Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-013-0159-4
ORIGINAL PAPER
Inhibition of murine cardiomyocyte respiration by amine local anesthetics Elhadi H. Aburawi • Abdul-Kader Souid
Received: 1 June 2013 / Accepted: 6 November 2013 Ó Springer-Verlag France 2013
Abstract The hydrophobic amino acyl amide-linked local anesthetics (e.g., lidocaine and bupivacaine) impose potent cardiac toxicity and direct mitochondrial dysfunction. To investigate these adverse events, an in vitro system was employed to measure their effects on O2 consumption (cellular respiration) by murine myocardium. Specimens were collected from the ventricular myocardium and immediately immersed in ice-cold Krebs–Henseleit buffer saturated with 95 % O2:5 % CO2. O2 concentration was determined as a function of time from the phosphorescence decay rates of Pd(II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Myocardial O2 consumption was linear with time (zero-order kinetics); its rate (k, in lM O2 min-1), thus, was the negative of the slope of [O2] vs. time. Cyanide inhibited O2 consumption, confirming the oxidation occurred in the respiratory chain. Lidocaine and bupivacaine produced immediate and sustained inhibition of cellular respiration at plasma concentrations of the drugs (low micromolar range). Bupivacaine was twice as potent as lidocaine. The inhibition was dose-dependent, saturating at concentrations C30 lM. At saturating doses, lidocaine produced *20 % inhibition and bupivacaine *40 % inhibition. Cellular ATP was also decreased in the presence of 30 lM lidocaine or bupivacaine. The studied amines inhibited myocardial cellular respiration. This effect is consistent with their known adverse events on mitochondrial function. The described approach allows accurate assessments and comparisons of the toxic effects of local anesthetics on heart tissue bioenergetics.
E. H. Aburawi (&) A.-K. Souid Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Ain, United Arab Emirates e-mail: [email protected]
Keywords Oxygen Respiration Mitochondria Cardiomyocytes Local anesthetics
1 Background Lidocaine and bupivacaine are commonly used amino acyl amide-linked local anesthetics. These agents also possess anti-arrhythmic and pro-arrhythmic actions and are potent inhibitors of the mitochondrial respiratory chain. The drugs produce cardiac depression. Owing to its higher lipophilicity, bupivacaine is more cardiotoxic than lidocaine (Heavner 2002). Lidocaine and bupivacaine stabilize the neuronal membrane by inhibiting voltage-gated sodium channels. As a result, the postsynaptic membrane fails to transmit action potentials, creating the anesthetic effect (Arlock 1988). Bupivacaine also binds to the intracellular domain of sodium channels, blocking Na? influx into nerve cells and preventing depolarization. At toxic levels, the drug invokes direct cardiac toxicity. The bupivacaine-induced blockade of voltage-gated sodium channels is more sustainable and less reversible than lidocaine, accounting for its more prominent impairments of myocardial contractility (Clarkson and Hondeghem 1985; Tanz et al. 1984; Eledjam et al. 1988). The negative inotropic effect of bupivacaine is also linked to drops in intra-cytoplasmic [Ca2?], resulting in disturbed mitochondrial function and impaired cellular bioenergetics (Chazotte and Vanderkooi 1981; Sztark et al. 1998; Dabadie et al. 1987). Moreover, bupivacaine decreases myocardial O2 consumption in isolated perfused guinea pig heart (Tanz et al. 1984). In principle, the same findings apply to lidocaine. Moreover, the amino acyl amide-linked local anesthetics are potent uncouplers of oxidative phosphorylation (uncoupling phosphorylation to e- and H? transfer); thus,
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these agents impair cellular bioenergetics (Sztark et al. 1994; Garlid and Nakashima 1983; Terada et al. 1990). The term ‘‘uncoupling oxidative phosphorylation’’ implies a state of high O2 consumption and low ATP synthesis due to increased leakage of H? across the inner mitochondrial membrane. Uncoupling agents (uncouplers) function as H? carriers (protonophores); they halt oxidative phosphorylation by dissipating the proton-motive force (DW). An analytical method of assessing murine tissue respiration in vitro is recently reported (Al Samri et al. 2010). The same approach is employed here to assess the inhibitory effects of lidocaine and bupivacaine on ventricular muscle respiration (cellular mitochondrial O2 consumption). The main objective of the study was to explore potential mechanisms for the cardiotoxicity of local anesthetics. The results show lidocaine and bupivacaine inhibit respiration of the myocardium (inhibits in vitro O2 consumption by non-beating ventricular specimens). Bupivacaine is more toxic than lidocaine.
*60 % relative humidity and a 12-h light/dark cycle. All mice had ad libitum access to standard rodent chow and filtered water. The protocols received approval from the Animal Ethics Committee, United Arab Emirates University, College of Medicine and Health Sciences.
