Electrophysiological Effects of Procainamide in Acute and Healed Experimental Ischemic Injury of Cat Myocardium

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1 386 Electrophysiological Effects of Procainamide in Acute and Healed Experimental Ischemic Injury of Cat Myocardium Robert J. Myerburg, Arthur L. Bassett, Kristina Epstein, Marion S. Gaide, Patricia Kozlovskis, Sam S. Wong, Agustin Castellanos, and Henry Gelband From the Departments of Medicine (Division of Cardiology), Pharmacology, and Pediatrics (Division of Pediatric Cardiology), University of Miami School of Medicine and the Miami Veterans Administration Hospital, Miami, Florida SUMMARY. We studied the effects of a membrane-active antiarrhythmic agent, procainamide (PA), on cellular electrophysiological consequences of ischemic injury to cat ventricular muscle. The left ventricles of 90- to 120-minute acute myocardial infarctions (AMI) (n = ), and 2- to 4-month healed myocardial infarctions (HMI) (n = ), were studied by microelectrode techniques in isolated tissue bath. Control action potential duration at 90% repolarization (APD90) recorded from ventricular muscle cells in AMI areas were short (1 ± 4 msec) compared to recordings from cells in normal areas (136 ± 6 msec) (P < 0.001). In contrast, APD90 of cells surviving ischemia in HMI preparations were longer than normals (159 ± 5 vs. 0 ± 5 msec, P < 0.001). After 60 minutes of exposure to PA, the APD90 of all cells was prolonged, but the absolute and relative magnitudes of prolongation were greater in (mean = +40 msec, +35%), than in (mean = +19 msec, +13%), P < The prolongation of APD90 of normal cells was intermediate. Local refractory period changes paralleled APD90 changes. In seven additional HMI preparations, sustained ventricular activity was induced by premature stimulation. APD90 of prolonged less than APD90 of normal cells during exposure to PA in these preparations, and decreased differences of APD90 between normal and was associated with loss of inducibility of sustained ventricular activity. The effect of tetrodotoxin (TTX) was compared to the effect of PA in four HMI preparations to determine whether impaired delivery of test substances caused only an apparent decreased responsiveness to PA in HMI zones. TTX caused nearly identical prolongations of conduction times in HMI zones and normal zones, whereas PA caused different effects on APD90 in the two zones. In conclusion, PA alters the time course of repolarization of more than that of, decreasing the dispersion of repolarization in a given AMI or HMI preparation. The decreased dispersion correlated with loss of ability to induce sustained ventricular activity. Finally, the decreased responsiveness of to PA does not appear to be due to impaired delivery to cell membranes, but, rather, appears to be a membrane difference persisting in cells which have survived ischemic injury. (Circ Res 50: , 1982) IN STUDIES of experimental myocardial infarction in cats, we have observed that the cellular electrophysiological abnormalities resulting from acute ischemic injury ( minutes) differ from those which persist long-term (2-4 months) after healing from acute injury (Myerburg et al., 1978; Myerburg et al., in press). Transmembrane action potentials recorded from endocardial cells overlying acute myocardial infarction areas had variable decreases in resting membrane potential, upstroke amplitude, and dv/dt m ax, and shortened action potential durations. In contrast, the majority of surviving endocardial ventricular muscle cells overlying healed myocardial infarction scars were characterized by normal resting transmembrane potentials and upstroke velocities, but action potential durations were prolonged. The present study was designed to compare the cellular electrophysiological effects of a membraneactive antiarrhythmic agent, procainamide, on ventricular muscle cells studied under these two experimental conditions. The data demonstrate quantitatively different effects of this drug on the time course of repolarization of ventricular muscle studied minutes after acute ischemic injury, compared to ventricular muscle studied 2-4 months after healing of acute ischemic injury. The net effect of these different responses was decreased dispersion of action potential durations and refractory periods. In isolated healed infarction preparations, decreased dispersion was associated with loss of inducibility of sustained ventricular activity. Methods Acute myocardial infarction was created by single stage ligation of two or three distal tributaries of the left anterior descending and left circumflex coronary arteries in conditioned domestic cats (Myerburg et al., 1977). This procedure predictably produces an infarction of 5-15% of the left ventricular muscle mass at the base of the anterior papillary muscle and adjacent areas of the apex, apical free wall, and lower septum. For acute myocardial infarction (AMI) studies, the injury was allowed to evolve for a period of minutes under pentobarbital anesthesia (30 mg/kg, ip), and the heart then was excised (see below) and mounted in a tissue bath. For the healed myocardial infarction (HMI) preparations, the chest was closed after coronary ligation, and a postoperative ECG recorded. The surviving cats were

