[HSP70 inhibits smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells].
OBJECTIVE
To observe whether HSP70 could protect against H2O2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria.


METHODS
HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33 258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence.


RESULTS
H2O2 ( 0.5 mmol/L ) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H2O2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H2O2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H2O2.


CONCLUSIONS
HSP70 inhibits Smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells.
Jiang
Bi-mei
BM
Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha 410078, China.
Xiao
Wei-min
WM
Shi
Yong-zhong
YZ
Liu
Mei-dong
MD
Xiao
Xian-zhong
XZ
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D002478:Cells, Cultured; D018840:HSP70 Heat-Shock Proteins / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D032446:Myoblasts / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D018840:HSP70 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D006861:Hydrogen Peroxide