Ropivacaine and articaine are relatively new local anaesthetics extensively applied in regional anaesthesia [1,2]. High concentrations of ropivacaine – similar to other local anesthetics – are known to evoke cardiodepressant side effects both in vitro[3–6] and in vivo[7–9], with sometimes fatal outcome in the latter case. Little is known about articaine in this regard. Although the fatal consequences of ropivacaine overdose were generally attributed to cardiac arrest [10–12], contribution of the negative inotropic effects of these high drug concentrations cannot be excluded either. Ropivacaine was shown to exert negative inotropic effects in ventricular myocardium of various mammalian species, including dogs [3,13], pigs , guinea pigs [15,16], rats [4,17] and rabbits [5,6]; however, the exact mechanism of action was not explored in these studies. The negative inotropic and lusitropic effects of ropivacaine were claimed to be related to the impaired Ca2+ handling of sarcoplasmic reticulum . Ropivacaine was shown to decrease the amplitude of [Ca2+]i transients in aequorin-injected ferret papillary muscle preparations suggesting also the involvement of altered sarcoplasmic reticulum function . Articaine and ropivacaine decreased action potential duration and maximal rate of depolarization in canine ventricular myocytes [19,20]. These effects were accompanied by suppression of a variety of transmembrane ion currents including L-type Ca2+ current [19,20]. In contrast to the detailed electrophysiological characterization, no data have been published on the action of articaine on [Ca2+]i transients and sarcoplasmic reticulum function.
In the present study, therefore, we aimed to study the concentration-dependent effects of articaine and ropivacaine on the chief determinants of intracellular calcium handling and contractility in canine ventricular myocardium, in order to reveal the possible contribution of the altered sarcoplasmic reticulum function in the cardiodepressant effects of these local anaesthetics. Canine preparations were chosen because their electrophysiological properties are believed to be the most similar to those of humans [21,22]. Our results suggest that articaine and ropivacaine fail to modify cardiac contractility when applied in therapeutically relevant concentrations; however, cardiodepression can be anticipated with both drugs in cases of accidental intravenous injection or overdose.
Adult mongrel dogs of either sex (n = 24) were anaesthetized with intravenous injections of 10 mg kg−1 ketamine hydrochloride (Calypsol, Richter Gedeon Rt., Budapest, Hungary) and 1 mg kg−1 xylazine hydrochloride (CP-Xilazin, CP-Pharma, Burgdorf, Germany) according to a protocol approved by the local ethics committee and conforming to the principles outlined in the Declaration of Helsinki. The hearts were quickly removed and placed in Tyrode solution. A wedge-shaped section of the left ventricular wall, supplied by the left anterior descending coronary artery, was dissected and cannulated for isolation of single myocytes. Trabeculae were excised for measurement of contractility, and the remaining left ventricular tissue was used for preparation of sarcoplasmic reticulum vesicles.
Isolation of single ventricular myocytes
Single myocytes were obtained by enzymatic dispersion using the segment perfusion technique . Briefly, the left anterior descending coronary artery was cannulated and perfused with oxygenated Tyrode solution containing: NaCl 144, KCl 5.6, CaCl2 2.5, MgCl2 1.2, HEPES 5, and dextrose 11 mM at pH = 7.4. Perfusion was maintained until the removal of blood from the coronary system and then switched to a nominally Ca2+-free Joklik solution (Minimum Essential Medium Eagle Joklik Modification, Sigma–Aldrich Co., St. Louis, Missouri, USA) for 5 min. This was followed by a 30 min perfusion with Joklik solution supplemented with 1 mg ml−1 collagenase (type II; Worthington Biochemical Co., Lakewood, New Jersey, USA) and 0.2% bovine serum albumin (Fraction V; Sigma-Aldrich Co.) containing 50 μmol l−1 Ca2+. Portions of the left ventricular wall were cut into small pieces and the cell suspension, obtained at the end of the procedure, was washed with Joklik solution. Finally, the Ca2+ concentration was gradually restored to 2.5 mM. The cells were stored in Minimum Essential Medium Eagle until use.
