Author + information
- Received July 8, 1996
- Accepted August 13, 1996
- Published online December 1, 1996.
- Charles Antzelevitch, PhD⁎ (, )
- Zhuo-Qian Sun, MD, PhD,
- Zi-Qing Zhang, MD and
- Gan-Xin Yan, MD, PhD
- ↵⁎Address for correspondence: Dr. Charles Antzelevitch, Masonic Medical Research Laboratory, 2150 Bleecker Street, Utica, New York 13501.
Objectives This study sought to elucidate the cellular and ionic basis for erythromycin-induced long QT syndrome.
Background Erythromycin is known to produce long QTU intervals on the electrocardiogram (ECG) and to be associated with the development of torsade de pointes (TdP). The mechanisms responsible for the adverse effects of this widely used antibiotic are not well defined.
Methods The present study used microelectrode and whole-cell patch-clamp techniques to assess the effects of erythromycin on epicardial, endocardial and M cells in transmural strips, arterially perfused wedges and single myocytes isolated from the canine left ventricle.
Results In isolated strips, erythromycin (10 to 100 Mg/ml) produced a much more pronounced prolongation of the action potential duration (APD) in M cells than in endocardial and epicardial cells, resulting in the development of a large dispersion of repolarization across the ventricular wall at slow stimulation rates. Erythromycin (50 to 100 Mg/ml) induced early afterdepolarizations (EADs) in cells in the M (20%) but not epicardial or endocardial regions in transmural strips of ventricular free wall. Erythromycin (100 Mg/ml) also caused APD prolongation and a transmural dispersion of repolarization, but not EADs, in intact arterially perfused wedges of canine left ventricle. These changes were attended by the development of a long QT interval on the transmural ECG. A polymorphic ventricular tachycardia closely resembling TdP was readily and reproducibly induced after erythromycin but not before. Whole-cell patch-clamp techniques, used to examine the effects of erythromycin on myocytes isolated from the M region, showed a potent effect of the drug to inhibit the rapidly activating component (IKr) but not the slowly activating component (IKs) of the delayed rectifier potassium current (IK). The inward rectifier current (IK1) was unaffected.
Conclusions Our data demonstrate a preferential response of M cells to the class III actions of erythromycin, due principally to the effect of the drug to inhibit IKr in a population of cells largely devoid of IKs. Our findings indicate that erythromycin thus produces long QT intervals as well as a prominent dispersion of repolarization across the ventricular wall, setting the stage for induction of TdP-like tachyarrhythmias displaying characteristics typical of reentry.
The clinical syndrome of acquired long QT occurs in association with various pharmacologic agents, electrolyte abnormalities and bradycardic states (1–5). Most pharmacologic agents capable of prolonging the QT interval appear to be capable of causing ventricular tachyarrhythmias, most notably a polymorphic ventricular tachycardia known as torsade de pointes (TdP). A recent addition to the group is the macrolide antibiotic erythromycin (6–11). A recent study by Daleau et al. (12) demonstrates the effect of erythromycin to inhibit the rapid component of the delayed rectifier current (IKr)in guinea pig myocytes of unknown transmural origin. Monophasic action potentials recorded from the endocardial surface of a Langendorff perfused guinea pig heart showed modest prolongation. A recent study by Rubart et al. (10) presented indirect evidence suggesting that erythromycin-induced prolongation of the action potential in canine Purkinje fibers is due to inhibition of the delayed rectifier potassium current (IK). Their study also showed that ventricular endocardium (papillary muscle) was little affected by the drug. Thus, the mechanism by which erythromycin prolongs ventricular repolarization and causes long QT intervals and TdP is not well defined.
Recent studies (5,13–15) have shown that M cells in the deep structures of ventricular myocardium are primary targets for most agents that prolong repolarization in the canine heart. Epicardial and endocardial cells were found to be little affected by concentrations of drug that cause marked prolongation, early afterdepolarization (EAD) and triggered activity in M cells. The present study tests the hypothesis that the ability of erythromycin to induce long QT intervals, EADs, triggered activity and TdP is due principally to its ability to delay repolarization of M cells in the deep structures of the canine heart through inhibition of one or both components of IK. The experimental protocols assess the differential responsiveness of the three principal cell types spanning the left ventricular wall to erythromycin using isolated tissue preparations, the development of polymorphic arrhythmias resembling TdP using an arterially perfused wedge of canine left ventricle and the ionic mechanism underlying the response to erythromycin using the whole-cell patch-clamp technique applied to myocytes isolated from the M region of the canine left ventricle. Our preliminary results have been reported in abstract form (16).
