MilliporeSigma
  • Home
  • Search Results
  • Impaired autophagosome clearance contributes to local anesthetic bupivacaine-induced myotoxicity in mouse myoblasts.

Impaired autophagosome clearance contributes to local anesthetic bupivacaine-induced myotoxicity in mouse myoblasts.

Anesthesiology (2015-01-16)
Rongrong Li, He Ma, Xiaojin Zhang, Chuanfu Li, Jingwei Xiong, Ting Lu, Yu Mao, Juncheng Dai, Li Liu, Zhengnian Ding
ABSTRACT

The current study examined the role(s) of autophagy in myotoxicity induced by bupivacaine in mouse myoblast C2c12 cells. 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). 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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium dodecyl sulfate, BioXtra, ≥99.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, ≥99.0% (GC), dust-free pellets
Sigma-Aldrich
Sodium dodecyl sulfate, ≥90%
Supelco
Sodium dodecyl sulfate, suitable for ion pair chromatography, LiChropur, ≥99.0%
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, for molecular biology, ≥99.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, ≥98.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, tested according to NF, mixture of sodium alkyl sulfates consisting mainly of sodium dodecyl sulfate
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, 92.5-100.5% based on total alkyl sulfate content basis
Sigma-Aldrich
Sodium dodecyl sulfate, ReagentPlus®, ≥98.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC), free-flowing, Redi-Dri
Sigma-Aldrich
Rapamycin, Ready Made Solution, 2.5 mg/mL in DMSO (2.74 mM), from Streptomyces hygroscopicus
Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
Supelco
Sodium dodecyl sulfate, dust-free pellets, suitable for electrophoresis, for molecular biology, ≥99.0% (GC)
Supelco
Rapamycin, VETRANAL®, analytical standard
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 10% in H2O
Sodium laurilsulfate, European Pharmacopoeia (EP) Reference Standard