2 Myerburg et al. /Acute and Healed Ischemic Injury 387 maintained in a colony for 2-4 months. On the day of terminal studies, the cats were anesthetized with pentobarbital (30 mg/kg, ip), and the heart was removed through a thoracotomy. The atria and right ventricle were excised, and the left ventricle opened by an incision through the free wall between the posterior papillary muscle and posterior paraseptal free wall (Myerburg et al., 1972). The mitral valve was excised and the aortic ring opened. After the left ventricular preparation had been weighed rapidly in a bottle of oxygenated Tyrode's solution, it was mounted in a Lucite tissue bath with small steel pins. The preparation was superfused with Tyrode's solution prepared as previously described (Myerburg et al., 1977), and the temperature was maintained at C. The preparation was allowed to equilibrate for a minimum of 1 hour, or until transmembrane action potential characteristics were stable for 30 minutes, before supervision studies were initiated. After equilibration, in control studies, electrophysiological characteristics remained stable for 4 or more hours, in the absence of interventions (Myerburg et al., in press). Surface electrograms were recorded through fine silver wire bipolar electrodes, 0.01" (0.25 mm) in diameter and triple-teflon coated except at the tips. These electrodes were used to map endocardial surface activation of the preparation during specific stimulation of the left bundle branch, which was achieved by setting stimulus strength at 1.2-times threshold current (Myerburg et al., 1972). The surface electrograms were used to delineate areas of electrophysiological abnormality, particularly in the AMI preparations in which it was not always possible to visually identify abnormal areas. In the HM1 preparations, a visible scar was almost always present; and surface electrograms from surviving cells over the region of the scar frequently appeared normal, although focal areas of abnormal electrograms also were identified. Transmembrane action potentials were monitored from endocardial cells in areas of acute and healed myocardial infarctions, and from normal areas of the same hearts. The cells monitored during intervention studies were determined to be representative of the respective areas from which they were recorded by evaluation of the grid maps constructed as reported in detail elsewhere (Myerburg et al., 1977; Myerburg et al., in press). The cells from HM1 preparations were selected from the center of the infarct zone, where action potential duration is uniformly prolonged at 2-4 months, rather than the border zone where the characteristics are more variable and very short action potentials may be recorded (Wong et al., 1981). Similar sites were selected for studies in AMI preparations; and in these preparations, we are reporting data only on cells that maintained resting membrane potentials and upstroke velocities in the range of normal. The major electrophysiological manifestation of ischemia in such cells is shortening of the duration of repolarization (Myerburg et al., in press). This selection process was carried out in order to have a population of which had resting potentials and upstroke velocities comparable to those of healed cells from HMI preparations. Standard microelectrode techniques, previously reported in detail, were used (Myerburg et al., 1970, 1973). Premature stimuli were delivered to various sites on the endocardial surface both to measure refractory periods and to initiate sustained ventricular activity before and during exposure to procainamide. Premature stimuli were delivered at 2.0 times late diastolic threshold strength, and diastole was scanned as previously reported (Myerburg et al., 1970, 1972, 1973). In those preparations in which it was not possible to initiate sustained ventricular activity by premature stimulation, we determined the effects of drug on action potential duration, local refractory period, upstroke velocity, and resting membrane potential. In those preparations with inducible sustained ventricular activity, action potential durations and local refractory periods often changed as a consequence of the periods of rapid activity itself, and therefore the experiments were focused on the influence of procainamide on inducibility of sustained ventricular activity. The action potential duration and refractory period data from the latter experiments were not pooled with the data from experiments without inducibility of sustained activity. Aqueous solutions of procainamide hydrochloride (Sigma Chemical Co.) were added to Tyrode's solution in amounts appropriate to achieve desired concentrations. The smallest possible aqueous volumes were used, and never exceeded 10 ml/liter of Tyrode's solution. Doseresponse