Proarrhythmic and Antiarrhythmic Effects of Bupivacaine in an In Vitro Model of Myocardial Ischemia and Reperfusion 

Care of the animals conformed to the recommendations of the Helsinki Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture.

Guinea pigs of either sex weighing 300–400 g were killed after brief anesthesia with ether. The hearts were quickly removed and placed in oxygenated Tyrode's solution at room temperature. A thin myocardial strip was dissected longitudinally from the free wall of the right ventricle and pinned with the endocardial surface upward in a special perfusion chamber. [22–25]This chamber (5 ml) is bisected by a thin latex membrane containing a centrally located hole that allowed the preparation to be passed carefully through and divided into two zones called the normal zone (NZ) and the altered zone (AZ)(Figure 1). The two compartments were perfused independently at the rate of 2 ml/min with Tyrode's solution oxygenated with 95% oxygen and 5% carbon dioxide. The Tyrode's solution was composed of 135 mM Na⁺, 4 mM K (⁺), 1.8 mM Ca²⁺, 1 mM Mg²⁺, 1.8 mM H²PO^4-, 25 mM HCO^3-, 117.8 mM Cl^-, and 5.5 mM glucose. The pH was 7.35 +/- 0.05, and the temperature was maintained at 36.5 [degree sign]C with thermostated water circulation (Polystat 5HP; Bioblock, Illkirch, France). At the end of each experiment, absence of leak between the two compartments was tested by injecting methylene blue dye into one of the two compartments.

The myocardial strips were stimulated at a frequency of 1 Hz by two bipolar Teflon-coated steel wire electrodes positioned in the NZ and the AZ. A commutator allowed us to apply the stimulation to the preparation by one or the other stimulating electrode. Stimuli were rectangular pulses lasting 2 ms and twice the diastolic threshold intensity delivered by a programmable stimulator (model SMP 310; Biologic, Grenoble, France). Preparations that needed pulses stronger than 5 V to elicit AP were discarded because there could be a conduction block at the level of the latex separating membrane. During the protocol, stimulation was stopped whenever sustained spontaneous arrhythmias occurred. An extrastimulus that lasted 2 ms and was twice the diastolic threshold amplitude was applied every four stimulations in an attempt to elicit extrastimulus-induced repetitive responses. The coupling time interval between the stimulus and the extrastimulus was divided into increments by 5-ms steps from the effective refractory period to the total repolarization duration. Transmembrane potentials were recorded simultaneously in both myocardial regions using glass microelectrodes filled with 3 M KCl, and the tip resistance ranged from 10–30 M Omega. The intracellular microelectrodes were coupled to the input stages of a homemade high-impedance capacitance-neutralizing amplifier. The transmembrane recordings were displayed on a memory dual-beam storage oscilloscope (Gould Instruments Systems, Cleveland, OH). The following AP characteristics (Figure 2) were automatically stored and measured by a cardiac AP automatic acquisition system and processing device (DATA-PAC; Biologic): resting membrane potential, action potential amplitude, action potential duration at 50% of repolarization (APD⁵⁰) and at 90% of repolarization (APD⁹⁰), and maximal upstroke velocity (V^max). Whenever possible, the same impalement was maintained throughout the experiment. When impalement was lost during measurement, readjustment was attempted. If the readjusted parameters deviated by no more than 10% from the previous ones, experiments were continued, otherwise they were terminated.

During a 60-min equilibration period, the two compartments were perfused with normal Tyrode's solution. Thereafter, simulated ischemia was induced and maintained for 30 min in one compartment (AZ) by superfusion with a modified Tyrode's solution, whereas the other compartment remained in normal conditions (NZ;Figure 1). The modified Tyrode's solution differed from normal by an elevated potassium concentration (from 4 to 12 mM); decreased bicarbonate concentration (from 25 to 9 mM), leading to a decrease in pH (from 7.35 +/- 0.05 to 7.00 +/- 0.05); a decrease in oxygen tension by replacement of 95% oxygen and 5% carbon dioxide with 95% nitrogen and 5% carbon dioxide; and withdrawal of glucose. As previously reported, [22–25]the present modifications, which combined hypoxia, hyperkalemia, acidosis, and lack of substrates, are similar to those reported by Morena et al., [26]who reproduced in vitro the electrophysiologic abnormalities induced in vivo by ischemia. At the end of the ischemia period, reperfusion was simulated by perfusing the AZ chamber with normal Tyrode's solution for 30 min (the reperfusion period).