2 Materials and methods
2.4 O2 consumption
2.1 Reagents
Phosphorescence O2 analyzer was used to monitor O2 consumption by heart specimens. O2 detection was performed with the aid of Pd phosphor, which had an absorption maximum at 625 nm, and a phosphorescence maximum at 800 nm (Lo et al. 1996). The samples were exposed to light flashes (600 min-1) from a pulsed lightemitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc., Wheeling, IL). Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube after passing through a wide-band interference filter centered at 800 nm. The amplified phosphorescence decay was digitized at 1.0 MHz by a 20-MHz A/D converter (Computer Boards, Inc., Mansfield, MA). A program was developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices; http://www.mccdaq.com/daq-software/ universal-library.aspx). It allowed direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board; http://www.mccdaq.com/pci-data-acquisition/PCIDAS4020-12.aspx) (Shaban et al. 2010). The phosphorescence decay rate (1/s) was characterized by a single exponential; I = Ae-t/s, where I = Pd phosphor phosphorescence intensity. The values of 1/s were linear with dissolved O2: 1/s = 1/so ? kq[O2], where 1/ s = the phosphorescence decay rate in the presence of O2, 1/so = the phosphorescence decay rate in the absence of O2, and kq = the second-order O2 quenching rate constant in s-1 lM-1.
Pd (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (Pd phosphor) was purchased from Porphyrin Products (Logan, UT). Glucose (anhydrous) and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO). Lidocaine (10 mg mL-1 or 43 mM; m.w. 234.34) and bupivacaine (5 mg mL-1 or 17.3 mM; m.w. 288.43) were purchased from Aguettant Pharmaceuticals (France). The lidocaine and bupivacaine solutions were diluted in 0.9 % NaCl. Krebs–Henseleit (KH) buffer (115 mM NaCl, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCl, 1.25 mM CaCl2, 1.18 mM MgCl2, and 6 mM glucose, pH 7.4) saturated with O2:CO2 (95:5) was prepared daily. Pd phosphor solution (2 mg mL-1 = 2 mM) was prepared in dH2O and stored in aliquots at -20 °C. NaCN solution (1.0 M) was prepared in dH2O; the pH was adjusted to *7.0 with 12 N HCl and stored at -20 °C. Glucose oxidase (10 mg mL-1) was prepared in dH2O and stored at -20 °C. 2.2 Mice Male Taylor outbred mice (8–12 week old; weight & 18–22 g) were maintained at the animal facility in compliance with the NIH guidelines (http://grants.nih.gov/ grants/olaw/references/phspol.htm). The mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were housed in rooms maintained at 22 °C with
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2.3 Sample collection Male mice were anesthetized using urethane (100 lL per 10 g body weight, from a 25 % solution, w/v, in 0.9 % NaCl). A piece of the ventricular myocardium (about 15 mg) was excised from the heart using special scissors (Moria Vannas Wolg Spring, cat. # ST15024-10). The tissue cut was performed, while the heart was beating and well perfused. The specimen was immediately immersed in ice-cold KH buffer saturated with 95 % O2:5 % CO2. The specimen was then placed in 1.0 mL KH buffer supplemented with 3 lM Pd phosphor and 0.5 % fat-free bovine serum albumin and processed for O2 measurement at 37 °C as described below.
Eur J Drug Metab Pharmacokinet
Fig. 1 Inhibitory effects of the amine local anesthetics on cardiomyocyte respiration. Representative runs of O2 consumption by specimens from the ventricular myocardium are shown. The rate of cellular mitochondrial O2 consumption (k, lM O2 min-1) was set as the negative of the slope of [O2] vs. time. The lines are linear fits. Panels a and b show the inhibitory effects of 100 and 77 lM
lidocaine, respectively. Panels c and d show the effects of 0.35 and 0.087 lM bupivacaine, respectively. Panel e shows the effect of injecting 5 lL 0.9 % NaCl, which lowered the value of k by 18 %. Panel f shows the effects of injecting 10 lM bupivacaine, 10 mM NaCN and 50 lg mL-1 glucose oxidase
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Respiration was measured at 37 °C in 1-mL sealed vials. Mixing was with the aid of parylene-coated stirring bars. In vials sealed from air, [O2] decreased linearly with time, indicating the kinetics of cellular mitochondrial O2 consumption was zero order. The rate of respiration (k, in lM O2 min-1) was, thus, the negative of the slope d[O2]/dt. NaCN inhibited respiration, confirming O2 was consumed in the mitochondrial respiratory chain. Different concentrations of lidocaine or bupivacaine were injected directly into the vial during the O2 runs. As a result, the change in the slope d[O2]/dt reflected drug effects. The calibration reaction contained phosphate-buffered saline (PBS) with 3 lM Pd phosphor, 0.5 % fat-free albumin, 50 lg mL-1 glucose oxidase and various concentrations of b-glucose. The values of 1/s were linear with [b-glucose]; the value of kq was the negative of the slope (kq = 101.1 s-1 lM-1) (Lo et al. 1996).