curves demonstrated concentration-dependent prolongation of action potential durations, refractory periods, and dv/dt m ai at procainamide concentrations from 10 to 120 /ig/ml. The data reported herein is based on observations at a concentration of 40 /ig/ml, since this was the concentration at and below which no alteration in resting potential was recorded, consistent with data previously reported by Rosen et al. (1973). In selected experiments, 1 mg of tetrodotoxin (Sigma Chemical Co.) was dissolved in 5 ml of water and added to 500 ml of Tyrode's solution. Data were recorded photographically from an oscilloscope screen, using techniques which we have previously reported (Myerburg et al., 1972,1973), and dv/dt max, action potential duration, and local refractory periods were measured on-line (Myerburg et al., 1970; Gelband et al., 1970). Statistical analyses were carried out using a two-tailed, twosample t-tests. When the same data were used in multiple t-tests, levels of significance were adjusted by the Bonferroni procedure to maintain an overall level of significance of p < 0.01 or better. This procedure was applied to the properties of vs. those of their controls, versus their controls, vs., and the controls for both groups vs. each other. To determine whether there was a significant difference between the characteristics of the responses of AMI and and their normal controls to procainamide, we calculated analyses of variance and covariance, using repeated measures, throughout the time courses of each of these studies. Results Comparison of Effects of Procainamide on Action Potential Duration and Local Refractory Periods in AMI and HMI Preparations Fourteen experiments were performed on ventricles isolated in tissue bath minutes after coronary ligation (AMI), and experiments were carried out on ventricles with healed myocardial infarction (HMI) 2-4 months after coronary ligation. Representative illustrations of the effects of procainamide (PA) on transmembrane action potentials recorded from cells in an AMI ventricle and from an HMI ventricle are shown in Figure 1. In both experiments, recordings from the normal and infarct zones were carried out simultaneously, and the impalements for these recordings were maintained throughout the experiments. In the AMI preparation (Panel A), action potential duration at 90% repolarization (APD90) of the cell in the AMI zone was shorter than the normal zone cell. After 45 minutes of exposure to PA (40 /ig/

3 388 Circulation Research/ Vol. 50, No. 3, March 1982 PANEL NORMAL ACUTE INFARCT PANEL B A 90 MINUTE ACUTE MYOCARDIAL INFARCTION CONTROL 45 min. PA 90 min. WASH APD 90 = 100 =162 msec (+ 62%) = 135 msec 2 MONTH HEALED MYOCARDIAL INFARCTION (+ 35%) CONTROL 45 min. PA 90 min. WASH two experiments suggest that, in AMI and HMI preparations, relative and absolute changes in response to superfusion of PA are quantitatively different in normal, AMI, and HMI tissues. The cumulative data on absolute and relative change in APD90 at a drive cycle length of 630 msec for the series of AMI preparations and HMI preparations are shown in Figures 2 and 3, and Table 1. The data in Figure 2 show that the APD90 of both the normal cells (longer control APDgo's) and acute infarct area cells (shorter control APD^'s) prolong during superfusion with PA, but that the prolongation is greater in the (mean = +40 msec or +35%, at 60 minutes) than in the normal cells (mean = +30 msec, or +22%, at 60 minutes). In Figure 3, based on the data recorded from HMI preparations, the control APD 90 's recorded from cells in the HMI zone were longer than those recorded from normal zones, with the having less responsiveness to PA (mean = +21 msec or +13%, at 60 minutes) than normal cells (mean = +29 msec, or +21%, at ACUTE MYOCARDIAL INFARCT HEALED INFARCT APD 90 = 203 = 213 msec =196 msec msec (+5%) (-3%) FIGURE 1. Comparison of effects of procainamide on transmembrane action potentials recorded from acvte and healed myocardial infarction areas. Each panel demonstrates simultaneous recordings of transmembrane action potentials from a normal zone and infarct zone prior to superfusion with procainamide (PA), 45 minutes after beginning PA superfusion at a concentration of 40 tig/ml, and after 90 minutes of wash with Tyrode's solution. Panel A demonstrates data recorded from a preparation isolated in tissue bath 90 minutes after acute coronary ligation, and panel B is a 2-month healed myocardial infarction. Action potential durations printed under each transmembrane action potential recording were measured at 90% repolarization (APDm) and the percentages in parenthesis under APDgo's compare APD m during and after PA superfusion to control for each recording. Horizontal calibration = 50 msec, vertical calibration 20mV. ml), the absolute and relative increase of APD90 of the cell in the AMI zone was greater than that in the normal zone. After a 90-minute wash with Tyrode's solution, the APD90 of these cells returned toward control values. In contrast to the AMI