarily due to the small decrease in resting potential but rather due to the marked decrease in membrane responsiveness ; the Vmax at any given level of
600,
450 tilox V/SW
300.
J .‘
150.
.
*
.
L.
l
Control
.A
~xIO-~M
L
A M
IxIO-~
.
A
. 0
60
70
90
80
MEMBRANE
VOLTAGE
100
mv
Figure 8. A typical example of the effect of bretylium tosylate on the membrane responsiveness of a canine Purkinje fiber. Transmembrane voltage at the time of activation (in millivolts) is plotted on the abscissa; the corresponding phase zero \jmax (in volts per second) is plotted on the ordinate. Each control experimental point (filled circles) repiesents the mean of at least 3 determinations. Control peak Vmax was 560 volts/set and transmembrane voltage at 50 percent peak \jmax (V,, was 72 mv. Using these 2 measured variables (peak \imax and V,,), the S-shaped curve was computed by the Weidmann modification of the HodgkinHuxley formula.” Thirty minutes after a bretylium concentration of 1 v lo-” M measurements were identical to the control values (the points obtained at 1 x lo-” M are not shown). After a concentration of 5 x 10 i M (open triangles), v,, shifted 1 to 2 mv rightward on the voltage axis and peak Vmax decreased by 50 volts/set (at 90 mv transmembrane voltage). Thirty minutes after a bretylium concentration of 1 y: 10-l M (closed triangles), V,, shifted 9 mv rightward on the voltage axis and peak Vmax decreased by 240 volts/set; this change represents a pronounced decrease in responsiveness.
transmembrane voltage is markedly reduced from the value obtained at the identical level under control conditions. The decrease in membrane responsiveness was not seen until high concentrations of bretylium were used, i.e., those which also reduced the transmembrane resting potential (presumably a toxic effect). There was also no tendency for bretylium to produce a rightward shift of the steep portion of the S-shaped curve on the voltage axis before the decrease in peak Vmax, in contrast to quinidine and procainamide. Effect of Bretylium on Conduction in Purkinje Fibers The velocity with which an action potential propagated down a linear strand of Purkinje fiber was measured in 5 preparations which were arranged in the manner shown in Figure 2 and stimulated at a constant rate of 75 ‘min. The distance between microelectrodes A and B was 0.8 -i 0.24 cm. The average conduction velocity
The
American
Journal
of
CARDIOLOGY
BRETYLIUM
EFFECT
OF BAETYLIUV
AND QUINIDINE
BRETYLIUM
CONTROL
ON AUTOMATIC
IxIO-~
PURKINJE
QUINIDINE
FIBER
1~10~~ 5 set
Figure 9. The effect of bretylium tosylate and quinidine sulfate on spontaneous firing of Purkinje fibers. In both A and B recordings were made from undriven preparations with a microelectrode located in site 3 of Figure 1. A, effect on a fiber which was firing spontaneously under control conditions. Each frame was recorded from the same Purkinje fiber site 30 minutes after exposure to the drug concentration listed below the frame. The control (upper row, left frame) spontaneous firing rate was 44/min. Note the typical diastolic (phase 4) depolarization. The sharp junction between action potential phase 4 and phase zero suggests that the pacemaker site is distant and that the impaled cell is depolarized by the propagating impulse. Exposure to a bretylium concentration of 5 x 10.” M produced an increased rate of firing during the initial 10 minute period of firing (not shown), but, after 30 minutes, the rate decreased to 3l/min (upper row, center frame). At a concentration of 1 x 10-l M (upper row, right frame), the firing rate increased to 40/min, the action potential prolonged in duration, maximal diastolic transmembrane voltage decreased (shifted toward zero), and terminal phase 4 depolarization became more concave upward. This change in the configuration of phase 4 suggests that (1) the pacemaker moved closer to the recording site or (2) more of the Purkinje fiber surface membrane participated in pacemaker activity. After exposure to quinidine, 1 x lo-” M, action potential configuration altered noticeably, but the firing rate remained unaltered at 45/min (lower row, left frame). Exposure to quinidine, 1 x lo-‘M, (lower row, right frame) caused a decrease in maximal diastolic voltage and action potential amplitude, decreased the slope of phase 4 depolarization and reduced the spontaneous firing rate to 25/min. B, effect on a fiber that was quiescent (not firing spontaneously) under control conditions. The fiber showed no evidence of spontaneous activity under control conditions (left frame). Spontaneous firing began 4 minutes after bretylium (1 x 10-l M) perfusion was begun. After 35 minutes, the spontaneous firing rate was 57/min (center frame) and remained stable during a subsequent 30 minute period of observation. Bretylium perfusion was then stopped, and spontaneous firing stabilized at 42/min in normal Tyrode solution. When quinidine (1 x lo-” M) was added, slowing began within 5 minutes and spontaneous activity ceased after 12 minutes (right frame).
