Since the observation of lipid rescue from bupivacaine (BPV)-induced cardiac arrest in rats by Weinberg et al,1 2 3–5 6 , 7 8 9 10 1 , 11
METHODS
To determine the time- and concentration-dependent effects of LE on contractility after achieving BPV toxicity, experiments were performed in isolated guinea pig myocardium, by applying LE in both the ongoing presence of BPV and during its washout. A broad range of concentration was applied to the covered clinical concentrations, as well as to those that might be achieved experimentally. To clarify the actions of LE, its time- and concentration-dependent effects were determined when LE was applied alone.
Preparation of Cardiac Muscles
According to a procedure approved by the Yonsei University College of Medicine Animal Research Committee, hearts were removed from 400 to 450 gm male guinea pigs after sevoflurane anesthesia. Right ventricular papillary muscles were excised, mounted horizontally in a tissue bath, and superfused (8 mL/min) at 37°C with modified normal Tyrode solution (Tyrode solution) (118 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1.2 mM MgSO4 , 25 mM NaHCO3 − , 11 mM glucose, and 0.10 mM EDTA). The solution was recirculated through a bath from a reservoir through which 95% O2 and 5% CO2 was bubbled (flow rate: 0.2 L/min) maintaining a pH of 7.4 ± 0.05. Tendinous end of the papillary muscle was attached to a Grass FT03 force transducer (Grass Instruments, Quincy, MA), while the other end of the muscle was pinned to the bottom of the tissue bath. For stabilization, muscles were field-stimulated using a Grass S44 stimulator (Grass Instruments, Quincy, MA) at 120% of the stimulus threshold at 0.5 Hz for 60 minutes. The forces were continuously recorded using a Powerlab Data Analysis System (Chart v.7.0; ADInstruments, Sydney, Australia).
Solution Preparation
Five hundred milliliters of commercial 20% LE contains 100 gm (107.9 mL) of soybean oil and 12 gm (11.6 mL) of lecithin, constituting the lipid phase (calculated 24% by volume). The 380-mL aqueous phase contains no electrolytes and 22.5 gm of glycerin. Therefore, the aqueous phase occupies approximately 75% of the volume in 20% LE. We added 10 mL of the 13.1-mL concentrated Tyrode solution (1.18 M NaCl: 10 mL, 0.5 M KCl: 1 mL, 200 mM CaCl2 : 1 mL, 120 mM MgSO4 : 1 mL, 210 mg NaHCO3 − , 198 mg glucose, and 100 μM EDTA: 0.1 mL) to 120 mL of 20% LE, so the aqueous volume became 100 mL because 90 mL of water was contained in the 120 mL of 20% LE, with approximately the same concentration of electrolytes and glucose concentration as in the Tyrode solution, and became an approximate 18% lipid concentration. To make 4% LE from 18% LE stock solution, the following calculation was utilized:
Thus, when 28.6 mL of 18% LE-Tyrode stock solution was added to 100 mL of Tyrode solution in a reservoir, it became a 4% LE concentration. The 20% Intralipid was purchased from Fresenius Kabi (Uppsala, Sweden). Oxfenicine and all other chemicals were purchased from Sigma-Aldrich Chemicals Company (St. Louis, MO).
Experimental Protocols
Recirculation Experiments.
After stabilization, a 1.2-Hz stimulation rate was applied for 15 minutes, followed by application of 500 μM-BPV (500 μL of 100 mM BPV stock solution was added to 100 mL of Tyrode solution in a reservoir). The 1.2-Hz stimulation rate was continuously applied during the entire experimental period after stabilization. If asystole did not develop for 20 minutes, an additional 100 μL of BPV were added. If asystole did not develop despite a 600-μM application for 20 minutes, we stopped the experiment. To determine the concentration level for inducing asystole in isolated guinea pig papillary muscles, we conducted a preliminary study to test various concentrations of BPV at a 1.2-Hz stimulation rate; 100-, 200-, 300-, and 500-μM concentrations were examined. In the experiments, using successive administration of BPV from 100 to 300 μM (n = 6), we found that 200 or 300 μM of BPV induced intermittent contractile responses (conduction block) and contractile depression; however, no conduction block was observed at the 100-μM concentration in all muscles. In another experiment using a 500-μM concentration (n = 5), we observed the development of asystole developed at 10–15 minutes after exposure to this level of concentration in all muscles. Therefore, we used this concentration to induce the BPV-induced asystole during the experiment.