- Abbreviations and Acronymns
- = action potential duration
- = action potential duration at 90% repolarization
- = basic cycle length
- = early afterdepolarizations
- = electrocardiogram, electrocardiographic
- = delayed rectifier K+ current
- = rapidly activating component of delayed rectifier K+ current
- = slowly activating component of delayed rectifier K+ current
- = inward rectifier K+ current
- = extracellular K+ concentration
- = torsade de pointes
Action potential studies. Transmural strips (~2.0 × 1.0 × 0.1 to 0.2 cm) were isolated from the left ventricular free wall of canine hearts removed from anesthetized (30 mg/kg body weight sodium pentobarbital) mongrel male dogs. The tissue preparations were obtained by razor blade shavings (Der-matome Power Handle no. 3293 with cutting head no. 3295, Davol Simon). The preparations were obtained near the base of the left ventricle, and shavings were made either parallel or perpendicular to the surface of the free wall.
The preparations were placed in a tissue bath and allowed to equilibrate for at least 2 h while superfused with an oxygenated (95% oxygen/5% carbon dioxide) Tyrode's solution (37 ± 0.5°C, pH = 7.35; composition: [mmol/liter] NaCl 129, KCl 4, NaH2PO4 0.9, NaHCO3 20, CaCl2 1.8, MgSO4 0.5, D-glucose 5.5).
The tissues were stimulated at a basic cycle length (BCL) ranging between 500 and 8,000 ms using field or point stimulation delivered to the endocardial side through silver bipolar electrodes insulated except at the tips. Transmembrane potentials were recorded from endocardial, epicardial and M region sites using glass microelectrodes filled with 2.7 mol/liter KCl (10 to 20-?O direct current resistance) connected to a high input impedance amplification system (World Precision Instruments). The signal was displayed on Tektronix oscilloscopes, amplified (model 1903A programmable amplifiers, Cambridge Electronic Design), digitally sampled at a rate of >7 kHz (Spike 2 acquisition module, Cambridge Electronic Design), stored on magnetic or other media (personal computer hard drives, magnetic tapes and CD-ROM) and analyzed (Spike 2 analysis module, Cambridge Electronic Design).
Arterially perfused wedge of canine left ventricle. Dogs weighing 20 to 25 kg received anticoagulation with heparin and were anesthetized with pentobarbital (30 mg/kg intravenously). The chest was opened by means of a left thorocatomy, and the heart was excised, placed in a cardioplegic solution consisting of cold (4°C) Tyrode's solution containing 8.5 mmol/liter extracellular K+ concentration ([K+]o) and transported to a dissection tray. Transmural wedges with dimensions of 0.8 × 0.9 × 0.8 cm to 1.2 × 2 × 1.1 cm (height × length × wall thickness) were dissected from the left ventricle. The tissue was cannulated through a small (-100 to 150-μπ? diameter) branch of the left anterior descending or other coronary artery and perfused with cardioplegic solution. The total period of time from excision of the heart to cannulation and perfusion of the artery was usually <4 min. The preparation was then placed in a small tissue bath and arterially perfused with Tyrode's solution of the following composition (mmol/liter): NaCl 129, KCl 4, NaH2PO4 0.9, NaHCO3 20, CaCl2 1.8, MgSO4 0.5, glucose 5.5, and 1 U/liter of insulin buffered with 95% oxygen and 5% carbon dioxide (temperature 36 ± 1°C). The perfusate was delivered to the artery by a roller pump (Cole Parmer Instrument Co.). Unperfused tissue, identified by its dark red appearance (erythrocytes not washed out) soon after the wedge was cannulated and perfused, was excised using a razor blade. In initial experiments with this preparation, we injected triphenyl tetrazolium chloride to identify ischemic regions (17) in wedge preparations that had been beating in the tissue chamber for a period of >5 h. No ischemic regions were detected.
Perfusion pressure was monitored with a pressure transducer (World Precision Instruments, Inc.) and maintained between 40 and 50 mm Hg by adjustment of the perfusion flow rate. This pressure is considered normal for small arteries (100 to 150μm diameter) (18). Perfusion pressures >50 mm Hg were difficult to maintain constant and were found to cause edema, whereas pressures <30 mm Hg sometimes caused mild ischemia in endocardium accompanied by perceptible action potential and ST segment changes. The preparations remained immersed in the arterial perfusate, which was allowed to rise to a level 2 to 3 mm above the tissue surface (temperature 36 ± 1°C).
The left ventricular wedges were allowed to equilibrate in the tissue bath until electrically stable, usually 1 h. The preparations were stimulated at BCLs ranging from 500 to 5,000 ms using bipolar silver electrodes insulated except at the tips and applied to the endocardial surface.
A transmural electrocardiographic (ECG) signal was recorded using extracellular silver chloride electrodes placed near the epicardial and endocardial surfaces of the preparation plugged into a differential direct current amplifier. Transmembrane action potentials were simultaneously recorded from the epicardial, endocardial and M regions using three separate intracellular floating microelectrodes (direct current resistance 10 to 20 MΩ) filled with 2.7 mol/liter KCl and connected to a high input impedance amplifier. Impalements were obtained from the cut surface of the preparation at positions approximating the transmural axis of the ECG recording. Amplified signals were digitized, stored on magnetic media and CD-ROM and analyzed using Spike 2 (Cambridge Electronic Designs, Cambridge, England, U.K.). The electrical stability of the preparation was assessed in a series of seven experiments in which action potential duration (APD) and QT interval were measured over a period of 4 h (Table 1).