During simulated ischemia and reperfusion, several myocardial conduction disturbances and arrhythmias were recorded:(1) myocardial conduction blocks between the two regions;(2) loss of responsiveness in the myocardial tissue, considered when the preparation failed to elicit AP regardless of the compartment stimulated, with a constant stimulation intensity;(3) extra-stimulus-induced repetitive responses, defined as one, two, or a salvo of spontaneous extrasystoles induced by a single extrastimulus; and (4) spontaneous repetitive responses such as sustained activities (fewer than 10 spontaneous APs) independent of the stimulation.

After the 60-min equilibration period, during the simulated ischemia and reperfusion phases, plain bupivacaine diluted in Tyrode's solution at 1 micro Meter, 5 micro Meter, or 10 micro Meter (each n = 12), or Tyrode's solution alone (n = 12) was perfused in random order in both compartments (NZ and AZ). Thus the electrophysiologic effects of bupivacaine on AP parameters and the incidence of arrhythmias were investigated simultaneously in normal (NZ) and altered (AZ) conditions.

All results were expressed as mean +/- SD. Categorical variables were compared using the chi-square test with Yates correction as appropriate. Multiple comparison of continuous variables was performed by two-way analysis of variance followed by comparison with control or initial values using Dunnett's test. Differences were considered significant when P < 0.05.

Accounting for losses on impalement during the experiments, data analysis was performed on n = 12 in the control group, n = 11 in the 1 micro Meter bupivacaine group, n = 10 in the 5 micro Meter bupivacaine group, and n = 9 in the 10 micro Meter bupivacaine group.

As summarized in Table 1, simulated ischemia rapidly induced alterations of AP time course parameters. After 5 min, simulated ischemia induced significant membrane depolarization (P < 0.01), V^maxand action potential amplitude reduction (P < 0.01), and APD⁵⁰and APD⁹⁰shortening (P < 0.01). The maximal electrophysiologic effects occurred and reached a plateau within the first 10 min. In the NZ, all these AP parameters remained unchanged after 30 min and even after 60 min (see Reperfusion -30 min, in Table 1) of superfusion of normal Tyrode's solution. Reperfusion of the AZ was associated with a rapid recovery of the AP parameters. Electrophysiologic effects induced by simulated ischemia were rapidly reversed within 10 min of reperfusion for resting membrane potential, V^max, and action potential amplitude and within 20 min for APD⁵⁰and APD⁹⁰(to 114 +/- 38 ms and 143 +/- 28 ms, respectively).

As shown in Table 2, there was no significant difference in initial AP parameter values for the four experimental groups. Figure 3shows the percentage variations of the AP parameters at 10 min of simulated ischemia in NZ and AZ with and without increased bupivacaine concentrations. Because of the high incidence of conduction block leading to inexcitability of the stimulated AZ, the results are shown at 10 min of the ischemic phase, at which time it was possible to measure AP parameters. In NZ, V^maxwas significantly reduced by bupivacaine at 5 and 10 micro Meter (respectively, -26 +/- 27% and -27 +/- 24% after 30 min, P < 0.05 compared with the control group). Unlike APD⁹⁰, which decreased in NZ in the presence of bupivacaine during the simulated ischemic period (Figure 3), resting membrane potential, action potential amplitude, and APD⁵⁰remained unchanged. In AZ, the V^maxdecrease induced by ischemia was worsened only in the presence of 10 micro Meter bupivacaine (-89 +/- 15% after 10 min compared with a decrease of -69 +/- 15% after 10 min of ischemia with no drug, P < 0.05). The time course of recovery of resting membrane potential, action potential amplitude, APD⁵⁰, and APD⁹⁰during reperfusion in the AZ was similar in the control and treated groups and reached initial values, with the exception of V^max, which remained significantly depressed after 30 min of reperfusion in the presence of 5 and 10 micro Meter bupivacaine (respectively, 234 +/- 60 V/s and 200 +/- 63 V/s after 30 min of reperfusion compared with 264 +/- 79 V/s and 309 +/- 105 V/s as initial values, P < 0.01). In NZ, the APD⁹⁰) shortening, measured during exposure to the altered conditions in the presence of 1, 5, and 10 micro Meter bupivacaine, was reversed during the reperfusion phase (respectively, APD⁹⁰, 166 +/- 20 ms, 153 +/- 21 ms, and 152 +/- 20 ms after 30 min of the reperfusion phase compared with 160 +/- 20 ms, 156 +/- 29 ms, and 157 +/- 17 ms as initial values).