preparation, the control recordings from the HMI preparation demonstrated a longer APD90 recorded from the cell within the HMI zone, compared to the normal zone cell. After 45 minutes of exposure to PA, the absolute and relative increase of the APD90 of the cell in the normal zone was greater than those of the cell in the HMI zone. After a 90-minute wash, both the normal cell and the HMI cell had returned to near control levels. These CONTROL TYROOES min I-TYROOESH FIGURE 2. Cumulative data from acute myocardial infarction experiments. The upper panel demonstrates APD90 (mean ± SD) recorded from normal cells (open bars) and surviving cells in the AMI area (cross-hatched bars). The first pair of bars demonstrate control data prior to procainamide (PA) superfusion, and the next two pairs of bars demonstrate the effect of procainamide on APD 90 of normal and at 30 and 60 minutes of superfusion. The mean control APDm for normal cells was 136 ± 6 msec and, for, 1 ± 4 msec. At 60 minutes of PA superfusion, normal cell APD90 increased to 166 ±8 msec (+22%), and AMI cell APD90 increased to 158 ± 8 msec) (+35%). The last two pairs of bars show data at 30 and 60 minutes of wash with Tyrode's solution. The lower panel demonstrates the mean percent change of APD 90 at 30 and 60 minutes of PA, and 30 and 60 minutes of Tyrode's wash, compared to control, for normal cells and (see text).

4 Myerburg et al. /Acute and Healed Ischemic Injury HEALED MYOCARDIAL INFARCT 180,, 389 fusion, at 30 and 60 minutes of PA superfusion, and at 30 and 60 minutes of Tyrode wash (P < for each r-test). Correcting the level of significance for multiple r-tests on this data gives an overall experimental level of significance of P < In all three comparisons, analysis of variance and covariance, with repeated measures, demonstated that the t-values were maximum before superfusion and minimum at 60 minutes of superfusion with PA (see Table 1). This statistical analysis suggests quantitative and relative differences between the responses to PA of vs. their controls, HMI vs. their controls, and AMI cells vs., throughout the overall time course of each group of studies. Figures 2 and 3 also show percent change of APD90 for and their corresponding normals (Fig. 2) and and their corresponding normals (Fig. 3). The control APDgo's for each experiment were the denominators, and percent changes were calculated at 30 and 60 minutes of PA superfusion and 30 and 60 minutes of Tyrode washout. The differences in percent change between and their corresponding normals CONTROL min TYRODES I PA 1 I-TYROOES-I FIGURE 3. Cumulative data from healed myocardial infarction experiments. The upper panel demonstrates APDm (mean ± SD) recorded from normal cells (open bars), and surviving cells in the HMI area (cross-hatched bars). The first pair of bars demonstrates control data prior to procainamide (PA) superfusion, and the next two pairs of bars demonstrate the effect of procainamide on APDm of normal and at 30 and 60 minutes of superfusion. The mean control APDm for normal cells was 0 ± 40 msec and for was 159 ± 5 msec. At 60 minutes of PA superfusion, normal cell APDm increased to 169 ± 7 msec (+21%), and HMI cell APD M increased to 180 ± 5 msec (+13%). The last two pairs of bars show data at 30 and 60 minutes of wash with Tyrode's solution. The lower panel demonstrates the mean percent change in APDm at 30 and 60 minutes of PA, and 30 and 60 minutes of Tyrode's wash, compared to control for normal cells and (see text). minutes). Thus, PA superfusion made the absolute values of APD90 less disparate, with mean APD90 differences between acutely ischemic and normal cells in AMI preparations decreasing from 22 msec before PA to 12 msec during PA (A = -45%); and in HMI preparations, from 19 msec before to 11 msec during PA superfusion (A = -42%). Statistical analyses of APD90 changes were carried out using 2-tailed, two-sample r-tests. There was a small but significant difference between APD90 of normal cells in AMI preparations (mean = 136 ± 6 msec) and normal cells in HMI preparations (mean = 0 ± 3 msec) prior to PA superfusion (F = 0.024); but there were no significant differences between the two normal cell groups at 30 and 60 minutes of PA superfusion and at 30 and 60 minutes of Tyrode's wash after superfusion. In contrast, the differences in APD90 between and their controls, HMI cells and their controls, and compared to, were highly significant before PA super- TABLE 1 Changes in APD90 during and after Procainamide Superfusion at Drive Cycle Length of 630 msec Control 30 min PA 60 min PA 30 min wash 60 min wash n APD90 (msec) (mean ± SD) 1 ± ± ±5 0 ± ± ± 5 6 ± ± ± ± ± ± ± ± ± ± ± 8 1 ± ±5 3 ±9 t-value Tailed probability n = number of observations; APD90 = action potential duration at 90% repolarization; control, 30-min wash, 60-min wash = superfusion with Tyrode's solution; 30 min PA, 60 min PA = superfusion with procainamide (40 ftg/ml) in Tyrode's solution; AMI = acute myocardial ischemia; HMI = healed myocardial infarction.

