Impaired Autophagosome Clearance Contributes to Local Anesthetic Bupivacaine-induced Myotoxicity in Mouse Myoblasts. (March 2015)

• Record Type: Journal Article
• Authors: Li, Rongrong; Ma, He; Zhang, Xiaojin; Li, Chuanfu; Xiong, Jingwei; Lu, Ting; Mao, Yu; Dai, Juncheng; Liu, Li; Ding, Zhengnian
• Is Part Of: Anesthesiology. Volume 122:Number 3(2015)
• Publication Date: 2015-03
• Subjects: Anesthesiology -- Periodicals; Anesthetics -- Periodicals; Anesthesia -- Periodicals; 617.9605
• DOI: 10.1097/ALN.0000000000000568
• Languages: English
• ISSNs: 0003-3022

Abstract

<abstract> <title> <x xml:space="preserve">Abstract</x> </title> <sec> <title>Background:</title> <p>The current study examined the role(s) of autophagy in myotoxicity induced by bupivacaine in mouse myoblast C2c12 cells.</p> </sec> <sec> <title>Methods:</title> <p>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).</p> </sec> <sec> <title>Results:</title> <p>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 <italic>vs.</italic> 2.1 ± 1.2) and light chain 3 conversion (2.16 ± 0.15 <italic>vs.</italic> 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 <italic>vs.</italic> 1.00 ± 0.15), suggesting that the drug impaired autophagosome clearance. Further examination revealed that bupivacaine interrupted autophagosome–lysosome fusion (10.87% ± 1.48% <italic>vs.</italic> 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.</p> </sec> <sec> <title>Conclusions:</title> <p>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.</p> </sec> </abstract>