Effects of lidocaine on the electrophysiological properties of subendocardial Purkinje fibers surviving acute myocardial infarction

Journal of Molecular aud Cellular Cardiology (1979) 11, 669-681

Effects

of Lidocaine Subendocardial

on the Electrophysiological Purkinje Fibers Surviving Myocardial Infarction

C. M. WANG,

C. A. JAMES

Properties Acute

of

AND R. A. MAXWELL

Department of Pharmacology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, U.S.A. (Received 31 October 1978, accegted 30 JVovember 1978) C. M. WANG, C. A. JAMES AND R. A. ~~AXWELL. Effects of Lidocaine on the Electrophysiological Properties of Subendocardial Purkinje Fibers Surviving Acute Myocardial Infarction. Journal of Molecular and Cellular Cardiology (1979) 11, 669-681. An electrophysiologic study was conducted in titro on the surviving subendocardial Purkinje fibers of the canine left ventricle 20 to 24 h after coronary occlusion. The surviving Purkinje cells displayed a low maximum diastolic potential, reduced action potential amplitude, depressed maximal rate of upstroke and a prolonged action potential duration. Spontaneous discharges of high frequency and induced repetitive activity are consistently observed in the infarcted zone but are rarely seen in the normal tissue. Superfbsion of lidocaine hydrochloride in concentrations of 1 x 10-s to 1 x 10-4~ slowed or abolished spontaneous and induced firings. The effective refractory period and effective refractory period relative to the total action potential duration were consistently prolonged by lidocaine in the ischemic fibers. Membrane response curves of ischemic fibers were shifted farther to the right than those of normal fibers. Lidocaine exhibited a differential effect on maximul rate of upstroke (dV/dt) of ischemic Purkinje fibers while causing minor change in the normal fibers. It is suggested that lidocaine exerts its ant&rhythmic effect by selectively depressing the already depressed conducting fibers in the infarcted heart. KJXY WORDS. Coronary ligation; Myocardial infarction; Ventricular arrhythmias; Subendocardial Purkinje fibers; Spontaneous rhythm; Transmembrane potentials; Differential depression.

1. Introduction The question was raised [1.2, 161 regarding the applicability of information obtained from studies on normal cardiac tissue to diseased or abnormal hearts. It was later suggested that the response of diseased myocardial fibers to actions of pharmacological agents may differ both quantitatively and qualitatively from the response of normal fibers [25]. Such a suggestion was well founded based on the fact that (1) cellular and electrolyte alteration are known to occur after experimental myocardial ischemias [14, 20,2q and (2) abnormalities in electrophysiological properties were evidenced in ischemic myocardial fibers and those surviving extensive infarction [S, 9, 17, 181. Since antiarrhythmic drugs exerted selective depressive effects on hypoxic and ischemic cardiac cells [13, 191, and on the heart in situ 0022-2828/79/070669

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following acute myocardial infarction [15], it seemed imperative that a genuine understanding of the mechanism of action of antiarrhythmics be achieved by studying their effects on both normal and diseased cardiac tissue. Based on this rationale, the electrophysiological actions of lidocaine were investigated in normal and in the surviving subendocardial Purkinje fibers 20 to 24 h after acute myocardial infarction in dogs. 2. Materials

