Apoptotic signaling induced by H2O2-mediated oxidative stress in differentiated C2C12 myotubes

Parco M. Siu, Yan Wang, Stephen Alway

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

Aims: Apoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H2O2 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. Main methods: Apoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots. Key findings: H2O2 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 H2O2. Treatment with 4 mM of H2O2 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 H2O2. AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H2O2 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 H2O2, respectively. Significance: These findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H2O2 in differentiated myotubes.

Original languageEnglish (US)
Pages (from-to)468-481
Number of pages14
JournalLife Sciences
Volume84
Issue number13-14
DOIs
StatePublished - Mar 27 2009
Externally publishedYes

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Oxidative stress
Skeletal Muscle Fibers
Caspases
Oxidative Stress
Apoptosis
Proteins
DNA
DNA Fragmentation
Apoptosis Regulatory Proteins
Mitochondria
Cell death
Nuclear Proteins
Cytochromes c
Caspase 3
AIDS-Related Complex
Therapeutics
Peptide Hydrolases
Chemical activation
Cytosol
Cell Death

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

Cite this

Apoptotic signaling induced by H2O2-mediated oxidative stress in differentiated C2C12 myotubes. / Siu, Parco M.; Wang, Yan; Alway, Stephen.

In: Life Sciences, Vol. 84, No. 13-14, 27.03.2009, p. 468-481.

Research output: Contribution to journalArticle

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abstract = "Aims: Apoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H2O2 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. Main methods: Apoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots. Key findings: H2O2 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 H2O2. Treatment with 4 mM of H2O2 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 H2O2. AIF measured in the nuclear protein fraction increased by 74{\%} and 352{\%} following treatment with 4 mM of H2O2 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 H2O2, respectively. Significance: These findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H2O2 in differentiated myotubes.",
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N2 - Aims: Apoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H2O2 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. Main methods: Apoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots. Key findings: H2O2 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 H2O2. Treatment with 4 mM of H2O2 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 H2O2. AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H2O2 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 H2O2, respectively. Significance: These findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H2O2 in differentiated myotubes.

AB - Aims: Apoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H2O2 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. Main methods: Apoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots. Key findings: H2O2 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 H2O2. Treatment with 4 mM of H2O2 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 H2O2. AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H2O2 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 H2O2, respectively. Significance: These findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H2O2 in differentiated myotubes.

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