Diazoxide Pretreatment Enhances L6 Skeletal Myoblast Survival and Inhibits Apoptosis Induced by Hydrogen Peroxide
Article first published online: 20 JAN 2012
Copyright © 2012 Wiley Periodicals, Inc.
The Anatomical Record
Volume 295, Issue 4, pages 632–640, April 2012
How to Cite
Shao, S.-X., Zhang, L., Chen, H.-X., Liu, Y., Zhang, J.-P., Chen, W. and Xue, G.-Y. (2012), Diazoxide Pretreatment Enhances L6 Skeletal Myoblast Survival and Inhibits Apoptosis Induced by Hydrogen Peroxide. Anat Rec, 295: 632–640. doi: 10.1002/ar.22410
- Issue published online: 12 MAR 2012
- Article first published online: 20 JAN 2012
- Manuscript Accepted: 17 NOV 2011
- Manuscript Received: 20 SEP 2011
- Science Technology Agency of Hebei Province. Grant Number: 10206130D
- skeletal myoblasts;
- hydrogen peroxide
Skeletal myoblast (SKM) transplantation is a promising approach to regenerate tissue and improve the function of the injured heart. However, the number of survival cells transplanted to host myocardium is quite poor due to high rate of apoptosis; diazoxide (DZ) is a highly selective mito-KATP channel opener that may reduce cell apoptosis by relieving reactive oxygen species (ROS) damage. The aim of this study is to explore the protective effects of DZ on L6 SKM damage induced by hydrogen peroxide (H2O2) in vitro. Different dose and time of H2O2 and DZ treatment were performed and only 24 hr of 1.00 mmol/L H2O2 treatment and 200 μmol/L DZ pretreatment for 30 min were used for further experiment. L6 SKMs were cultured and divided into control group (no treatment), H2O2 group (24 hr of 1.00 mmol/L H2O2 treatment) and DZ + H2O2 group (pretreated with 200 μmol/L DZ for 30 min before 24 hr of 1.00 mmol/L H2O2 treatment). Compared with control group, H2O2 treatment caused cell damage, increased lactate dehydrogenase release, cell apoptosis, and bax gene expression, while reduced cell proliferation and decreased bcl-2 expression. DZ pretreatment may protect cells from damage induced by H2O2 and reduce cell apoptosis by increasing bcl-2 and decreasing bax expression. DZ pretreatment may also promote cell proliferation measured by both PCNA expression and flow cytometry method. These results suggest that DZ may protect L6 SKMs from damage induced by H2O2 by maintaining integrity of cell membrane, reducing apoptosis and increasing proliferation in vitro. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.
Ischemic heart disease is a life-threatening disease, which has high morbidity and mortality, although large progress has been made in its therapy in recent years. Cell transplantation is a promising approach to regenerate tissue and improve function for injured heart by promoting angiogenesis, regenerating new myocardium, improving myocardial blood supply, and enhancing cardiac function (Guo et al., 2008; Cui et al., 2010; Gmeiner et al., 2011; Oguz et al., 2011).
Skeletal myoblasts (SKMs) have been widely studied in transplantation. However, the number of survival cells transplanted to host myocardium is quite poor. Studies have shown that there are about 70%–80% or even more of the SKMs died within 3 days after transplantation (Qu et al., 1998; Toma et al., 2002; Pagani et al., 2003), which might be caused by apoptosis. Therefore, to reduce cell apoptosis of transplanted SKMs is important for improving therapeutic effects.
Local microenvironments of transplantation, such as persistent ischemia and inflammation, may cause cell apoptosis, leading to poor therapy effects (Hodgetts et al., 2000; Frangogiannis et al., 2002; Zhu et al., 2006). Therefore, the reduction of cell apoptosis by increasing the tolerance ability and improving the transplantation microenvironment can all be promising. Studies conducted in many cell types have found that, pretreatment cells with heat shock, hypoxic, and diazoxide (DZ), may reduce cell apoptosis and improve cell survival under some oxidative stress (Riederer et al., 2008; Rosova et al., 2008; Cui et al., 2010). During cardiac infarction, reactive oxygen species (ROS) was increased in both infarct and noninfarct area, which may induce apoptosis of transplanted cells. H2O2 is one of ROS that may cause cell damage.
