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The Influence of Age on Bupivacaine Cardiotoxicity

Kiuchi, Marcio G. MD*; Zapata-Sudo, Gisele MD, PhD*; Trachez, Margarete M. MD, PhD; Ririe, Douglas MD; Sudo, Roberto T. MD, PhD*

doi: 10.1213/ANE.0b013e31820a9178
Anesthetic Pharmacology: Research Reports
Chinese Language Editions

BACKGROUND: The susceptibility of children and newborns to cardiotoxicity from racemic bupivacaine, RS(±)-bupivacaine, is controversial. Some studies indicate that newborns can sustain higher bupivacaine plasma levels than adults, without severe toxicity. In this study, we compared the influence of age on cardiotoxicity from RS(±)-bupivacaine and S(−)-bupivacaine in rats. The effects of these local anesthetics (LAs) on the regulation of intracellular Ca2+ concentrations in cardiac fibers were also investigated.

METHODS: The lethal dose was determined in ventilated male Wistar rats at 2, 4, 8, and 16 weeks of age by monitoring when cardiac electrical activity stopped after infusion of RS(±)-bupivacaine and S(−)-bupivacaine (4 mg · kg−1 · min−1). The effects on cardiac muscle contraction were investigated by in vitro measurement of papillary muscle twitches in the presence and absence of RS(±)-bupivacaine or S(−)-bupivacaine. Skinned ventricular fibers were used to investigate the intracellular effects on Ca2+ regulation induced by both LAs.

RESULTS: The lethal dose for RS(±)-bupivacaine and S(−)-bupivacaine in 2-week-old animals (46.0 ± 5.2 and 91.3 ± 4.9 mg · kg−1, respectively) was higher than in 16-week-old animals (22.7 ± 1.3 and 22.0 ± 2.7 mg · kg−1, respectively). Papillary muscle twitches were reduced in a dose-dependent manner, with significant difference between young and adult hearts. In adults, the muscle twitches were reduced to 8.6% ± 0.8% of control by RS(±)-bupivacaine, and to 18.1% ± 2.7% of control by S(−)-bupivacaine (100 μM). S(−)-bupivacaine had a positive inotropic effect at <10 μM, but only in 2-week-old animals. In chemically skinned ventricular fibers, RS(±)-bupivacaine and S(−)-bupivacaine induced similar increases in Ca2+ release from the sarcoplasmic reticulum (SR) preactivated with caffeine (1 mM), and this effect was greater in younger rats than adults. In 16-week-old rats, caffeine-induced tension was 53.9% ± 1.7% of the maximal fiber response with RS(±)-bupivacaine, and 54.1% ± 3.2% with S(−)-bupivacaine. The caffeine response in 2-week-old rats was 81.1% ± 3.7% of the maximal response with RS(±)-bupivacaine, and 78.1% ± 4.5% with S(−)-bupivacaine. The Ca2+ sensitivity of contractile proteins was equally increased at both ages tested, with RS(±)-bupivacaine or S(−)-bupivacaine. Ca2+ uptake from the SR was not altered by the LA or by age.

CONCLUSIONS: Differences in the mechanisms for regulating intracellular SR Ca2+ may contribute to the decreased susceptibility of young animals to cardiodepression induced by RS(±)-bupivacaine and S(−)-bupivacaine.

Published ahead of print February 8, 2011 Supplemental Digital Content is available in the text.

From the *Programa de Desenvolvimento de Farmacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Serviço de Anestesiologia, Departamento de Cirurgia, Universidade Federal Fluminense, Rio de Janeiro, Brazil; Department of Anesthesia, Wake Forest University Winston-Salem, North Carolina.

Supported by Cristália Produtos Químicos e Farmacêuticos Ltda, São Paulo, Brazil; Conselho Nacional de Desenvolvimento Cientifico e Tecnológico; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Fundação Universitária Jose Bonifácio; and Fundação Carlos Chagas Filho de Amparo á Pesquisa do Estado do Rio de Janeiro.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Roberto T. Sudo, MD, PhD, Universidade Federal do Rio de Janeiro, Rua Republica do Peru 114/802 Rio de Janeiro, Brazil 22021-040. Address e-mail to

