DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells

Free Radical Biology & Medicine 39 (2005) 658 – 667 www.elsevier.com/locate/freeradbiomed

Original Contribution

Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells Bimei Jiang, Weimin Xiao, Yongzhong Shi, Meidong Liu, Xianzhong Xiao* Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha 410078, China Received 19 December 2004; revised 14 April 2005; accepted 21 April 2005

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 (H2O2)-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) H2O2 (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 H2O2-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 H2O2 ( P < 0.05). These data demonstrate that H2O2 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 H2O2-induced apoptosis in C2C12 myogenic cells. D 2005 Elsevier Inc. All rights reserved. Keywords: Apoptosis; C2C12 myogenic cells; Hydrogen peroxide; Smac/DIABLO

Introduction Apoptosis is an evolutionarily conserved process that plays an important role in the cardiovascular system in

Abbreviations: DIABLO, direct IAP-binding protein with low PI; H2O2, hydrogen peroxide; IAPs, inhibitors of apoptosis proteins; Smac, the second mitochondria-derived activator of caspases; ROS, reactive oxygen species; COX II, cytochrome oxidase subunit II; DTT, dithiothreitol; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phospate-buffered saline; BSA, bovine serum albumin. * Corresponding author. E-mail address: [email protected] (X. Xiao). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.04.018

development, homeostasis, and disease pathology [1]. Apoptosis of cardiac myocytes has recently been recognized as a cellular mechanism of ischemic injury in the heart. While prolonged ischemia appears to result primarily in cardiac myocyte necrosis [2], limited ischemia and tissue reperfusion in rodents, rabbits, and humans [3– 5] induces apoptosis. Several factors, including ATP depletion, acidosis, calcium fluxes, and reactive oxygen species have been proposed to cause apoptosis and/or cytochrome c release in myocytes [6– 10]. Cell death mediated by reactive oxygen species is a significant component of ischemia – reperfusion injury in tissues, such as myocardium [11,12]. While ischemia causes some cell death on its

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own, the reintroduction of oxygen during reperfusion is associated with accelerated apoptotic cell death and necrosis. This likely results, in part, from a burst of free radical production. H2O2 and its reactive by-products are potential mediators of cell death induced by diverse stimuli [13,14]. Despite convincing evidence that apoptosis occurs, the mechanism and signaling pathway by which reactive oxygen species (ROS) lead to apoptosis in cardiac cells are as yet unknown. Recent studies have advanced our understanding of the molecular mechanisms of apoptosis under normal and pathological conditions. During apoptosis, a family of cysteine proteases (caspases) is activated. Caspases are key components of mammalian apoptosis [15], which are expressed in cells as inactive precursors, and activated by proteolytic processing [16,17]. In mammals, two main caspase cascades have been identified.The first pathway links caspase-8 to death receptors expressed at the cell surface [18]. In the second pathway, mitochondrial damage induced by diverse extracellular stresses, such as UV irradiation, hyperosmolality, and chemotherapeutic drugs, causes release of cytochrome c from mitochondria into the cytoplasm [19]. In the cytosol, cytochrome c associates with Apaf-1. In the presence of dATP/ATP, Apaf-1 then binds to and activates caspase-9 [20]. Activation of initiator caspases-8 or -9 causes proteolytic activation of a common set of downstream proteases, including caspases-3 and -7, and induces cell death [19]. The activation of initiator caspases is thought to irreversibly trigger the caspase cascade, necessitating that caspase activation be tightly regulated by layered control mechanisms. Among the growing number of cellular proteins that have been shown to regulate caspase activation and activity are the IAPs which are found in a range of organisms and are characterized by one or more baculovirus IAP repeats and responsible for their antiapoptotic activity [21,22]. IAPs suppress apoptosis by directly binding to and inhibiting caspases [21,23]. For example, XIAP, c-IAP-1 , and c-IAP-2 bind to procaspase-9 and prevent its activation [24], thereby blocking the downstream apoptosis-related events such as proteolytic cleavage of caspases-3, -6, and -7 [25]. Recently, a novel mitochondrial protein, Smac, and its murine homologue, DIABLO, were found to be released into the cytosol in response to apoptotic stimuli, including UVB irradiation, etoposide, or glucocorticoids and to promote caspase activation by eliminating IAP inhibition of caspases [26,27]. After mitochondria import, the N terminus of precursor Smac/DIABLO is removed by limited proteolysis to generate a mature form of the molecule. Mature Smac/DIABLO could promote caspase activation by binding and neutralizing the IAPs, sush as XIAP, CIAP-1, and CIAP-2 [26 –28]. A number of reports confirmed that mature Smac/DIABLO interacted with BIR2 and BIR3 of XIAP, and the N terminus of mature Smac/DIABLO was absolutely required for this interaction [29 –31]. Smac/DIABLO has been shown to serve as a new