Measurement of contractility
Thin ventricular trabeculae, having diameters less than 1 mm, were mounted in a plexiglas chamber allowing continuous superfusion (10 ml min−1) with Krebs solution containing: NaCl 120, KCl 5.4, CaCl2 2.7, MgCl2 1.1, NaH2PO4 1.1, NaHCO3 24 and glucose 5.5 mM. The solution was equilibrated with 95% O2 and 5% CO2 at a temperature of 37°C, and the pH was adjusted to 7.4 ± 0.05. Developed force was recorded under isometric conditions using a capacitive mechano-electronic transducer fixed to a micromanipulator. Each preparation was stretched to the length at which maximum developed force was evoked, and was allowed to equilibrate for 60 min while pacing at a constant cycle length of 2 s. Articaine (n = 7) and ropivacaine (n = 6) were applied at cumulatively increasing concentrations (each for 20 min) followed by a 60 min period of washout. Records were digitized at 1 kHz using Digidata 1200 A/D card (Axon Instruments, Foster City, California, USA) and stored for later analysis.
Recording of cytosolic Ca2+ transients
Changes in intracellular free Ca2+ concentration were assessed by the ratiometric technique using the calcium-sensitive fluorescence dye fura-2 . Isolated myocytes were loaded at room temperature in Tyrode solution containing 3 μmol l−1 of the acetoxymethyl ester of fura-2 in the presence of Pluronic F-127 for 10 min. After loading, a period of at least 30 min was allowed for de-esterification of the dye, then cells were stored at 15°C until use. Loaded cells were placed in a superfusion chamber fixed to the stage of an inverted microscope (Eclipse TE2000-U, Nikon, Japan) and viewed using a 40× oil immersion objective (CFI S-Fluor 40x oil, Nikon, Japan). Measurements were performed at 37°C. Cells were field-stimulated at a constant cycle length of 1 s through a pair of platinum wires. Rectangular pulses, having durations of 1–2 ms and amplitudes of twice the diastolic threshold, were generated by an electronic stimulator (DS-R3; Főnixcomp Ltd, Hungary) and delivered at a frequency of 1 Hz. Articaine (n = 6) and ropivacaine (n = 5) were applied at cumulatively increasing concentrations (each for 3 min) followed by a 10 min period of washout.
Excitation wavelengths of 340 and 380 nm were obtained from a xenon arc lamp (Ushio Deutshland GmbH, Steinhöring, Germany) by a dual-wavelength excitation monochromator and an on-line connected microcomputer (DeltaScan, Photon Technology International, New Brunswick, New Jersey, USA). Fluorescence emission was monitored at 510 nm using a R1527P photomultiplier tube (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany) at an acquisition rate of 500 Hz. Changes in intracellular free Ca2+ levels were approximated by the ratio of the fluorescence intensity obtained at 340 and 380 nm excitation after correction for nonspecific background fluorescence and recorded using FeliX32 Software and BryteBox Interface (Photon Technology International). Ten consecutive [Ca2+]i transients were averaged and analysed off-line.
Measurement of L-type Ca2+ current
Myocytes were superfused with oxygenated Tyrode solution, supplemented with 3 mM 4-aminopyridine, 1 μmol l−1 E 4031 and 30 μmol l−1 chromanol 293B in order to block K+ currents. Suction pipettes, fabricated from borosilicate glass, had a tip resistance of 2 MΩ after filling with pipette solution containing KCl, 110; KOH, 40; HEPES, 10; EGTA, 10; TEACl, 20; K-ATP, 3 mM. The pH of this solution was adjusted to 7.2 with KOH. Membrane currents were recorded at 37°C with an Axopatch-2B amplifier (Axon Instruments) using the whole cell configuration of the patch clamp technique . After establishing a high (1–10 GΩ) resistance seal by gentle suction, the cell membrane beneath the tip of the electrode was disrupted by further suction or by applying 1.5 V electrical pulses for 1 ms. The series resistance was typically 4–8 MΩ before compensation (usually 50–80%). Experiments were discarded when the series resistance was high or substantially increased during the measurement. Outputs from the clamp amplifier were digitized at 100 kHz under software control (pClamp 6.0; Axon Instruments). ICa was measured at a rate of 0.2 Hz using 200 ms long voltage pulses clamped to +5 mV from the holding potential of −40 mV. ICa was normalized to cell capacitance, determined in each cell using short hyperpolarizing pulses from −10 to −20 mV. Articaine (n = 5) and ropivacaine (n = 5) were applied at cumulatively increasing concentrations (each for 3 min) followed by a 10 min period of washout.