Five minutes after asystole, 2%, 4%, 8%, 12%, or 16% LE was applied for 60 minutes (n = 47). The time after adding LE to the appearance of the first contractile response (T1stR ) and to the first appearance of regular contraction (TRR ), which responded regularly to a stimulation rate of 1.2 Hz after the T1stR were noted. The peak force and the maximum rate of force development (dF/dt-max) were also recorded. Noncontractile muscles, as well as muscles showing conduction block to a 1.2-Hz stimulation rate at various times, were excluded from the calculation of contractile forces (Table ). As a control, Tyrode solution was used.
Table.: Changes of dF/dt-max Following LE Treatment After Bupivacaine-Induced Asystole in Recirculation Conditions
We also determined whether the dilution of 500-μM BPV in Tyrode solution by added LE volume had altered the recovery of contractions (n = 11). After 5 minutes of asystole with 500 μM-BPV in 100 mL of Tyrode solution, 154 mL of Tyrode solution, equivalent to the aqueous component of added 12% LE, was added to the reservoir and recirculated for 60 minutes.
As small increases in either resting tension or contracture developed in some asystolic muscles after exposure to 16% LE, we examined the effect of 16% LE itself to exclude it as a cause (n = 7).
Washout (Nonrecirculation) Experiments.
Using the same experimental protocol as in recirculation experiments, 500 μM of BPV was applied and Tasystole was noted. Five minutes after asystole, 0.05%, 0.1%, 0.5%, 1%, 2%, 4%, 8%, or 12% LE were superfused in a nonrecirculating manner for 60 minutes (n = 51). T1stR and TRR were determined. As a control, Tyrode solution was used (n = 7).
To evaluate the reversal effect of LE on the metabolic inhibition of BPV in mitochondria, 2-mM oxfenicine, an inhibitor of carnitine-palmitoyl-transferase I in heart mitochondria,12 , 13
To examine the effect of LE alone on contractility, 0.05%, 0.1%, 0.5%, 1%, 2%, 4%, 8%, or 12% LE were applied after achieving stable contractions for 15 minutes with the 1.2-Hz stimulation rate (n = 55). Based on instantaneous and linear relationship between dF/dt and [Ca2+ ]i during the rise of tension in cardiac twitch contraction,14
Measurements of BPV Concentrations in the Cardiac Tissues or Solutions
At 60 minutes following application of each concentration of LE in 500-μM BPV-treated muscles, stimulation and superfusion were stopped, and the solution was suctioned from the tissue bath. The tendinous part of the muscle was removed. Samples were stored at −80°C. The muscles were obtained with control solution (n = 5) and after being treated with 2%, 4%, 8%, or 12% LE in the recirculation condition (n = 5 at each), and control solution (n = 5) with 2%, 4%, 8%, and 12% LE in the nonrecirculation condition (n = 5 at each). In addition, the BPV concentrations were measured in the recirculating condition at 3 minutes after applying 500-μM BPV, 5 minutes after obtaining asystole, and after 15, 30, 45, and 60 minutes in either the control solution (n = 3) or 8% LE-treated groups (n = 5). A 1-mL solution was obtained at each time period. In LE-treated groups, 1 mL of LE was centrifuged at 10,000g for 10 minutes to separate the aqueous phase from the lipid phase. After discarding the lipid phase, it was centrifuged once more to obtain the lower clear aqueous phase. Samples were stored at −80°C. BPV concentrations in the Tyrode solution and muscles were measured using a liquid chromatography-tandem mass spectrometry (LC/MS/MS) system. The LC/MS/MS system consisted of an Agilent 1200 (HPLC; Agilent Technologies, Santa Clara, CA) and a 4000 Qtrap (Mass spectrometry; AB SCIEX, Concord, ON, Canada). Lidocaine was used as the internal standard.