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important regulator of apoptosis induced by apoptotic stimuli in a variety of cancer cells. However, whether Smac/ DIABLO plays an important role in apoptosis of myocytes induced by oxidative stress is poorly understand. In the present study, we examined the role of Smac/DIABLO in hydrogen peroxide (H2O2)-induced cell death in C2C12 myogenic cells. It was demonstrated that H2O2 was able to trigger apoptosis significantly in C2C12 myogenic cells, resulting in the activation of cellular caspase-9 and caspase-3 and the release of Smac/DIABLO from the mitochondria. C2C12 myogenic cells that. overexpressed Smac/DIABLO ectopically were more sensitive to death-inducing effects of H2O2. While it had no activity in the absence of H2O2, ectopic expression of the full-length Smac/DIABLO was able to significantly enhance H2O2-induced activation of caspase-9 and caspase-3 and subsequent apoptosis.

Materials and methods Cell culture and treatment C2C12 myogenic cells were cultured in growth medium (Dulbecco’s modified Eagle’s medium [DMEM] supplemented with 10% heat-inactivated fetal bovine serum [FBS]) at 37-C in the presence of 5% CO2 under a humidified atmosphere. H2O2 was diluted in phosphatebuffered saline (PBS: 137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4) and further diluted in culture medium. The final H2O2 concentration in medium was 0.5 mmol/L. Quantitation of apoptotic cells After H2O2 treatment, adherent cells were released from tissue culture plates with trypsin-EDTA or cell scrapers, combined with the nonadherent cells, and. sedimented at 500g for 5 min. The cells were fixed for 30 min at room temperature in 4% paraformaldehyde and then washed with PBS. Fixed cells were incubated for 30 min at room temperature with Hoechst 33258 (50 ng/ml) and then washed with PBS. Cells were mounted onto glass slides and examined by fluorescence microscopy. Apoptotic cells were identified by the condensation and fragmentation of their nuclei. The percentage of apoptotic cells was calculated from the ratio of apoptotic cells to total cells counted. A minimum of 500 cells were counted for each treatment. Western blotting analysis Cells were washed with PBS and resuspended with 5 vol of cold lysis buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5% Nonidet P40) supplemented with protease inhibitor mixture (Roche Applied Science). The cell lysate was incubated on ice for

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30 min and then centrifuged at 10,000g for 10 min at 4-C. The protein content of the supernatant was determined by the Bradford assay (Bio-Rad). Equal amounts of proteins (10 – 20 Ag) were loaded and separated by SDSPAGE and stained with Ponceau S as a loading control, the resolved proteins were transferred to nitrocellulose membrane. The blot was blocked with 2% albumin in TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 % Tween 20) overnight at 4-C and then probed with rabbitanti-Smac (Research and Development), anti-cytochrome oxidase subunit II (A-6404; Molecular Probes), rabbitanti-caspase-9, and rabbit-anti-caspase-3 (Santa Cruz Biotechnology). The immunoreactive bands were visualized with an HRP-conjugated secondary antibody and DAB (Boster Biological Technology). Caspase activity assay The caspase fluorescent assay kits specific for caspase-3 or caspase-9 (Biovision, Mountain View, CA) were used to detect caspase activation by measuring the cleavage of a synthetic fluorescent substrate. In brief, cells were cultured in 60-mm dishes and treated with 0.5 mmol/L H2O2 for the indicated periods. Cell lysates were prepared with the lysis buffer provided by the assay kit and centrifuged at 10,000g for 1 min, and the supernatants were collected. With bovine serum albumin (BSA) as the standard, equal amounts of proteins were reacted with synthetic fluorescent substrates at 37-C for 1.5 h and read at 405 nm on a Microplate reader. Fold increase in caspase-3 activity over control was determined. Preparation of mitochondrial and cytosolic fractions Methods used for isolation of subcellular fractions were essentially as described [32]. Untreated and H2O2-treated cells were harvested by centrifugation at 1000g for 10 min at 4-C. Adherent cells were removed by trypsinization in 0.25% trypsin at 37-C for 5 min. When cells were treated with H2O2, floating cells were also collected in the same tubes. After being washed once with ice-cold PBS, the cell pellet was suspended in 5 vol of buffer A [20 mM Hepes-KOH (pH 7.5), 10 MM KCl, 1.5 MM MgCl2, 1 mM Na-EDTA, 1 mM NaEGTA, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride containing 250 mM sucrose] supplemented with protease inhibitors (5 Ag/ml pepstatin A, 10 Ag/ml leupeptin, 2 Ag/ml aprotinin, and 25 Ag/ml N-acetyl-leu-leu-norleucine (ALLN)). After incubation on ice for 15 min, the cell suspension was gently homogenized with a glass Dounce homogenizer (20 –30 strokes) and cell lysates were checked by trypan blue staining. After centrifugation twice at 750g for 10 min at 4-C, the supernatant was collected and centrifuged at 10,000g for 15 min at 4-C, and the resulting mitochondrial pellets were resuspended in buffer A. S-100 cytosolic fractions were prepared by further centrifugation at 100,000g at 4-C, for 1 h.