Whole-cell patch-clamp studies. Myocytes were isolated by enzymatic dissociation as previously described (19). Briefly, adult male and female mongrel dogs were anesthetized with sodium pentobarbital (30 mg/kg intravenously); the hearts were quickly removed and placed in normal Tyrode's solution. A wedge consisting of that part of the left ventricular free wall supplied by the left circumflex coronary artery was excised. The artery was cannulated and flushed with Ca2+-free Krebs buffer supplemented with 0.1% bovine serum albumin (fraction V, Sigma) and gassed with 95% oxygen/5% carbon dioxide for 5 min at a rate of 12 ml/min. Perfusion was then switched to 75 ml of calcium-free Krebs buffer containing 75 mg of bovine serum albumin and 37.5 of mg collagenase (CLS 2,171 U/mg, Worthington) for 15 to 20 min at 37°C (95% oxygen/5% carbon dioxide, with recirculation). After perfusion, thin slices of tissues were dissected from epicardium (<1.5 mm from the epicardial surface), M region (2 to 7 mm from the epicardial surface) and endocardium (<2 mm from the endocardial surface) using a dermatome. Shavings were made parallel to the surface of the left ventricular free wall midway along the apicobasal axis. Tissues from each region were placed in separate beakers; minced and incubated in fresh Krebs buffer containing 0.5 mg/ml of collagenase, 3% bovine serum albumin and 0.3 mmol/liter CaCl2; and agitated with 95% oxygen/5% carbon dioxide. Incubation was repeated three to five times at 15-min intervals with fresh enzyme solution. The supernatant from each digestion was filtered (220-μm mesh) and centrifuged (200 to 300 rpm for 2 min). Cells were then stored in a HEPES-buffered Tyrode's solution (see below) supplemented with 0.5 mmol/liter Ca2+ at room temperature for later use.
The Krebs buffer used in the cell dissociation procedure contained (mmol/liter): NaCl 118.5, KCl 2.8, NaHCO3 14.5, KH2PO4 1.2, MgSO4 1.2, glucose 11.1. The composition of the HEPES-buffered Tyrode's solution was (mmol/liter): NaCl 132, KCl 4, CaCl2 2, MgSO4 1.2, HEPES 20, glucose 11.1; pH was adjusted with NaOH to 7.35. The Na+-, K+- and Ca2+- free external solution contained (mmol/liter): choline chloride 140, MgCl2 2.0, HEPES (free acid) 20, glucose 11.1; pH was adjusted to 7.35 with LiOH. Ethyleneglycol-bis-betaaminoethylether- N,N'-tetraacetic acid (EGTA [0.5 mmol/ liter]) was added in some experiments. The pipette solution contained (mmol/liter): potassium aspartate 125, KCl 20, MgCl2 1, adenosine triphosphate (Mg salt) 5, HEPES 5, EGTA 10; pH was adjusted to 7.1 to 7.2 with KOH.
Myocytes were superfused with a HEPES-buffered Tyrode's solution (aerated with 100% oxygen) at a flow rate of 2 to 3 ml/min. Only relaxed, quiescent cells displaying clear cross striations were used. All experiments were performed at 35 to 37°C (±0.5°C).
The delayed rectifier current was measured using standard whole-cell patch-clamp techniques. For this study, an Axopatch-1D amplifier with a CV-4 1/100 headstage (Axon Instruments) was used. Suction pipettes made of borosilicate glass (1.5 mm outer diameter and 1.1 mm inner diameter, Becton, Dickinson) were pulled on a Flaming-Brown type pipette puller (Sutter Instrument Co.) and heat polished before use. Pipette tip resistances measured in Tyrode's solution (passed through a 0.22-μm sterile filter, Millipore Corp.) were 2 to 4 MΩ when filled with pipette solution. The junction potential between the pipette solution and Tyrode's solution was zeroed before the formation of the membrane-pipette seal in normal Tyrode's (~10 mV, pipette negative). This zeroing created an offset equal to the junction potential, but of opposite sign, that remained after the establishment of wholecell recording. All voltages in the patch-clamp experiments were corrected for this offset. Once the suction pipette made a gigaohm seal with the cell, the pipette capacitance was partially neutralized. The membrane was ruptured by applying additional negative pressure.
The delayed rectifier current was measured by holding the cells at -40 mV, a potential at which Na+ current is inactivated, and stepping to more positive voltages (-20 to -60 mV). The Ca2+ current was inhibited by the addition of 4.0 mmol/liter nifedipine to the superfusate. Because 0 mmol/liter [K+]o has been shown to increase IKs and greatly diminish IKr (20–22), these conditions were used to assess the possible effects of erythromycin on IKs. In another series of experiments designed to assess the relative effect of erythromycin on IKr versus IKs, we exposed the cells to erythromycin in the presence and absence of E-4031, a specific IKr blocker (4 mmol/ liter [K+]o and 4 μmol/liter nifedipine) (21,23). The transient outward current was not blocked, but it had little influence on our measurement of IK because of its fast inactivation kinetics (24).