Accepted November 16, 2010

Published ahead of print February 8, 2011

RS(±)-bupivacaine, introduced in 1963, is the most widely used local anesthetic (LA) because of its longer effectiveness than lidocaine1,2 and selective action on different neuronal fibers. However, RS(±)-bupivacaine is 6- to 10-fold more cardiotoxic than lidocaine.35 Cardiotoxicity induced by bupivacaine after accidental IV administration led to a plasma concentration of 0.5 to 5 μg · mL−1 (1.7–17 μM), which depressed myocardial conduction68 and contractility.5,9 The cardiotoxicity of RS(±)-bupivacaine and its isomers is attributed to blocking sodium,10 potassium,11 and L-type calcium channels12,13 in the sarcolemma of cardiomyocytes. In addition, LAs can either decrease9,14 or increase the Ca2+ mobilization15 from the sarcoplasmic reticulum (SR) and also decrease the sensitivity of myofilaments to Ca2+.16 The stereoselective effects induced by the bupivacaine isomers, R(+)- and S(−)-bupivacaine, have been intensively studied to determine which of the 2 isomers causes less cardiotoxicity while still providing potent nerve blockade. Laboratory and clinical studies showed that R(+)-bupivacaine and S(−)-bupivacaine have similar LA durations17; however, R(+)-bupivacaine is associated with a more intense negative chronotropism10 and a more arrhythmogenic effect than the S(−) isomer.1820 This cardiotoxic effect has been investigated in pediatric patients for several decades with controversial results. Some studies suggest that neonates and children may support higher blood levels of bupivacaine after IV administration than adults,21,22 but other studies demonstrated an increased neonatal heart susceptibility to RS(±)-bupivacaine.2325 In this study, we investigated how age may change the toxicity induced by RS(±)-bupivacaine and S(−)-bupivacaine by determining the maximal supporting dose (lethal dose [LD]) after systemic administration and by measuring the papillary muscle and ventricular skinned fiber contraction in rats from 2 to 16 weeks of age.

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The Universidade Federal do Rio de Janeiro Animal Care and Use Committee approved the protocols used in this work. Animals were housed in a temperature, humidity, and 12-hour day/dark controlled room. Water and food were offered ad libitum.

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Determination of RS(±)-Bupivacaine and S(−)-Bupivacaine LD

The LD was determined in male Wistar rats at 2, 4, 8, and 16 weeks of age. Under anesthesia with sodium pentobarbital (50 mg · kg−1, intraperitoneally), a plastic tube was introduced into the trachea and connected to a Harvard Apparatus model 681 (Harvard Apparatus, Holliston, MA) for controlled pulmonary ventilation.The pattern of ventilation was adjusted to a volume of 15 mL · kg−1 and 40 to 60 breaths per minute to maintain the pH, PO2, and PCO2 in the normal range. A pair of electrodes was fixed to the chest for electrocardiographic recording. RS(±)-bupivacaine or S(−)-bupivacaine was continuously infused via the external jugular vein at a dose of 4 mg · kg−1 · min−1 through a micrometric Harvard pump (model 1100). This dose and rate of infusion were experimentally adjusted in preliminary experiments. The pump infusion was immediately stopped when absence of cardiac electrical activity, the variable used to indicate death, was observed. Amount of drug injected was calculated from the time and rate of infusion and converted to LD for each animal. LD was expressed as mean ± SEM for each LA. The estimated lethal free serum concentration was estimated considering a 90% protein binding to LA.

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Preparation and Investigation of Papillary Muscle

Male Wistar rats at 2, 4, 8, and 16 weeks of age were killed under anesthesia with sodium pentobarbital (60 mg · kg−1, intraperitoneally). Hearts were quickly removed and papillary muscles dissected, positioned in a vertical chamber filled with a solution of (in mM) NaCl, 130; KCl, 5; MgCl2, 1; CaCl2, 2.5; NaH2PO4, 0.5; NaHCO3, 24; glucose, 5.6; pH 7.4, and prepared for isometric tension recording. The solution was continuously oxygenated with a carbogen mixture (95% O2/5% CO2) keeping temperature at 37°C ± 0.2°C. Each muscle was mounted between 2 hooks with one end attached to a fixed clamp and the other to a force transducer (Grass model FT03; Grass Technologies, West Warwick, RI). A Cyberamp model 380 (Axon Instruments, Inc., Union City, CA) was used to condition the signal generated by the transducer. Muscle twitches were induced by field electrical stimulation (40–50 V, 2-ms duration, and 1-Hz frequency) and analyzed with Axoscope software (Axon Instruments, Inc.). After 30 minutes of adaptation, the muscle was exposed to incrementally increasing concentrations (1–100 μM) of RS(±)-bupivacaine or S(−)-bupivacaine added to the solution. This range of LA concentration was based on previous experiments performed in isolated heart.26 Twitch amplitude of papillary muscles was measured for the control and in the absence or presence of RS(±)-bupivacaine or S(−)-bupivacaine. Data were presented as percentage of control twitches measured at the start of each experiment.