Detection of DNA fragmentation Floating and adherent cells (5  107) were combined, centrifuged, pelleted at 400g for 5 min, and washed twice with PBS. The pellet was resuspended in 200 Al lysis buffer [10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% Triton X100, and 0.1 mg/ml RNase A] and incubated for 1 h at 37-C. The cell lysates were then treated with Protease K (0.2 mg/ml) at 54-C for 30 min. The genomic DNA was extracted by two separations, with phenol/chloroform and then with chloroform only. The DNA pellet was then washed in 70% ethanol and resuspended in 1 mM EDTA, 10 mM Tris-HCl (pH 8.0) at a final concentration of 20 Ag/ml. Aliquots were electrophoresed on 1.5% agarose gel containing ethidium bromide, and photographed under ultraviolet illumination. A GeneRuler 100-bp DNA ladder (MBI Fermentas, Hanover, MD) was utilized as DNA size markers. Lipofectamine-mediated gene transfection Transfection of C2C12 myogenic cells was carried out according the manufacturer’s instructions (Lipofectamine 2000, Invitrogen). Briefly, about 5  105 cells per bottle containing 5 ml appropriate complete growth medium were seeded and incubated at 37-C in a CO2 incubator until the cells were 70 to 80% confluent (24 h). After the cells were rinsed with serum-free and antibiotic-free medium, the cells were transfected separately with pcDNA3.1-Smac (kindly provided by Dr Xiaodong Wang of the University of Texas, Southwestern School of Medicine) 8 Ag/lipofectamine 20 AL (experimental group), pcDNA3.1 8 Ag/lipofectamine 20 AL (vector control), followed by incubation at 37-C in a CO2 incubator for 6 h. The medium was replaced by DMEM culture medium containing 20% FBS. After 48 h, two samples in each group were used to detect the transient expression of Smac by Western blotting. Transfection of anti-sense Smac/DIABLO and control oligonucleotides To delineate the role of Smac/DIABLO as a positive regulator of H2O2-induced apoptosis in C2C12 myogenic cells, we transfected the cells with fully phosphorothioated single-stranded anti-sense oligonucleotide (asODN), complementary to mouse Smac/DIABLO mRNA (for sequence see GenBank Accession No. BC054109), directed against the Smac/DIABLO translation initiation codon (sequence, 5VTACCGCCGAGACTCTTCAACCCACTGAGCC-3V) or control (scrambled) phosphorothioate oligodeoxynucleotide (sequence, 5V-CACGGTCTGAGG ATGGATGACA CCTCAGCG-3V). FITC-labeled oligonucleotides were manufactured by Bioasia Biotech (Shanghai, China). C2C12 myogenic cells were plated in 6-well plates and transfected with the help of Lipofectamine 2000, (Invitrogen). Twentyfour hours later, H2O2 (0.5 mmol/L) was added to appropriate

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wells and the cells were incubated for additional times as indicated. Statistical analysis Data are expressed as means T SEM of the indicated number of separate experiments. Statistical comparison between experimental group and control.was performed using unpaired two-tailed Student’s t tests. P values less than 0.05 were considered significant.

Results

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morphological changes characteristic of apoptotic cells, such as nuclear fragmentation and condensation (Fig. 1A). A significant time-dependent increase of apoptotic cells was observed after H2O2 treatment (Fig. 1B), reaching about 50% of the total C2C12 myogenic cells 24 h after H2O2 treatment (0.5 mmol/L). Furthermore, H2O2 treatment resulted in internucleosomal DNA fragmentation in C2C12 myogenic cells, as evidenced by the formation of a DNA ladder in agarose gel (Fig. 1C). These results demonstrated that H2O2 (0.5 mmol/L) induced apoptotic cell death in C2C12 myogenic cells in a time-dependent manner. H2O2 induced Smac/DIABLO release from mitochondria in C2C12 myogenic cells

H2O2 induce typical apoptosis in C2C12 myogenic cells We challenged C2C12 myogenic cells with H2O2 (0.5 mmol/L) and examined the resulting morphological changes. Nuclear staining with the dye Hoechst 33258 demonstrated