5 390 Circulation Research/Vol. 50, No. 3, March 1982 were significant at 30 minutes of PA superfusion (P < 0.01) and 60 minutes of superfusion (P < 0.001). In HMI experiments, the differences were significant at 60 minutes of PA superfusion (P < 0.001) and at 30 minutes of subsequent washout (P < 0.001). In nine of the AMI experiments and 10 of the HMI experiments, it was possible to measure local RP's by premature stimulation in the regions of the recording microelectrodes. Local refractory periods paralleled APDgo's in both groups before and during superfusion with PA (see Table 2). Delivery of PA to HMI Cells during Superfusion One possible explanation for the lesser responsiveness of to procainamide is failure of delivery of this test substance, due to the interstitial fibrosis which occurs as a consequence of the ischemic injury (Myerburg et al., 1977). We therefore compared the effects of PA and tetrodotoxin (TTX) on the same in four experiments. Figure 4 demonstrates the response of a normal zone cell and an HMI zone cell to PA superfusion, followed by TTX after PA washout. PA caused a greater prolongation of APD90 of the normal cell compared to the HMI cell (Fig. 4, left panel), as observed in the other experiments, but there was little change in time from the stimulus to the response of either cell during PA superfusion. In contrast, during exposure to TTX, 2 jug/ml, both cells showed equivalent prolongation of conduction time from the site of stimulation to the recording microelectrodes. Both values returned to control after washout with Tyrode's solution for 60 minutes. In two of the other three experiments comparing TTX and PA, exposure to the two agents was carried out in reverse order and the results were the same. In all four experiments, PA had different effects on APD90 of normal cells vs., and TTX had the same effects on conduction time and dv/dtmax- Effect of Procainamide on Inducibility of Sustained Ventricular Activity in HMI Ventricles We have previously reported inducibility of sustained ventricular activity by premature stimulation TABLE 2 LRP's before and during Superfusion with PA Acute preparations Normal zone AMI zone Healed preparations Normal zone HMI zone n Control LRP (msec) (mean ± SD) 1 ± 126 ± 11 4 ± ± min PA LRP (msec) (mean ± SD) 5 ± ± ± ± 20 Percent change AMI = acute myocardial ischemia/infarction; HMI = healed myocardial infarction; LRP = local refractory period; PA = procainamide. 2 MONTH HEALED MYOCARDIAl INFARCTION NORMAL HEALED M.I. APD90= 3 msec = 7 msec APD90 = 4 msec = 201 msec (+ 22%) (+ 54) APD90 z 44 msec ' (+ < 1%) =8 msec 1+ < 1%) TTX 2ug/ml NORMAL MEALED M.,1. = 152 msec =180 msec = 0 msec msec (- 8%) (- 13%) - 0 msec I- 8% FIGURE 4. Comparison of effectiveness of procainamide and tetrodotoxin on normal and. The left panel demonstrates effects of procainamide (PA), 40 fig/ml, on a cell from a normal zone and an HMI zone in an isolated preparation. The normal zone cell has a shorter control APD90 and a greater percent response to PA. In the panel on theright,exposure of the same preparation to tetrodotoxin (TTX) shows equivalent prolongation of conduction time from stimulus site to the recording site in both the normal zone and HMI zone cells (+31 msec in the normal zone, +30 msec in the HMI zone). These data suggest that delivery of test substances to the cell membranes is equivalent in a normal zone and HMI zone, and that the differential response to PA is due to a membrane change rather than impaired delivery. of surviving subendocardial ventricular muscle cells overlying scars in isolated HMI ventricles (Myerburg et al., 1978; Myerburg et al., in press). In the present series of studies, we evaluated the influence of superfusion with PA on inducibility of sustained ventricular activity in isolated HMI ventricles. In seven experiments, sustained ventricular activity was reproducibly inducible (>2 inductions) in tissue bath before PA superfusion and was not inducible (>2 tests for inducibility) during PA superfusion. In five of the seven, inducibility returned after wash with drug-free solution. The loss of inducibility during PA superfusion was associated with prolongation of APDgo's and local RP's in both HMI zones and normal zones, with the greater absolute and relative prolongation in the normal cells (see Fig. 5). The dispersion of local refractory periods decreased from 28 ± 12 msec before PA to 11 ± 7 msec during PA superfusion. Before loss of inducibility of sustained ventricular activity was achieved, the cycle length of induced sustained activity prolonged by an average of 43 ± msec. Pre-PA cycle length of sustained activity averaged 115 ± 12 msec. Figure 5 illustrates a representative sustained ventricular activity experiment. Prior to exposure to PA (Fig. 5, top row), the cell from the HMI zone had a longer APD90 than the normal zone cell, and sustained ventricular activity at a cycle length of 108 msec was initiated by premature stimulation. After exposure to PA at a concentration of 25 /ig/ml for 30 minutes, the