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Cardiac Skinned Fiber Preparation

Fascicles approximately 0.30 mm in diameter and 1 to 2 mm long were excised from the subendocardium of the left ventricular wall from Wistar rats (2 and 16 weeks old) at room temperature in oxygenated (95% O2 and 5% CO2), nominally Ca2+-free buffered saline (in mM): NaCl, 130; KCl, 5; MgCl2, 1; NaH2orally4, 0.5; NaHCO3, 24; glucose, 5.6; pH 7.0. Fascicles were transferred to a 1-mL internal volume chamber filled with relaxation (R) solution (mM) (K propionate, 185; Mg acetate, 2.5; imidazole propionate, 10; K2Na2ATP, 5; and K2-ethyleneglycol-bis[β-aminoethylether]-N,N,N′,N′-tetraacetic acid [EGTA], 5; pH 7.0) and the ends were attached to 2 hooks, one connected to a force transducer (model FT03; Grass Technologies) and the other to a micromanipulator (Narishige model 3, East Meadow, NY). Fascicles were stretched to 120% of resting length using a binocular stereomicroscope and exposed to solution R containing the detergent saponin (0.5% v/v; Merck Chemical Co., Darmstadt, Germany) over 5 minutes.26 This procedure created a sarcolemma lesion that permitted free access of solution without damaging the functionality of the contractile protein and SR.27 To investigate the direct effects of RS(±)-bupivacaine and isomers on the Ca2+ sensitivity of contractile proteins, SR membranes were further disrupted by 60 minutes of exposure to solution R containing the nonionic detergent octylphenoxy polyethoxyethanol (Triton X-100, 1% v/v; Sigma Chemical Co., St. Louis, MO).28 Temperature was maintained at 22.0°C ± 0.5°C.

After the skinning procedure, the maximal contractile response of the fibers was determined by exposure to a solution containing 15.85 μM Ca2+ (pCa 4.8), which was the amount of free Ca2+ required to induce the maximal contraction response (not shown). Fiber relaxation was induced by exposure to solution R during the Ca2+-activated contraction plateau. Isometric tension generated by the fibers was recorded on a chart recorder (Grass model 7400). After measuring the maximal contraction response, the SR was depleted of Ca2+ using 20 mM caffeine dissolved in solution R and the SR Ca2+ loading cycle induced by 3 minutes of exposure to pCa 6.8 (0.16 μM Ca2+) prepared by adding K2EGTA and CaK2EGTA to maintain a total EGTA concentration of 5 mM. Association constants for K2EGTA/CaK2EGTA ratios required to achieve the desired pCa were from Orentlicher et al.29 For other ligands, constants were from Fabiato and Fabiato.30 The efficiency of the SR Ca2+ loading procedure was evaluated by measuring the contractile response induced by caffeine (20 mM) in wash solution (solution R without EGTA).

To investigate the effect of RS(±)-bupivacaine and its isomer on Ca2+ release from the SR, after the loading cycle, each LA (0.001–10 mM) was added to solution R containing 1 mM caffeine, which was insufficient to promote complete release of stored Ca2+ from the SR. Thus, the tension amplitude was smaller than with 20 mM caffeine. Additional increases in the contraction amplitude were used to measure the effect of LA on Ca2+ release from the SR.

Effects on SR Ca2+ uptake were evaluated by repeating the loading cycle with pCa 7.4 over 1 minute in the presence or absence of RS(±)-bupivacaine or S(−)-bupivacaine (0.1, 0.5, 1.0, and 5.0 mM). In this protocol, submaximally loaded Ca2+ was obtained by reducing the concentration in the solution and the loading time. Procaine (40 mM) was added during loading to prevent Ca2+ leak. Tension response to caffeine (20 mM) was used as an indicator of Ca2+ accumulation.