As illustrated in Figure 4, conduction changes observed during simulated ischemia in the presence of bupivacaine were characterized by the occurrence of unidirectional conduction block in AZ (Figure 4(A, B)) and loss of responsiveness of the AZ to stimulation (Figure 4(C, D)), followed by their removal during reperfusion (Figure 4(E, F)).

During simulated ischemia, the mean occurrence time of conduction block was evaluated in the presence of 1, 5, and 10 micro Meter bupivacaine. Conduction blocks occurred after 17 +/- 6 min in the control group, 14 +/- 7 min (NS) in the presence of 1 micro Meter bupivacaine, and after 9 +/- 3 min (P < 0.05) with both 5 micro Meter and 10 micro Meter bupivacaine. Further, as shown in Figure 5, 1, 5, and 10 micro Meter bupivacaine enhanced the incidence of loss of responsiveness to stimulation in the AZ. At the end of the ischemic period in the control group, 17% of preparations were unexcitable in their AZ (control), whereas the incidence of responsiveness loss reached 55% in the 1 micro Meter bupivacaine group and 100% in the presence of 5 and 10 micro Meter bupivacaine. As shown in Figure 6, reperfusion of the AZ induced a rapid recovery of responsiveness that was similar in the four experimental groups.

As Figure 7shows, two types of arrhythmias occurred during simulated ischemia and reperfusion: repetitive responses induced by an extrastimulus (Figure 7(A)) and spontaneous repetitive responses (Figure 7(B)). During simulated ischemia, superfusion with bupivacaine (5 and 10 micro Meter) drastically suppressed extrastimulus-induced and spontaneous repetitive responses (Figure 5). However, in the presence of the lowest concentration of bupivacaine (1 micro Meter), the number of preparations exhibiting spontaneous repetitive responses was significantly higher (8 of 11) than in the control group (3 of 12; P = 0.02). During reperfusion, the extrastimulus-induced arrhythmias observed in the control group were inhibited by the concentrations of bupivacaine, and the spontaneous arrhythmias were inhibited by the two highest bupivacaine concentrations (Figure 6).

This study had several results:(1) Ischemic conditions enhanced the V^maxdecrease induced by bupivacaine (10 micro Meter), (2) bupivacaine (5 and 10 micro Meter) shortened the mean time to the occurrence of conduction block during ischemia, (3) bupivacaine cardiotoxicity was enhanced by ischemia, resulting in loss of excitability of the ischemic myocardium, (4) 5 and 10 micro Meter bupivacaine decreased the incidence of ischemia-induced arrhythmias, whereas 1 micro Meter of the drug enhanced them, and (5) 1, 5, and 10 micro Meter bupivacaine decreased the occurrence of reperfusion-induced arrhythmias.

To simulate ischemic conditions, we used a modified Tyrode's solution and combined acidosis, hyperkalemia, hypoxia, and the lack of substrates because, as previously described in detail, [23–26]the association of these components can reproduce the electrical alterations on cardiac APs observed in more complex in vivo animal models during acute myocardial ischemia. [18]The electrical modifications measured in this study, namely resting membrane depolarization, decrease in AP amplitude and V^max, AP shortening, and lengthening of myocardial conduction times, are comparable to those found in other in vitro studies using similar ischemic-like solutions [18,26,27]and also to in vivo investigations during coronary artery occlusion. [28]Further, the reliability of the double-bath technique, as demonstrated by the constancy of the AP parameters in the NZ adjacent to the AZ submitted to simulated ischemia, made it possible to investigate the electrophysiologic effects of bupivacaine simultaneously in normoxic and ischemic-reperfused myocardium, as might occur in vivo during ischemia.