Preparation of heavy sarcoplasmic reticulum vesicles
Heavy sarcoplasmic reticulum vesicles, containing vesicles formed from membrane fragments of the terminal cisternae of the sarcoplasmic reticulum, were isolated from canine left ventricular muscle samples (obtained from 10 animals) according to Lai and Meissner  with slight modifications . Homogenization in 100 mM NaCl, 20 mM EGTA, 20 mM Na-HEPES, pH = 7.5, was followed by centrifugation at 4500g and the crude microsomes were collected by centrifugation at 40 000g from the supernatant. Actomyosin contamination was removed by solubilization in 600 mM KCl, the microsome fraction was collected at 109 000g. The pellet was resuspended and loaded onto a 20–45% linear sucrose gradient. Heavy sarcoplasmic reticulum (HSR) vesicles were extracted from the 36–38% region of the continuous sucrose gradient, collected by centrifugation, and resuspended in 0.4 mol l−1 sucrose, 10 mM K-PIPES, at pH = 7.0. Protein concentration was measured by the Biuret method.
Determination of ATPase activity
ATPase activity was determined at 37°C by a coupled enzyme assay in a medium containing 100 mM KCl, 20 mM Tris–HCl, 5 mM MgCl2, 5 mM ATP, 0.42 mM phosphoenolpyruvate, 1 μmol l−1 calcimycin (A23187, a Ca2+ ionophore), 0.2 mM NADH, 7.5 U ml−1 pyruvate kinase and 18 U ml−1 lactate dehydrogenase (pH 7.5). Total hydrolytic activity was measured as the decrease of optical density at the NADH absorbance wavelength (340 nm) and expressed in micromoles of inorganic phosphate per milligram of protein per minute (indicated as IU). The total hydrolytic activity of the HSR vesicles ranged from 2.71 to 2.82 IU, and Ca2+-dependent ATPase activity was identified as the portion of total hydrolytic activity inhibited by 5 mM thapsigargin. The drug was added to the preparation 5 min before starting the measurement of enzyme activity. The assay was performed in the presence of Ca2+ and EGTA mixtures, producing the estimated free Ca2+ concentration (2.4 mM) found to induce maximum SERCA activation.
Measurement of Ca2+ uptake and release in heavy sarcoplasmic reticulum vesicles
Ca2+ release from, and Ca2+ uptake into, cardiac HSR vesicles was measured using the Ca2+-sensitive dye antipyrylazo III. The absorbance was monitored at 710 nm by a SPEX spectrophotometer (Fluoromax, Jobin-Yvon Ltd., Longjumeau, France).
When measuring Ca2+ uptake, the vesicles were actively loaded with Ca2+ in the presence of articaine or ropivacaine at 37°C in a buffer containing 100 mM KCl, 7.5 mM Na-pyrophosphate, 20 mM MOPS, 1 mM ATP/MgCl2 and 0.25 mM antipyrylazo III at pH = 7.0. Ca2+ loading was initiated by addition of 100 μmol l−1 CaCl2 to the medium, and the rate of uptake was monitored as a decrease of extravesicular Ca2+ concentration. The absorbance signal was calibrated by consecutive additions of CaCl2. Total recording time in each experiment was 3–8 min.
When measuring Ca2+ release, the vesicles were passively loaded with Ca2+ during an overnight incubation in the presence of 200 μmol l−1 CaCl2. The sample was diluted so as to have a protein concentration of 10 mg ml−1. Ca2+ release was induced by addition of 0.3 mM thymol 5 min after preincubation with the given concentration of articaine or ropivacaine. The initial rate of Ca2+ uptake was determined from the increase of extravesicular Ca2+ concentration.
