Bupivacaine and ropivacaine are potent local anesthetics (LAs) frequently used clinically. Because of their high analgesic potency and long-lasting effects, both substances are commonly used for intraoperative regional anesthesia and postoperative pain management. All clinically used LAs inhibit specific sodium channels and prevent electrical activity in peripheral nerves. On their way from the place of injection to the intended treatment, LAs have to diffuse various structures like muscle tissue and cause specific dose-dependent local cytotoxic effects. Prior studies described myotoxic and neurotoxic properties in experimental animal models and revealed that lipophile LAs like bupivacaine have the highest myotoxic potential.1–4
Various mechanisms of LA-induced myotoxicity have been examined and discussed. An increase of intracellular calcium induced by LAs is suggested to be responsible for acute cytotoxic effects. Intracellular calcium seems to accumulate because of an increased calcium release from the sarcoplasmic reticulum via the type 1 ryanodine receptor. Simultaneously, calcium reuptake into the sarcoplasmic reticulum via the sarcoplasmic calcium ATPase is inhibited. Increased intracellular calcium-stimulated, energy-consuming processes can result in a breakdown of cellular homeostasis.5–8 In addition, inhibition of mitochondrial function can aggravate the deranged energy balance.9,10 Further studies also indicated an induction of apoptosis as relevant in LA-induced myotoxicity.11 Although the exact mechanisms are still not fully understood, a combination of all mechanisms described earlier seems to be responsible for skeletal muscle fiber damage.12
To further examine mechanisms of myotoxicity, in vitro models were established, and myotoxicity was studied in cell cultures.13 In general, 2 different types of muscle cell cultures can be distinguished. For primary cell cultures, tissue is harvested and placed in a proliferation medium. In muscle tissue, myoblasts as resting progenitor cells start to divide. With a change of medium, the growing myoblasts can be forced to differentiate into myocytes. In addition to tissue harvesting and dissection, another disadvantage is that primary cell cultures cannot proliferate indefinitely. Because of the physiological cell cycle, only a limited amount of cell divisions is possible until apoptosis occurs. Despite the fact that primary cell cultures are more complex to establish, they are suggested to be a realistic model.14,15
For most in vitro studies, however, immortalized cell lines are used because of feasibility reasons. Embryonic myocyte progenitor cells are proliferated through various passages to select immortal cells. Fusion with tumor cells is another option to immortalize cells. Immortalized cells, in general, are capable of dividing indefinitely and therefore simpler to culture.16 With a change of medium, differentiation into myocytes can be achieved.13
In theory, immortalization only has an influence on proliferation. Differentiated immortalized cells should have the same physiologic properties as differentiated cells of a primary cell culture. However, it cannot be excluded that selection of potential abnormal cells or fusion with tumor cells has any influence on basic cell physiology.
It was the aim of this study to compare the myotoxic effects of LAs on immortalized and primary cells in culture. Furthermore, we investigated whether different myotoxic properties of bupivacaine and ropivacaine can be reproduced in vitro and whether incubation time affects myotoxicity. Two different time points to measure survival were chosen to evaluate possible differences in acute necrosis or delayed cell death by apoptosis.
For the C2C12 cell line, cells (CLS-Cell Lines Service, Eppelheim, Germany) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and L-glutamine 2 mM (Life Technologies GmbH, Darmstadt, Germany). For the actual study, cells were removed from the primary flask with Trypsin-EDTA (Sigma-Aldrich Chemie GmbH) diluted 1:4 with phosphate-buffered saline (PBS) without calcium and magnesium chloride (Sigma-Aldrich Chemie GmbH) and 150,000 cells per well were transferred to 6-well plates (neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany).
For primary muscle cell culture, with consent of the local committee for Laboratory Animal Care, muscle cells of Mm. extensor digitorum longus, soleus, and tibialis anterior of BALB/c AnNcrl mice were extracted. Therefore, the thigh was skinned, and the muscles were prepared and washed in PBS. Intact muscles were incubated for 1.5 hours with 8 mL 0.2% Collagenase Typ 1 (GIBCO® Cell Culture, Invitrogen GmbH, Darmstadt, Germany) in DMEM, 1% penicillin-streptomycin (Sigma-Aldrich Chemie GmbH) and 1% L-glutamine (Sigma-Aldrich Chemie GmbH) in Petri dishes (Sigma-Aldrich Chemie GmbH). Six-well plates were prepared with 0.5 mL Matrigel (Becton Dickinson GmbH, Bedford, MA) and 4.5 mL DMEM without adjuvant for 30 minutes 37°C and 5% CO2 in incubator (Heraeus Holding GmbH, Hanau, Germany) and afterwards carefully washed twice with DMEM devoid of adjuvant. Eight milliliters of DMEM with 10% horse serum (Invitrogen GmbH) were put onto sterile Petri dishes which were primary flushed with pure horse serum. Skeletal muscles were carefully dissolved, and myofibers were separated. About 20 intact single fibers were brought onto Matrigel and DMEM-coated 6-well plates. After 3 minutes, 0.5 mL of plating medium (Table 1) was added. After 3 days, plating medium was replaced by proliferation medium (Table 1).