The bupivacaine concentrations of 1, 5, and 10 micro Meter used in our in vitro study were chosen to account for clinically observed bupivacaine plasma concentrations and drug concentrations used during in vivo investigations. After intravenous injection of bupivacaine in awake, unanesthetized sheep, the clinical toxicity was observed with a whole-blood concentration that ranged from 3–11 micro gram/ml. [6]Assuming a blood-to-plasma concentration ratio of 0.73, [29]this should correspond to a plasma dose of 4–15 micro gram/ml. Because bupivacaine is 66–88% bound to plasma proteins for this concentration range, [30]the free form of the drug should be approximately 0.5–5 micro gram/ml (1.5–15 micro Meter). These concentrations of free bupivacaine can easily be achieved after accidental intravascular injections in humans. To clarify the mechanisms implicated in cardiotoxic effects of an ischemia-bupivacaine interaction, we used 5 and 10 micro Meter of the drug, which corresponds to the clinically relevant range for toxicity, and 1 micro Meter, which is slightly less than the range of toxic doses (1.5–15 micro Meter).

In normoxic conditions, our results showed a significant (P <0.05) slowing of V^maxinduced by 5 and 10 micro Meter bupivacaine but not by the 1-micro Meter dose. These effects can be explained by bupivacaine's ability to block sodium channels, particularly in their inactivated state, as reported by Clarkson and Hondeghem. [9,31]In our in vitro model of myocardial ischemia, bupivacaine affected V^maxonly slightly in AZ during simulated ischemia compared with its effects under normoxic conditions. A likely explanation might be the ischemic conditions used, which mimicked acute myocardial ischemia and already dramatically depressed V^max, even in the absence of bupivacaine. Thus 10 micro Meter bupivacaine only significantly worsened the V^maxdecrease induced by simulated ischemia. These effects of the ischemia-bupivacaine interaction on V^maxmay be due to voltage-dependent inactivation of sodium channels at depolarized membrane potentials. Using a single sucrose gap voltage-clamp technique in guinea pig ventricular muscle, Clarkson and Hondeghem [9]clearly showed that 1 micro gram/ml bupivacaine (3.5 micro Meter) shifted the voltage dependence of V^maxavailability toward hyperpolarized potentials by 10.7 +/- 2.6 mV (P < 0.01). This shift of sodium channel availability, when combined with ischemia-induced depolarization, may substantially increase the fraction of sodium channels in the inactivated state, and it probably explains the larger reduction of V (^max) with ischemia in the presence of bupivacaine.

We also observed a shortening of APD⁹⁰on normoxic tissue in preparation treated with bupivacaine while ischemia was simulated on the adjacent myocardial zone. It seems unlikely that this APD⁹⁰decrease was a result of the action of bupivacaine alone on the AP duration because the APD⁹⁰shortening observed in normoxic tissue was suppressed when reperfusion was performed on the adjacent compartment; in other study, [9]APD⁹⁰decreases only with concentrations higher than those used in the current study. The APD⁹⁰modifications, observed in the “normal” zone of our preparations treated with bupivacaine, could result as a consequence of the anatomic continuity between “ischemic” and “normoxic” myocardial regions. Kupersmith et al. [32]recently reported that AP durations and membrane potential inhomogeneities in sheep Purkinje fibers led to electronic transmission of an injury current to border zones adjacent to zones of abnormal APD changes. The cable properties of the myocardial tissue altered during simulated ischemia might be implicated in changes in APD and in the emergence of arrhythmias, particularly those involving reentry mechanisms.

During simulated myocardial ischemia, bupivacaine (5 and 10 micro Meter) dramatically decreased the mean occurrence time of conduction blocks. This marked depressant effect of bupivacaine on conduction led to the excitability loss of the ischemic myocardial tissue. In previous in vivo and in vitro studies, bupivacaine induced significant increases in atrial and ventricular conduction times with no conduction block. [33–35]In these investigations, the authors used moderate doses of bupivacaine (2 micro gram/ml plasma concentration, leading to an estimated free form of bupivacaine of 0.5–1.5 micro Meter). In these studies, investigations were performed in healthy animals or isolated hearts, whereas in our in vitro model we studied the cardiotoxic effects of bupivacaine during simulated ischemia. When compared with results obtained on healthy myocardium, our findings suggest that myocardial ischemia reinforces certain cardiotoxic effects of bupivacaine at a “nontoxic” 1 micro Meter concentration. However, care should be taken when extrapolating these results to the clinical setting.

