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

Abstract

Aims: 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.

Main 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.

Key findings: 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.

Significance: 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.

Fig. 1

DNA fragmentation of myotubes in response to different doses of H₂O₂. DNA fragmentation was quantitatively examined by cell death ELISA in 6-day differentiated C2C12 myotubes treated with 0, 0.2, 0.5, 1, 2 and 4 mM of H₂O₂ for 48 h. *P<0.05, data are significantly different from the corresponding 0 mM H₂O₂; *P<0.05, data are significantly different from 1 mM H₂O₂; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.

Fig. 2

Verification of protein extract purity. The purity of protein fractions was verified by immunoblotting with anti-histone H2B, anti-β-tubulin, anti-CuZnSOD, and anti-MnSOD antibodies. An equal amount of total cytosolic (Total CP), mitochondria-free cytosolic (Mito-free CP), and nuclear (NP) protein was used in the immunoblot.

Fig. 3

Morphology C2C12 Myotubes. (A). 6-day differentiated C2C12 myotubes were incubated with a mouse monoclonal MF20 antibody against sarcomeric myosin heavy chains (MF20, Developmental Studies Hybridoma Bank, University of Iowa, IA). Red fluorescence was developed by incubation with Cy3 following MF20 primary antibody incubation. Nuclei were counterstained with DAPI. These data show the high proportional ratio of myotubes to myoblasts in our used C2C12 culture. Objective, 20×. Bar, 100 μm. (B). The C2C12 myotubes were treated with 0, 1, 2, and 4 mM of H₂O₂ for 48 h and with 4 mM of H₂O₂ for 24 h and 96 h. The morphological health condition and survival of cells was apparently declined with increasing dose of H₂O₂ treatment or with prolonging period of treatment. Objective, 40×. Bar, 100 μm.

Fig. 4

DNA laddering and apoptotic DNA fragmentation. Apoptotic DNA fragmentation was qualitatively analyzed by DNA gel electrophoresis (A). The extracted DNA was loaded on 1.5% agarose gel and was stained with ethidium bromide. The extent of apoptotic DNA fragmentation was quantitatively examined by measuring the cytosolic mono- and oligo-nucleosomes with ELISA (B). The data are presented as means±standard error of mean (SE) of percent relative to 0 mM H₂O₂ (i.e., 0 mM refers to 100% of the level of DNA fragmentation in control samples). *P<0.05, data are significantly different from the corresponding 0 mM H₂O₂; ^#P<0.05, data are significantly different from 1 mM H₂O₂; ⁺P<0.05, data are significantly different from 2 mM H₂O₂. Quantitative data of TUNEL labeling in myotubes treated with H₂O₂ for 24 h (C). The staining results were analyzed under a Nikon eclipse E800 fluorescence microscope. For each of the four treatment groups (H₂O₂ 0, 1, 2, 4 mM), 3 observation fields were randomly chosen. The following three parameters were counted: a) TUNEL-positive nuclei located within MHC-positive myotubes, b) TUNEL-positive nuclei located outside of MHC-positive myotubes, and c) total nuclei in each observation field were counted; the percentage of a/c and b/c was calculated. The data are presented as means±standard deviation. * P<0.05, data are significantly different from the TUNEL-positive/MHC-negative group.

Fig. 5

Bcl-2 and Bax. The protein content of Bcl-2 (A) and Bax (B) was determined by Western analysis. Insets show representative blots. The ratio of Bax/Bcl-2 protein content (C) was estimated according to the data of Western analysis. The data are presented as means±SE of percent relative to 0 mM H₂O₂ (i.e., 0 mM refers to 100% of the level of Bcl-2 and Bax protein content in control samples). *P<0.05, data are significantly different from the corresponding 0 mM H₂O₂; ^#P<0.05, data are significantly different from 1 mM; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.

Fig. 6

Mitochondrial cytochrome c release. The release of mitochondrial cytochrome c was estimated by examining the cytochrome c protein content in the extracted mitochondria-free cytosolic fraction with an ELISA. The data are presented as means±SE of percent relative to 0 mM H₂O₂ (i.e., 0 mM refers to 100% of the cytochrome c protein content in control samples). *P<0.05, data are significantly different from the corresponding 0 mM H₂O₂; ^#P<0.05, data are significantly different from 1 mM; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.

Fig. 7

Caspase-3 protease activity. The change in fluorescence during a 2 h incubation of a fluorometric assay specific for caspase-3 protease activity is normalized to mg protein used in the assay. The normalized data are presented as mean±SE of percent relative to 0mMH₂O₂ (i.e., 0 mM refers to 100% of the level of caspase 3 protease activity in control samples). *P<0.05, data are significantly different from the corresponding 0 mM H₂O₂; ^#P<0.05, data are significantly different from 1 mM H₂O₂.

Fig. 8

XIAP, ARC, and Smac/DIABLO protein. The protein content of XIAP (A), ARC (B), and Smac/DIABLO (C), was measured by Western immunoblot in the total cytosolic (XIAP and ARC) and mitochondria-free cytosolic fractions (Smac/DIABLO). The insets show representative blots. The data are presented as means±SE of percent relative to 0 mM H₂O₂ (i.e., 0 mM refers to 100% of the level of the respective protein content in control samples). *P<0.05, data are significantly different from the corresponding 0 mM; ^#P<0.05, data are significantly different from 1 mM H₂O₂; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.

Fig. 9

AIF protein. The AIF protein content was determined in mitochondria-free cytosolic (A) and nuclear protein fraction (B) by Western analysis in order to indicate the extent of mitochondrial release and nuclear translocation of AIF, correspondingly. The insets show representative blots. The data are presented as means±SE of percent relative to 0 mM of H₂O₂ (i.e., 0 mM refers to 100% of the level of AIF protein content in control samples). *P<0.05, data are significantly different from the corresponding 0 mM H₂O₂; ^#P<0.05, data are significantly different from 1 mM; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.

Fig. 10

Cleaved PARP. The p85 cleaved PARP fragment was detected by Western immunoblot in the nuclear protein fraction obtained from cells followed 4 mM of H₂O₂ treatment for 48 h and 96 h.

Fig. 11

p53 protein. The p53 protein content was determined in nuclear (A) and total cytosolic protein fraction (B) by Western analysis. The insets show representative blots. The data are presented as means±SE of percent relative to 0 mM H₂O₂ (i.e., 0 mM refers to 100% of the level of p53 protein content in control samples). *P<0.05, data are significantly different from the corresponding 0 mM H₂O_2; ^#P<0.05, data are significantly different from 1 mM H₂O₂; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.

Fig. 12

HSP70, MnSOD, and CuZnSOD protein. The protein content of HSP70 (A), MnSOD (B), and CuZnSOD (C) was determined in the total cytosolic fraction by Western immunoblot. The insets show representative blots. The data are presented as means±SE of percent relative to 0 mM H₂O₂ (i.e., 0 mM refers to 100% of the respective protein content level in control samples). *P<0.05, data are significantly different from the corresponding 0 mM; ^#P<0.05, data are significantly different from 1 mM H₂O_2; ⁺P<0.05, data are significantly different from 2 mM H₂O₂.