mean = 16 %); this change was due to deviation of the [O2] vs. t curve from linearity as a result of using tissue instead of isolated cells. Nevertheless, the use of tissue is superior to isolated cardiomyocytes, which are subjected to physical and chemical (e.g., collagenase digestion) treatments. Percent of inhibition by the drug was corrected for the 16 % drift (artifact, e.g., for Fig. 1a, the percent of inhibition was 4216 % = 26 %). Similarly, 77 lM lidocaine produced 29 % inhibition (Fig. 1b), 10 lM bupivacaine 43 % inhibition (Fig. 1e), 0.35 lM bupivacaine 44 % inhibition (Fig. 1c) and 0.087 lM bupivacaine 0 % inhibition (Fig. 1d). Cyanide inhibited respiration, confirming the oxidation mainly
2.5 Cellular ATP Tissue fragments were homogenized in 0.5 mL of ice-cold 2 % trichloroacetic acid for 2 min. The supernatants were collected by centrifugation (1,0009g at 4 °C for 5 min) and stored at -20 °C. The samples were neutralized immediately before ATP measurements with 0.5 mL 100 mM Tris– acetate, 2 mM EDTA (pH 7.75). [ATP] was determined using the Enliten ATP Assay System (Bioluminescence Detection Kit, Promega, Madison, WI); 5 lL of the supernatant was added to 50 lL of the luciferin/luciferase reagent. The luminescence intensity was measured using GloMax Luminometer (Promega, Madison, WI). 2.6 Statistical analysis Data were analyzed using SPSS statistical package (version 20). Data were presented as mean ± SD (n). The Mann– Whitney test was used for nonparametric values; p \ 0.05 was considered significant.
3 Results Representative runs of myocardial respiration are shown in Fig. 1a–f. As expected, O2 concentration decreased linearly with time, confirming the zero-order kinetics of cellular O2 consumption. This finding also confirms the feasibility of using a fragment of the ventricular myocardium for accurate determination of the rate of respiration (k, in lM O2 min-1). Lidocaine (100 lM, final concentration in the vial) resulted in a drop of the value of k from 1.3 before the injection to 0.76 after the injection (*42 % difference, Fig. 1a). Injection of 5 lL of 0.9 % NaCl, however, also resulted in a milder change in the value of k (Fig. 1f; typically 14–18 %;
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Fig. 2 Inhibitory effects of the amine local anesthetics on cardiomyocyte respiration as a function of drug concentration. Panel a, percent inhibition is plotted as a function of drug concentration. The lines are logarithmic fits. Panel b, natural log of percent inhibition is plotted as a function of drug concentration. The lines are linear fits
Eur J Drug Metab Pharmacokinet Fig. 3 Effects of the amine local anesthetics on cardiomyocyte ATP content. Specimens from the ventricular myocardium were incubated in oxygenated KH buffer at 37 °C with and without the addition of 30 lM lidocaine or bupivacaine. At t = 15, 30 and 60 min, samples were removed from the incubation medium and processed for ATP measurement as described in Sect. 2. Values are means and error bars are standard deviations (n = 3). The standard curve (insert) included 1.0, 10 and 100 nM ATP
occurred in the respiratory chain. Glucose oxidase (catalyzes the oxidation of b-D-glucose into D-glucono-1,5-lactone) then depleted the remaining O2 in the solution (Fig. 1e). Figure 2a–b compares the dose-dependent inhibition of respiration by lidocaine vs. bupivacaine. The drug effects were saturated at about 30 lM, yielding about 20 % maximum inhibition by lidocaine and about 40 % by bupivacaine (Fig. 2a). The natural log of inhibition was somewhat linear with dosing; yielding a slope that was three times larger for bupivacaine (Fig. 2b). Thus, both drugs produced similar immediate inhibition of heart muscle cellular respiration, but bupivacaine was more potent than lidocaine. Figure 3 shows changes in cellular ATP as a function of time in the presence and absence of 30 lM lidocaine or bupivacaine. Consistently, as compared to untreated samples, cellular ATP contents were lower in the treated samples for 15 B t B 60 min.