Results are expressed as mean ± SEM values. Statistical significance of differences was evaluated by using one-way ANOVA followed by Dunnett's test. Differences were considered significant when P was less than 0.05. Concentration–response curves were obtained by fitting data to the Hill equation using Microsoft Origin 6.0 software. Half-effective concentrations (EC50 values) and Hill coefficients were determined from these Hill fits.
Articaine (Ultracain ampoules, 5 ml, 2%) was obtained from Aventis Pharma Deutschland GmbH (Frankfurt, Germany), whereas ropivacaine (Naropin ampoules, 20 ml, 7.5 mg ml−1) was purchased from AstraZeneca AB (Söderstalje, Sweden). Both drugs were freshly diluted with Tyrode solution to final concentration on the day of experiment. Other drugs were obtained from Sigma–Aldrich Co.
Effects of articaine and ropivacaine on contractility
Both articaine and ropivacaine caused a concentration-dependent negative inotropic action on ventricular trabeculae paced at a constant frequency of 0.5 Hz (Fig. 1). This effect was statistically significant (P < 0.05) at the concentration of 10 μmol l−1, and suppression of the contractile force was practically complete at 1000 μmol l−1. These effects of articaine and ropivacaine were partially reversible (to 73 ± 21 and 85 ± 10% of the respective predrug control values) after 60 min superfusion with drug-free Tyrode solution. The morphology of the contraction curve was slightly modified by high concentrations of the compounds. There was a small, but statistically significant, lengthening in half-relaxation time induced by 300 μmol l−1 articaine (from 74 ± 4 to 85 ± 6 ms) and ropivacaine (from 75 ± 2 to 88 ± 4 ms, P < 0.05). No change in the time required to reach peak tension was observed (100 ± 5 and 102 ± 10 ms before and after 300 μmol l−1 articaine, the respective values with the same concentration of ropivacaine were 91 ± 6 and 95 ± 4 ms). Suppressive effects of the drugs on contractile force were characterized using Hill plots, yielding EC50 values of 73.7 ± 10.4 and 72.8 ± 14.1 μmol l−1 for articaine (n = 7) and ropivacaine (n = 6), respectively, with the corresponding Hill coefficients of 0.81 ± 0.08 and 1.08 ± 0.19. In summary, no statistically significant differences between the effects of the two drugs on contractile parameters were observed.
Effects of articaine and ropivacaine on [Ca2+]i transients
In isolated canine ventricular cells articaine and ropivacaine displayed a concentration-dependent reduction of systolic [Ca2+]i, with a concomitant decrease in the amplitude of [Ca2+]i transients (Fig. 2). Diastolic [Ca2+]i was not altered by the drugs. This suppressive effect was statistically significant at concentrations of 10 μmol l−1 or higher. Suppression of [Ca2+]i transients was statistically significant at concentrations of 10 μmol l−1 or higher, and was almost fully reversible upon washout. Fitting [Ca2+]i amplitude data to the Hill equation EC50 values of 87.4 ± 12 and 99.3 ± 17 μmol l−1, Hill coefficients of 0.87 ± 0.09 and 0.96 ± 0.15 were estimated for articaine (n = 6) and ropivacaine (n = 5), respectively. Both drugs resulted in a moderate prolongation of relaxation of the [Ca2+]i transient at high concentrations. Whereas 300 μmol l−1 articaine and ropivacaine increased the monoexponential decay time constant (from 200 ± 22 to 284 ± 40 ms, and from 199 ± 20 to 326 ± 39 ms, respectively, P < 0.05 in both cases), this effect was not significant at the 100 μmol l−1 concentration (the respective decay time constants were 222 ± 31 and 228 ± 20 ms). Again, differences between the effects of the two drugs were not statistically significant.