To investigate the mechanisms of H2O2-induced apoptosis in C2C12 myogenic cells, we examined whether H2O2induced apoptosis in C2C12 myogenic cells was associated with the release of Smac/DIABLO from mitochondria by Western blotting and immunofluorescence analysis. C2C12 myogenic cells were treated with H2O2 (0.5 mmol/L) for various times, and cytosolic and mitochondrial extracts were analyzed for the levels of Smac/DIABLO. We detected Smac/DIABLO predominantly within the mitochondrial fractions prior to treatment with H2O2. By 1 h of treatment, there was a significant loss of Smac/DIABLO from the mitochondrial fractions and a concomitant increase of Smac/ DIABLO in the cytosolic fractions. After 2 h, the majority of Smac/DIABLO were detected in the cytosolic fractions (Fig. 2). The mitochondrial inner membrane protein cytochrome oxidase subunit II (COX II) was retained in the mitochondria-rich fractions in all of samples, indicating that the H2O2 exposure did not lead to general mitochondrial destruction. Ponceau staining of the same lysates confirmed an equal amount of proteins, indicating that the results were not due to differential loading of samples. Moreover, similar to Western blotting analysis, immunofluorescence analysis showed that Smac displayed a dot-like distribution in untreated control cells. In contrast, H2O2 treatment resulted in a diffused distribution of Smac, indicating that H2O2 could induce Smac release from mitochondria into the cytosol (data not shown). The results confirmed that H2O2 induced Smac/DIABLO release from mitochondria in C2C12 myogenic cells. H2O2-induced activation of caspase-9 and caspase-3

Fig. 1. H2O2 induced typical apoptosis in C2C12 myogenic cells. (A) After being fixed, the cells were stained for the nuclear marker Hoechst 33258. Apoptotic cells were identified by their typical nuclear appearance. a, Control C2C12 cells cultured in regular medium; b, C2C12 cells exposed to H2O2 (0.5 mmol/L for 24 h); (B) percentages of apoptotic cells in each culture condition. At least 10 fields per dish and four independent cultures were examined. (* vs control group, P < 0.05). (C) C2C12 cells were treated with H2O2 (0.5 mmol/L) for the time indicated. DNA samples were extracted from cells, subjected to agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. M, DNA marker; 1, untreated C2C12 cells; 2, C2C12 cells treated with 0.5 mmol/L H2O2 for 24 h; 3, C2C12 cells treated with 0.5 mmol/L H2O2 for 36 h.

Caspase activation is believed to be a key factor during apoptosis. Caspase-9 is a proenzyme in the cells and can be processed to smaller fragments during apoptosis mainly through the mitochondrial apoptosis pathway, and caspase-3 is a biochemical hallmark of apoptosis. To examine whether H2O2-induced Smac/DIABLO release and apoptosis were associated with processing of caspase-9 and caspase-3, we analyzed the cleavage pattern of caspase-9 and caspase-3. The cytosolic extracts from H2O2-treated cells were subjected

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fectamine Plus reagent ranged between 30 and 50%. Fourtyeight hours after transfection, cell lysates were subjected to Western blotting with an anti-Smac/DIABLO antibody. Fig. 4A shows Smac/DIABLO overexpression in the cells transfected with Smac/DIABLO cDNA (C2C12/Smac) but not in the vector control cells (C2C12/Ctrol). The full-length cDNA of Smac/DIABLO has been shown to contain an Nterminal, 55-residue mitochondrial targeting sequence, which is cleaved after its mitochondrial import to generate the mature mitochondrial Smac/DIABLO. Therefore, we determined whether the ectopically overexpressed Smac/ DIABLO was localized to the mitochondria. Immunoblot analyses of the heavy membrane (mitochondria fractions) versus the S-100 cytosolic fractions revealed that the

Fig. 2. Western blotting showing that H2O2 induced the release of Smac/ DIABLO from mitochondria. C2C12 myogenic cells were treated with H2O2 (0.5 mmol/L) and harvested at the indicated times. Cytosolic and mitochondrial extracts were separated by 12% SDS-PAGE and analyzed by immunoblotting with anti-Smac and an antibody against cytochrome oxidase subunit II (COX II), a mitochondrial inner membrane protein. Ponceau staining was used as a loading control. The bottom panel is ratio of density of Smac bands to their loading control bands and statistic analysis (* vs Cyto of control group, P < 0.05; # vs Mit of control group, P < 0.05). Ctrol, Control; Cyto, cytoplasm; Mit, mitchondria. Equal loading of proteins was measured by using the Bradford assay (Bio-Rad). The blot presented is representative of three independent experiments.