4 Discussion In vitro studies on the effects of amine local anesthetics on ventricular myocardium are limited. In one report, the
highly lipophilic drug bupivacaine stimulated O2 consumption by isolated mitochondria from rat heart, while lidocaine did not. The maximum effect was observed at about 1.0 mM of bupivacaine. This effect was attributed to uncoupling oxidative phosphorylation, thus, accelerating the rate of oxidation. Moreover, a direct inhibition of complex I of the respiratory chain was also noted. The authors concluded that the effect of bupivacaine on mitochondrial bioenergetics is complex (uncoupling plus direct inhibition of NADH dehydrogenase) (Sztark et al. 1998). In mitochondria isolated from rat liver, bupivacaine collapsed the proton-motive force (an uncoupling effect that stimulates respiration) only in the presence of hydrophobic anions, such as tetraphenyl borate. The effect was postulated to be cooperative interactions between the molecules, producing protonophores (Terada et al. 1990). The amine local anesthetics are not protonophores, but can form lipophilic anion pairs to invoke uncoupler-like activity (Dabadie et al. 1987; Garlid and Nakashima 1983). The adverse effects of lidocaine and bupivacaine on mitochondrial bioenergetics are mostly inhibitory to the electron transport, which results in decreasing respiration (as shown in Figs. 1, and 2). The data here (Figs. 2, and 3),
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however, do not rule out the possibility that lidocaine and bupivacaine reduce cellular energy demands (expenditures). Inhibition of respiration was noted at plasma concentrations of the drugs, which could be an adverse event or a mode of action (see below). The effect of bupivacaine was also studied in cultured fibroblasts. The drug produced dose-dependent inhibition of O2 consumption; 50 % inhibition was at 1.5 mM. Consistent with the results in Figs. 1, 2, and 3, submillimolar concentrations were also inhibitory and the effect was immediate (Sztark et al. 1994). The studied amino acyl amide-linked local anesthetics are associated with measurable adverse effects on the mitochondrial O2 consumption. The finding of compromised cellular O2 consumption by these agents may partially explain their well-known clinical adverse events (Muravchick and Levy 2006). For example, bupivacaine is toxic to the brainstem (Bourne et al. 2010). In isolated perfused guinea pig Langendorff heart, both lidocaine (43–128 lM) and bupivacaine (1–10 lM) inhibited myocardial O2 consumption in a concentrationdependent manner; bupivacaine was more cardiotoxic than lidocaine (Tanz et al. 1984). Our study demonstrates the feasibility of using the phosphorescence-based oxygen instrument to investigate the effects of drugs on ventricular muscle mitochondrial O2 consumption (cellular respiration). The amino acyl amidelinked local anesthetics lidocaine and bupivacaine are potent inhibitors of cardiac mitochondrial function. The rational to investigate bupivacaine is related to its potent cardiac toxicity. Consistently, the results show bupivacaine is a more potent inhibitor of cellular bioenergetics than lidocaine (Figs. 2, 3). Plasma concentrations of lidocaine range from 1.5 to 5 lg mL-1 (6.4–21.3 lM) and toxic levels exceed 6 lg mL-1 (17.8 lM) (Schulz and Schmoldt 2003; Dawling et al. 1989). For bupivacaine, plasma levels following local or epidural infusion are 0.5–1.5 lg mL-1 (1.7–5.2 lM) and toxic levels are C2.0 lg mL-1 (6.9 lM) (Schulz and Schmoldt 2003; Dawling et al. 1989; Kratochwil et al. 2002). Because the phosphorescence O2 probe is suspended in 0.5 % albumin, the effective concentrations of lidocaine and bupivacaine in the O2 measuring solution are likely lower than the values shown in Figs. 1, and 2. The in vitro observations in Figs. 1, 2, and 3 may not directly extrapolate to the known in vivo cardiac toxicity of local anesthetics. For example, the effects of lidocaine and bupivacaine on cellular respiration are demonstrated here in non-contracting heart tissue. The clinical effects of the drugs, however, occur in contracting hearts. Of note, the uncoupling effect of the drugs is not noted since the measurements are performed without hydrophobic anions, such as tetraphenyl borate. As discussed above, the
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uncoupling effect (accelerated respiration) results from cooperative interactions between the local anesthetics and the hydrophobic anions, producing protonophores (Terada et al. 1990).
5 Conclusion The data show lidocaine and bupivacaine inhibit respiration of non-beating myocardium in vitro. This effect is consistent with reported inhibition of the mitochondrial respiratory chain by amine local anesthetics. Bupivacaine was more toxic than lidocaine. Acknowledgments The authors thank Mr. Jose Kochiyil and Dr. Sheela Benedict for their experimental assistance. Conflict of interest There are no potential, perceived, or real conflicts of interest. No financial or non-financial interests.
References Al Samri MT, Al Shamsi M, Al-Salam S, Marzouqi F, Mansouri A, Al-Hammadi S, Balhaj G, Al Dawaar SKM, Al Hanjeri RSMS, Benedict S, Sudhadevi M, Conca W, Penefsky HS, Souid AK (2010) Measurement of oxygen consumption by murine tissues in vitro. J Pharmacol Toxicol Methods 63:196–204 Arlock P (1988) Actions of three local anesthetics: lidocaine, bupivacaine and ropivacaine on guinea pig papillary muscle sodium channels (Vmax). Pharmacol Toxicol 63:96–104 Bourne E, Wright C, Royse C (2010) A review of local anesthetic cardiotoxicity and treatment with lipid emulsion. Local Reg Anesth 3:11–19 Chazotte B, Vanderkooi G (1981) Multiple sites of inhibition of mitochondrial electron transport by local anesthetics. Biochim Biophys Acta 636:153–161 Clarkson CW, Hondeghem LM (1985) Mechanism for bupivacaine depression of cardiac conduction: fast block of sodium channels during the action potential with slow recovery from block during diastole. Anesthesiology 62:396–405 Dabadie P, Bendriss P, Erny P, Mazat JP (1987) Uncoupling effects of local anesthetics on rat liver mitochondria. FEBS Lett 226:77–82 Dawling S, Flanagan RJ, Widdop B (1989) Fatal lignocaine poisoning: report of two cases and review of the literature. Hum Toxicol 8:389 Eledjam JJ, de la Coussaye JE, Bassoul B, Brugada J (1988) Mechanisms of the cardiac toxicity of bupivacaine. Ann Fr Anesth Reanim 7:204–210 Garlid KD, Nakashima RA (1983) Studies on the mechanism of uncoupling by amine local anesthetics. J Biol Chem 258:7974–7980 Heavner JE (2002) Cardiac toxicity of local anesthetics in the intact isolated heart model: a review. Reg Anesth Pain Med 27:545–555 Kratochwil NA, Huber W, Mu¨ller F, Kansy M, Gerber PR (2002) Predicting plasma protein binding of drugs: a new approach. Biochem Pharmacol 64:1355–1374 Lo L-W, Koch CJ, Wilson DF (1996) Calibration of oxygendependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application