and Methods

Thirty-three adult male mongrel dogs weighing between 10 to 15 kg were used in this study. Dogs were first anesthetized with pentobarbital sodium 30 mg/kg, i.v. for control study and surgery. The occlusion of the anterior descending branch of the left coronary artery was performed in 21 dogs by the two-stage method of Harris [IO]. Twenty to 24 h post-occlusion, the animals were re-anesthetized with pentobarbital sodium 20 mg/kg and the right thorax was re-opened to remove the heart for in vitro study. Normal, non-infarcted left ventricle from dogs without prior surgery and the tissues surrounding the infarcted region were also studied for comparison. The method of dissection and excision of infarcted myocardium and adjacent tissue was essentially the same as described previously by Friedman et al. [8]. The procedure yielded a block of 2 in x 2 in ventricular tissue which was then mounted in the 50 ml tissue bath. Both normal and infarcted tissues were superfused at a rate of 8 ml/min with Tyrode’s solution oxygenated with 95% 0s + 5% COs. The Tyrode’s solution contained (mM/l) Na+, 149.4; K+, 4; Casf, 2.7; Cl-, 147.4; Mgs+, 0.5; HCOs-, 12; HsPOh-, 0.4; dextrose, 5.5. The bath temperature was maintained constant at 37 f 0.5%. Both normal (N) and infarcted ventricular tissues were equilibrated with bathing solution for 60 min before testing. Figure 1 schematically illustrates the relative position of stimulating and recording electrodes (glass) on the pale-colored infarcted zone (stippled) and the adjacent red-colored control region. Microelectrode A was positioned at the center of free running false tendon. A single recording electrode was used to probe one of the three sites a, b, and c within the infarcted zone. Occasionally, a third electrode B was introduced to the mid-section of anterior papillary muscle. Bipolar platinum electrodes were placed on the proximal region of the free-running false tendon. The stimulation pulses had a duration of 1 to 3 ms and strength of 1.5 times threshold voltage. Because of high frequency of spontaneous firing in the infarcted zone, a stimulation frequency of 2 Hz was maintained throughout the experiment. To determine the effective refractory period (ERP) of the subendocardial Purkinje fibers, fine bipolar stimulating electrodes were placed on the fibers imbedded in the endocardium. A premature pulse with twice threshold voltage was delivered from Grass S88 stimulator every eighth basic drive. A conventional microelectrode technique was used to record transmembrane action potentials of normal fibers and those from infarcted zone. All microelectrode implements were

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FIGURE 1. Schematic illustration of relative position of stimulating and recording electrodes on the infarcted (stipple-d) and the undamaged control regions of left ventricle. A, the electrode is positioned at the midsection of free-running false tendon. B, electrode at the subendocardial Purkinje branch in the papillary muscle. Sites a, b, c are within the infarcted area.

maintained throughout the experiment. The output of the DC amplifier was led into the R-C circuit of an operational amplifier to electronically differentiate the rate of upstroke of an action potential (dV/dt). Electrical signals were displayed on the Tektronix 5 100 oscilloscope and the tracings on the screen were photographed with Nihon Koden recording camera. Lidocaine hydrochloride (Astra) was directly dissolved into Tyrode’s solution and later diluted to desired concentrations for superfiision. Tissues were exposed to lidocaine in successive applications from low to high concentrations. Comparison of the difference between actual mean data from pre-treatment and post-treatment was made by using the grouped Student ‘Y-test. 3. Results

Purkinje fibers in the endocardial surface of infarcted zone were found to be electrically active 24 h after coronary occlusion. Electrophysiological measurement of these surviving fibers revealed certain changes which were quantitatively and qualitatively different from fibers of the normal non-infarcted heart. (a) Spontaneousrhythm The frequency of spontaneous firings in the fibers from infarcted zone was greatly enhanced in all 15 successful experiments. The highest frequency was recorded

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immediately after the excision in which the firing pattern became rapid and oscillatory at the rate of 250 to 300 beats/min. The firing rate gradually decreased and stabilized at 70 to 80 beats/min after mounting in the tissue bath for 45 to 60 min. With repeated impalements in each preparation, only spontaneous rapid beats were recorded but not spontaneous diastolic depolarization in the infarcted region. In all eight preparations, lidocaine suppressed the spontaneous firings in a dosedependent manner with a detectable effect at 1 x 10-s M. Figure 2 shows that lidocaine at 5 x 10-s M markedly slowed the frequency of firing and depressed the potentials from the infarcted area. Lidocaine at 1 x lo-4 M either further reduced the frequency or completely abolished such activity depending upon the severity of depression of individual fiber caused by infarction. The action of lidocaine was partially reversible after washing. CONTROL

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FIGURE 2. Effect of lidocaine (5 x 10%~) on the spontaneous rapid discharges of subendocardial Purkinje fibers. Top trace, recorded at control non-infarcted region (C) of papillary muscle. Middle trace, recorded at infarcted zone (I). Bottom trace, dV/dt of fiber within infarcted zone. CONTROL = untreated.