to immunoblot analysis with anti-caspase-9 and anti-caspase3. The results, as shown in Fig. 3A, demonstrated that treatment of C2C12 myogenic cells with H2O2 resulted in the activation of caspase-9 and caspase-3 as evidenced by the appearance of the characteristic 10-kDa fragment (P10) and 11-kDa fragment (P11), respectively. Moreover, caspase activity assays revealed that caspase-9 and caspase-3-like activities began to increase at 12 h after H2O2 treatment (Fig. 3B). Taken together, these findings demonstrated that treatment of C2C12 myogenic cells with H2O2 was associated with activation of caspase-9 and caspase-3 and suggest the involvement of a mitochondrial-dependent apoptosis pathway. Ectopic overexpression of Smac/DIABLO sensitized C2C12 myogenic cells to H2O2-induced apoptosis In the above experiment, we found that treatment with H2O2 could result in the release of Smac/DIABLO from mitochondria (Fig. 2); it is therefore rational to propose that released cytosolic Smac/DIABLO may increase the susceptibility of C2C12 myogenic cells to H2O2-induced apoptosis. To test this hypothesis, we transiently transfected C2C12 myogenic cells with full-length human Smac/DIABLO cDNA (pcDNA3.1-Smac) or the vector control (pcDNA3.1) (Fig. 4A). The transfection efficiency utilizing the Lipo-

Fig. 3. H2O2 induced activation of caspase-9 and caspase-3. (A) C2C12 myogenic cells were treated with H2O2 (0.5 mmol/L) and harvested at the indicated times. Cytosolic extracts were separated by 12% SDS-PAGE and analyzed by immunoblotting with anti-caspase-9 (Cas-9) (upper panel) or anti-caspase-3 (Cas-3) (lower panel). P10 and P12 represent 10- and 12kDa fragments produced during the activation of caspase-9 and caspase-3, respectively. Ponceau staining was used as a loading control. The lower panel shows the ratio of density of P12 or P10 bands to their loading control bands and statistic analysis (* vs Ctrol group, P < 0.05). (B) C2C12 myogenic cells were treated with H2O2 (0.5 mmol/L) and harvested at the indicated times. Cytosolic extracts were assayed for protease activity of caspase-9 and caspase-3 using the caspase fluorescent assay kits. The experiments were performed as described under Materials and methods. Data were obtained from four independent experiments (*P < 0.05).

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lysates confirmed equal amounts of proteins, indicating that the results were not due to differential loading of samples. Consistent with this, the ectopic overexpression of Smac/ DIABLO was not associated with apoptosis of C2C12/Smac

Fig. 4. Ectopic overexpression of Smac/DIABLO. C2C12 myogenic cells were transiently transfected with either a control vector (C2C12/Ctrol) or a vector containing the full coding sequence of Smac/DIABLO (C2C12/ Smac). (A) After transfection for 36 or 48 h, cells were harvested and Smac/ DIABLO expression was determined by immunoblot analysis of the cell lysates by using anti-Smac antibody. Ratio of density of Smac bands to their loading control bands and statistical analysis (* vs C2C12/Ctrol group, P < 0.05). (B) Immunoblot analyses of the mitochondrial versus the S-100 cytosolic fractions of the untreated transfectants revealed the localization of ectopically expressed Smac/DIABLO. The lower panels show the ratio of density of Smac bands to their loading control bands and statistical analysis (* vs Mit of C2C12/Ctrol group, P < 0.05). Cyto, cytoplasm; Mit, mitchondria. Ponceau staining as a loading control. The results are representative of three separate experiments.

ectopically expressed Smac/DIABLO was localized only in the heavy membrane fraction containing the mitochondria in the absence of H2O2 (Fig. 4B). The mitochondrial inner membrane protein cytochrome oxidase subunit II was retained in the mitochondria-rich fractions in all samples, indicating that the transfection did not lead to general mitochondrial destruction. Ponceau staining of the same

Fig. 5. Ectopic overexpression of Smac/DIABLO sensitized C2C12 myogenic cells to H2O2-induced apoptosis. C2C12 myogenic cells were transiently transfected with either a control vector (C2C12/Ctrol) or a vector containing the full coding sequence of Smac/DIABLO (C2C12/Smac). (A) Forty-eight hours after transfection, the cells were treated with 0.5 mmol/L H2O2 for the indicated times, and the percentage of apoptotic cells was determined by Hoechst 33258 staining followed by fluorescence microscopy. Values represent the mean T SE (* vs C2C12/Ctrol group, P < 0.05; # vs C2C12/Ctrol + H2O2 group, P < 0.05, n = 5). (B) The cells were treated with 0.5 mmol/L H2O2 for 24 h, cytosolic DNA was separated by electrophoresis in 1.5% agarose gels and stained by ethidium bromide. A typical result of three independent experiments is shown. M, 100-bp marker; 1, C2C12/Ctrol without. H2O2 treatment; 2, C2C12/Smac without H2O2 treatment; 3, C2C12/Ctrol were treated with 0.5 mmol/L H2O2 for 24 h; 4, C2C12/Smac were treated with 0.5 mmol/L H2O2 for 24 h.