It has been suggested that, temporary ischemic pretreatment may cause ATP-regulated potassium channel (mito-KATP channel) open and make heart more tolerant to lethal damage of ischemia (Maslov et al., 2010). These effects can be mimicked by DZ treatment (Djordjevic et al., 2008; Otani, 2008; Cui et al., 2010; Maslov et al., 2010), a highly selective mito-KATP channel opener in mitochondria. DZ may reduce cell apoptosis by decreasing calcium overload in mitochondria, relieving ROS injure, maintaining mitochondrial intramembrane steady, reducing caspase-3 activation and inhibiting cytochrome c release and therefore reduce apoptosis (Otani, 2008).
In this study, we will explore the protective effects of DZ on L6 SKM damage induced by H2O2in vitro and try to find an effective way to improve the survival of transplanted cells, which may enhance the efficiency of clinical transplantation therapy.
MATERIALS AND METHODS
L6 SKMs Culture
L6 SKMs (cell line CRL-1458™, American Type Culture Collection, USA) were cultured in DMEM, which containing 15% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C at presence of 5% CO2. To maintain their undifferentiated state, L6 SKMs were transferred when they were grown to 60%–70% confluence and experiments were conducted after three to five times transferring.
Part 1: The Effect of H2O2 or DZ on L6 SKMs at Different Dosage and Time
The H2O2 treatment on L6 SKMs
L6 SKMs were cultured at a density of 4 × 103 cells per well of 96-well plate for 2 days and then were divided into control (only DMEM) and H2O2 groups (supplemented with 0.10, 0.50, 1.00, 1.2, and 1.5 mmol/L H2O2) for 12, 24, and 36 hr separately. Cell viability was detected by methyl thiazolyl tetrazolium (MTT, Sigma, USA) method as follows: 20 μL of MTT(5 mg/mL) were added in the medium and cells were cultured for 3 hr afterward. Then the medium was discarded and 150 μL dimethyl sulfoxide was added. The optical density (OD) of the plate was measured at 490 nm after 10 min shaking and the percentage of cell survival was calculated by the formula:
The Effect of DZ on L6 SKM Damage Induced by H2O2
L6 SKMs were cultured for 2 days as described above. Then cells were divided into control group (no treatment), H2O2 group (1.00 mmol/L H2O2 treatment for 24 hr), and DZ pretreatment group. To choose the appropriate concentration of DZ, different concentration of DZ (Sigma, USA) including 50, 100, 200, and 400 μmol/L were used for 30 min before H2O2 treatment. Cells were collected after 24 hr treatment and MTT method was used to check cell viability.
Cells were cultured and divided into control group (no treatment), H2O2 group (1.00 mmol/L H2O2 treatment for 24 hr) and DZ + H2O2 group (pretreated with 200 μmol/L DZ for 30 min before 24 hr of 1.00 mmol/L H2O2 treatment). Then cells were collected for further analysis.
Inverted Phase-Contrast Microscopy Analysis
The morphological changes of the cells in different groups were observed and photographed directly by inverted microscope.
Lactate Dehydrogenase Measurement
Lactate dehydrogenase (LDH) is an intracellular enzyme and its leakage is widely used as a marker for the integrity of the cell membrane. LDH activities in medium were quantified using LDH assay kit (Genmed Scientifics, USA) according to the manufacturer's instructions. The OD was measured at 490 nm by Elx800 microplate reader (BioTek, USA). Results are expressed as the percentage of total releasable LDH.
