title: Acetylcholine Attenuated TNF-α-Induced Apoptosis in H9c2 Cells: Role of Calpain and the p38-MAPK Pathway.
abstract: BACKGROUND
  Previous studies have shown that inflammation is associated with excessive activation of calpains. Acetylcholine (ACh) has been reported to inhibit pro-inflammatory cytokine release and protect against cardiomyocyte injury. However, there is no direct evidence regarding whether ACh can regulate calpains to exert cardioprotection. To this end, we investigated the effect of ACh on tumour necrosis factor alpha (TNF-α)-induced cardiomyocyte injury and further explored the underlying mechanism.
  
  
  METHODS
  Flow cytometry and transmission electron microscopy were performed to evaluate apoptosis and cellular ultrastructure. Western blotting was performed to assess changes in protein expression. siRNA was employed to silence specific proteins.
  
  
  RESULTS
  TNF-α treatment increased the expression of cleaved caspase-3, calpain-1 and p38-mitogen-activated protein kinase (p38- MAPK). The calpain inhibitor PD150606 and the p38-MAPK inhibitor SB203580 inhibited apoptosis induced by TNF-α. Moreover, SB203580 decreased the expression and activity of calpain-1, possibly related to the up-regulation of calpastatin. ACh significantly inhibited TNF-α-induced cell apoptosis, as evidenced by decreases in caspase-3 cleavage, p38-MAPK phosphorylation, and calpain-1 expression and activity as well as increases in calpastatin expression. These beneficial effects of ACh were abolished by atropine or M2AChR siRNA.
  
  
  CONCLUSIONS
  Our results suggest that ACh ameliorated TNF-α-induced calpain activation by decreasing p38-MAPK phosphorylation and enhancing calpastatin expression, indicating that calpain may be an important link between inflammatory factors and myocardial cell apoptosis.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Xueyuan
authors.initials: X
authors.lastname: Bi
authors.forename: Xiaojiang
authors.initials: X
authors.lastname: Yu
authors.forename: Hanghuan
authors.initials: H
authors.lastname: Jia
authors.forename: Huanle
authors.initials: H
authors.lastname: Fang
authors.forename: Weijin
authors.initials: W
authors.lastname: Zang
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000502:physiology*; D002460:Cell Line; D020935:MAP Kinase Signaling System; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D002135:Calcium-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; C033668:calpastatin; D051057:Proto-Oncogene Proteins c-akt; D002154:Calpain; D000109:Acetylcholine