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cells (Fig. 5). Only the mature cytosolic form of Smac/ DIABLO could counter the inhibitory activity of IAPs and promote apoptosis induced by various apoptotic stimuli. We then determined whether overexpressed Smac/DIABLO promoted apoptosis induced by H2O2. As shown in Figs. 5A and B, elevated expression of full-length Smac/DIABLO significantly increased H2O2-induced apoptosis, as indicated by DNA ladder formation and nuclear condensation. At 24 and 36 h after H2O2 exposure, the percentages of apopotic cells detected by fluorescence microscopy were 70.3 and 75.2%, respectively, in C2C12/Smac cells, versus 46.8 and 55.7% in C2C12/Ctrol cells. Ectopic overexpression of Smac/DIABLO increased H2O2-induced caspase-9 and caspase-3 activation in C2C12 myogenic cells To determine whether transient expression of the fulllength Smac/DIABLO in C2C12 myogenic cells enhanced H2O2-induced caspase activity, we analyzed caspase-9 and caspase-3 activity. Caspase activity was determined by an in vitro substrate-cleaving reaction and Western blotting analysis. Fig. 6A shows that the ectopic overexpression of Smac/ DIABLO was not associated with activation of caspase-9 and caspase-3 in C2C12/Smac cells in the absence of H2O2. After 8 h of exposure to H2O2, there was a 1.65-fold increase of caspase-9 activity and a 2.01-fold increase of caspase-3 activity from control transfected cells (C2C12/Ctrol). Ectopic overexpression of Smac/DIABLO significantly increased H2O2-induced activation of caspase-9 (2.61-fold) and caspase-3 (3.02-fold). Consistent with this, Western blotting analysis showed that the activities of caspase-9 and caspase-3 in Smac-transfected cells C2C12/Smac) were significantly higher than those from the vector control cells (C2C12/Ctrol) as revealed by the appearance of the characteristic p10- and p11-kDa fragments (Fig. 6B). These data indicate that ectopic overexpression of Smac/DIABLO could further increase the activities of caspase-9 and caspase-3 induced by H2O2 in C2C12 myogenic cells. Transfer of phosphorothioate ODN against Smac/DIABLO inhibited apoptosis and activation of caspase-3 and caspase-9 induced by H2O2 in C2C12 myogenic cells To further characterize the role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells, antisense phosphorothioate oligonucleotides against Smac/DIABLO were transferred into the cells. Using Lipofectamine 2000 as a transfer reagent, FITC-labeled phosphorothioate ODNs were successfully transferred into cultured C2C12 myogenic cells and. the transfection efficacy was about 80% of all cultured C2C12 myogenic cells. Morever, transfer of antisense oligonucleotide was able to selectively inhibited Smac/DIABLO protein content by more than 75% after 24 h, compared to the tubulin, while gene transfer of scrambled control oligonucleotides (scrODN) did

Fig. 6. Ectopic overexpression of Smac/DIABLO further increased H2O2induced activation of caspase-9 and caspase-3 in C2C12 myogenic cells. C2C12 myogenic cells were transiently transfected with either the control vector (C2C12/Ctrol) or a vector containing the full coding sequence of Smac/DIABLO (C2C12/Smac). Forty-eight hours after transfection, cells were either treated with 0.5 mmol/L H2O2 for 8 h or not. The cells were harvested and cell lysates were either assayed for protease activity of caspase-9 and caspase-3 by using the caspase fluorescent assay kits (A) or immunoblotted with anticaspase-9 (Cas-9) and anticaspase-3 antibodies (Cas-3) (B). Ponceau staining was used as a loading control. The results are representative of three separate experiments. The bottom panel shows the ratio of density of P12 or P10 bands to their loading control bands and statistical analysis (* vs C2C12/Ctrol group, P < 0.05; # vs C2C12/Ctrol + H2O2 group, P < 0.05, n = 3). Data of caspase fluorescent assays were obtained from four independent experiments (* vs C2C12/Ctrol group, P < 0.05; # vs C2C12/Ctrol + H2O2 group, P < 0.05, n = 8).