(b) Induced repetitive rhythm The Purkinje fibers from the infarcted zone generated unstimulated impulses and rapid repetitive activity after a single premature stimulation delivered at the freerunning false tendon in the control zone (four out of ten preparations). As shown in Figure 3 [(A) and (B)] w h en a single premature stimulus (arrows) was coupled at 240 ms from the previous regular pacing (triangles), one or two unstimulated depolarizations were recorded from Purkinje fibers at the control zone. In five preparations, however, a rapid repetitive activity was induced by a single premature stimulation [Figure 3(C)]. These unstimulated depolarizations and sustained rapid repetitive activity were considered to be the re-entrant impulses propagating from the infarcted to non-infarcted zone [9]. Such activities could result in a predisposition to ventricular tachyarrhythmias. Figure 4 depicts the effect of lidocaine on rapid repetitive activity induced by a single premature stimulation. The induced repetitive beats were gradually slowed as concentrations of lidocaine

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FIGURE 3. The induction of re-entrant heats and repetitive firings in the suhendccardial Purkinje network 20 to 24 h after the acute myocardial infarction. Top trace, recorded from Purkinje cell of free-runnin g false tendon. Middle trace, recorded at infarcted zone. Bottom trace, dV/dt of fiber from infarcted area. (A), (IS), (C) are recorded from three different sites from the same preparation roughly corresponding to positions a, b, c in Figure 1. Triangle denote a regular pacing at cycle length 500 ma. Arrow represents premature stimulation.

increased until a total depression at 1 x IO-4 M. It was also found that any concentration diminishing sustained repetitive activity was also effective in suppressing the ectopic beat. The activity reappeared after washing with Tyrode’s solution. Although induced repetitive activity was not seen in five normal ventricular preparations under similar experimental conditions, spontaneous beats at the rate of 15 to 25/min were observed. These spontaneous rhythms from normal tissues were also slowed by lidocaine (not shown). (c) Tram-membrane action potential (AP)

The transmembrane electrical properties fibers are generally characterized by a potential, a prolonged action potential upstroke (dV/dt). F’g 1 ures 5 and 6 show from

these moderately

depressed

fibers

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maximum

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duration (APD) and a reduced rate of the action potentials and dV/dt recorded (I) and AP from

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FIGURE 4. Effect of lidocaine on the induced repetitive discharges of Purkinje network after acute myocardial infarction. Top trace, recorded from Purkinje cell of free-running false tendon. Middle trace, recorded at the infarcted zone. Bottom trace, dV/dt of Purkinje cell from infarcted area. Arrow represents premature stimulation which was coupled at 260 ms into the regular pacing (triangle). Tissue was exposed to each dose for about 45 min. CONTROL = untreated.

action potential and dV/dt and slightly shortened APD in fibers from infarcted zone while APD from false tendon was markedly abridged by the same concentration of lidocaine. The corresponding subendocardial Purkinje fibers from normal non-infarcted ventricle were subjected to identical treatment for comparison. The result showed little change in APD and dV/dt except lidocaine at the high concentration of 1 x 1O-4 M (Figure 7). An analysis of AP characteristics from normal fibers of non-infarcted heart and moderately depressed fibers from infarcted heart is shown in Table 1. The Purkinje cells from infarcted heart have an average 5 mV less in maximum diastolic potential, 100 ms longer in total APD and 250 V/s smaller in dV/dt compared to those of normal hearts. Lidocaine slightly depolarized the maximum diastolic potential and exerted at least 2 times greater depression on dV/dt in infarcted tissues than in the normal counterparts. Although total duration of action potential (APDIoo) was not altered by lidocaine in either fiber, total refractoriness of Purkinje fibers was significantly prolonged in both tissues as seen from AERP/APDrm after the