In another set of experiments, steady state current-voltage (I-V) relations were determined using 2-s hyperpolarizing and depolarizing voltage steps applied from a holding potential of -40 mV. Nifedipine (4.0 μmol/liter) and ouabain (3.0 μmol/liter) were used throughout the course of these experiments to block the Ca2+ and Na+/K+ pump currents, respectively; the Na+ current was inactivated by the holding potential at - 40 mV. Cell capacitance was calculated by integrating the area under the uncompensated capacitance transient produced by a 10-mV hyperpolarizing step from 0 mV and dividing this area by the voltage step. The average access resistance (the sum of the pipette resistance and the residual resistance of the ruptured patch) was 5.1 ± 0.91 Mil (mean ± SD, n = 21), estimated by dividing the time constant tau of the decay of the capacitance transient by the calculated cell membrane capacitance (25). The membrane currents recorded in the present study were <500 to 600 pA in most cases. Thus, the maximal voltage error caused by the series resistance would be expected to be on the order of 2 to 3 mV.
All measurements of IK reported were obtained between 5 and 12 min after rupture of the plasma membrane. In previous studies (19), we demonstrated that rundown of IK is not observed for at least 15 min after membrane rupture.
A personal computer equiped with 12-bit analog-digital/digital-analog converters (DIGIDATA 1200, Axon Instruments, Inc.) was used for data acquisition and generation of pulse template and command potentials for both current- and voltage-clamp modes (pCLAMP software, Axon Instruments, Inc.). Currents were filtered with a four-pole Bessel filter at 0.5 to 1 kHz and digitized at 1 kHz.
Statistics. Statistical analysis of the data was performed using one-way analysis of variance coupled with the Scheffé or Tukey procedure or with a Student ttest (SigmaStat software package, Jandel Scientific).
Drugs. Erythromycin HCl (Sigma) was prepared fresh daily as a stock solution of 10 or 50 mg/ml. It was added to the Tyrode's perfusate to obtain final concentrations of 10 to 100 μg/ml. Nifedipine (Calbiochem-Novabiochem Corp.) and E-4031 (Eisai Co., Ltd., Tokyo, Japan) were prepared fresh before each use.
Action potential experiments.Figure 1 to 5 illustrate the characteristics of the three principal cell types encountered in the free wall of the canine left ventricle and their responsiveness to erythromycin. Figure 1 shows the time course of development of the actions of erythromycin on transmembrane activity recorded in a transmural strip isolated from the canine left ventricle. Shown are recordings obtained from epicardial, endocardial and deep subepicardial (M region) sites before and during a 2-h period of exposure to 50 μg/ml of erythromycin. At slow stimulation rates, the effects of erythromycin to prolong the APD are greater in cells from the M region than in those from the epicardial or endocardial regions of the left ventricular free wall. The APD prolongation reaches a quasisteady state after 30 min of exposure to the drug, although a very slow progressive increase is observed in the succeeding 90-min period. Qualitatively similar results were obtained in two other experiments. On the basis of these results, we designed the protocols for assessing dose-response relation (Fig 2).
Figure 2 illustrates the dose dependence of the actions of erythromycin to prolong the APD in the three cell types recorded along the length of a transmural left ventricular preparation. Transmembrane recordings were obtained before and 30 min after each increase in the concentration of the drug (10 to 100 μg/ml). The data demonstrate a clear dose-dependent effect of erythromycin to prolong the APD in the M cell but little effect of the drug on the APD in epicardial and endocardial tissues.
The effect of erythromycin to delay repolarization of the action potential is a sensitive function of rate. As Figure 3 illustrates, the dependence of the APD on rate is greatly exaggerated in the presence of erythromycin. This effect of the drug is barely noticeable in epicardial cells, modest in endocardial cells and profoundly exaggerated in M cells. Erythromycin prolonged the APD of the M cell to 370 ms at a BCL of 1,000 ms and to 1,800 ms at the slowest rate tested (BCL of 8,000 ms).
Summary data collected from preparations in which APD-rate relations were examined in the absence and presence of erythromycin (10 and 100 μg/ml) are presented in Figure 4. The effect of erythromycin to markedly prolong the APD in the M cell is clearly reverse rate (or use) dependent, whereas the much more modest effect of the drug to delay repolarization in epicardial and endocardial cells appears to be rate independent. These distinctions are maintained even when a single premature beat is used to examine the effects of erythromycin on the restitution of the APD at epicardial and M region sites (data not shown).
High concentrations of erythromycin (50 to 100 μg/ml) induced EADs in cells recorded from the M region (2 of 10 preparations) but not in those from the epicardial or endocardial regions of the canine left ventricular free wall. Figure 5 A illustrates an example of the differential effects of erythromycin (100 μg/ml) on the three cell types in a transmural preparation. At slow stimulation rates, the erythromycin-induced EADs in the M cells gave rise to triggered responses that often propagated to neighboring epicardial and endocardial regions, thus generating extrasystolic activity. The rate dependence of erythromycin-induced triggered activity and the ability of the triggered response originating in the M cell to reexcite ventricular epicardium are illustrated in Figure 5B.