VOLUME
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JANUARY
1971
TOSYLATE
under control conditions was 3.1 -+ 0.51 meters/ sec. During exposure to a bretylium concentration of 1 x lo-” M, the conduction velocity increased to 3.4 f 0.62 meters;/sec, a change which was not statistically significant (P < 0.1) ; no change in resting potential or peak vmax was seen at concentration in these experiments. When these preparations were exposed to a bretylium concentration of 1 x 10-I M, the conduction velocity fell to 2.4 +- 0.89 meters ‘see, a 22 percent decrease (P < 0.05) ; resting potential decreased by 3.0 + 0.7 mv and peak vimax by 48 + 19 volts/see in these experiments. Effect of Bretylium on Purkinje Fiber Automaticity Automaticity is a property of Purkinje fibers which is responsible for in vivo escape rhythms and which may also be involved in the genesis of cardiac arrhythmias. All previously studied antiarrhythmic drugs have shown a significant ability to suppress automaticity in Purkinje fibers. The effects of bretylium tosylate on Purkinje fiber automaticity were studied in 14 preparations. Seven preparations showed stable spontaneous firing during a 1 to 2 hour period of control observation. The average control spontaneous firing rate was 39 i: 2.0 clepolarizat.ions ‘min. Preparations were then perfused with Tyrode solution containing bretylium. The initial concentrations used were 5 X 10msM in 4 experiments ; preparations that were initially exposed to a concent.ration of 5 x 10~~ M were all subsequently exposed to a concentration of 1 >( 10-l M. Two preparations were finally perfused with solution containing a bretylium concentration of 5 >( lOed M. All fibers studied showed an initial increase in rate when exposed to bretylium. This increase in rate peaked 10 to 15 minutes after adding bretylium, at which time the average rate was 47 -+ 2.4 depolarizations ‘min, an increase of 20 percent (P < 0.05). The rat.e then declined to 36 -i- 6.7 minutes and stabilized by 30 to 40 minutes after bretylium was begun; at this time, the spontaneous firing rate was not significantly different from the rate found under control conditions (Fig. 9A). This was even true in the 2 preparations exposed to a bretylium concentration of 5 S 10-j M. Bretylium, in all the concentrations employed in this series of experiments, did prolong the action potential duration in these unstimulated preparations and at concentrations of > 1 X lo--’ M caused a decrease in the maximal cliastolic potential. In 3 preparations, bretylium changed the voltage-time course of the terminal portion of phase 4 so that the transmembrane voltage-time course became more concave upward (Fig. 9A). This change suggests that (1) the site of the pacemaker in the preparation shifted
89
BIGGER AND JAFFE
closer to the microelectrode recording site or (2) more of the Purkinje fiber surface membrane area became automatic. Bretylium compared with quinidine: Kretylium’s inability to suppress automaticity in Purkinje fibers was unexpected. We, therefore, compared the response of 5 of the preparations to quinidine sulfate (Fig. 9A). After the response to bretylitim had been observed, these 5 preparations were perfused for an hour with drug-free Tyrode solution. Then Tyrode solution containing a quinidine concentration of either 5 c: 10 Z or 1 v 10 ’ M was used to perfuse the preuasation. The average spontaneous firing rate of the 5 preparations at the end of the drug-free perfusion period was 41 +- 2.2 depolarizations./min, a value not significantly different from the original control observations. After quinidine, the mean firing rate fell to 14 f~ 4/min, a 66 percent decrease (P < 0.01) ; in 1 preparation, spontaneous firing ceased altogether. It is thus evident that in comparable concentrations quinidine had a more powerful antiautomatic effect than bretylium and that the lack of significant antiautomatic eff’ect was not due to unusual resistance to suppressive drugs in the Purkinje fibers studied. Effects of pretreatment with reserpine: A known early effect of bretylium is release of norepinephrine from the adrenergic nerve terminals. It seems reasonable to propose that the initial increase in rate seen during perfusion with bretylium in our experiments was due to release of catecholamines, To provide further information on this point, experiments were performed on obtained from dogs previously preparations treated with reserpine. The protocol directed that 5 dogs be given 0.5 mgfkg of reserpine on 3 consecutive days and sacrificed on the fourth. Only 2 dogs survived to the time of sacrifice. Four preparations were obtained from these 2 animals, but only 2 showed spontaneous firing; these had rates of 14 and 20 depolarizations ‘min. Each preparation was first perfused with Tyrode solution containing a bretylium concentration of 5 Y 10Ws M and then with a solution containing a concentration of 1 x lO- I M. The significant increase in rate seen in unreserpinized preparations was absent in the 2 preparations pretreated with reserpine. After a 30 minute exposure to a bretylium concentration of 1 >c; IO--' M, the rate of 1 preparation was unaltered, and spontaneous activity ceased in the other. In addition, bretylium, in concentrations of 5 .Y 1O-S and 1 x 10-q M, did not initiate firing in the 2 quiescent reserpinized preparations ; application of norepinephrine did.