The cells were stained by 4′,6-diamidino-2-phenylindole (DAPI, 20 μg/mL, Serva, Germany) for 2 hr and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were then dehydrated gradiently after PBS washing and covered by neutral gum. Slices were visualized by fluorescent microscope at excitation wave 360 nm.
Transmission Electron Microscopy Analysis
Cells from all groups were collected after treatment and used for transmission electron microscopy (TEM) analysis according to the protocol. In brief, after 0.01 M PBS washing, cells were digested by 0.25% trypsogen and then centrifuged at 800g for 10 min. The cell pellet was fixed in 2.5% glutaraldehyde for 24 hr. After washing with 0.01 M PBS twice, cell pellets were postfixed with 1% osmium tetroxide aqueous solution for 2 hr and then were dehydrated in acetone and embedded in Epon 812. A total of 0.1 μm sections were cut by Leica UCT ultramicrotome and were double stained with uranyl acetate and lead citrate and then observed by transmission electron microscope (Hitachi H-7500, Japan).
Scanning Electron Microscopy Analysis
The cells were cultured on coverslips and treated as described above. After 0.01 M PBS washing, cells were fixed in 2% glutaraldehyde at 4°C for 2 hr and postfixed with 1% osmium tetroxide aqueous solution for 1 hr after PBS. After dehydration, cells were dried, gold sputtered and observed by scanning electron microscopy (SEM; Hitachi-S3500N, Japan) at 15 kV.
Cells were digested by 0.25% trypsogen and then harvested. Cell suspension was prepared after twice washing with 0.01 M PBS. Two milliliters of ice-cold 75% ethanol was added in cell suspension and stored at 4°C overnight. The ethanol was removed after centrifugation and cell pellets were re-suspended in PBS. RNase A and then propidium iodide was added into the cell suspension with final concentration of 10 μg/μL and 50 μg/mL separately. The cell mixtures were kept in dark for >30 min until analysis of cell cycle and cell apoptosis rate by a flow cytometer (Epics-XLII, Beckman Coulter, USA). Cell proliferation index (PI) was calculated by the formula:
Cells were cultured on coverslips and treated as described above. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Cells were then incubated with rabbit anti-bax (1:200, Santa Cruz, USA), rabbit anti-bcl-2 (1:200, Santa Cruz, USA), mouse anti-cytochrome C (1:200, Santa Cruz, USA), mouse anti-PCNA (1:400, Santa Cruz, USA) primary antibodies at 4°C for 24 hr. Slices were then incubated with biotin-labeled goat anti-rabbit IgG or biotin-labeled goat anti-mouse IgG (Santa Cruz, USA) for 1 hr at room temperature and then incubated with peroxidase-conjugated streptavidin for 1 hr after rinsing. Peroxidase activity was developed by using 3,3′-diaminobenzidine tetrachloride for 10 min. Eight fields were chosen randomly in each slice and average OD was analyzed with Image-Pro Plus 6.0 system (USA).
Protein was extracted from cells by cold lysis buffer supplemented with protease inhibitor. The protein concentration was measured by BCA Kit (Novagen, Germany).
Fifty-microgram protein were electrophoresed in a denaturing 12% polyacrylamide gel and then transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked with TBST containing 5% nonfat milk. Membranes were incubated in a rabbit anti-bax (1:200 Santa Cruz, USA), rabbit anti-bcl-2 (1:200, Santa Cruz, USA), mouse anti-GAPDH (1:200) antibody at 4°C overnight, respectively. Then membranes were incubated in fluorescent-conjugated anti-rat or anti-mouse secondary antibody (1:3,000, Santa Cruz, USA) for 1 hr at room temperature. The integral optical density (IOD) of each band was measured using Odyssey infrared imaging system (LI-COR, USA). GAPDH were used as a house-keeping gene and relative gene expression on protein level was calculated.