Long QT intervals and TdP. The arrhythmogenic potential of these disparate cellular actions of erythromycin was further evaluated using an arterially perfused wedge of canine left ventricle in which we are able to simultaneously record transmembrane responses from several intramural sites along with a transmural ECG. Floating microelectrodes were used to record action potentials from subendocardial Purkinje, M region and epicardial cells. Figure 6 graphically illustrates the effect of erythromycin (100 μg/ml) to prolong the APD measured at 90% repolarization (APD90) in the three cell types. As with the results obtained using tissue slices, the response of the M cell action potential is much greater than that of epicardium. The drug-induced prolongation of the Purkinje action potential is greater than that of the M cell, consistent with the results previously reported by Rubart et al. (10). Not unexpectedly, the QT interval changes parallel those of the M cell action potential. Much of the difference is attributable to impulse conduction time to the M region and the difference between APD90 and the APD at 100% repolarization. These results indicate that the erythromycin-induced long QT interval is attended by the development of a marked dispersion of repolarization across the ventricular wall, even in intact wedges of left ventricle where the myocardial cells are electrically well coupled. As expected, electronic forces in the wedge serve to damp the dispersion of repolarization across the wall. Comparison of APD90 values presented in Figures 4 and 6 (BCL 2,000 ms) indicate that under control conditions, the average APD90 of the M cell is 37 ms shorter in the wedge than in the tissue slices (279 ± 9 vs. 316 ± 11 ms), whereas the APD90 of the epicardial cell is nearly 19 ms longer in the wedge than in the tissue slices (231 ± 10 vs. 212 ± 9 ms) and that after erythromycin (100 μg/ml), the average APD90 of the M cell is 131 ms shorter in the wedge than in the tissue slices (357 ± 11 vs. 488 ± 107 ms), whereas the APD90 of the epicardial cell is nearly 52 ms longer in the wedge than in the tissue slices (280 ± 18 vs. 228 ± 24 ms).
In a series of four experiments we used programmed stimulation to assess the arrhythmogenic potential of these actions of erythromycin. Figure 7 presents an example of induction of an episode of polymorphic ventricular tachycardia in which the QRS complex is seen to twist about the isoelectric line, typical of TdP. A premature beat elicited at an S1-S2 interval of 220 ms is observed to initiate what can best be described as an intramural reentry showing a 4:3 Wenckebach conduction of the impulse from epicardium to the M region.
Figure 8 illustrates an example of a longer run of TdP recorded in the same preparation. Erythromycin (100 μg/ml) produced a marked dispersion of repolarization across the wall, with the M cell manifesting much longer APDs than epicardium. A premature stimulus applied to epicardium at an S1-S2 of 220 ms initiates a run of TdP that persists for -7 s before self-terminating. Similar results were obtained in three of four preparations exposed to erythromycin (100 μg/ml). In all cases, basic stimuli were applied to the endocardial surface at a BCL of 2,000 ms, and the premature beats were applied to the epicardial surface at S1-S2 intervals of 200 to 270 ms. Although TdP was inducible at shorter BCLs (e.g., 1,500 ms), we did not systematically evaluate the stimulation criteria in this study. In the three experiments in which TdP was readily induced with an S2 delivered to the epicardium, TdP could not be induced when the S2 was delivered to the endocardial or M region (twice diastolic threshold intensity). When the stimulus intensity was increased to 5 to 10 times diastolic threshold, TdP could be observed after endocardial stimulation (one of three). The only preparation that failed to manifest TdP was the smallest of the four. Prominent EADs or EAD-induced triggered activity were never observed. Programmed stimulation applied during the predrug control period or in our time controls (4 h of perfusion) failed to induce TdP (0 of 11 preparations).
Ionic current experiments. The mechanism underlying the effect of erythromycin to delay repolarization and to induce EAD and triggered activity in canine ventricular M cells was probed in a series of experiments using whole-cell patch-clamp techniques applied to myocytes isolated from the M region of the canine left ventricular free wall. The primary focus of these studies was on the outward potassium currents IKr and IKs and the inward rectifier current (IK1).
Effect of erythromycin on IK. IK was initially recorded in cells bathed in Tyrode's solution containing 4 mmol/liter [K+]o and 4 μmol/liter nifedipine using either 5,000- or 250-ms depolarizing pulses applied from a holding potential of -40 mV to progressively more positive potentials (-20 to +60 mV). Figure 9 shows the effect of erythromycin on IK, recorded using 5,000-ms pulses. Figure 9A shows typical currents recorded before and after the addition of erythromycin (100 μg/ml). The drug significantly inhibits both the developing (Fig 9B) and tail currents (Fig 9C). Similar results were obtained in another group of cells in which IK was measured using 250-ms pulses (Fig 10). With both protocols, erythromycin-induced inhibition of the tail current is greater than that of the developing current. These results are consistent with a predominant effect of erythromycin to inhibit IKr.