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FIGURE 5. Effect of lidocaine on the transmembrane potentials of the Purkinje cell from freerunning false tendon (C) and the subendocardial Purkinje fiber (I) of infarcted zone. Bottom trace, d Y/dt of infarcted tissue. Tissue was exposed to each dose for 40 to 45 min at which time effect had stabilized. Cycle length = 500 ms. CONTROL = untreated.

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FIGURE 6. Blocking action of lidocaine on a moderately infarcted subendocardial fiber (I). Top trace, recorded from free-running false tendon in control zone. Bottom trace, dV/dt for infarcted tissue. A total blockade occurred in 37 mm after drug administration. Cycle length = 500 ms. CONTROL = untreated.

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FIGURE 7. Effect of lidocaine on the transmembrane potentials of a normal subendocardial Pnrkinje fiber from non-infarcted heart. The site of recording corresponded to that of infarcted heart. Tissue was exposed to each dose for 40 to 45 min. Cycle length = 500 ms. CONTROL = untreated. TABLE 1. Effectoflidocaine on the action potential characteristics of normal subendocardial Purkinje fibers and those surviving acute myocardial infarction (mean f s.E.) Maximum Action diastolic potential potential amplitude APDss ERP ERP/ d V/dt ADPloo APDloo (V/s) (-mv) (mv) b-4 (4 (4 .Normal PF (n = 9) Control 89 & 1 119 5 2 142 f 8 259 4 10 213 f 9 0.819 566 f 22 1 x 10-s, 89 f 2 122 f 1 123 h 5 244 * 6 199 f 6 0.816 579 f 41 % mange 0 $2 -14 -6 -6 0 3 X 10-5M 87 f 2 120 h 1 108 +33t 237 f8 209 +5 0.881.t 474+:52 % Change -2 $1 -24 -9 -2 +7 -16 1 x lo-4M 86 & 2 113 * 2* 98 3 5t 235 5 8 320 f 17t 1.364 427 & 37* +68 -25 o/OChange -4 -6 -31 -10 + 50 Infarcted PF Control 84 f 3 114 + 3 175 * 7 351 & 8 275 rt 12 0.781 311 & 28 (n = 10) 1 X 10-5M 80 + 4 110&3 148f5* 33038 277&11 0.840 259 -J=25 (n = 10) % mange -5 -3 -16 -6 +1 ff-3 -17 3 x 10-s, 82 & 3 111 f 3 138 f 7* 326 f 12 300 & 15 0.9257 190 f 32* o/o Change -2 -3 -21 -7 +9 +18 -39 (n = 10) 1 X 10-4M 76 f 2* 101 + 5* 128 f 13* 338 f 10 411 f 17t 1.222.t 124 f 26t (n = 8) O/cChange -10 -11 -26 -4 +50 +56 -60 * StatisticalIy signilicant at P < 0.05. 7 Statistically signiikant at P < 0.01.

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application of 3 x 10-5 M and 1 x 10-4 M. The most noticeable effect of lidocaine on the transmembrane electrical properties of Purkinje fibers was that d V/dt of the infarcted tissues was more selectively affected than the normal ones in the range of 1 x 10-5 M to 1 x IO-4 M. However, little or no effect on dV/dt was observed at concentrations below 1 x 10-5 M in these moderately depressed fibers.