Effects of RS(±)-bupivacaine and the isomer on Ca2+ sensitization of contractile proteins were studied in SR-disrupted preparations. The pCa versus isometric tension curves were performed in the absence (control) and presence of 5.0 mM RS(±)-bupivacaine or S(−)-bupivacaine. The pCa value that induced 50% of maximal tension (pCa50) was calculated using the Hill equation. Six experiments were performed for each LA for both the 2- and 16-week-old groups.

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Statistical Analysis

All data were expressed as means ± SEM. Differences between LD means were evaluated by Student t test with statistical significance at P < 0.05. To analyze LA effects on papillary muscle isometric tension, one-way analysis of variance (ANOVA) was followed by the Dunnett test. ANOVA followed by Dunn's method was used to compare responses between age groups. Tension amplitudes of skinned fibers were expressed as percentage of maximal fiber response. Comparison between RS(±)-bupivacaine and S(−)-bupivacaine on SR Ca2+ release was evaluated using ANOVA and the Bonferroni test for critical difference. The Kruskal-Wallis test followed by the Student-Newman-Keuls test was used to compare pCa50 values. Ca2+ concentration-response curves were fitted to the equation y = ymax · Ca2+n (Ca2+n + k0.5), where y is the percentage of isometric tension, n the Hill coefficient, and k0.5 the Ca2+ concentration producing 50% of the maximal tension. Differences were considered significant at P < 0.05.

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Lethal Dose

To determine the LD for mechanically ventilated rats, RS(±)-bupivacaine and S(−)-bupivacaine were infused at 4 mg · kg−1 · min−1 until cardiac electrical activity stopped. As shown in Figure 1, the LD for RS(±)- and S(−)-bupivacaine was greater in 2-week-old rats than in older groups. The LD for RS(±)-bupivacaine in 2-week-old rats was 46.0 ± 5.2 mg · kg−1, which was significantly higher (P < 0.01) than in 4-week-old rats (24.0 ± 3.4 mg · kg−1), 8-week-old rats (23.3 ± 2.2 mg · kg−1), or 16-week-old rats (22.7 ± 1.3 mg · kg−1). For 2-week-old animals, the LD for infusion of S(−)-bupivacaine was 91.3 ± 4.9 mg · kg−1, significantly higher (P < 0.01) than for animals at 4 weeks (24.0 ± 4.3 mg · kg−1), 8 weeks (28.7 ± 3.2 mg · kg−1), or 16 weeks (22.0 ± 2.7 mg · kg−1). Also, in the 2-week-old group, the LD for S(−)-bupivacaine was higher (P < 0.01) than for RS(±)-bupivacaine.

Figure 1

Figure 1

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Effects of RS(±)-Bupivacaine and S(−)-Bupivacaine on Papillary Muscle Contraction

Figure 2 shows the effects of RS(±)- and S(−)-bupivacaine (25 μM) on twitches from electrically stimulated papillary muscles of rats aged 2, 4, 8, and 16 weeks. At 2 weeks, twitches were not significantly decreased after exposure to RS(±)-bupivacaine (88.7% ± 3.4% control) or S(−)-bupivacaine (104.9% ± 5.6% control). However, they were significantly reduced in an age-dependent manner for both RS(±)- and S(−)-bupivacaine. The maximal depression effect was observed in 16-week-old animals with 28.2% ± 6.0% of the control for RS(±)-bupivacaine, and 53.4% ± 7.8% for S(−)-bupivacaine. Importantly, RS(±)-bupivacaine caused a more significant reduction (P < 0.05) in papillary muscle depression than S(−)-bupivacaine in 16-week-old rats (Fig. 2).

Figure 2

Figure 2

When papillary muscles of 2- and 16-week-old rats were exposed to increasing concentrations (2–100 μM) of RS(±)- and S(−)-bupivacaine, several different effects were observed (Fig. 3). Cardiac depression was more prominent in adults (16 weeks). In the presence of 10 μM RS(±)-bupivacaine, twitches in 2-week-old rats were 95.5% ± 2.1% of control, and in 16-week-old rats they were 35.1% ± 5.7% of the control (P < 0.01). In the adult group, the twitches were 8.6% ± 0.8% of control for 100 μM RS(±)-bupivacaine, and 18.1% ± 2.7% for S(−)-bupivacaine (Fig. 3). At the same concentration, RS(±)- and S(−)-bupivacaine reduced the twitches to 58.7% ± 2.9% and 56.0% ± 7.0% of the control in the young group (2 weeks old) (Fig. 3). Although not observed in adults, an interesting biphasic response to S(−)-bupivacaine occurred in 2-week-old rats with increased contractility at 5 to 10 μM (twitch amplitude at 10 μM was 119% ± 7% of control, P < 0.05), returning to control value at 20 μM and dose dependently decreasing with higher concentrations.