Eur J Drug Metab Pharmacokinet for measuring oxygen concentration in biological systems. Anal Biochem 236:153–160 Muravchick S, Levy RJ (2006) Clinical implications of mitochondrial dysfunction. Anesthesiology 105:819–837 Schulz M, Schmoldt A (2003) Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics. Pharmazie 58:447–474 Shaban S, Marzouqi F, Al Mansouri A, Penefsky HS, Souid AK (2010) Oxygen measurements via phosphorescence. Comput Methods Programs Biomed 100:265–268 Sztark F, Tueux O, Erny P, Dabadie P, Mazat JP (1994) Effects of bupivacaine on cellular oxygen consumption and adenine nucleotide metabolism. Anesth Analg 78:335–339
Sztark F, Malgat M, Dabadie P, Mazat JP (1998) Comparison of the effects of bupivacaine and ropivacaine on heart cell mitochondrial bioenergetics. Anesthesiology 88:1340–1349 Tanz RD, Heskett T, Loehning RW (1984) Comparative cardiotoxicity of bupivacaine and lidocaine in the isolated perfused mammalian heart. Anesth Analg 63:549–556 Terada H, Shima O, Yoshida K, Shinohara Y (1990) Effects of the local anesthetic bupivacaine on oxidative phosphorylation in mitochondria. J Biol Chem 265:7837–7842
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ORIGINAL PAPER
Inhibition of murine cardiomyocyte respiration by amine local anesthetics Elhadi H. Aburawi • Abdul-Kader Souid
Received: 1 June 2013 / Accepted: 6 November 2013 Ó Springer-Verlag France 2013
Abstract The hydrophobic amino acyl amide-linked local anesthetics (e.g., lidocaine and bupivacaine) impose potent cardiac toxicity and direct mitochondrial dysfunction. To investigate these adverse events, an in vitro system was employed to measure their effects on O2 consumption (cellular respiration) by murine myocardium. Specimens were collected from the ventricular myocardium and immediately immersed in ice-cold Krebs–Henseleit buffer saturated with 95 % O2:5 % CO2. O2 concentration was determined as a function of time from the phosphorescence decay rates of Pd(II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Myocardial O2 consumption was linear with time (zero-order kinetics); its rate (k, in lM O2 min-1), thus, was the negative of the slope of [O2] vs. time. Cyanide inhibited O2 consumption, confirming the oxidation occurred in the respiratory chain. Lidocaine and bupivacaine produced immediate and sustained inhibition of cellular respiration at plasma concentrations of the drugs (low micromolar range). Bupivacaine was twice as potent as lidocaine. The inhibition was dose-dependent, saturating at concentrations C30 lM. At saturating doses, lidocaine produced *20 % inhibition and bupivacaine *40 % inhibition. Cellular ATP was also decreased in the presence of 30 lM lidocaine or bupivacaine. The studied amines inhibited myocardial cellular respiration. This effect is consistent with their known adverse events on mitochondrial function. The described approach allows accurate assessments and comparisons of the toxic effects of local anesthetics on heart tissue bioenergetics.
E. H. Aburawi (&) A.-K. Souid Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Ain, United Arab Emirates e-mail: [email protected]
Keywords Oxygen Respiration Mitochondria Cardiomyocytes Local anesthetics
1 Background Lidocaine and bupivacaine are commonly used amino acyl amide-linked local anesthetics. These agents also possess anti-arrhythmic and pro-arrhythmic actions and are potent inhibitors of the mitochondrial respiratory chain. The drugs produce cardiac depression. Owing to its higher lipophilicity, bupivacaine is more cardiotoxic than lidocaine (Heavner 2002). Lidocaine and bupivacaine stabilize the neuronal membrane by inhibiting voltage-gated sodium channels. As a result, the postsynaptic membrane fails to transmit action potentials, creating the anesthetic effect (Arlock 1988). Bupivacaine also binds to the intracellular domain of sodium channels, blocking Na? influx into nerve cells and preventing depolarization. At toxic levels, the drug invokes direct cardiac toxicity. The bupivacaine-induced blockade of voltage-gated sodium channels is more sustainable and less reversible than lidocaine, accounting for its more prominent impairments of myocardial contractility (Clarkson and Hondeghem 1985; Tanz et al. 1984; Eledjam et al. 1988). The negative inotropic effect of bupivacaine is also linked to drops in intra-cytoplasmic [Ca2?], resulting in disturbed mitochondrial function and impaired cellular bioenergetics (Chazotte and Vanderkooi 1981; Sztark et al. 1998; Dabadie et al. 1987). Moreover, bupivacaine decreases myocardial O2 consumption in isolated perfused guinea pig heart (Tanz et al. 1984). In principle, the same findings apply to lidocaine. Moreover, the amino acyl amide-linked local anesthetics are potent uncouplers of oxidative phosphorylation (uncoupling phosphorylation to e- and H? transfer); thus,
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these agents impair cellular bioenergetics (Sztark et al. 1994; Garlid and Nakashima 1983; Terada et al. 1990). The term ‘‘uncoupling oxidative phosphorylation’’ implies a state of high O2 consumption and low ATP synthesis due to increased leakage of H? across the inner mitochondrial membrane. Uncoupling agents (uncouplers) function as H? carriers (protonophores); they halt oxidative phosphorylation by dissipating the proton-motive force (DW). An analytical method of assessing murine tissue respiration in vitro is recently reported (Al Samri et al. 2010). The same approach is employed here to assess the inhibitory effects of lidocaine and bupivacaine on ventricular muscle respiration (cellular mitochondrial O2 consumption). The main objective of the study was to explore potential mechanisms for the cardiotoxicity of local anesthetics. The results show lidocaine and bupivacaine inhibit respiration of the myocardium (inhibits in vitro O2 consumption by non-beating ventricular specimens). Bupivacaine is more toxic than lidocaine.
