Changes Of Autophagy And Its Effect In Bupivacaine-induced Myotoxicity

BackgroundLocal anesthetics (LAs) are widely used during regional blocks for improving postoperative analgesia and postoperative rehabilitation in patients. However, LAs solution during continuous blocks comes into contact with muscles and can provoke muscle injury including postoperative iatrogenic muscle pain, dysfunction and degeneration. Though the potential myotoxicity of LAs has been investigated for more than 50 years, the mechanisms by which they induce muscle cell injury are not fully understood. Therefore, identification of these mechanisms will help us to develop effective clinical strategies for preventing adverse outcomes after the administration of LAs.Autophagy is an evolutionarily conserved process by which cytoplasmic materials, including damaged proteins and organelles, are sequestered for lysosome-dependent degradation. Thus, the autophagy machinery is responsible for the maintenance of cellular homeostasis. Three types of autophagy occur in mammalian cells:macroautophagy, microautophagy, and chaperone-mediated autophagy.Macroautophagy (referred to as autophagy hereafter) is a dynamic process comprising two consecutive stages:(1) autophagosome formation, which begins with the formation of isolation membranes that engulf substrates; and (2) autophagosome clearance, which involves autolysosome formation via autophagosome-lysosome fusion, followed by degradation of the inner membrane together with its luminal contents. Evidence suggests that inhibition of autophagy flux may either result in cell death directly or sensitize cells to stimuli-induced damage.Recent evidence suggests a possible involvement of autophagy in LAs-induced cytotoxicity. Morissette and colleagues reported that smooth muscle cell death induced by two LAs, bupivacaine and lidocaine, was accompanied by increases in LC3-Ⅱ levels and GFP-LC3 puncta. In another study, Peropadre et al showed that autophagosomes accumulated in cells treated with dibucaine. These findings suggest that LAs can increase the number of autophagosomes within cells. And autophagy was involved in LAs-induced cytotoxicity. Thus what are the role and mechanisms of autophagy in bupivacaine-induced myotoxicity?ObjectiveIn the present study, we investigated the role and mechanisms of autophagy in bupivacaine-induced myotoxicity.Materials and Methods1. Bupivacaine provokes myotoxicity and increases autophagosome formation1.1 Bupivacaine provokes myotoxicityAfter stimulation with bupivacaine, cell viability was measured by MTT assy. Cell death rate was determined by live/dead staining. Cell morphology was observed using phase-contrast light microscope with a magnification of 200×. Gastrocnemius muscle was harvested for Histological examination by HE staining.1.2 Bupivacaine increases autophagosome in C2cl2 cells.After transfection with the pEGFP-LC3 plasmid,LC3 punctuation was observed under a confocal microscope. After treatment with bupivacaine, LC3-Ⅱ protein level was measured by Western blot, Autophagosome structure was observed under electron microscope.2. Mechanisms involved in autophagosome increase by bupivacaine2.1 Measurement of autophagy activationExpression of p-Akt, p-p70S6K and p-mTOR was measured by Western blot.2.2 Measurement of autophagy degradationp62 protein level was evaluated by Western blot.2.3 Analysis of lysosomal functionLysosomal abundance was evaluated by examining LysoTracker fluorescence intensity. Lysosomal pH was measured using LysoSensor fluorescence. The activity of cathepsin B was examined using the Magic RedTM Cathepsin B Assay Kit. (LAMP-1)lysosomal-associated membrane protein 1 levels were evaluated by Western blot.2.4 Measurement of autophagosome-lysosome fusionColocalization of pEGFP-LC3 puncta and LysoTracker staining.3. Impaired autophagosome-lysosome fusion contributes to bupivacaine-induced myotoxicity3.1 Rapamycin attenuates bupivacaine-induced myotoxicity by promoting the clearence of autophagosomesAfter pretreatment of Rapamycin, Protein levels of LC3-Ⅱ and p62 were measured by Western blot. Autophagosome-lysosome fusion was determined by colocalization of pEGFP-LC3 puncta and LysoTracker staining. Cell death rate was determined by live/dead staining. Cell morphology was observed using phase-contrast light microscope with a magnification of 200×3.2 Autophagy inhibition by Atg5 knockdown aggravated the bupivacaine-induced