Effect of erythromycin on IKr versus IKs. As a test of this hypothesis, we performed an envelope of tails test in the presence and absence of erythromycin (100 μg/ml). If IK is due to conductance of a single type of channel, the envelope of tails test (26) predicts that the magnitude of tail currents recorded after depolarizing pulses of progressively longer duration should increase parallel to the time course of activation of the developing outward current recorded during the pulse. In other words, the ratio of tail current to time-dependent developing current should be constant, regardless of the pulse duration. The data presented in Figure 11 are from midmyocardial cells depolarized from a holding potential of - 40 to + 40 mV for pulse durations ranging between 200 and 5,000 ms. The interpulse interval was 20 s, sufficiently long to permit complete deactivation of the current. In the absence of erythromycin, the ratio of the tail and developing currents is nearly constant (0.46) for pulse durations > 1,000 ms but increases progressively with shorter pulses. In the presence of erythromycin (100 μg/ml), the ratio of the tail and developing currents was constant (0.38) at all pulse durations. This value is close to the predicted ratio (0.4) calculated from the ratio of the driving force at + 40 and - 40 mV for a nonrectifying, K+-selective outward current. Thus, the envelope of tails test was satisfied only in the presence of 100 μg/ml of erythromycin. Once again the data point to a predominant effect of erythromycin to reduce IKr.
To assess the effect of erythromycin on IKs, we measured the effect of erythromycin on IK in the presence of the methanesulfonamide E-4031 (5 μmol/liter) and in Tyrode's solution containing 0 mmol/liter [K+]o. Both interventions are known to largely eliminate IKr (19,21,27). Figure 12 (A and D) illustrates representative current tracings recorded in cells bathed in Tyrode's solution containing 5.0 μmol/liter E-4031 (Fig 12A) or 0 mmol/liter [K+]o (Fig 12D) in the absence and presence of erythromycin. From a holding potential of - 40 mV, the cells were depolarized to progressively more positive potentials for 5,000 ms and then returned to - 40 mV. The developing current as a function of test potential before and after erythromycin (100 μg/ml) in the presence of 5.0 μmol/ liter E-4031 (n = 7) or 0 mmol/liter [K+]o (n = 6) is plotted in Figure 12, B and E, respectively; the cumulative data for the tail currents are plotted in Figure 12, C and F. These results indicate that 100 μg/ml of erythromycin exerts no effect on IKs. The absence of IKr was substantiated by the fact that the envelope of tails test is satisfied under these conditions (not shown).
Effect of erythromycin on steady state I-V relation. The effect of erythromycin on IK1 was assessed in another series of six experiments. Steady state I-V relations were constructed by plotting the current levels recorded at the end of 2-s pulses from a holding potential of - 40 mV to test potentials ranging between -100 and 0 mV. Measurements were made in the presence of nifedipine (4.0 μmol/liter) to inhibit inward Ca2+ current and ouabain (3.0 μmol/liter) to inhibit Na+/K+ pump current before and after erythromycin (100 μg/ml). The Na+ current was inactivated by a holding potential of -40 mV. Figure 13 A shows the representative current tracings recorded before and after exposure of the cell to erythromycin. Figure 13B shows cumulative data of current density as a function of test voltages before and after erythromycin. Clearly, the drug exerts no effect on steady state I-V relations, suggesting that erythromycin has no effect on IK1.
Our findings demonstrate for the first time an effect of erythromycin, a widely prescribed macrolide antibiotic, to produce prominent action potential prolongation and EAD-induced triggered activity in M cells but not epicardial or endocardial cells in the free wall of the canine left ventricle. The marked dispersion of repolarization created across the ventricular wall is shown to set the stage for the development of polymorphic ventricular arrhythmias displaying the ECG manifestation of TdP. Our voltage-clamp results point to erythromycin-induced block of IKr as a prominent mechanism contributing to the prolongation of repolarization in the M cells.
In the canine heart, endocardial, epicardial and M region action potentials differ principally with respect to repolarization characteristics. Epicardial and M cell action potentials commonly display a spike and dome configuration due to the presence of a distinct early repolarization phase (phase 1). In addition, M cell action potentials differ from those of epicardial and endocardial cells with respect to phase 3, showing a delayed repolarization phase, especially at slow stimulation rates. A smaller contribution of IKs has been shown (19) to participate in determining this unique repolarization properties of the M cell. This ionic distinction is also thought to contribute to the exceptional sensitivity of the M cell to a wide variety of agents with class III actions (5,13,14). Our findings indicate that erythromycin, like other APD-prolonging agents, targets the M cells in the deep structures of the ventricular myocardium. The effects of erythromycin on the action potential of the M cell are similar to those previously described (10) in canine Purkinje fibers. In both isolated Purkinje and M cell tissues, erythromycin produces a major dose-dependent prolongation of the APD, as well as EADs and EAD-induced triggered activity. The EADs were observed in only 20% of M cell tissue slices. In contrast, erythromycin in concentrations as large as 100 μg/ml never produced EAD or triggered activity and only a modest prolongation of APD in canine ventricular epicardial and endocardial tissue slices. Because EAD activity was observed in only 20% of M cell tissues and in 0% of endocardial or epicardial preparations, it is not surprising that the drug produced no EADs in the arterially perfused left ventricular wedge, where the electrotonic influences of epicardium and endocardium would be expected to diminish the extent of APD prolongation and prevent the appearance of EADs in the M region. The higher levels of interstitial [K ]0 expected in the intact wall preparation may have also contributed to the absence of erythromycin-induced EAD activity in the arterially perfused left ventricular wedge preparation (28).