Statistical Analysis
Alterations in contractile function were expressed as a percentage of the baseline, due to variations in baseline values from one muscle to another. T1stR , TRR , and myocardial BPV concentrations among treatment groups were analyzed using one-way analysis of variance, followed by Bonferroni test. Repeated measures of analysis of variance were used to test for differences among baseline and time-dependent changes of dF/dt-max in 16% LE alone-treated group in the recirculation condition. A linear mixed model was used to analyze the changes of dF/dt-max during recovery among LE-treated groups and between 1% LE- and oxfenicine-treated group following BPV-induced asystole in both recirculation and washout conditions. In addition, changes of dF/dt-max for 60 minutes in LE alone-treated groups in washout condition as well as changes of BPV concentrations in solution during recovery in recirculation condition were analyzed using linear mixed model. In this model, we analyzed the interaction between treatment groups and time. Additionally, we performed post hoc analysis to estimate the time points at which treatment effects differed between groups. In the post hoc analysis, the two-tailed P values adjusted by Bonferroni correction were used to control the significance level. The overall significance level (α) for each hypothesis was .05. All results were presented as mean ± standard deviation. All data were analyzed using SAS for Microsoft Windows (version 9.2; SAS Institute Inc, Cary, NC). Sample sizes were determined in accordance with our previous experimental protocols; an a priori power analysis was not performed in this study. Instead, post hoc power analysis was conducted for the predetermined sample sizes to determine the study’s power. Based on standard deviations, we calculated significant differences with which we could detect using the number of samples employed. Power Analysis and Sample Size 12.0 (NCSS, LLC, Kaysville, Utah) was used to conduct the analysis, with α = .05. Mean ± standard deviation values of the contractile forces (dF/dt-max) for control (0%, n = 5) and 8% LE treatment (61% ± 31%, n = 9) at 60 minutes after asystole were assessed in the recirculation condition, which showed a 99.9% power with which to detect significant differences. In the washout condition, based on the mean ± standard deviation values of the contractile forces for control (52% ± 22%, n = 7) and 2% LE treatment (113% ± 20%, n = 7) at 60 minutes following asystole, 73.9% power was obtained. The LE concentrations at each condition were chosen based on maximum recovery of the contractile forces at 60 minutes following asystole.
RESULTS
Recirculation Experiments
Effects of LE on BPV-Induced Asystole.
In all muscles, asystole developed 10–15 minutes after exposure to 500 μM of BPV. LE at high concentrations (4%–12%) restored stimulated contractile responses and contractility in muscles during continuous exposure to 500 μM (Figure 1A and A’ ); however, no restoration of these parameters was observed with the control solution (Figure 1B ). Two-percent LE led to a response to stimulation in only 2 of the 8 muscles; intermittent contractile response to 1.2-Hz stimulation (conduction block) was shown and sustained for the 60-minutes recirculation. In 4% LE-treated muscles, a contractile response occurred in 5 of the 9 muscles; however, regular contractile responses were restored in only 3 of the 5 muscles; 2 muscles had sustained conduction block for 60 minutes. In contrast, regular stimulated contractile responses were restored in the majority of 8% or 12% LE-treated muscles (Table ). The times to the appearance of the T1stR (from 4.6 ± 4.3 to 6.8 ± 3.8 minutes) and TRR (from 12.3 ± 9.0 to 23.0 ± 19.4 minutes) did not differ among the 4%, 8%, and 12% LE groups (Table ). Modest to moderate levels of recovery in contractile forces were observed in 4%, 8%, and 12% LE-treated muscles during the entire experimental period (Table ). There was no significant interaction between the treatment group (8% and 12% LE) and time in contractile forces. The contractile forces did not show differences between 8% and 12% LE-treated groups at each time point. As regular contractile responses were restored in few muscles in 4% LE-treated group, we did not include such data for statistical analysis.
Figure 1.: A, Typical pattern of recovery of contractile responses and peak force (PF) following bupivacaine (BPV)-induced (500 μM) asystole in recirculating experiment. Application of 12% lipid emulsion (LE; Intralipid) led to a stimulated contractile response at approximately 3 minutes, intermittent contractile responses, and after a short period, restoration of regular contractions at a 1.2-Hz stimulation rate. Partial recovery, approximately 70% of the baseline, was shown following a 60-minute recirculation. B, Typical pattern of sustained asystole after BPV-induced (500 μM) asystole, followed by recirculation with control (modified normal Tyrode, NT) solution for 60 minutes in recirculating experiment. A’, Enlarged view of the period between the 2 down arrows of A, which shows asystole induced by 500-μM BPV and the restoration of regular contractile responses following application of 12% LE in recirculating condition.