*60 % relative humidity and a 12-h light/dark cycle. All mice had ad libitum access to standard rodent chow and filtered water. The protocols received approval from the Animal Ethics Committee, United Arab Emirates University, College of Medicine and Health Sciences.
2 Materials and methods
2.4 O2 consumption
2.1 Reagents
Phosphorescence O2 analyzer was used to monitor O2 consumption by heart specimens. O2 detection was performed with the aid of Pd phosphor, which had an absorption maximum at 625 nm, and a phosphorescence maximum at 800 nm (Lo et al. 1996). The samples were exposed to light flashes (600 min-1) from a pulsed lightemitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc., Wheeling, IL). Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube after passing through a wide-band interference filter centered at 800 nm. The amplified phosphorescence decay was digitized at 1.0 MHz by a 20-MHz A/D converter (Computer Boards, Inc., Mansfield, MA). A program was developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices; http://www.mccdaq.com/daq-software/ universal-library.aspx). It allowed direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board; http://www.mccdaq.com/pci-data-acquisition/PCIDAS4020-12.aspx) (Shaban et al. 2010). The phosphorescence decay rate (1/s) was characterized by a single exponential; I = Ae-t/s, where I = Pd phosphor phosphorescence intensity. The values of 1/s were linear with dissolved O2: 1/s = 1/so ? kq[O2], where 1/ s = the phosphorescence decay rate in the presence of O2, 1/so = the phosphorescence decay rate in the absence of O2, and kq = the second-order O2 quenching rate constant in s-1 lM-1.
Pd (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (Pd phosphor) was purchased from Porphyrin Products (Logan, UT). Glucose (anhydrous) and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO). Lidocaine (10 mg mL-1 or 43 mM; m.w. 234.34) and bupivacaine (5 mg mL-1 or 17.3 mM; m.w. 288.43) were purchased from Aguettant Pharmaceuticals (France). The lidocaine and bupivacaine solutions were diluted in 0.9 % NaCl. Krebs–Henseleit (KH) buffer (115 mM NaCl, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCl, 1.25 mM CaCl2, 1.18 mM MgCl2, and 6 mM glucose, pH 7.4) saturated with O2:CO2 (95:5) was prepared daily. Pd phosphor solution (2 mg mL-1 = 2 mM) was prepared in dH2O and stored in aliquots at -20 °C. NaCN solution (1.0 M) was prepared in dH2O; the pH was adjusted to *7.0 with 12 N HCl and stored at -20 °C. Glucose oxidase (10 mg mL-1) was prepared in dH2O and stored at -20 °C. 2.2 Mice Male Taylor outbred mice (8–12 week old; weight & 18–22 g) were maintained at the animal facility in compliance with the NIH guidelines (http://grants.nih.gov/ grants/olaw/references/phspol.htm). The mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were housed in rooms maintained at 22 °C with
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2.3 Sample collection Male mice were anesthetized using urethane (100 lL per 10 g body weight, from a 25 % solution, w/v, in 0.9 % NaCl). A piece of the ventricular myocardium (about 15 mg) was excised from the heart using special scissors (Moria Vannas Wolg Spring, cat. # ST15024-10). The tissue cut was performed, while the heart was beating and well perfused. The specimen was immediately immersed in ice-cold KH buffer saturated with 95 % O2:5 % CO2. The specimen was then placed in 1.0 mL KH buffer supplemented with 3 lM Pd phosphor and 0.5 % fat-free bovine serum albumin and processed for O2 measurement at 37 °C as described below.