(d) Membrane rtsponsiveness In response to low concentration (1 x IO-5 M) of lidocaine, the membrane response curve of normal subendocardial Purkinje fiber (Figure 8) was slightly shifted to the left at higher membrane potentials and to the right at lower membrane potentials, the curve was then shifted to the right after perfusing with concentrations higher than 1 x 10-5 M. A curve along with voltage axis was not drawn for the high concentration due to the fact that ERP was greater than APDrm after perfusion with 1 x lo-4 M lidocaine. The fibers from the infarcted zone present a dissimilar curve in membrane responsiveness (Figure 9) to that of normal fibers.

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Membrane potential (mV) FIGURE 8. The membrane response curve of a subendocardial Purkinje fiber from normal, non-infarcted heart. The displacement of curve results from lidocaine application. The site of recording corresponded to that of infarcted heart. (0) Control (untreated); (@) 1 x 10-s M lidocaine; (A) 3 x IO-5 M lidocaine.

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FIGURE 9. A representative membrane response curve of a subendocardial Purkinje fiber from infarcted zone. The displacement of curve to the right of voltage axis is caused by lidocaine superfusion. ( 0) Control (untreated) ; (0) 1 x 10-5 M lidocaine; (A) 3 x IO-5 M lidocaine.

The slope of the untreated control curve was smaller, and under the influence of lidocaine, the curve was consistently shifted further to the right. Such an action on infarcted fibers by lidocaine resembled those of “quinidine-like” antiarrhythmics in affecting the membrane response curve of normal free-running Purkinje fibers. 4. Discussion Lidocaine is one of the most frequently used drugs in cardiac care units to control ventricular arrhythmias resulting from acute myocardial infarction. The target organ of lidocaine therapy is undoubtedly the diseased or abnormal heart rather than the normal healthy one. Therefore it seems more logical to define the mechanism of action of lidocaine with abnormal tissues. Hypoxia is not the sole factor directly causing the aberrant electrical activity in the surviving subendocardial Purkinje fibers [17]. The offending factors may also include reduced intracellular pH, energy deficiency, efflux of [K+], catecholamine release, accumulation of metabolic products, electrolyte imbalance and other deleterious factors. It is still speculative which is the primary cause for the induction of observed electrophysiological changes. A greater spontaneous firing was recorded in the depressed fibers within the

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infarcted zone than the normal Purkinje cells and from the undamaged control zone dissociated from infarcted heart suggesting that rapid activity is originated from infarcted area. The possibility exists that the border between infarcted and surrounding control regions may also initiate impulses without electrical stimulation [II]. Although spontaneous phase 4 depolarizations were reported in ischemic Purkinje fiber after myocardial infarction [9], we failed to record them after multiple impalements in each preparation. Thus it could not be ascertained whether re-entrant impulses or the existing ectopic foci or both contribute to the spontaneous rapid firing recorded from the depressed Purkinje fiber. Lidocaine, in a dose-dependent fashion, depressed the spontaneous firing of infarcted tissue. As in normal Purkinje fiber, the depression of spontaneous depolarization could be a manifestation of the ability of lidocaine to increase potassium conductance [g. A single unstimulated depolarization and the rapid repetitive activity were induced in the infarcted zone but rarely seen in the healthy normal ventricle. These impulses, however, exhibited different characteristics from the rapid spontaneous firing; the former need to be induced and abruptly terminates itself after a few milliseconds. It was suggested that the induced ectopic beat and rapid repetitive discharges observed in infarcted zone are the re-entrant impulses propagating from the infarcted to the neighboring control zone [9]. Since the re-entrant impulses could be facilitated by depressed Purkinje fibers through one or several pathways [.5] and by the participation of myocardial cells in the ischemic heart [19], the abolishment of such induced activities by lidocaine can be attributed to its ability to disrupt re-entrant pathway(s). This action by lidocaine was further substantiated by the study of infarcted heart in situ [7]. In the infarcted region of left ventricle, the severity of infarction differs from one site to the other, which is revealed by the variation of transmembrane potentials recorded in the surviving Purkinje cells [8]. In this study the severely depressed fibers generated low magnitudes of both action potential and maximal rate of rise (150 V/s or less) whereas slightly depressed fibers displayed a near normal action potential (AP) and dI’/dt (400 V/ s or more). Lidocaine at the concentration of 1 x 10-S M would often block membrane excitation in severely depressed fibers while exerting little or no effect on the transmembrane potentials in the slightly depressed fibers. Since it was our attempt to compare the pharmacological responses of both abnormal and normal Purkinje fibers to lidocaine action, and secondly because of the inherent complexity of infarction, we selected only those moderately depressed fibers (dI’/dt ranging from 150 to 400 V/s) for comparative study of transmembrane potentials (Table 1). Despite this, selective effects of lidocaine could still be demonstrated in this study. The two distinct effects of lidocaine on AP characteristics of Purkinje cells from infarcted area are (a) consistent increase in effective refractory period and (b) marked depression of dV/dt [24]. In the normal fibers the effective refractory period (ERP) was slightly decreased by lidocaine, while the ERP and ratio of