Figure 3

Figure 3

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Effect of LA on Ca2+ Loading and Release from SR in Skinned Cardiac Muscle

A sustained and maximal contraction response was induced in skinned ventricular fibers by a high concentration of Ca2+ (pCa 4.8; 15.85 mM Ca2+). All ordinate data in Figures 4 and 5 are expressed relative to maximal response. Figure 4 shows concentration-response curves for RS(±)- and S(−)-bupivacaine in fibers from 2- and 16-week-old animals. RS(±)-bupivacaine and S(−)-isomer did not change the caffeine-induced contraction at any tested concentrations (0.001–10 mM) in adult animals. In contrast, both LAs increased caffeine-induced contraction in the younger groups, in which the caffeine-induced response increased from 53.7% ± 3.2% to 81.1% ± 3.6% of the maximal response for RS(±)-bupivacaine, and from 50.9% ± 2.7% to 78.1% ± 4.5% for S(−)-bupivacaine (Fig. 4). These results demonstrate that either racemic or S(−)-bupivacaine can enhance caffeine-induced SR Ca2+ release in young animals with no difference between both LAs.

Figure 4

Figure 4

Figure 5

Figure 5

The effect of RS(±)-bupivacaine and the isomer on Ca2+ uptake from the SR was tested on ventricular skinned fibers loaded with pCa 7.4 solution (0.039 μM Ca2+) for 1 minute in the absence or presence of the LAs at 0.1, 0.5, 1.0, or 5.0 mM. The accumulation of Ca2+ in the SR was evaluated by measuring the isometric tension after addition of caffeine (20 mM). No significant changes were observed in the caffeine-induced contraction when RS(±)-bupivacaine or S(−)-bupivacaine was present during SR loading in ventricle fibers from 2- or 16-week-old animals. This finding suggested that the SR Ca2+ loading procedure was not affected by RS(±)- or S(−)-bupivacaine at either age.

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Effect of LA on Ca2+ Sensitivity of Contractile Proteins in Skinned Cardiac Fibers

Saponin-skinned ventricular fibers from 2- and 16-week-old rats were exposed to Triton X-100 for 60 minutes to disrupt SR membranes. To investigate the effects of LA on myofibril Ca2+ sensitivity, contraction was induced by increasing Ca2+ in the solution in the absence and presence of LA (5 mM). The Ca2+-induced contractions were altered by RS(±)- and S(−)-bupivacaine in both 2- and 16-week-old rats, with both RS(±)-bupivacaine and the isomer shifting the Ca2+-response curves to the left (Fig. 5). In adult animals (16 weeks), the Ca2+ concentration that caused 50% of maximal response ([Ca2+]50) decreased from 1.62 ± 0.07 to 0.71 ± 0.03 μM with RS(±)-bupivacaine (P < 0.01) and from 1.48 ± 0.02 to 0.66 ± 0.05 μM with S(−)-bupivacaine (P < 0.01). The [Ca2+]50 decreased from 1.58 ± 0.01 to 0.072 ± 0.03 μM with RS(±)-bupivacaine (P < 0.01) and from 1.70 ± 0.01 to 0.71 ± 0.03 μM with S(−)-bupivacaine (P < 0.01) in young animals (2 weeks) (Fig. 5). These results demonstrated that both LAs increased the Ca2+ sensitivity of contractile proteins, but this was not affected by age.