In 12 muscles treated with 16% LE, intermittent and regular contractile responses were observed in 1 and 4 muscles, respectively. On the contrary, a contracture or small increase in resting tension following LE exposure was observed in 5 and 2 muscles, respectively. A total of 600 μM-BPV was required in one muscle in the 16% LE-treated group.
In matching the dilutional effect of LE by adding an equivalent volume of Tyrode solution (n = 6), following asystole induced by 500 μM-BPV, there were no responses to electrical stimuli during the 60-minute perfusion period. The baseline value of dF/dt-max in this group was 5.77 ± 5.26 mN/s.
In muscles treated with 16% LE alone (n = 7), neither a contracture nor increased resting tension was observed. Immediately after exposure to 16% LE, contractions were enhanced to a maximum at approximately 2 minutes (2.24 ± 0.73), after which the contraction gradually decreased, stabilizing at approximately 20 minutes (18.94 ± 2.14) after reaching the maximum contraction. Contractions remained mildly depressed for 45 minutes (30 minutes: 86% ± 13% [P < .019]; 45 minutes: 82% ± 13% [P = .002]) compared with baseline. Baseline value of dF/dt-max was 24.77 ± 18.36 mN/s.
Washout (Nonrecirculation) Experiments
Effects of LE on BPV-Induced Asystole. Figure 2.: A, Typical pattern of recovery of contractile responses and peak force (PF) following bupivacaine (BPV)-induced (500 μM) asystole in the washout (nonrecirculating) experiment. Subsequent BPV washout with application of 2% lipid emulsion (LE; Intralipid) led to a stimulated contractile response at approximately 5 minutes, intermittent contractile responses, and, after a short period, restoration of regular contractions at a 1.2-Hz stimulation rate. B, Recovery pattern of contractile responses and PF after BPV-induced (500 μM) asystole, followed by washout with control (modified normal Tyrode, NT) solution for 60 minutes. A’, Enlarged view of the period between the 2 down arrows of A, which showed asystole induced by 500-μM BPV and the restoration of regular contractile responses following application of 2% LE and BPV washout.
Figure 3.: Changes of contractile forces (dF/dt-max) following lipid emulsion (LE; Intralipid) treatment after bupivacaine-induced asystole in the washout (nonrecirculation) experiments. All data are presented as mean ± SD. The recovery of dF/dt-max revealed only a significant effect between the treatment group and time (control versus 0.5% LE, P = .011). Regarding dF/dt-max from 30 to 60 minutes compared with 15 minutes, P values were as follows: .1%: 15 vs 45 minutes, P = .019; 15 vs 60 minutes, P = .001; 0.5%: 15 vs 45 minutes, P = .010; 15 vs 60 minutes, P < .006; 1%: 15 vs 45 minutes, P = .011; 15 vs 60 minutes, P < .001. 2%: 15 vs 45 minutes, P = .013; 15 vs 60 minutes, P < .006; 4%: 15 vs 60 minutes, P < .046; 8%: 15 vs 45 minutes, P = .017; 15 vs 60 minutes, P = .003. *Difference (P < .05) compared with 15 minutes at each time period at each concentration of LE. The baseline values of dF/dt-max (mN/s) are as follows: control, 4.41 ± 3.25; 0.05%, 7.85 ± 4.95; 0.1%, 5.50 ± 2.84; 1%, 12.10 ± 7.78; 2%, 7.91 ± 4.42; 4%, 8.38 ± 3.56; 8%, 7.64 ± 4.25; 12%, 11.95 ± 4.25. Control indicates modified normal Tyrode solution-treated group; N, number of papillary muscles.
In all muscles superfused with either a concentration of LE or the control solution, asystole developed in 10–15 minutes after exposure to 500-μM BPV. Application of either LE or a control solution permitted the return of intermittent contractions, followed shortly by regular contractions (Figure 2A and A’ ). Times to the appearance of the T1stR (from 2.1 ± 0.9 to 5.3 ± 1.9 minutes) and TRR (from 4.9 ± 1.5 to 12.1 ± 6.0 minutes) did not differ among the control and different LE concentrations. Recovery of dF/dt-max only revealed a significant effect between the treatment group and time (control versus 0.5% LE, P = .011). In the LE-treated muscles, complete recovery of contractile forces was observed after a 45-minute superfusion with LE concentrations from 0.1% to 8%, while partial recovery and contractile depression, approximately 60% of initial contractile force, were observed in 0.05% and 12% LE-treated muscles, respectively (Figure 3 ). Superfusion with control solution for 60 minutes restored contractility to approximately 60% of baseline values (Figure 3 ). A total of 600-μM BPV was used in 3, 2, and 1 muscles in the 0.05%, 0.1%, and 8% LE groups, respectively.