Action potential prolongation and EADs are known to result from 1) a reduction in the availability of K+ currents that contribute to repolarization (IK and IK1); 2) an increase in the availability of the inward Ca2+ current; 3) a delay in Na+ current inactivation, giving rise to an increase in late Na+ current (5); or 4) an increase in the contribution of electrogenic current generated by the Na+-Ca2+ exchanger (29). Rubart et al. (10) reported that in Purkinje fibers, erythromycin (100 μg/ml) dramatically prolongs the APD but does not affect maximal rate of rise of the action potential upstroke (Vmax), action potential amplitude or developed tension. They concluded that the effects of erythromycin on APD are unlikely to be mediated through actions of the drug on fast Na+ and Ca2+ inward currents. Augmentation in the level of tetrodotoxin-sensitive slowly inactivating Na+ current (late Na+ current or window current) (30–33) was also discounted by Rubart et al. (10) on the basis of the effects of erythromycin after tetrodotoxin. Pretreatment of the Purkinje fibers with erythromycin was shown to antagonize the effect of dofetilide, an IKr blocker. On the basis of this observation, Rubart et al. suggested that the effect of erythromycin to prolong the action potential is through block of IK. In the present study, we demonstrated a similar effect of erythromycin on the action potential of M cells and provide a direct test of the hypothesis that IK block contributes to these changes.
In some species, the delayed rectifier potassium current is composed of two components or currents (IKr and IKs) (19,21,23). The two components are distinguished by their activation kinetics, rectification and sensitivity to methanesulfonamide agents displaying class III actions, such as E-4031. Daleau et al. (12) recently showed that erythromycin blocks IKr but not the inward Ca2+ current or IK1 in guinea pig ventricular myocytes (unknown ventricular origin). The two components of IK are larger in guinea pig than in dog ventricular myocytes under control conditions (19). Our data show that erythromycin (100 μg/ml) inhibits IK elicited by stepping from a holding potential of - 40 mV to progressively more positive potentials in M cells (Figure 9 and Figure 10). The envelope of tails test is satisfied only in the presence of erythromycin (Figure 11), suggesting that erythromycin is a potent IKr blocker in the canine ventricle. Under conditions designed to eliminate IKr (5.0 μmol/liter E-4031 or 0 mmol/liter [K+]o), erythromycin (100 μg/ml) exerts no effect on IK or the envelope of tails test (Figure 12), indicating that IKs is unaffected by the drug. It is well known that IK1 also plays an important role in the regulation of the APD. A lack of an effect of erythromycin on the steady state I-V relations (Figure 13), indicates that inhibition of IK1 does not contribute to the effects of the drug to prolong the APD in the canine ventricle. In summary, our whole-cell patch-clamp data point to IKr block as a mechanism responsible for the effect of erythromycin to delay repolarization and induce EAD activity, although they do not exclude the possible participation of other mechanisms.
Physiologic and clinical implications. Our findings show that the actions of erythromycin to markedly prolong APDs and induce EAD and triggered activity in a select population of cells (M cells) in ventricular myocardium can lead to the development of a prominent dispersion of repolarization and refractoriness within the ventricle, setting the stage for the induction of tachyarrhythmias such as TdP. Our results obtained using the arterially perfused left ventricular wedge suggest circus movement reentry as the basis for the maintenance of TdP. There are several lines of evidence that point to reentry as a likely mechanism: 1) the presence of a marked dispersion of repolarization and refractoriness after erythromycin; 2) the ability to most easily induce the arrhythmia using a single premature beat introduced at the site of earliest repolarization (epicardium); and 3) the occurrence of maintained arrhythmic activity only in larger preparation. These are all well established hallmarks of circus movement reentry. The absence of any other obvious arrhythmogenic sources such as EAD-induced triggered activity, delayed afterdepolarization (DAD)-induced triggered activity or abnormal automaticity (based on direct recording or expected behavior) also leaves reentry as the most likely mechanism.