Discussion Electrophysiologic properties of antiarrhythmic agents : The electrophysiologic properties of quinidine,22,2U procainamide,“” diphenylhydan-
90
toin,Z’: pr0pt~10101” attd litlocaincYJ all have lien studied in canine Purkinje fiber and ventricular muscle; each of these agents possesses ;I significant antiarrhythmic effect against ventricular arrhythmias in man. Many difl’ercnces harTe 1~11 identified amon% these antiarrhythmic agents. Quinidine, procainamide ant1 p~op~-uJJ0101 decreiise diastolic escit,:Lbility, whereas comparable concentraiions of’ diphenylhgdantoin and lidocnine have minimal effects.“” Therapeutic ~on~cnli.~~iions of quiniditlc, procainamide and prop~*anolol also decrease peak <7mW nX3?-tbJ~arK’ I-espoltSjveMW :lJld coJldll~~ioJ1 velocity in Purkinje fibers; comparable conccntrations of diphenylhydantoin and lidocaine either do not alter these properties, or under some circumstances may increase each of these variablesL.X1.“-D.:NI Quinidine and procainamide cause small but definite increases in action potential duration and effective refractory period in ventricular muscle and Purkinje fibers”‘; diphenylpropranolol” and lidocaine”” cause hydantoin,‘:’ a decrease in both action potential duration and effective refractory period in each cell t.ype. There are, of course, other electrophysiologic differences among these agents. Quinicline’” and procainamidti” prolong atria1 action potential tluration. These agents and propranolol:‘” decrease peak vmax and slow conduction in rabbit atria1 fibers ; diphenylhydantoin”” and lidocaine:‘:: do not significantly affect atria1 action potential duration, either increase or do not change peak Qmax and do not slow conduction in atrium. Quinidine, procainamide and propranolol prolong atrioventricular refractory periods and conduction time in a variety of animals including man ; diphenylhydantoin and lidocaine do not prolong these variables and often even shorten the atrioventricular refractory period and conduction time, particularly when digitalis has been previously administered.:‘-’ Quinidine and procainamide produce unreliable results when used to combat arrhythmias caused by digitalis excess, while propranolol, diphenylhydantoin and lidocaine are highly effective.:‘f Two properties were uniformly concordant in studies of these 5 drugs in ventricuIar muscle-Purkinje fiber preparations. First, all 5 agents had a significant ability to depress automaticity in Purkinje fibers. Second, all 5 caused t.he effective refractory period of Purkinje fibers (and when studied, ventricular muscle fibers, as well) to become long relative to action potential duration; this was true although some of the drugs (quinidine and procainamide) prolong both action potential duration and effective refractory period and others (propranolol, diphenylhydantoin and lidocaine) shorten both. Because these two electrophysiologic changes were produced by all 5 antiarrhythmic drugs in concentrations comparable to in viva therapeutic concentration, they have
The American
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BRETYLIUM
been considered of great importance. Indeed, it has been proposed that changes in these 2 properties can largely explain the antiarrhythmic properties of these agents.29 Comparison of antiarrhythmic effects of bretylium and other agents: Bretylium stands out in bold relief against the background of the 5 previously studied agents. Although bretylium resembled quinidine and procainamide in prolonging action potential duration and effective refractory period, bretylium primarily affects phase 2 of action potential duration, whereas quinidine and procainamide primarily lengthen phase 3. Furthermore, action potential lengthening caused by bretylium was not accompanied by significant change in Purkinje fiber peak iimax, membrane responsiveness or conduction velocity in concentrations thought to be comparable to therapeutic doses in man. The sequence of alterations in membrane responsiveness during perfusion with high (toxic) concentrations of bretylium was also different from that seen with quinidine or procainamide. With bretylium, peak iimax decreased concomitantly with a rightward shift of the S-shaped responsiveness relation on its voltage axis whereas quinidine and procainamide shifted the S-shaped curve rightward on the voltage axis and increased V,, (that