myotoxicityAfter pretreatment of Atg5 siRNA transfection, Atg5 messenger RNA levels were evaluated by Real-time quantitative polymerase chain reaction. Protein levels of LC3-Ⅱ and p62 were measured by Western blot. Cell viability was measured by MTT assy.3.3 Autophagy inhibitor CQ aggravated the bupivacaine-induced myotoxicityAfter pretreatment of CQ, expression levels of LC3-Ⅱ and p62 were measured by Western blot. Autophagosome-lysosome fusion was evaluated by colocalization of pEGFP-LC3 puncta and LysoTracker staining. Cell viability was determined by MTT assy. Cell death rate was measured by live/dead staining. Cell morphology was observed under phase-contrast light microscope with a magnification of 200×Results1. Bupivacaine provokes myotoxicity and increases autophagosome formation1.1. Bupivacaine provokes myotoxicity1.1.1 Dose-response of bupivacaine on the viability in C2cl2 cellsCells were treated with different doses of bupivacaine for 24 hr.100 μM of bupivacaine had no significant effect on cell survival. However, doses of 300,600, 900 and 1,200 μM led to significant reductions in cell viability by 16.0,49.0,65.0, and 76.8%, respectively, compared with untreated controls (P< 0.01).1.1.2 Bupivacaine induces morphological changes in C2cl2 cellsCells were treated with 600 μM bupivacaine for 24 hr. Compared with the untreated controls, cells treated with bupivacaine were round and shrunken, and most cells lost their cellular integrity.1.1.3 Bupivacaine increases death rate of C2cl2 cellsCells were treated with 600μM bupivacaine for 36 hr. Cell survival and cell death were further evaluated in a live/dead assay. The number of dead cells in bupivacaine-treated cultures was 18-fold higher than that in untreated cultures (P 0.01).1.1.4 Bupivacaine injection induces mouse gastrocnemius muscle injuresGastrocnemius muscle was harvested from mice at 72 hr post-bupivacaine (0.5%, 20μ) injection. Muscle was paraffin-embedded, sectioned, stained with hematoxylin and eosin. There is only mild edema and inflammatory cell infiltration in saline-treated group. However there is significant interstitial edema, inflammatory cell infiltration,muscle fiber rupture and karyolysis in bupivacaine-treated group.1.2. Bupivacaine increases autophagosome in C2cl2 cells.1.2.1 Bupivacaine increases EGFP-LC3 punctation in C2cl2 cellsC2cl2 cells transfected with pEGFP-LC3 were exposed to 600 μM bupivacaine for 6 hr. The number of EGFP-LC3 puncta in bupivacaine-treated cells was significantly higher by 34-fold than that in untreated controls (P< 0.01).1.2.2 Bupivacaine increases LC3-Ⅱ protein level in C2cl2 cellsC2cl2 cells were exposed to 600μM bupivacaine for 6 hr. Western blot analysis was performed to examine the changes in LC3 conversion, a marker for autophagosome formation. The ratio of LC3-Ⅱ/LC3-Ⅰ was significantly higher by 5.5-fold in bupivacaine-treated cells than that in untreated controls (P< 0.01).1.2.3 Bupivacaine increases LC3-Ⅱ protein level in muscle tissueGastrocnemius muscle was harvested from mice at 18 hr post-bupivacaine (0.5%, 20μl) injection. Western blot analysis was performed to examine the changes in LC3 conversion, a marker for autophagosome formation. The ratio of LC3-Ⅱ/LC3-Ⅰ was significantly higher by 4.3-fold in bupivacaine-treated group than that in saline group (P<0.01).1.2.4 Bupivacaine increases autophagosome formation in C2cl2 cellsC2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. The autophagosomes were then examined under an electron microscope. Autophagosomes (a double-membraned structure containing undigested cytoplasmic contents) accumulated in bupivacaine-treated cells but not in untreated controls. Autophagosome frequency was significantly increased by 5.6-fold in bupivacaine group compared with control group (P< 0.01).1.2.5 Bupivacaine increases autophagosome formation in muscle tissueGastrocnemius muscle was harvested from mice at 18 hr post-bupivacaine (0.5%, 20μl) injection. The autophagosomes were then examined under an electron microscope. Autophagosomes accumulated in bupivacaine-treated group but not in saline group. Autophagosome frequency was significantly increased in bupivacaine group compared with control group.2. Mechanisms involved in autophagosome increase by bupivacaine2.1 Bupivacaine Suppresses Akt/mTOR/p70S6K signaling, leading to the increase in autophagosome generationC2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Western