not alter Smac/DIABLO expression (Fig. 7A). Having established that Smac/DIABLO asODN effectively inhibited Smac/DIABLO protein expression, we next sought to determine the effects of Smac/DIABLO ablation on apoptosis induced by H2O2. For these studies, Smac/DIABLO-directed asODNs or control scrODNs were introduced into C2C12 myogenic cells for 24 h before the cells were exposed to H2O2. As shown in Figs. 7B and C, ablated expression of Smac/DIABLO significantly inhibited H2O2-induced apop-

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tosis, as indicated by the percentage of apoptotic nuclei ( P < 0.05) and DNA ladder formation. In addition, we analyzed the activities of caspase-9 and caspase-3 by an in vitro substrate-cleaving reaction. Fig. 7D showed that Smac/ DIABLO ablation (asODN) markly inhibited activation of caspase-9 and caspase-3 after 8 h of exposure to H2O2 as compared with scrODN treatment and there was about an 1.9fold increase of caspase-9 activity and a 2.3-fold increase of caspase-3 activity from control scrODNs cells (scrODNs) and only 1.3-fold increase of caspase-9 activity and a 1.5-fold increase of caspase-3 activity from asODNs cells ( P < 0.05). Toxic effects after transfection with ODNs could not be observed.

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Discussion Under physiological conditions, the toxic effects of ROS are removed by antioxidant systems, but during biotic or abiotic stresses, the concentration of ROS in the cells can rise significantly, reaching the threshold that can trigger apoptosis [33]. Increased production of ROS has been implicated in stress induction of apoptosis in cardiac myocytes. Increasing evidence suggests that apoptosis is a major mechanism in the development of a number of cardiovascular diseases, such as hypertension, acute myocardial infarction, ischemia-reperfusion injury, and heart failure [34 – 36]. However, the detailed signal pathways responsible for ROS-induced apoptosis are not fully examined. Here, we try to demonstrate the pivotal role of mitochondria and pay special attention to the role of a newly found mitochondrial protein, Smac/DIABLO, in hydrogen peroxide-induced apoptosis in myocytes. Our results showed that hydrogen peroxide at a concentration of 0.5 mmol/L could lead to apoptosis of C2C12 myogenic cells in a time-dependent manner. From the results of Hoechst 33258 staining and DNA laddering, apoptosis could be considered one of the main mechanisms for the oxidative injury. These data suggests that the in vitro model of hydrogen peroxide-stimulated C2C12 myogenic cells used in the present study could be used to investigate the apoptosis of myogenic cells. Mitochondria are thought to play a central role in the activation of apoptosis induced by multiple stimuli. Within the past few years, the involvement of mitochondria in the Fig. 7. Transfer of phosphorothioate ODN against Smac/DIABLO, but not scrambled control ODN, into C2C12 myogenic cells inhibited H2O2induced apoptosis. C2C12 myogenic cells were transferred with Smac/ DIABLO antisense ODN (asODN) or scrambled control ODN (scrODN) as described under Materials and methods. (A) After incubation for 24 h, cells were harvested, lysed, and Western-blotted (WB) with the corresponding antibodies indicated on the right. Smac/DIABLO asODN were able to block Smac/DIABLO protein expression by about 75% compared to untreated cells (Ctrl) or stimulated cells using transfection reagent without ODNs (Lip) or cells transferred with scrambled control ODN (scrODN). Ratio of density of Smac bands to tubulin control bands was calculated and analyzed statistically (* vs scrODN group, P < 0.05). (B) Twenty-four hours after transfer, the cells were treated with 0.5 mmol/L H2O2 for 24 h, and the percentage of apoptotic cells was determined by Hoechst 33258 staining followed by fluorescence microscopy. Values represent the mean T SE (* vs untreated control group, P < 0.05; # vs scrODN + H2O2 group, P < 0.05, n = 5). (C) The cells were treated with 0.5 mmol/L H2O2 for 24 h, cytosolic DNA was separated by electrophoresis in 1.5% agarose gels and stained by ethidium bromide. A typical result of three independent experiments is shown. 1, C2C12 cells without H2O2 treatment; 2, ScrODN without H2O2 treatment; 3, AsODN without H2O2 treatment; 4, C2C12 cells treated with H2O2 for 24 h; 5, C2C12 cells transferred with ScrODN and treated with H2O2 for 24 h; 6, C2C12 cells transferred with AsODN and treated with H2O2 for 24 h. D, Twenty-four hours after transfection, cells were either treated with H2O2 for 8 h or not. The cells were harvested and cell lysates were assayed for protease activity of caspase-9 and caspase-3 by using the caspase fluorescent assay kits. Data were obtained from three independent experiments (* vs Ctrl group, P < 0.05; # vs scrODN + H2O2 group, P < 0.05, n = 5).