is, made V,, more negative) before peak Vmax was altered. Most disturbing was the failure of bretylium to exert a significant effect on either of the 2 electrophysiologic properties that are so strikingly modified by previously studied antiarrhythmic drugsautomaticity and the relation between effective refractory period and action potential duration. First, bretylium failed to show powerful effects, if any, in suppressing automaticity in Purkinje fibers. In fact, initially this drug caused a significant increase in the spontaneous firing rate of in vitro Purkinje fibers (Fig. 9). Second, bretylium did not significantly lengthen the effective refractory period relative to action potential duration in either Purkinje or ventricular muscle fibers. High (toxic) concentrations of bretylium did not alter the relation or actually made action potential duration slightly long relative to effective refractory period. seen in this study Only 2 actions of bretylium might. reasonably be considered to have antiarrhythmic effects by acting directly on ventricular myocardial cells. First, bretylium prolongs the action potential ancl lengthens refractory period without slowing conduction. This would alter the wave length in a reentrant pathway and might, thus. abolish reentrant arrhythmias. Second, in partially depolarized fibers, bretylium caused a prompt, if mild, increase in resting transmembrane voltage and, thus, in phase zero amplitude and jimas. This effect may be due to the early
VOLUME
27,
JANUARY
1971
TOSYLATE
catecholamine releasing action of bretylium, since catecholamines hyperpolarize partially depolarized Purkinje fibers and thereby increase phase zero amplitude and vmax.:s8 This effect could enhance conduction and abolish reentrant rhythms.s0 Although it remains to be demonstrated conclusively that bretylium is an effective antiarrhythmic drug in man, an increasing number of observations in intact animals and man lend credence to the hypothesis that its action is antiarrhythmic, particularly in cases of ventricular arrhythmia.34~1”~“” The findings of our study suggest that the effects of brktylium on the electrophysiologic properties of the cell membrane of Purkinje and ventricular muscle fibers do not entirely explain its antiarrhythmic effects in terms of present concepts of antiarrhythmic drug action. Indeed, the predominant mechanism through which bretylium acts as a cardiac antiarrhythmic agent may not involve, to a major degree, its effects on cell membranes of cardiac muscle. Adrenergic neural blocking action of bretylium : The role of the autonomic nerves and nerve terminals in the genesis of cardiac arrhythmias has been stressed recently in the case of arrhythmias occurring after cardiac coo1ing,3’i hypoxiaXG and excessive doses of digitalis.:‘i It has been suggested that drugs that depress neural function exert antiarrhythmic effects, whereas those that do not depress neural function lack antiarrhythmic activity.:” This hypothesis receives its strongest support in the instance of beta adrenergic agents used to treat digitalis-induced arrhythmias : at essentially equipotent blocking doses, the agents that are neural depressants are antiarrhythmic; those without this effect are not.“’ Han et aPR have shown that stimulation of cardiac sympathetic nerves, as well as hypothermia, myocardial ischemia and ouabain intoxication, increase the temporal dispersion of the refractory period in the canine ventricle and suggested that this would promote reentrant rhythms and fibrillation. Yanowitz et al. showed that unilateral increases or decreases in cardiac sympathetic nerve activity can lead to marked alterations in the duration of electrical systole and markedly change the sequence of repolarization in the canine ventricle.:“’ Roberts et al.:“’ have suggested that in so far as each cardiac axon or nerve terminal has the capacity for independent activity, the cardiac neural system may become discoordinated. The discordant cardiac neural activity could fractionate the conductivity, automat,icity and excitability of the ventricles and lead to arrhythmias.ZG It is reasonable to suppose that when such discordant cardiac neural activity plays a role in the genesis of cardiac arrhythmias, bretylium might have a beneficial effect through its adrenergic neural blocking action.
91
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