Having established a confluent monolayer in both cell lines after 3 days, medium was changed into differentiation medium (Table 1) to force myoblasts to differentiate into myotubes. After 24 hours, bupivacaine hydrochloride (Sigma-Aldrich Chemie GmbH) in clinically relevant concentrations of 0.05%, 0.1%, 0.175%, 0.25%, and 0.5% (1.6 mM, 3.2 mM, 5.5 mM, 7.9 mM, and 15.8 mM or 500 ppm, 1000 ppm, 1750 ppm, 2500 ppm, and 5000 ppm, respectively) was added to one part of the plates of each cell line. The other part was treated with ropivacaine hydrochloride (FAGRON GmbH & Co KG, Barsbüttel, Germany) in the same concentrations (1.7 mM, 3.3 mM, 5.8 mM, 8.3 mM, 16.5 mM, respectively). After incubation for 1 and 2 hours, the LA was removed. We used 6 C2C12 cell samples or 8 primary muscle cell samples for each condition tested (concentration, incubation time, and recovery time). Cells were washed with PBS and cultured with growth medium, described earlier, for another 24 and 48 hours recovery time. Using flow cytometry, cells without intact cell membrane were marked with propidium iodide (Invitrogen GmbH) and selected as death cells. Flow cytometry was performed with FACS Calibur™ (Becton Dickinson, Heidelberg, Germany), and the fraction of dead cells in relation to total number of cells was analyzed by counting 5000 events per well. Identification was possible due to size and scattering (forward scatter/side scatter). Quantitative assignment, after marking with propidium iodide, was detected by alteration of intenseness of the emitting fluorescence signals.
Statistical analyses was done with Microsoft Excel (Microsoft Corporation, Redmond, WA) and IBM SPSS Statistics Version 20 (IBM Deutschland GmbH, Ehningen, Germany). Significance was tested through 1-way analysis of variance corrected with post hoc Dunnett T3 test. Pairwise comparison of means was tested by T test.
For pharmacodynamic modeling of the 50% effective concentration (EC50) values, a simple inhibitory sigmoid Emax model predefined in Phoenix™ WinNonlin® 6.2 (Pharsight Certara, St. Louis, MO) was used.
Myoblasts extracted from mouse muscle and C2C12 myoblasts are able to proliferate in vitro, and differentiation into myocytes can be provoked by changing the medium. Because of their specific appearance, microscopic identification is possible. Treatment of myocytes with bupivacaine and ropivacaine in vitro leads to cell destruction in a dose- and drug-dependent manner.
The amount of dead cells increases in primary murine skeletal muscle cells after incubation with bupivacaine for 1 and 2 hours. Higher concentrations of the drug cause an increased number of dead cells (Fig. 1). After 2 hours of incubation, lower concentrations of bupivacaine are required for the same effects than after 1 hour (Tables 2 and 3). Ropivacaine has the same effects on primary muscle cells as does bupivacaine but on a higher concentration level with increased EC50 values (Fig. 2, Table 2), which means that higher concentrations are needed to kill 50% of cells.
Also in C2C12 cells, 1-hour incubation with LA causes fewer dead cells than 2 hours. The fraction of dead cells increases when treated with higher concentrations of bupivacaine (Fig. 3, Tables 3 and 4); however, in comparison with primary muscle cells, EC50 values are more than twice as high (3203 ppm in C2C12 cells vs 1054 ppm in primary muscle cells). The same effect is demonstrated after 2 hours of incubation.
Similar to primary muscle cell culture, ropivacaine has an analog impact as does bupivacaine but higher concentrations are needed (Figs. 3 and 4, Tables 4 and 5). In both cell types, the time of recovery has no effect on survival of cells (Table 5). Significance versus mean living cells at 0 ppm and 24 hours recovery time is shown in Table 3.