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ETAL.

ERP to total duration of AP (ERP/APD UJOratio) are consistently increased by the same concentration in infarcted tissues. This observation is in agreement with the results of in situ study [15] which demonstrated the specific prolongation of ERP by lidocaine in infarcted canine heart. Lidocaine, at therapeutic concentrations, has a slight depressant effect of dV/dt and membrane responsiveness in normal fibers [Z]. However, a selective depression was consistently observed in infarcted tissues in response to lidocaine treatment (1 x 10-s to 1 x 10-4 M). The increased sensitivity to depressant activity of lidocaine in the fibers from infarcted zone could be attributed to two factors. First, Purkinje fibers within infarcted zone are characterized by a low maximum diastolic potential which is indicative of slow rate of upstroke and the deficiency in Na+ carrier system. Since lidocaine was suggested to depress only fast sodium channels [3], which contribute to the fast upstroke of AP, a greater depression of dV/dt by lidocaine should result from the partially depolarized fibers of infarcted tissue [19] and depolarized myocardial cells [fl. Secondly the intracellular pH of mammalian cardiac muscle was estimated to be near 7.0 [S] but that value is frequently reduced after an ischemia and myocardial cellular injury [Pg. With a pX% value of near 7.9, lidoCaine exists as both charged and uncharged molecules in the physiological pH range. If the charged molecule is the active form of lidocaine [22] the reduction of pH in infarcted tissue should invariably result in an increase in the proportion of charged lidocaine molecules and culminate in a greater blocking potency. It was suggested that a drug might abolish re-entrant arrhythmias either by improving a condition in a depressed area or by further depressing conduction to the complete blockade [23]. Present results show that lidocaine exerts an antiarrhythmic effect through the depressant activity in the existing depressed Purkinje fibers as manifested by the reduced dV/dt, and in some instances, a complete block. Lidocaine may eventually render unidirectional block to bidirectional block that disrupts re-entrant pathway(s). The differential effect of hdocaine on both depressed and normal cardiac cells is a desirable feature which is also shared by several antiarrhythmics [13]. These facts strongly suggest that ischemic and diseased

tissues may prove more suitable for the study of the mechanism of action of antiarrhythmic

agents.

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ARNSDORF,

2.

W. J. Effect of lidocaine on the electrophysiological muscle and Purkinje fibers. Journal of Clinical Investigation 49,

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properties of ventricular 63-77 (1970). 3.

of lidocaine hydrochloride on membrane cardiac F’urkinje fibers. Journul of Clinical Investigation 51,

M. F. & BIGGER, J. T., JR Effect

conductance in mammalian 2252-2263 (1972).

BRENNAN, F. J., CRANEFIELD, P. F. & WIT, A. L. Effects of lidocaine on slow response and depressed fast response action potentials of canine cardiac Purkinje fibers. 3ournal of Phurmacologvand Experimental lhapeutics 204, 3 12-324 ( 19781.

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25.

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