Effects of articaine and ropivacaine on L-type Ca2+ current
Articaine and ropivacaine caused concentration-dependent suppression of peak ICa in isolated canine ventricular cells (n = 5 for each drug). Peak ICa was defined as a difference between the peak value of ICa and its pedestal measured at the end of the voltage pulse. This suppressive effect was statistically significant at concentrations of 30 μmol l−1 and above. The magnitude of reduction was largely comparable in the case of articaine and ropivacaine, having respective EC50 values of 327 ± 56 μmol l−1 and 263 ± 67 μmol l−1, and Hill coefficients of 0.91 ± 0.23 and 1.27 ± 0.15. These differences are not statistically significant. Effects of both drugs developed within 1–2 min and were partially reversible during 10-min period of washout (Fig. 3).
Effects of articaine and ropivacaine in heavy sarcoplasmic reticulum vesicles
Articaine and ropivacaine caused a moderate, but statistically significant, reduction in both Ca2+ release and Ca2+ uptake of the HSR vesicles at concentrations of 300 μmol l−1 and above (Fig. 4). No significant differences were seen between these effects of ropivacaine and articaine. The decreased Ca2+ uptake was accompanied by proportional inhibition of the sarcoplasmic reticulum Ca2+ ATPase (significant from 200 μmol l−1). Although these effects were relatively small in amplitude, increasing the articaine concentration above 200 μmol l−1 failed to further suppress Ca2+ uptake or ATPase activity. In contrast, the effect of ropivacaine on ATPase activity was not saturated at the highest applied concentration of 1 mM.
The study is the first to analyse the effects of articaine and ropivacaine on intracellular calcium handling in canine ventricular cardiomyocytes. The major finding of the present study was to show that articaine and ropivacaine reduced contractility and [Ca2+]i transients in canine ventricular cells. Regarding articaine this is the first study, whereas ropivacaine, similarly to bupivacaine, has been previously shown to decrease [Ca2+]i transients in ferret papillary muscles in concentrations comparable to the present results obtained in canine myocytes . No significant differences were obtained between the suppressive effects of articaine and ropivacaine on contractility, [Ca2+]i transients and ICa, suggesting that cardiodepressant side effects of the two drugs may also be similar. In comparison with bupivacaine, less cardiodepressant effects are anticipated with articaine since bupivacaine was shown to cause a stronger suppression of ICa than articaine or ropivacaine [19,20,27] in canine ventricular myocytes. In line with these results, the negative inotropic action of ropivacaine was found to be significantly weaker than that of bupivacaine in canine , rabbit [5,6] and guinea pig  cardiac preparations.
Mechanism of the negative inotropic action of articaine and ropivacaine
In our study suppression of contractility and [Ca2+]i transients was statistically significant at the 10 μmol l−1 concentration, and displayed very similar concentration dependences: the respective EC50 values were 74 and 87 μmol l−1 for articaine, whereas they were 73 and 99 μmol l−1 for ropivacaine. It can be concluded, therefore, that the decreased contractility is due to the reduction in the [Ca2+]i transient. Suppression of ICa required somewhat higher concentrations: it was significant from 30 μmol l−1, and half maximal block occurred close to 300 μmol l−1. The magnitude of ICa inhibition, induced by articaine and ropivacaine in the present study, is in good agreement with previous results obtained in canine and guinea pig ventricular cells [19,20,28,29].
Suppression of [Ca2+]i transients can be caused by altered kinetics of Ca2+ release and/or Ca2+ reuptake in the sarcoplasmic reticulum, or, alternatively, by changes in trans-sarcolemmal Ca2+ fluxes. Since at concentrations lower than 200 μmol l−1 both drugs failed to modify Ca2+ release and Ca2+ uptake in HSR vesicles, and also left SERCA ATPase activity unaltered, changes in transmembrane Ca2+ movements seem to be the underlying mechanism. This is congruent with the reduction of ICa observed in the presence of articaine and ropivacaine. However, at the concentration of 10 μmol l−1 no significant reduction in ICa was observed in contrast to the significant suppression of [Ca2+]i transients and contractility. This discrepancy can probably be ascribed to inhibition of Na+ current by these local anaesthetics. Indeed, both articaine [19,30] and ropivacaine [20,29,31,32] were shown to decrease the maximum velocity of action potential upstroke by blockade of cardiac Na+ channels at low concentrations. This, in turn, leads to a reduction in [Na+]i, resulting in increased Ca2+ efflux and decreased Ca2+ influx through the Na+/Ca2+ exchanger . In summary, the decreased Ca2+ content of the sarcoplasmic reticulum due to a reduction of net trans-sarcolemmal Ca2+ influx may be the reason for the reduced amplitude of [Ca2+]i transients, and the concomitantly diminished contractility.