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control of apoptosis has been well documented [37,38]. Several proteins possessing proapoptotic functions are localized to mitochondria, including cytochrome c [32], Smac/DIABLO [26,27], AIF [39], and endonuclease G [40]. Additionally, it has been reported that stores of procaspases are present in mitochondria [41 –45]. A death-inducing signal propagates to the mitochondria, which releases several harmful proteins such as cytochrome c. Cytochrome c, the adaptor protein Apaf-1, the precursor procaspase-9, and dATP form the apoptosome death complex [46]. Procaspase-9 is cleaved to form the active enzyme, caspase-9, which activates caspase-3, -6, or -7 in the same way. It is well known that mitochondria are both a target and a source of ROS [47]. It was previously reported that cytochrome c was released from the mitochondria of H2O2treated cells, leading to the activation of caspase-9 [48]. We wished to determine whether additional mitochondrial molecules were also involved in H2O2-induced apoptosis. Two groups [26,27] have recently identified a novel mitochondrial factor, Smac/DIABLO, with a central role in the modulation of apoptosis, which unblocks one step in the cell death pathway. Smac/DIABLO remains associated with the mitochondrial membrane and is released simultaneously with cyt c in some cancer lines. Once released into the cytoplasm, Smac/DIABLO binds to IAPs, allowing caspases at the. apoptosome to be activated. ROS has been proposed to cause cytochrome c release and apoptosis during ischemia and reperfusion injury in myocytes. However, the role of Smac/DIABLO in ROS-induced apoptosis in myocytes is yet unknown. Smac/DIABLO is synthesized as an apoprotein on cytosolic ribosomes and then translocates to mitochondrial intermembranous space. It is rarely detectable in cytosol, but can be released to the cytosol by apoptotic stimuli such as TRAIL, UV irradiation, and etoposide [49,50]. Results from our study demonstrate that, for the first time, H2O2 exposure causes the release of Smac/DIABLO from mitochondria to cytosol in C2C12 myogenic cells. Further, we noted that Smac/DIABLO release was a much earlier event (1– 2 h) than caspase-9 and caspase-3 activation (4 h) (Fig. 3B). These findings are consistent with observations in human melanoma that Smac/ DIABLO release into the cytosol precedes caspase activation and other cellular changes [51,52]. Therefore, Smac/ DIABLO detected in the cytosol can be regarded as an early and sensitive apoptotic indicator in H2O2-induced apoptosis in C2C12 myogenic cells. However, whether Smac/DIABLO plays an important role in myocytes is poorly understood. Therefore, we transfected a plasmid containing full-length Smac/DIABLO (pcDNA3.1-Smac) or antisense oligonucleotide against Smac/DIABLO (asODN) to investigate whether it was possible to regulate the mitochondrial pathway of caspase activation in C2C12 myogenic cells. In the present study we showed that, consistent with the previous resports, the.ectopically expressed Smac/DIABLO containing a mitochondrial targeting sequence was localized

only in the heavy membrane fraction, and it had no activity in the absence of H2O2. In addition, we demonstrated for the first time that the ectopic overexpression of Smac/DIABLO sensitized C2C12 myogenic cells to H2O2-induced processing and activation of caspase-9 and caspase-3 and apoptosis. On the contrary, antisense oligonucleotide against Smac/ DIABLO (asODN) inhibited the Smac/DIABLO protein content by more than 75%, activation of caspase-9 and caspase-3, and apoptosis induced by H2O2. These findings are consistent with the reports that Smac/DIABLO can neutralize the repressive influence of IAPs on the processing and activation of caspases-9 and -3 [53,54]. In conclusion, we have demonstrated that Smac overexpression promoted apoptosis induced by H2O2 in C2C12 myogenic cells, which is associated with enhanced activation of caspase-9 and caspase-3, and ablated expression of Smac/DIABLO significantly inhibited apoptosis induced by H2O2, which is associated with enhanced activation of caspase-9 and caspase-3. Given that abnormalities in the mitochondrial pathway of caspase activation are known to occur during myocardial ischemia-reperfusion injury and cardiomyocytes possess abundant mitochondria, we suggest that the inhibition of Smac/DIABLO release from mitochondria, or downregulation of Smac/DIABLO activity, may have genuine potential in the treatment of this disease. Acknowledgments This study was supported by grants from The National Natural Science Foundation of China (30000069; 30270533), The Special Funds for Major State Basic Research of China (G2000056908), and The Special Funds for Ph D Training from The Ministry of Education of China (20020533032). The authors thank Dr. Xiaodong Wang (University of Texas Southwestern Medical Center at Dallas) for providing the Smac/DIABLO plasmid and Dr. Randy McMillan (University of Texas Southwestern Medical Center at Dallas) for critical comments on the study.

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