Effects of Oxfenicine on LE Reversal of BPV-Induced Asystole Figure 4.: Effects of oxfenicine on lipid emulsion (LE; Intralipid) reversal of bupivacaine-induced asystole. All data are presented as mean ± SD. The recovery of contractile forces (dF/dt-max) did not exhibit a significant interaction effect between treatment group and time. The contractile forces at each time point for 60 minutes following application of oxfenicine-added 1% LE was similar to that observed after treatment with 1% LE alone. N indicates number of papillary muscles.
Before application of 500-μM BPV, pretreatment with 2-mM oxfenicine for 10 minutes did not alter contractility. Following application of 1% LE, recovery of contractile forces did not show a significant interaction between the treatment group and time. Recovery of contractile forces at each time period for 60 minutes did not differ in the presence (n = 6) or the absence of oxfenicine (n = 7) (Figure 4 ). To further confirm this effect, 4-mM oxfenicine was used (n = 2), and the results were similar to that of 2-mM oxfenicine (data not shown). The baseline value of dF/dt-max in 2-mM oxfenicine-added 1% LE group was 13.87 ± 8.73 mN/s.
Effects of LE Alone on Contractility Figure 5.: A, Effect of 1% lipid emulsion (LE; Intralipid) on peak force (PF) in the nonrecirculating condition. Contractile enhancement reached a peak approximately 3 minutes after wash, and gradually decreased at approximately 20 minutes reaching a plateau that was sustained for 60 minutes. B, Changes of contractile forces (dF/dt-max) following LE treatment alone in the washout (nonrecirculation) conditions. All data are presented as mean ± SD. The changes of dF/dt-max from 30 to 60 minutes did not exhibit a significant interaction effect between treatment group and time. Regarding dF/dt-max values from 30 to 60 minutes at each time period, P values are as follows: 30 minutes: 0.05%–4% vs 8% or 12%, P < .05; 45 minutes: 0.05%–4% vs 8% or 12%, P < .05; 60 minutes: 0.05%–2% vs 4%, 8%, or 12%, P < .05. *Difference (P < .05) compared with 15 minutes from 30 to 60 minutes. The baseline values of dF/dt-max (mN/s) are as follows: 0.05%, 11.0 ± 6.3; 0.1%, 7.0 ± 3.5; 0.5%, 5.6 ± 1.3; 1%, 5.6 ± 1.3; 2%, 18.8 ± 19.7; 4%, 8.2 ± 2.2; 8%, 7.8 ± 7.1; 12%, 11.7 ± 5.6. N indicates number of papillary muscles.
LE enhanced contractions at all concentrations after about 3 minutes (2.83 ± 0.70). Contractility gradually declined and then reached a plateau at approximately 20 minutes (17.43 ± 6.49) (Figure 5A ). From the beginning of the plateau, contractile forces were sustained at baseline levels at low and intermediate concentrations; however, the forces were depressed at high concentrations (8% and 12%) (Figure 5B ). Among each concentration of LE, the times to maximal contraction and to subsequent beginning of the plateau were similar. Although the maximal contractions appeared to be approximately 2-fold greater than the baseline values in 0.05%, 0.1%, 0.5%, 1%, 2%, and 4% LE-treated groups, the maximal contractions were approximately 30% greater than the baseline values in 8% and 12% LE-treated groups. From the beginning of the plateau to the end of experiment, changes of contractile forces did not exhibit a significant interaction effect between treatment groups (n = 55) and time. At 30, 45, and 60-minute time periods, contractile forces were sustained at the baseline levels in 0.05%, 0.1%, 0.5%, 1%, 2%, and 4% LE-treated groups, while contractions in 8% and 12% LE-treated groups appeared to be depressed by approximately 15% and 50%, respectively.