Effects of local anesthetics on sarcoplasmic reticulum Ca2+ handling
Although neither articaine nor ropivacaine influenced Ca2+ release and Ca2+ uptake at concentrations lower than 300 μmol l−1 in our canine HSR vesicles, at higher concentrations both drugs significantly suppressed Ca2+ release and uptake with the concomitant reduction in SERCA ATPase activity. From this point of view actions of the two drugs were somewhat different. The articaine-induced inhibition saturated around 0.5 mM and its maximal magnitude was approximately 20%, whereas the inhibitory effect of ropivacaine increased progressively with increasing concentrations of the drug, similar to the action of lidocaine on canine sarcoplasmic reticulum ATPase . As could be expected, inhibition of ATPase activity showed good correlation with the reduction in Ca2+ uptake. There is no explanation, however, for the apparently similar actions on the Ca2+ release and Ca2+ uptake observed with high concentrations of articaine or ropivacaine. In the absence of relevant data in the literature on the effects of articaine and ropivacaine on RyR2, our results can be compared only with similar actions of other local anaesthetics. Conductance of the RyR2 channel of the sheep was reduced by procaine and QX222 , and ryanodine binding was decreased by bupivacaine and tetracaine in porcine sarcoplasmic reticulum vesicles . Except for tetracaine, which had an EC50 value of 100 μmol l−1, all these effects were evident only in millimolar concentrations, suggesting unspecific interactions between RyR2 and the local anaesthetic compound. It is interesting to note that Ca2+ release from rat sarcoplasmic reticulum vesicles was significantly increased by bupivacaine . This result is different from the observations obtained with local anaesthetics in other species, suggesting that rat may be an inappropriate species for local anaesthetic studies.
The clinical relevance of the present electrophysiological data can be evaluated only when comparing the concentrations used in our experiments to the plasma levels of articaine and ropivacaine measured in patients during anaesthesia. The typically found peak plasma levels were 6–7 μmol l−1 for both articaine [38,39] and ropivacaine [40–42], although peak concentrations of 10–12 μmol l−1 were also measured after administration of high doses of ropivacaine [43,44]. Since the lowest concentrations of articaine and ropivacaine that caused a statistically significant reduction in the amplitude of [Ca2+]i transients and force of contraction was 10 μmol l−1 in our study, neither articaine nor ropivacaine is likely to alter cardiac Ca2+ handling and contractility markedly at plasma levels typically obtained during neuraxial or regional anaesthesia. However, in cases of overdose or intoxication caused by accidental intravenous injection, plasma levels may increase above 100 μmol l−1 for a short period of time. These concentrations may strongly compromise the mechanical performance of the heart in addition to development of life-threatening cardiac arrhythmias .
Limitations of the study
Although canine ventricular preparations are believed to be the best human model for electrophysiological studies, it is not safe to directly extrapolate the present results to humans. However, in the absence of relevant human data, the best predictions can be based on observations made in these animal models.
Recordings of ICa and [Ca2+]i transients were obtained in isolated cardiac cells. The cell digestion procedure is based on the application of proteolytic enzymes, which may be harmful to integral membrane proteins (e.g. ion channels, transporters and receptors) as well. This must always be considered as an additional source of uncertainty when evaluating single cell results.
Financial support for the studies was provided by grants from the Hungarian Ministry of Health (ETT-060/2006), the Hungarian Research Fund (OTKA-K68457, OTKA-K73160, OTKA-K61442) and the Medical and Health Science Centre of University of Debrecen (MEC-14/2008).
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