title: Impaired autophagosome clearance contributes to local anesthetic bupivacaine-induced myotoxicity in mouse myoblasts.
abstract: BACKGROUND
  The current study examined the role(s) of autophagy in myotoxicity induced by bupivacaine in mouse myoblast C2c12 cells.
  
  
  METHODS
  C2c12 cells were treated with bupivacaine. Myotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (n = 3 to 30), live/dead assay (n = 3 to 4), and morphological alterations (n = 3). Autophagosome formation was reflected by microtubule-associated protein light chain 3 conversion (n = 4 to 12) and light chain 3 punctation (n = 4 to 5). Autophagosome clearance was evaluated by p62 protein level (n = 4) and autolysosomes generation (n = 3).
  
  
  RESULTS
  Bupivacaine induced significant cell damage. Notably, there was a significant increase in autophagosome generation as evidenced by light chain 3 puncta formation (72.7 ± 6.9 vs. 2.1 ± 1.2) and light chain 3 conversion (2.16 ± 0.15 vs. 0.33 ± 0.04) in bupivacaine-treated cells. Bupivacaine inactivated the protein kinase B/mammalian target of rapamycin/p70 ribosomal protein S6 kinase signaling. However, cellular levels of p62 protein were significantly increased upon bupivacaine treatment (1.29 ± 0.15 vs. 1.00 ± 0.15), suggesting that the drug impaired autophagosome clearance. Further examination revealed that bupivacaine interrupted autophagosome-lysosome fusion (10.87% ± 1.48% vs. 32.94% ± 4.22%). Administration of rapamycin increased autophagosome clearance and, most importantly, improved the survival in bupivacaine-treated cells. However, knockdown of autophagy-related protein 5 (atg5) exacerbated bupivacaine-induced impairment of autophagosome clearance and myotoxicity.
  
  
  CONCLUSIONS
  The data suggest that autophagosome formation was induced as a stress response mechanism after 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.
authors.affiliation: From the Departments of Anesthesiology (R.L., H.M., J.X., T.L., Z.D.) and Geriatrics (X.Z., Y.M., L.L.), First Affiliated Hospital with Nanjing Medical University, Nanjing, China; Department of Surgery, East Tennessee State University, Johnson City, Tennessee (C.L.); and Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China (J.D.).
authors.forename: Rongrong
authors.initials: R
authors.lastname: Li
authors.forename: He
authors.initials: H
authors.lastname: Ma
authors.forename: Xiaojin
authors.initials: X
authors.lastname: Zhang
authors.forename: Chuanfu
authors.initials: C
authors.lastname: Li
authors.forename: Jingwei
authors.initials: J
authors.lastname: Xiong
authors.forename: Ting
authors.initials: T
authors.lastname: Lu
authors.forename: Yu
authors.initials: Y
authors.lastname: Mao
authors.forename: Juncheng
authors.initials: J
authors.lastname: Dai
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Zhengnian
authors.initials: Z
authors.lastname: Ding
mesh_terms: D000779:Anesthetics, Local / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D002045:Bupivacaine / Q000378:metabolism / Q000633:toxicity*; D002478:Cells, Cultured; D051379:Mice; D032446:Myoblasts / Q000187:drug effects* / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000779:Anesthetics, Local; D002045:Bupivacaine