Effect of LE on Solution and Myocardial BPV Concentrations
Recirculation Experiments. Figure 6.: A, Changes of bupivacaine (BPV) concentrations at each time point in the control (modified normal Tyrode) solution (n = 3) and with 8% lipid emulsion (LE; Intralipid) (n = 5) treatment groups following asystole induced by 500 μM-BPV. All data are presented as mean ± SD. Myocardial BPV concentrations following superfusion with control solution (n = 5 at each group) and various concentrations of LE (n = 5 at each group) for 60 minutes after BPV-induced asystole in either the recirculating (B) or washout condition (C). The BPV concentrations in solution (A) revealed a significant interaction effect between the treatment group (control versus 8% LE) and time (P < .0001). Regarding BPV concentrations at each time point between the 2 groups, P values are as follows: 15 and 30 minutes: P < .006; 45 minutes: P = .001; 60 minutes: P = .003. Although the BPV concentrations in cardiac tissues did not show differences among groups in the nonrecirculation condition, concentration-dependent decrease was shown in the recirculation condition (control versus 4%, 8%, or 12%, P < .05; 2% vs 4%, 8%, or 12%, P < .05; 4% vs 8% or 12%, P < .05).
In the control solution, BPV concentration gradually decreased over 60 minutes. However, in the 8% LE-containing solution, it decreased to a much greater extent at 15 minutes and was sustained at lower value compared to the control group (P < .0001) (Figure 6A ). BPV concentrations in solutions revealed a significant interaction between the treatment group and time, indicating that the LE-treated group had a larger decrease in BPV concentrations compared to the control solution group (P < .0001). Myocardial BPV concentrations showed a LE-concentration-related decrease at 60 minutes (Figure 6B ).
Washout (Nonrecirculation) Experiments.
At 60 minutes after the washout of BPV, myocardial BPV concentrations was notably, but similarly, decreased in both control and LE-treated groups (Figure 6C ). In LE-treated groups, the myocardial BPV concentrations in 8% and 12% LE-treated muscles appeared to be approximately 40%–50% lower than those in recirculating condition.
DISCUSSION
LE induced a time- and concentration-dependent recovery of initial contractile force following BPV-induced asystole.
Our work differs from previous studies in that we attempted to better define differences in the time course of treatment that occur with various concentrations of LE after BPV-induced asystole, as well as the importance of BPV washout in recovery. We also observed the effects of various concentrations of LE itself over long durations to define its direct contribution on contractility. Using both recirculation (BPV-present) and washout conditions, we defined the mechanisms related to contractile recovery after BPV-induced asystole, using isolated guinea pig papillary muscles. We found that lower concentrations of LE (below 1%) were not effective and higher concentrations (above 8%) were detrimental on the recovery of contractile function after BPV-induced asystole, whereas intermediate concentrations were effective and progressively increased recovery to the initial contractile force over treatment time course.
Comparing the progressive increase in contractility to initial contractile forces, Chen et al’s10 , 15 2 + and Ca2+ concentrations) would have been proportionally diluted by the aqueous component of LE, potentially resulting in decreased contractility. Although the causes remain unclear regarding the deterioration of cardiac recovery by higher concentrations of LE, dilution may, in part, be a contributing factor.
Isolated rat heart models have been used as a powerful tool for studying the pathophysiology, mechanisms, and treatment of BPV-related cardiac toxicity.2 + channel block and its block of a conducted contractile response. In addition, since our model does not involve perfusion, direct effect of a drug on myocardial contractility can be evaluated. Another advantage is that both recirculation and washout condition can be used in our model, while isolated heart preparation is used only in nonrecirculation condition. Via closed recirculation condition, we could definitely provide evidence on lipid sink effect based on spontaneous recovery of stimulated contractile responses (heart rate), as well as the changes of BPV content by LE in comparison with those by Tyrode solution. We could also investigate the detrimental effects of high concentration of LE in the presence of carnitine-acylcarnitine translocase inhibition by BPV, such as contracture development by 16% LE application. However, an important limitation of our model is its use of superfusion instead of perfusion. The use of superfused papillary muscles results in longer diffusion distances than using blood-vessel perfused myocardium. Based on the capillary density in guinea pig with 2300 capillaries per mm,2 ,16
In the present study, 2 different circulation conditions were used. Recirculation condition was used to demonstrate the recovery of stimulated contractile responses as BPV declined, due to its uptake by LE liposomes. On the other hand, BPV washout was used to determine whether LE could enhance or accelerate the time-dependent recovery of contractions, in addition to predict the metabolic effect or other undefined mechanisms. With BPV washout, the tissue concentration will be quickly lowered due to continuous wash. However, in spite of quickly lowered BPV in tissues, the remained BPV in tissues seems to cause a long-term injury. In this regard, our results showed that the recovery of contractile forces was approximately 60% of baseline values at 60 minutes after wash with control solution, while LE completely recovered the contractile forces to baseline level. As the tissue BPV concentration was quickly lowered by continuous wash, LE may provide a metabolic fuel or other undefined mechanisms, which contribute to improvement of contractility.
