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Updated: Aug 16, 2026
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells
Bimei Jiang1, Weimin Xiao, Yongzhong Shi
1Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha 410078, China.
Abstract:
Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.
Insights
Hydrogen peroxide induces apoptosis in C2C12 cells by releasing Smac/DIABLO from mitochondria. This protein then activates caspases, promoting programmed cell death in these myogenic cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Smac/DIABLO is a mitochondrial protein that promotes apoptosis by inhibiting apoptosis inhibitors.
- Its role in hydrogen peroxide (H2O2)-induced apoptosis in C2C12 myogenic cells is not well understood.
- Smac/DIABLO is implicated in sensitizing cancer cells to apoptosis.
Purpose of the Study:
- To investigate the role of Smac/DIABLO in H2O2-induced apoptosis of C2C12 myogenic cells.
- To determine if Smac/DIABLO release from mitochondria is involved in this process.
- To elucidate the involvement of caspase activation in H2O2-induced apoptosis mediated by Smac/DIABLO.
Main Methods:
- Hoechst 33258 staining for apoptotic nuclei and morphological changes.
- Agarose gel electrophoresis for DNA fragmentation analysis.
- Western blotting to detect Smac/DIABLO translocation and caspase activation.
- Colorimetric assays for caspase activity.
- Transient transfection of Smac/DIABLO cDNA and antisense oligonucleotides.
Main Results:
- H2O2 treatment caused Smac/DIABLO release from mitochondria, followed by caspase-3 and caspase-9 activation and apoptotic morphological changes.
- Overexpression of Smac/DIABLO enhanced H2O2-induced apoptosis and caspase activation.
- Inhibition of Smac/DIABLO synthesis using antisense oligonucleotides reduced H2O2-induced apoptosis and caspase activation.
Conclusions:
- H2O2 induces apoptosis in C2C12 myogenic cells.
- The release of Smac/DIABLO from mitochondria to the cytoplasm is a key event in H2O2-induced apoptosis.
- Smac/DIABLO activation of caspase-9 and caspase-3 plays a critical role in this apoptotic pathway.