Eur J Drug Metab Pharmacokinet
Fig. 1 Inhibitory effects of the amine local anesthetics on cardiomyocyte respiration. Representative runs of O2 consumption by specimens from the ventricular myocardium are shown. The rate of cellular mitochondrial O2 consumption (k, lM O2 min-1) was set as the negative of the slope of [O2] vs. time. The lines are linear fits. Panels a and b show the inhibitory effects of 100 and 77 lM
lidocaine, respectively. Panels c and d show the effects of 0.35 and 0.087 lM bupivacaine, respectively. Panel e shows the effect of injecting 5 lL 0.9 % NaCl, which lowered the value of k by 18 %. Panel f shows the effects of injecting 10 lM bupivacaine, 10 mM NaCN and 50 lg mL-1 glucose oxidase
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Respiration was measured at 37 °C in 1-mL sealed vials. Mixing was with the aid of parylene-coated stirring bars. In vials sealed from air, [O2] decreased linearly with time, indicating the kinetics of cellular mitochondrial O2 consumption was zero order. The rate of respiration (k, in lM O2 min-1) was, thus, the negative of the slope d[O2]/dt. NaCN inhibited respiration, confirming O2 was consumed in the mitochondrial respiratory chain. Different concentrations of lidocaine or bupivacaine were injected directly into the vial during the O2 runs. As a result, the change in the slope d[O2]/dt reflected drug effects. The calibration reaction contained phosphate-buffered saline (PBS) with 3 lM Pd phosphor, 0.5 % fat-free albumin, 50 lg mL-1 glucose oxidase and various concentrations of b-glucose. The values of 1/s were linear with [b-glucose]; the value of kq was the negative of the slope (kq = 101.1 s-1 lM-1) (Lo et al. 1996).
mean = 16 %); this change was due to deviation of the [O2] vs. t curve from linearity as a result of using tissue instead of isolated cells. Nevertheless, the use of tissue is superior to isolated cardiomyocytes, which are subjected to physical and chemical (e.g., collagenase digestion) treatments. Percent of inhibition by the drug was corrected for the 16 % drift (artifact, e.g., for Fig. 1a, the percent of inhibition was 4216 % = 26 %). Similarly, 77 lM lidocaine produced 29 % inhibition (Fig. 1b), 10 lM bupivacaine 43 % inhibition (Fig. 1e), 0.35 lM bupivacaine 44 % inhibition (Fig. 1c) and 0.087 lM bupivacaine 0 % inhibition (Fig. 1d). Cyanide inhibited respiration, confirming the oxidation mainly
2.5 Cellular ATP Tissue fragments were homogenized in 0.5 mL of ice-cold 2 % trichloroacetic acid for 2 min. The supernatants were collected by centrifugation (1,0009g at 4 °C for 5 min) and stored at -20 °C. The samples were neutralized immediately before ATP measurements with 0.5 mL 100 mM Tris– acetate, 2 mM EDTA (pH 7.75). [ATP] was determined using the Enliten ATP Assay System (Bioluminescence Detection Kit, Promega, Madison, WI); 5 lL of the supernatant was added to 50 lL of the luciferin/luciferase reagent. The luminescence intensity was measured using GloMax Luminometer (Promega, Madison, WI). 2.6 Statistical analysis Data were analyzed using SPSS statistical package (version 20). Data were presented as mean ± SD (n). The Mann– Whitney test was used for nonparametric values; p \ 0.05 was considered significant.
3 Results Representative runs of myocardial respiration are shown in Fig. 1a–f. As expected, O2 concentration decreased linearly with time, confirming the zero-order kinetics of cellular O2 consumption. This finding also confirms the feasibility of using a fragment of the ventricular myocardium for accurate determination of the rate of respiration (k, in lM O2 min-1). Lidocaine (100 lM, final concentration in the vial) resulted in a drop of the value of k from 1.3 before the injection to 0.76 after the injection (*42 % difference, Fig. 1a). Injection of 5 lL of 0.9 % NaCl, however, also resulted in a milder change in the value of k (Fig. 1f; typically 14–18 %;
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Fig. 2 Inhibitory effects of the amine local anesthetics on cardiomyocyte respiration as a function of drug concentration. Panel a, percent inhibition is plotted as a function of drug concentration. The lines are logarithmic fits. Panel b, natural log of percent inhibition is plotted as a function of drug concentration. The lines are linear fits
Eur J Drug Metab Pharmacokinet Fig. 3 Effects of the amine local anesthetics on cardiomyocyte ATP content. Specimens from the ventricular myocardium were incubated in oxygenated KH buffer at 37 °C with and without the addition of 30 lM lidocaine or bupivacaine. At t = 15, 30 and 60 min, samples were removed from the incubation medium and processed for ATP measurement as described in Sect. 2. Values are means and error bars are standard deviations (n = 3). The standard curve (insert) included 1.0, 10 and 100 nM ATP
occurred in the respiratory chain. Glucose oxidase (catalyzes the oxidation of b-D-glucose into D-glucono-1,5-lactone) then depleted the remaining O2 in the solution (Fig. 1e). Figure 2a–b compares the dose-dependent inhibition of respiration by lidocaine vs. bupivacaine. The drug effects were saturated at about 30 lM, yielding about 20 % maximum inhibition by lidocaine and about 40 % by bupivacaine (Fig. 2a). The natural log of inhibition was somewhat linear with dosing; yielding a slope that was three times larger for bupivacaine (Fig. 2b). Thus, both drugs produced similar immediate inhibition of heart muscle cellular respiration, but bupivacaine was more potent than lidocaine. Figure 3 shows changes in cellular ATP as a function of time in the presence and absence of 30 lM lidocaine or bupivacaine. Consistently, as compared to untreated samples, cellular ATP contents were lower in the treated samples for 15 B t B 60 min.