In the recirculation condition, we observed recoveries of T1stR and TRR in LE-treated groups; however, no recovery was observed in the control group. This suggests that LE reduced the tissue BPV concentration sufficiently to permit restoration of conducted action potentials, consistent with restored Na+ channel function. Although 12% LE restored the regular contractile responses in most muscles, no appearance of contractile responses were observed when perfusate was diluted with only the aqueous component of 12% LE, suggesting that, indeed, the LE was necessary to reduce tissue BPV concentration and restore response to stimulation. These results suggest that the LE micelles appear to take up sufficient BPV to reduce the BPV-induced block of the Na+ channels. The accelerating effect of LE to reverse the Na+ channel blocking action of BPV has also been demonstrated in rat myocytes17 18 19 + and Ca2+ channels.
Our results on BPV concentrations in solution and myocardial tissues also demonstrated the ability of LE to decrease BPV in solution and in the tissue, due to its presumed diffusion from tissue into micelles. In recirculation experiments, BPV concentrations decreased gradually in the control solution, possibly due to ongoing tissue uptake of BPV. When 8% LE was applied, BPV concentration decreased by approximately 80% within 15 minutes and, thereafter, sustained until 60 minutes and decreased the tissue BPV concentration by approximately 70% at 60 minutes, when compared with the control. These results suggest partitioning of BPV into LE and are in agreement with the notion of strong positive correlation between myocardial BPV content and the aqueous plasma concentration.20 21 22 23 24
During acute local anesthetic toxicity, BPV has been shown to inhibit the activity of carnitine-acylcarnitine translocase,25 26 , 27
Recently, Fettiplace et al28
In terms of metabolic actions, our results of blocking of carnitine-palmitoyl-transferase I and inhibiting utilization of fatty acyl-CoA with oxfenicine (4-hydroxy-l -phenylglicine)12 , 13 29
The recommended dosage of lipid bolus to treat local anesthetic overdose is 1.5 mL/kg of 20% LE,8 22 9
In conclusion, our study demonstrated that LE caused time- and concentration-dependent recovery of contractile function after severe acute BPV toxicity. Based on the restoration of regular stimulated contractions together with the concentration-related reduction of tissue BPV concentration, the lipid uptake of BPV by LE liposomes explains much of the restoration of cardiac function. The metabolic effect of the LE on myocardial metabolism is unlikely to have played a prominent role at the concentration (500 μM) of BPV employed in our experiment.
ACKNOWLEDGMENTS
The authors thank Hayan Kim, a biostatistician in the Biostatistics Collaboration Unit, for statistical analysis and Changhun Park, MS, in the Office for Analytical Support in the Clinical Trial Center in Yonsei University Health System in Seoul, Korea, for measurement of bupivacaine concentrations.
DISCLOSURES
Name: Wyun Kon Park, MD.
Contribution: This authorhelpedconceive and design the study, performed the data and statistical analysis, interpreted the results of experiment, and prepared the manuscript.
Name: Hyun Soo Kim, PhD.
Contribution: This author helped perform the experiments and interpreted the results of the experiment.
Name: Soo Hwan Kim, MD.
Contribution: This author helped perform the experiments.
Name: Ja Rang Jung, MS.
Contribution: This author helped perform the experiments and analyze the data.
Name: Carl Lynch III, MD, PhD.
Contribution: This author helped interpret the results of the experiment, analyze the data, and prepare the manuscript.
Name: Nar Hyun Min, MD.
Contribution: This author helped perform the experiments.
This manuscript was handled by: Markus W. Hollmann, MD, PhD.
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