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In this study, we investigated age dependency of toxicity induced by RS(±)-bupivacaine and its S(−)-isomer. A significant difference in LD was observed in young animals (2 weeks) compared with older animals (>4 weeks) for both RS(±)- and S(−)-bupivacaine. Two weeks of age seemed to be the limit for decreased susceptibility to the toxic effect of bupivacaine because no difference in LD was observed among animals older than 4 weeks. However, differences in susceptibility to the drugs were detected in the younger group. The LD in 2-week-old animals compared with older animals was approximately 4-fold higher for S(−)-bupivacaine and approximately 2-fold higher for RS(±)-bupivacaine. This is in agreement with reports that human neonates and children support a higher plasma concentration of bupivacaine after accidental IV administration.21,22 No data have been reported comparing toxicity between bupivacaine isomers in human neonates or children. A combination of several mechanisms of respiratory depression and cardiac toxicity may explain the decreased susceptibility of young animals to bupivacaine. Because our experiments to determine the LD were conducted in artificially ventilated and convulsion-controlled conditions, our results suggest that direct cardiac toxicity may be involved.

Experiments from papillary muscles with RS(±)- and S(−)-bupivacaine demonstrated a decrease in twitch amplitude that was age- and LA-concentration dependent. RS(±)- and S(−)-bupivacaine gave significantly different results, with S(−)-bupivacaine showing less potency. Papillary muscles from 16-week-old animals were much more susceptible to depression induced by RS(±)- and S(−)-bupivacaine than muscles from 2-week-old animals (approximately 3 years old in humans). At <10 μM (specifically 2.9 μg · mL−1), which represents the clinical range, RS(±)-bupivacaine did not cause any significant effects in 2-week-old animals. However, a >60% reduction in contraction was observed in 16-week-old animals. In this tissue, S(−)-bupivacaine did not decrease twitches, but increased them by approximately 20%, an observation that could be of clinical significance.

The cardiac depression induced by LAs could be related to the alterations in the regulation of intracellular Ca2+ concentration that gradually changes with age. In adults, the Ca2+ source needed for myofibril activation and cardiac contraction depends on a Ca2+-induced–Ca2+-release (CICR) process that is triggered by a small amount of Ca2+ entry to the cell via an L-type Ca2+ channel. In newborns, CICR is not so efficient, probably because of immaturity of the T tubule,31 lower density of RyR2,32 and decreased SERCA2 activity.33 The main sources responsible for cardiac contraction in newborns are Ca2+ influx via the L-type Ca2+ channel3437 and Na+/Ca2+ exchanger, which is reversely activated by a decreased intracellular Na+ concentration.33 There is no information concerning the effects of bupivacaine and its S(−)-isomer on the L-type Ca2+ channel or in the Na+/Ca2+ exchanger in newborn rats. The negative inotropic effect observed with RS(±)-bupivacaine could be explained by a decrease in Ca2+ influx into the cell, caused by direct interaction with L-type Ca2+ channels.12,13 Thus, we suggest that RS(±)- and S(−)-bupivacaine promote a more significant cardio-depressant effect in older animals because the intracellular Ca2+ concentration depends entirely on the activation of L-type Ca2+ channels, and these LAs produce a nonstereoselective blockade.13 As a consequence, the CICR from intracellular stores could be impaired in these animals.

The LD varied from 20 to 90 mg · kg−1, which corresponded to a range of 61 to 277 μM. The free drug concentration would be approximately 6.1 μM (1.9 μg · mL−1) to 27.7 μM (8.1 μg · mL−1) considering the protein binding. However, because of several differences in young animals such as drug distribution, blood protein concentration, and rate of metabolism, the free drug concentration should be higher than expected. Pharmacokinetic data show that the lower plasma concentrations of albumin and α1-acid glycoprotein in young animals and humans compared with adults38 may result in less RS(±)- and S(−)-bupivacaine binding. Consequently, higher serum levels of these substances are found in children after injection,39 which may contribute to more severe cardiotoxic effect in young patients. The immaturity of isoforms CYP3A4 and CYP1A2 of the cytochrome P450 in young patients may also contribute to the increased cardiotoxicity of RS(±)- and S(−)-bupivacaine in pediatric patients40 because of reduced intrinsic clearance.39 Nonetheless, despite factors that favor higher toxicity of RS(±)- and S(−)-bupivacaine in the young, our results did not show the predicted increase in cardiotoxicity in younger animals.