4 Discussion In vitro studies on the effects of amine local anesthetics on ventricular myocardium are limited. In one report, the
highly lipophilic drug bupivacaine stimulated O2 consumption by isolated mitochondria from rat heart, while lidocaine did not. The maximum effect was observed at about 1.0 mM of bupivacaine. This effect was attributed to uncoupling oxidative phosphorylation, thus, accelerating the rate of oxidation. Moreover, a direct inhibition of complex I of the respiratory chain was also noted. The authors concluded that the effect of bupivacaine on mitochondrial bioenergetics is complex (uncoupling plus direct inhibition of NADH dehydrogenase) (Sztark et al. 1998). In mitochondria isolated from rat liver, bupivacaine collapsed the proton-motive force (an uncoupling effect that stimulates respiration) only in the presence of hydrophobic anions, such as tetraphenyl borate. The effect was postulated to be cooperative interactions between the molecules, producing protonophores (Terada et al. 1990). The amine local anesthetics are not protonophores, but can form lipophilic anion pairs to invoke uncoupler-like activity (Dabadie et al. 1987; Garlid and Nakashima 1983). The adverse effects of lidocaine and bupivacaine on mitochondrial bioenergetics are mostly inhibitory to the electron transport, which results in decreasing respiration (as shown in Figs. 1, and 2). The data here (Figs. 2, and 3),
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however, do not rule out the possibility that lidocaine and bupivacaine reduce cellular energy demands (expenditures). Inhibition of respiration was noted at plasma concentrations of the drugs, which could be an adverse event or a mode of action (see below). The effect of bupivacaine was also studied in cultured fibroblasts. The drug produced dose-dependent inhibition of O2 consumption; 50 % inhibition was at 1.5 mM. Consistent with the results in Figs. 1, 2, and 3, submillimolar concentrations were also inhibitory and the effect was immediate (Sztark et al. 1994). The studied amino acyl amide-linked local anesthetics are associated with measurable adverse effects on the mitochondrial O2 consumption. The finding of compromised cellular O2 consumption by these agents may partially explain their well-known clinical adverse events (Muravchick and Levy 2006). For example, bupivacaine is toxic to the brainstem (Bourne et al. 2010). In isolated perfused guinea pig Langendorff heart, both lidocaine (43–128 lM) and bupivacaine (1–10 lM) inhibited myocardial O2 consumption in a concentrationdependent manner; bupivacaine was more cardiotoxic than lidocaine (Tanz et al. 1984). Our study demonstrates the feasibility of using the phosphorescence-based oxygen instrument to investigate the effects of drugs on ventricular muscle mitochondrial O2 consumption (cellular respiration). The amino acyl amidelinked local anesthetics lidocaine and bupivacaine are potent inhibitors of cardiac mitochondrial function. The rational to investigate bupivacaine is related to its potent cardiac toxicity. Consistently, the results show bupivacaine is a more potent inhibitor of cellular bioenergetics than lidocaine (Figs. 2, 3). Plasma concentrations of lidocaine range from 1.5 to 5 lg mL-1 (6.4–21.3 lM) and toxic levels exceed 6 lg mL-1 (17.8 lM) (Schulz and Schmoldt 2003; Dawling et al. 1989). For bupivacaine, plasma levels following local or epidural infusion are 0.5–1.5 lg mL-1 (1.7–5.2 lM) and toxic levels are C2.0 lg mL-1 (6.9 lM) (Schulz and Schmoldt 2003; Dawling et al. 1989; Kratochwil et al. 2002). Because the phosphorescence O2 probe is suspended in 0.5 % albumin, the effective concentrations of lidocaine and bupivacaine in the O2 measuring solution are likely lower than the values shown in Figs. 1, and 2. The in vitro observations in Figs. 1, 2, and 3 may not directly extrapolate to the known in vivo cardiac toxicity of local anesthetics. For example, the effects of lidocaine and bupivacaine on cellular respiration are demonstrated here in non-contracting heart tissue. The clinical effects of the drugs, however, occur in contracting hearts. Of note, the uncoupling effect of the drugs is not noted since the measurements are performed without hydrophobic anions, such as tetraphenyl borate. As discussed above, the
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uncoupling effect (accelerated respiration) results from cooperative interactions between the local anesthetics and the hydrophobic anions, producing protonophores (Terada et al. 1990).
5 Conclusion The data show lidocaine and bupivacaine inhibit respiration of non-beating myocardium in vitro. This effect is consistent with reported inhibition of the mitochondrial respiratory chain by amine local anesthetics. Bupivacaine was more toxic than lidocaine. Acknowledgments The authors thank Mr. Jose Kochiyil and Dr. Sheela Benedict for their experimental assistance. Conflict of interest There are no potential, perceived, or real conflicts of interest. No financial or non-financial interests.
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