Cell death occurs in primary skeletal myocytes as well as C2C12 myocytes when treated with LAs. As prior studies show, bupivacaine has the most myotoxic effects.1–3 The results of this study indicate that in both types of cell culture, primary muscle cells and immortalized muscle cells, myotoxic effects of bupivacaine and ropivacaine can be observed. With ropivacaine, higher concentrations were needed to cause the same amount of dead cells as with bupivacaine independently from incubation or recovery time.
In vivo bupivacaine seems to be more myotoxic than ropivacaine. After IM injection, bupivacaine resulted in more tissue damage. Histological, necrotic, and apoptotic cells can be seen.1,2,11,12 In vitro different myotoxic properties of bupivacaine and ropivacaine could be reproduced. Detailed reasons for this behavior are not fully understood. However, an intracellular effect is most likely responsible for LA-induced myotoxicity. Bupivacaine is more lipophilic than ropivacaine and can therefore diffuse more easily into the cytoplasm. Within the cell, LA induces an increase of intracellular calcium which is assumed to be responsible for acute myotoxic effects. A higher affinity to intracellular receptors is also possible and subject to further studies.
Concentrations of 1.6 to 15.8 mM bupivacaine or 1.7 to 16.5 mM ropivacaine were chosen to imitate clinical use. Because of local dilution effects, experimental in vitro concentrations might be increased compared with in situ LA levels. Furthermore, because of differing lipophilicity, bupivacaine and ropivacaine could accumulate variable in vivo, and the real concentration remains unknown.
Incubation time has an influence on cell survival. Longer duration of noxious stimulus results in more dead cells. With longer exposure, more LAs can diffuse though the sarcolemma and reach intracellular targets.
The extent of the myotoxic effects in this study depended on the cell culture used. Primary muscle cells in culture seem to be more vulnerable to bupivacaine and ropivacaine. The C2C12 cells we used are immortalized to divide infinitely and to grow faster in culture. However, immortalized C2C12 cells seem to be more resistant to myotoxic effects although they are the same type of cells as primary skeletal muscle cells. Apparently, immortalization has an effect on proliferation. How immortalization can alter basic cell physiology is not clear. A possible explanation might be the potency of immortalized cells to avoid apoptosis. Prior studies found that apoptosis is 1 pathway of cell death in LA-induced myotoxicity.1,12,17 In primary cells, apoptosis can occur while immortalized cells seem to be resistant. Apoptosis as a form of delayed cell death usually occurs within hours after the noxious stimulus. To possibly differentiate these delayed effects, survival was determined after another 24 hours (48 hours after the stimulus). During recovery time, with a relevant amount of apoptotic cells, a decrease in survival would have to been seen. The results, however, indicate that survival did not change with an extended observation period. LA-induced apoptosis might occur very early, and an observation period of 48 hours might be too long. A shorter observation period and direct measurement of apoptosis might explain the different behavior of immortalized cells and should be the subject of future studies.
How other pathways of LA-induced myotoxicity, such as intracellular calcium handling and mitochondrial function, are influenced by immortalization is not clear. Immortalization of cells is common and intended for different in vitro models13,18 but might falsify results of studies examining external influences. Therefore, results of some studies might not be comparable with others. To evaluate the exact intracellular mechanisms of in vitro studies is probably more reliable, because in vivo there could be numerous susceptible coeffects. Whether results from primary cell culture models are better related to results from in vivo models has to be studied further.
Primary muscle cells are more vulnerable to the cytotoxic effects of bupivacaine and ropivacaine than immortalized cells of the same tissue and species. Further studies are needed to explain this behavior. Understanding the mechanisms of this difference may further our understanding of degenerative muscle diseases or muscle tumors and help us improve treatment.
Different myotoxic properties of bupivacaine and ropivacaine can be reproduced in vitro and allow a detailed analysis of this behavior. These results are important to optimize clinical care. Quality of regional anesthesia and the rate of myotoxic side effects could be improved by adjustment of the type of LAs. Clinically, the concentration of LAs and administration time could be optimized. Additional substances could be found to prevent myotoxicity.
Name: Petra Hofmann, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Petra Hofmann has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Thomas Metterlein, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Thomas Metterlein has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Gabriele Bollwein.
Contribution: This author helped conduct the study.
Attestation: Gabriele Bollwein has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Michael Gruber, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Michael Gruber 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.
Name: Christoph Plank, MD.
Contribution: This author helped analyze the data.
Attestation: Christoph Plank has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Bernhard M. Graf, MD, MSc.
Contribution: This author helped design and conduct the study.
Attestation: Bernhard M. Graf has seen the original study data and approved the final manuscript.
Name: Wolfgang Zink, MD, DEAA.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Wolfgang Zink has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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