blot analysis was performed. Bupivacaine treatment led to a significant reduction in the phosphorylation of Akt by 46.9%, p70S6K by 36.4%, and mTOR by 46.6%, respectively, compared with that in untreated controls (P< 0.01).2.2. Bupivacaine Increases p62 protein level, suggesting that bupivacaine impairs the clearance of autophagosomes2.2.1 Bupivacaine Increases p62 protein level in C2cl2 cellsC2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Western blot analysis was performed. Bupivacaine treatment significantly increased p62 protein level in C2cl2 cells by 29.1% compared with that in the untreated controls (P<0.01).2.2.2 Bupivacaine Increases p62 protein level in muscle tissueGastrocnemius muscle was harvested from mice at 18 hr post-bupivacaine (0.5%, 20μl) injection. Western blot analysis was performed. Bupivacaine treatment significantly increased p62 protein level in muscle tissue by 91.6% compared with that in saline groups (P<0.01).2.3. Bupivacaine-induced impairment of autophagosome clearance is not likely contributed by dysregulation of lysosomal abundance and activity2.3.1 Bupivacaine increases lysosomal abundance in C2cl2 cellsC2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Lysosomal abundance was evaluated by examining LysoTracker fluorescence intensity. LysoTracker fluorescence intensity was 15.2% greater in bupivacaine-treated cells than that in control cells (P<0.01)2.3.2 Bupivacaine does not affect lysosomal activity by neutralizing lysosomal pHC2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Lysosomal pH was measured using LysoSensor fluorescence. Cells treated with CQ served as positive controls for the neutralization of lysosomal pH. The fluorescence was not changed by bupivacaine but was effectively quenched by CQ in comparison with the untreated controls.2.3.3 Bupivacaine increases cathepsin B activityC2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Cathepsin B activity was 23.7% higher in bupivacaine-treated cells than that in untreated controls(P<0.01).2.3.4 Bupivacaine increases the level of lysosomal-associated membrane protein 1(LAMP-1)C2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Western blot analysis was performed.lysosomal-associated membrane protein 1 levels were significantly higher by 36.4% after bupivacaine challenge (P<0.01).2.4. Bupivacaine interferes with autophagosome-lysosome fusion, resulting in impairment of autophagosome clearence C2cl2 cells were exposed to 600 μM bupivacaine for 6 hr. Autophagosomes were visualized via EGFP-LC3 puncta (green fluorescence), and lysosomes were visualized using LysoTracker (red fluorescence, an acidic pH marker for lysosomes). The red and green signals merged in control cells; however, in bupivacaine-treated cells, the most green and red signals were not colocalized. The colocalization rate of EGFP-LC3 puncta with LysoTracker signals was significantly decreased in bupivacaine group by 67.0% compared with control group (P<0.01).3. Impaired autophagosome-lysosome fusion contributes to bupivacaine-induced myotoxicity3.1. Rapamycin attenuates bupivacaine-induced myotoxicity by promoting the clearence of autophagosomes3.1.1 Rapamycin increases the level of LC3-Ⅱ and decreases the level of p62 proteinC2cl2 cells were pretreated with 500nM rapamycin for 30min and then exposed to 600 μM bupivacaine for 6 hr. The ratio of LC3-Ⅱ/LC3-Ⅰ was significantly higher by 60.8% in bupivacaine-treated cells after exposure to rapamycin than that in cells treated with bupivacaine alone (P<0.01). The p62 protein level in bupivacaine-treated cells was reduced by 57.6% in the presence of rapamycin compared with that in the absence of rapamycin (P<0.01). Also, compared with untreated controls, there was a significant increase in the LC3-II/LC3-I ratio and a reduction in p62 protein levels in cells treated with rapamycin alone (P<0.01).3.1.2 Rapamycin promotes the fusion of autophagosomes with lysosomesC2cl2 cells were pretreated with 500nM rapamycin for 30min and then exposed to 600 μM bupivacaine for 6 hr. Compared with the cells solely treated with bupivacaine, rapamycin increased the colocalization (yellow) of EGFP-LC3 puncta (green) and LysoTracker (red) in bupivacaine-treated cells. The colocalization rate was significantly increased in bupivacaine group by rapamycin by 312.5% compared with that in the cells treated solely with bupivacaine (P<0.01). 