6 Myerburg ef al. /Acute and Healed Ischemic Injury CONTROL 5 MIN. AFTER PA. CONTROL 30 MIN. AFTER P.A. INFARCT NORMAL _ S, S 2' 50, FlGURE 5. Effect of procainamide on transmembrane action potentials and response to premature stimulation in a healed myocardial infarction preparation. The left column demonstrates the response of cells in the healed myocardial infarction zone to procainamide (PA), 25 ng/ml, and the right column shows effects on a cell in the normal zone. The center column demonstrates the response to premature stimulation close to the refractory period before, during, and after superfusion with procainamide. The APD90 of the infarct zone cell was 125 msec before PA compared to 95 msec in the normal zone cell. Prolongation of APD90 during PA exposure was much greater in the cell from the normal zone (+80 msec to APD90 = 5msec) than the infarct zone (+12 msec to APDgo = 135 msec), and, in addition, the ability to induce sustained ventricular activity disappears during superfusion with PA and reappears after wash. Repetitive ventricular responses still occur during superfusion with procainamide, however. The impalements of the two monitored cells were lost during sustained ventricular activity induced after PA fourth row. See text for further details. APD90 of the cell recorded from the HMI zone prolonged 12 msec (to 135 msec), while the APD90 of the normal zone cell prolonged 80 msec (to 5 msec). At this time, S2 resulted in a repetitive response, but no sustained activity could be induced. Thirty minutes after cessation of PA superfusion, sustained ventricular activity was again inducible. The lesser relative and absolute effects of PA on APD90 of, compared to normal cells, occurred in parallel with loss of inducibility of sustained ventricular activity. Discussion During the past 10 years, there has been increasing use of experimental myocardial infarction models to study the arrhythmias related to myocardial ischemia and its consequences. A natural extension of these experiments has been study of the effects of antiarrhythmic agents on the models, including characterization of cellular electrophysiological effects of these drugs. Most of the studies to date have focused on drug effects on acutely ischemic or early healing tissue (Brennan and Wit, 1973; Sasyniuk and Kus, 1973; Hondeghem et al., 1974; Sasyniuk et al., 1974; Kupersmith et al., 1975; Kus et al., 1975; El-Sherif et al., ; Allen et al., 1978; Brennan et al., 1978; Cardinal and Sasyniuk, 1978; El-Sherif and Lazzara, 1978; Lazzara et al., 1978; Wang et al., 1979; Guse et al., 1980). Our data in the present study demonstrate different effects of a membrane-active antiarrhythmic agent (procainamide) on acutely injured ventricular muscle cells compared to cells which have survived prior ischemic injury and overlie the scar of a healed myocardial infarction. These observations are consistent with differences in patterns of repolarization in AMI vs. (Myerburg et al., 1978; Myerburg et al., in press). Since the process of depolarization returns to normal after acute ischemic injury with healing, and the process of repolarization often shows persistent abnormalities manifested by prolongation of APD90, we carried out the TTX experiments based on the hypothesis that unimpaired superfusion of the superficial cells would result in similar effects of TTX on depolarization of both HMI and normal cells. The results of these experiments (Fig. 4), showing comparable TTX effects on cells in the HMI and normal zones, suggest that the differences recorded are indeed due to underlying abnormalities specifically in the process of repolarization, rather than to failure of delivery of test substances. However, the possibility of a differential responsiveness to