Tension recording in the chemically skinned ventricular myocytes technique demonstrated the effects of RS(±)- and S(−)-bupivacaine on intracellular Ca2+ handling at different ages. We investigated the influence of RS(±)- and S(−)-bupivacaine on Ca2+ release and uptake from the SR, and the effect on the Ca2+ sensitivity of myofibrils in the SR after disruption with Triton X-100. At 2 weeks of age, but not at 16 weeks, RS(±)- and S(−)-bupivacaine caused an increase in Ca2+ release from SR Ca2+–loaded myocytes preactivated by 1 mM caffeine in a concentration-dependent manner. This effect in young cardiac myocytes was seen at Ca2+ concentrations >10 μM, and the intracellular Ca2+ concentration may reach the mM range during tension development in physiological conditions. Thus, higher Ca2+ release may be a significant mechanism for cardiac protection against bupivacaine toxicity in young animals.

The mechanism of SR Ca2+ release is not clear. Several studies of adult animals suggest that RS(±)-bupivacaine may activate SR Ca2+ release in skeletal and cardiac cells. RS(±)-bupivacaine increases the likelihood of RyR1 channel opening in vesicles from rabbit skeletal muscle,41 and this effect was completely inhibited by procaine, an LA known to block SR Ca2+ release.4244 Similar results were reported in skinned skeletal muscle fibers of rats, in which RS(±)-bupivacaine (1–15 mM) promoted SR Ca2+ release.45 RS(±)-bupivacaine also increases ryanodine binding in both skeletal and cardiac pig microsomes, suggesting activation of the RyR1 or RyR2.14 RS(±)-, S(−)-, and R(+)-bupivacaine also promoted Ca2+ release from the SR trough RyR2 of cardiac myocytes and this effect was stereoselective, with S(−)-bupivacaine more effective than R(+)-bupivacaine.15

An increase in SR Ca2+ uptake or a decrease in Ca2+ leakage could result in an additional mechanism that causes differences in the cardiac contractile response to bupivacaine. However, our data using skinned myocytes showed that RS(±)- or S(−)-bupivacaine during SR loading did not change the amplitude of caffeine-induced tension in either 2- or 16-week-old animals. Previous studies demonstrated that the diastolic Ca2+ concentration in spontaneously activated intact myocytes increased15 in the presence of RS(±)-, R(+)-, and S(−)-bupivacaine. These data suggest that bupivacaine may impair the mechanisms for removal of Ca2+ from the cytoplasm such as SERCA2, the Na+/Ca2+ exchanger, or the ATP-dependent Ca2+ pump, which are located in the sarcolemma.46

The change in Ca2+ sensitivity of contractile proteins was investigated in skinned cardiac myocytes, in which the SR was completely destroyed. Exposure to RS(±)- and S(−)-bupivacaine shifted the pCa versus isometric tension curve to the left in both 2- and 16-week-old animals, suggesting an increase in Ca2+ sensitivity of myofibrils. However, no difference was observed in the response to RS(±)- or to S(−)-bupivacaine. Thus, we concluded that the Ca2+ threshold for activation of myofibrils is reduced by RS(±)- and S(−)-bupivacaine, but this effect is not age dependent.

In conclusion, we demonstrated that the toxicity induced by RS(±)- and S(−)-bupivacaine in rats is age dependent. The LD for young animals (<2 weeks) is 2- to 4-fold higher than for 4-week-old or older animals. The decreased susceptibility of young animals to these LAs is probably related to differently regulated intracellular SR Ca2+ concentrations, but does not result from increasing the Ca2+ sensitivity of contractile protein. Thus, this study supported the hypothesis that young patients (younger than 4 years) are less susceptible to the systemic toxic effect of bupivacaine, especially to the S(−)-isomer.

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Name: Marcio G. Kiuchi, MD

Role: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Marcio G. Kiuchi has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Gisele Zapata-Sudo, MD, PhD

Role: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Gisele Zapata-Sudo has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Margarete M. Trachez, MD, PhD

Role: This author helped design the study, conduct the study, and analyze the data.

Attestation: Margarete M. Trachez has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Douglas Ririe, MD

Role: This author helped design the study and write the manuscript.

Attestation: Douglas Ririe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Roberto T. Sudo, MD, PhD

Role: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Roberto T. Sudo has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

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The authors are grateful to Cristália Produtos Químicos e Farmacêuticos Ltda, São Paulo, Brazil, for providing RS(±)- and S(−)-bupivacaine, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Fundação Universitária Jose Bonifácio (FUJB, Brazil), and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil) for financial support and fellowships from CNPq (to GZS and RTS).

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