3.1.3 Rapamycin attenuates death rate induced by bupivacaineC2cl2 cells were pretreated with 500nM rapamycin for 30min and then exposed to 600 μM bupivacaine for 36 hr. Rapamycin markedly attenuated bupivacaine-induced cell death by 48.6% compared with that in cells treated with bupivacaine alone (P<0.01). Compared with untreated controls, rapamycin alone did not induce significant cell death.3.1.4 Rapamycin attenuates morphological changes induced by bupivacaineC2cl2 cells were pretreated with 500nM rapamycin for 30min and then exposed to 600 μM bupivacaine for 24 hr. Rapamycin attenuated morphological abnormalities in bupivacaine-treated cells.3.2. Autophagy Inhibition by Atg5 Knockdown Aggravated the Bupivacaine-induced Myotoxicity3.2.1 Atg5 siRNA decreased Atg5 messenger RNA levels by Atg5 KnockdownC2cl2 cells were transfected with Atg5 siRNA for 24hr. Atg5 siRNA decreased Atg5 messenger RNA levels by 83.8% compared with negative controls (P<0.01).3.2.2 Atg5 siRNA changes levels of autophagy-related proteins by Atg5 knockdownC2cl2 cells were transfected with Atg5 siRNA for 48hr. Atg5 protein levels were decreased by 91.1% compared with negative controls (P<0.01). After Atg5 knockdown by siRNA, the LC3-Ⅱ/LC3-Ⅰ ratio was decreased by 80.8%, whereas p62 level increased by 144.2%, respectively, compared with negative controls (P<0.01).3.2.3 Atg5 siRNA aggravates Bupivacaine-induced reductions in cell viability by Atg5 knockdownC2cl2 cells were exposed to 600 μM bupivacaine for 24 hr after 48hr transfection of Atg5 siRNA. The cell viability in bupivacaine-treated cells in the presence of Atg5 knockdown were measured by MTT assay.Bupivacaine-provoked cell injury was aggravated by 26.4% by Atg5 knockdown (P<0.01).3.3. Autophagy inhibitor CQ aggravated the bupivacaine-induced myotoxicity3.3.1 CQ changes levels of autophagy-related proteinsC2cl2 cells were pretreated with 10μM CQ for 30min and then exposed to 600 μM bupivacaine for 6 hr. The ratio of LC3-II/LC3-Ⅰ was significantly higher by 1.4 fold in bupivacaine-treated cells after exposure to CQ than that in cells treated with bupivacaine alone (p<0.01). Also, compared with untreated controls, the ratio of LC3-II/LC3-Ⅰ was significantly higher by 4.3 fold in cells treated with CQ alone (P<0.01). The p62 protein level in bupivacaine-treated cells was increased by 10.5% in the presence of CQ compared with that in the absence of CQ.3.3.2 CQ aggravates impairment of autophagosome-lysosome fusion by inhibition of lysosomal functionC2cl2 cells were pretreated with 10μM CQ for 30min and then exposed to 600 μM bupivacaine for 6 hr. Compared with the cells solely treated with bupivacaine, CQ decreased the colocalization of EGFP-LC3 puncta and LysoTracker in bupivacaine-treated cells. The colocalization rate was significantly decreased in bupivacaine group by CQ compared with that in the cells treated solely with bupivacaine. Also, the size of autophagosome was bigger in bupivacaine group by CQ compared with that in the cells treated solely with bupivacaine.3.3.3 CQ aggravates bupivacaine-induced reductions in cell viabilityC2cl2 cells were pretreated with 10μCQ for 30min and then exposed to 600 μM bupivacaine for 24 hr. The cell viability in bupivacaine-treated cells in the presence of CQ were measured by MTT assay. Cell viability was significantly decreased by 24.3% in bupivacaine group by CQ compared with that in the cells treated solely with bupivacaine(P<0.01).3.3.4 CQ aggravates morphologial injury induced by bupivacaineC2cl2 cells were pretreated with 100μM CQ for 30min and then exposed to 600 μM bupivacaine for 24 hr. CQ aggravated morphological abnormalities in bupivacaine-treated cells. Morphology of cells treated solely with CQ has no difference from untreated controls.3.3.5 CQ increases death rate induced by bupivacaineC2cl2 cells were pretreated with 10μQ for 30min and then exposed to 600 μM bupivacaine for 24 hr. CQ markedly increased bupivacaine-induced cell death by 20.3% compared with that in cells treated with bupivacaine alone (P<0.01). Compared with untreated controls, CQ alone did not induce significant cell death.ConclusionsThese data suggest that autophagosome formation was induced as a stress response mechanism following bupivacaine challenge; however, autophagosome clearance was impaired due to inadequate autophagosome-lysosome fusion. Therefore, impairment of autophagosome clearance appears to be a novel mechanism underlying bupivacaine-induced myotoxicity.