different amounts of test substances delivered cannot be completely excluded. The mechanism by which patterns of repolarization are impaired in the two experimental settings, AMI and HMI, have not yet been elucidated. Two likely possibilities are different impairments of membrane function in the two experimental settings, or different patterns of uncoupling of the superficial endocardial cells from deeper cells. Permutation of these two factors, membrane changes and uncoupling, are also possible. The analysis of the data on the effect of PA on normal and abnormal cells in AMI and HMI preparations suggests quantitative differences in the responses of AMI, HMI, and normal cells to PA. These quantitative differences may reflect underlying fundamental qualitative differences in the cell membranes, present before exposure to PA. Since, in clinical settings, it is common to find present tissue which has healed after ischemic injury, with co-existent superimposed acute ischemia, the tendency for PA to show a greater prolonging effect on the shortest action potential durations and a lesser prolonging effect on the longer action potential durations, with resultant tendency toward uniformity of APD90, is a theoretically desirable effect. This may, in part, explain its stabilizing influence on arrhythmias. Han and co-workers (Han, 1961; Han and Moe, 1964) have demonstrated the importance of dispersion of action potential durations and refractory periods in arrhythmogenesis. The data in the present study suggest that PA may have the capability of decreasing dispersion when co-existence of AMI and HMI cause bidirectional deviation of APDgo's and local refractory pe-

7 392 Circulation Research/Vol. 50, No. 3, March 1982 riods. In another study, we observed that exposure to P." did in fact decrease the dispersion of refractoriness between normal, AMI, and in the same heart when AMI was superimposed upon HMI, and this was associated with loss of ability to induce sustained ventricular activity (Myerburg et al., in press). Each of the tentative conclusions about the relationships between the observations in this study, and ischemic arrhythmias and their prevention by antiarrhythmic agents, must be interpreted cautiously because of the design of this study. As noted earlier, these studies required selection of a specific group of with good resting potentials and exclusion of with significant partial depolarization. The latter certainly may play a role in the genesis of AMI arrhythmias, and their response to PA may be different than the response of the cells studied in this project. Several of our experiments suggest that the experimental antiarrhythmic effects of PA may be two-fold: (1) prolongation of refractory periods resulting in loss of ability to induce sustained activity by premature stimulation, and (2) slowing of the rate of established sustained ventricular activity, presumably due to a change in conduction properties in re-entrant circuits. The two independent, but presumably interacting, effects may explain some observations on dissociation of effects of membrane-active drugs on various forms of clinical arrhythmias (Myerburg et al., 1981). Finally, the observation that effects of PA on normal tissue is greater than on surviving tissue from infarct zones in HMI preparations may be of fundamental importance to concepts of experimental cardiac electropharmacology. In recent years there has been a tendency to focus less attention to the cellular electrophysiological effects of membrane-active drugs on normal tissue, based upon the hypothesis that the significant drug effects must be on abnormal tissue, and might differ qualitatively from effects on normal tissue. Our present data support the latter point; but also suggest that effects of drugs on normal tissue might be equally important as effects on abnormal tissue for action ag.-.inst sustained ventricular activity. We are grateful to Thelma L Gottlieb for secretarial and administrative support. Supported in part by Grants-in-Aid from the NIH, National Heart, Lung, and Blood Institute HL and HL NIH Training Grant HL (Or. Kozlovskis), and the American Heart Association, Greater Miami Affiliate. Address for reprints: Robert ]. Myerburg, M.D., Professor of Medicine and Physiology, Director, Division of Cardiology, University of Miami School of Medicine, P. O. 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