The ability to induce TdP in the wedge using programmed electrical stimulation (PES) applied to epicardium is consistent with similar observations made in in vivo models of TdP. Programmed electrical stimulation-induced TdP is the rule rather than the exception in recently developed in vivo models of acquired long QT syndrome and TdP (34,35). In the clinic, the onset of TdP has long been known to follow a short-long-short cycle length sequence (36,37). Recent clinical reports indicate that a sudden moderate acceleration from an initially slow heart rate when followed by an intrinsic or extrinsic extrasystole holds the highest risk for induction of TdP in patients with long QT syndrome (38) as well as in animal models with acquired long QT syndrome (34,39–41). The behavior of our preparation is consistent with both clinical and experimental observations.
The absence of prominent EADs or EADs-induced triggered activity in the left ventricular wedge preparations in which TdP was readily and reproducibly inducible argues against the hypothesis that EAD-mediated triggered activity is responsible for the maintenance of TdP (5). Our results provide support for the hypothesis that TdP is maintained through a reentrant mechanism but leaves open the possibility that the arrhythmia may be induced (precipitated) by an EAD-triggered response, as suggested by Rubart et al. (10) or by an extrasystole from some other source (intrinsic or extrinsic).
Elucidation of the specific mechanism or mechanisms underlying the induction and maintenance of TdP requires further studies in which a greater number of intracellular, extracellular or monophasic action potentials are simultaneously recorded from the arterially perfused wedge preparation. Alternatively, voltage-sensitive dye techniques can be used to probe the complexities of TdP in this preparation. Both approaches are currently underway. Preliminary results using voltage-sensitive dye recording techniques to generate high resolution maps indicate that intramural reentry is responsible for the maintenance of TdP in wedge preparations pretreated with IKr blockers (Yan G-X, Rosenbaum D, Akar F, Antzelevitch C. Unpublished observations).
The disproportionate prolongation of the M cell action potential in response to erythromycin may underlie the prolongation of the QTU interval on the ECG and the development of TdP in some patients given the drug (6–9,11,42–44), especially those with the congenital long QT syndrome (45).
Summary. We examined the effects of erythromycin in isolated myocytes, tissues and intact left ventricular wall preparations so as to integrate information at these various levels. The results demonstrate how inhibition of an outward current can lead to important heterogeneities of repolarization in tissues spanning the left ventricular wall, thus setting the stage for arrhythmias with all the characteristics of TdP. The study provides a direct link between the degree of IKr inhibition (similar to that found in patients with LQTS with the chromosome 7 or HERG[human ether-à-go-go-related gene] defect) and arrhythmogenesis.
Limitations of the study. The concentrations of erythromycin used in our study (10 to 100 μg/ml) are higher than those normally measured in human plasma (46). This fact notwithstanding, use of these levels in our experimental protocols is not without clinical relevance for the following reasons: 1) Plasma concentrations of erythromycin have not been measured in the vast majority of cases of erythromycin-induced long QT intervals and TdP in the acquired long QT syndrome; 2) serum concentrations in healthy volunteers with normal liver function have been shown (46) to reach levels as high as 30 μg/ml; 3) in the dozen or more cases described recently (47–49), long QT syndrome usually develops in patients also taking disopyramide, terfenadine and amiodarone and immunosuppressants, such as pentamidine, each individually capable of prolonging the QT interval; 4) the effect of IKr blockers, of which erythromycin is one, to produce long QT intervals and to precipitate TdP is generally greatly enhanced by hypokalemia and hypomagnesemia as well as by various forms of heart disease, including congenital long QT syndrome.
The effect of these agents and disease states to potentiate the QT-prolonging effects of erythromycin is largely anecdotal because of the small number of reported clinical cases. However, numerous clinical and animal studies have demonstrated the ability of similar drugs, electrolyte imbalances and disease states to predispose patients or animals receiving IKr blockers to the development of long QT intervals and TdP.
Although we have interest in assessing these interactions, we considered it more important to establish a baseline for the dose-response relation and to gain an understanding of the mechanisms responsible. The experimental protocols used in this study are in keeping with these objectives.
Our study demonstrates significant prolongation of the canine M cell action potential with an erythromycin concentration as low as 10 μg/ml (the lowest concentration studied [Fig 2]), with dramatic prolongation of the action potential at concentrations of 50 and 100 μg/ml. Rubart et al. (10) observed a similar response of isolated canine Purkinje fibers over a range of 20 to 200 μg/ml; whereas Daleau et al. (12) observed a much more modest increase in endocardial monophasic APD in the guinea pig heart using an erythromycin concentration of 75 μg/ml (100 μmol/liter).
Erythromycin inhibition of the hepatic P450 metabolic pathway may further predispose some patients to long QTU intervals and TdP because many agents that cause long QTU intervals are metabolized by this route (e.g., terfenadine, astemizole and disopyramide) (50–53).
This study was supported by Grants HL37396 and HL47678 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; by a fellowship grant from the New York Affiliate of the American Heart Association; and by grants from Dolgeville Lodge 796, Dolgeville, New York, and the Sixth and Seventh Manhattan Masonic Districts, New York, New York. E-4031 was kindly donated by EISAI Co., Ltd., Tokyo, Japan.
- Received July 8, 1996.
- Accepted August 13, 1996.
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