During ischemia, bupivacaine decreased the incidence of extrastimulus-induced arrhythmias at all three concentrations and spontaneous arrhythmias at 5 and 10 micro Meter bupivacaine. On awake or anesthetized animals, hypoxia and acidosis increased the likelihood that bupivacaine would induce arrhythmias. [14,15]The antiarrhythmic effects of 5 and 10 micro Meter bupivacaine observed in our model of acute ischemia [18]differ from these latter findings. This might be explained primarily by the different in vivo and in vitro models and the type of ischemic conditions used, as Rosen et al. and Heavner et al. studied bupivacaine cardiotoxicity in acidotic-hypoxic [14]and hypoxic [15]conditions, respectively.

In addition to these reasons, we also hypothesized that there are differences in the mechanisms that may underlie the occurrence of arrhythmias. As previously discussed, [23]in our in vitro model of ischemic-reperfused myocardium, repetitive responses induced by an extrastimulus are likely a result of reentrant mechanisms between normal and ischemic myocardium. First, the representative arrhythmia induced by an extrastimulus illustrated in Figure 7(A) suggests reentry. Second, it is well established that, to occur, reentry movements require a site of unidirectional block and slow retrograde conduction. Thus, at all three concentrations, bupivacaine, which reduced the mean occurrence time to the onset of myocardial conduction blocks and led to the loss of excitability in the ischemic myocardium, might block pathways involved in reentry movements and impair reexcitation in healthy tissue. Spontaneous repetitive responses observed in our model are probably not related to early and delayed depolarizations, which were not observed in our experiments, but they may also be based on reentry or merely be associated with abnormal automaticity, perhaps induced by injury current occurring between myocardial zones with different electrical properties. [32]The loss of excitability induced in all experiments by 5 and 10 micro Meter bupivacaine might explain their antiarrhythmic effects, thus inhibiting the emergence of spontaneous arrhythmias. The promoting effect of 1 micro Meter bupivacaine on ischemia-induced spontaneous arrhythmias was accompanied by loss of ischemic tissue excitability in only 55% of preparations compared with 100% of preparations treated with 5 and 10 micro Meter of the drug. Although in normoxic conditions, De la Coussaye et al. [13]used epicardial mapping to show that bupivacaine prolongs longitudinal and transverse conduction velocity and facilitates induction of reentrant ventricular arrhythmias in isolated rabbit hearts. All these results suggest that, in our ischemic conditions, myocardial conduction with 1 micro Meter bupivacaine was not yet completely blocked, as it is in the presence of 5 or 10 micro Meter of the drug, but was sufficiently slowed to allow the emergence of spontaneous arrhythmias.

The mechanisms involved in arrhythmias that occur during reperfusion and were nearly prevented by bupivacaine in our experimental model are not yet well defined but might involve depletion of high-energy phosphates, sodium, or calcium overload and implication of reactive oxygen species. It cannot be ruled out that loss of responsiveness in the ischemic myocardium, induced by bupivacaine, may preserve high-energy phosphates in cells, and thus prevent the occurrence of certain arrhythmias during reperfusion. Although our investigations were performed on isolated ventricular walls, an adrenergic stimulation by cardiac catecholamines present in the myocardial strips cannot be excluded during the reperfusion phase, thereby encouraging the emergence of abnormal automatic activities. In support of this, we recently showed that the two beta-blocking agents propranolol and dl-sotalol exhibit anti-arrhythmic efficacy on the reperfusion-induced spontaneous arrhythmias in this in vitro model. [36]On the other hand, Kulier et al. [37]recently found that bupivacaine antagonizes epinephrine dysrhythmogenicity in conscious dogs susceptible to ventricular tachycardia and in anesthetized dogs with spontaneous postinfarct dysrhythmias, thus suggesting a possible interaction between bupivacaine and the adrenergic activity.

In conclusion, our in vitro study provided evidence of differential electrophysiologic effects of bupivacaine under simulated acute ischemic conditions in regard to the concentrations used. Loss of excitability of the myocardial tissue was observed in the presence of the two highest concentrations of bupivacaine (5 and 10 micro Meter), arising from a dramatic myocardial conduction slowing that resulted in conduction blocks. On the other hand, during simulated ischemia, a significant proarrhythmic effect occurred, with the lowest concentration of bupivacaine (1 micro Meter) considered as noncardiotoxic under normoxic conditions.

The authors thank Veronique Sabalos and Michel Morel for help in the computer analysis of the data, and Herve Tombette for technical assistance.