Apoptotic signaling induced by H2O2-mediated oxidative stress in differentiated C2C12 myotubes.
OBJECTIVE
Apoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H(2)O(2) activates both caspase-dependent and caspase-independent apoptotic proteins in differentiated C2C12 myotubes. We hypothesized that oxidative stress would decrease anti-apoptotic protein levels in C2C12 myotubes.


METHODS
Apoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21(Cip1/Waf1), c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots.


RESULTS
H(2)O(2) induced apoptosis in myotubes as shown by DNA laddering and an elevation of apoptotic DNA fragmentation. Cell death ELISA showed increase in the extent of apoptotic DNA fragmentation following treatment with H(2)O(2). Treatment with 4 mM of H(2)O(2) for 24 or 96 h caused increase in Bax (56%, 227%), cytochrome c (282%, 701%), Smac/DIABLO (155%, 260%), caspase-3 protease activity (51%, 141%), and nuclear and cytosolic p53 (719%, 1581%) levels in the myotubes. As an estimate of the mitochondrial AIF release to the cytosol, AIF protein content measured in the mitochondria-free cytosolic fraction was elevated by 65% after 96 h treatment with 4 mM of H(2)O(2). AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H(2)O(2) for 24 and 96 h, respectively. Bcl-2 declined in myotubes by 61% and 69% after 24 or 96 h of treatment in 4 mM H(2)O(2), respectively.


CONCLUSIONS
These findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H(2)O(2) in differentiated myotubes.
Siu
Parco M
PM
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China. htpsiu@inet.polyu.edu.hk
Wang
Yan
Y
Alway
Stephen E
SE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000473:pathology*; D032448:Myoblasts, Skeletal / Q000187:drug effects / Q000473:pathology*; D018384:Oxidative Stress / Q000187:drug effects*
D006861:Hydrogen Peroxide; D053148:Caspase 3