Data were described as mean ± SDs. Student's t-test and one-way ANOVA were performed by SPSS 13.0 to check the differences between groups. P value <0.05 was considered significant.
The Dose- and Time-Dependent Effect of H2O2 on L6 SKM Injury
Cell survival rate was calculated under different dose and time of H2O2 treatment. Our data have shown that, except 0.1 mmol/L H2O2 treatment for 12 hr group, other H2O2 treatments could significantly reduce L6 SKM viability when compared with control group, and this effect was almost dose and time dependent (Fig. 1). From this experiment, we found that treatment with 1.00 mmol/L H2O2 for 24 hr may induce almost 50% cell death. Therefore, this treatment was used for further analysis.
Effects of DZ on H2O2 Induced L6 SKM Injury
L6 SKMs were pretreated with DZ under different dosage for 30 min before 24 hr 1.00 mmol/L H2O2 treatment. Results from our experiment have indicated that, compared with only H2O2 treated group, 50 μmol/L DZ could not increase cell vitality, indicating little protective effect. A total of 100 and 200 μmol/L of DZ may increase cell viability significantly and these seemed also dose dependent. In addition, the maximal protective effect of DZ was observed at dose of 200 μmol/L, and no more increase in cell viability was observed under the dose of 400 μmol/L (Fig. 2). Therefore, the concentration of 200 μmol/L was applied in further experiments.
The Effect of DZ Pretreatment on L6 SKM Morphology
Under inverted microscope, H2O2 treatment caused some cells detachment. Some cells were contracted and the gaps between cells were widened. Cells with DZ pretreatment almost had no micromorphological alterations. The cells were fusiform shaped with clear boundary and arranged tightly (Fig. 3A–C).
DAPI is a blue fluorescence dye and may combine with DNA. In control group, we observed that the nuclei were big and regular with well-distributed chromatin. H2O2 treatment may cause cell damage, which was characterized by condensed and fragmented chromatin in shrunk nuclei. These pathological alterations can be largely improved by DZ pretreatment (Fig. 3D–F).
Under TEM, L6 SKMs in control group were round shaped and there were some microvilli on the cell membrane. The nucleus was elliptical with well-distributed euchromatin and the nucleolus was clear. The rough endoplasmic reticulum (RER) in cytoplasm consisted of parallel stacks of flattened cisternae. After H2O2 treatment, the cell membrane of the L6 SKMs was shriveled, and nuclei were shrunk with condensed chromatin. Some heterochromatins were crescent shaped and condensed and bound to the nuclear membrane, even fragmented. We also observed dilated RER in this group of cells. While cells in DZ pretreatment group had almost normal morphology, which was similar to those observed in control cells (Fig. 4A–C). Similar results had also been observed by SEM. Cells in control group were fusiform shaped with clear boundary and arranged tightly. H2O2 treatment may result in cell shrunk and crinkly shaped, and apoptotic bodies were found in some fields. No such alterations had been found in both control and DZ groups (Fig. 4D–F).
The Effect of DZ Pretreatment on LDH Leakage Induced by H2O2 in L6 SKMs
LDH leakage is a good marker for cell integrity. In this study, we have found that H2O2 treatment caused significantly increased LDH level compared with control (P < 0.05) and this increase was partly blocked by DZ pretreatment (Fig. 5).
The Effect of DZ Pretreatment on Proliferation
Cell proliferation, which was calculated by PI, was significantly decreased under H2O2 treatment compared with control (66.21% ± 4.65% vs. 41.07% ± 5.02%, P < 0.05). DZ pretreatment could significantly increase PI from 41.07% ± 5.02% to 72.50% ± 8.93% when compared with H2O2 treatment. This significance was also observed when DZ + H2O2 compared with control (P < 0.05) (Fig. 6E).
The Effect of DZ on L6 SKM Apoptosis Induced by H2O2
Apoptosis rate was measured by flow cytometry method (FCM). In this study, compared with control group, the apoptosis rate was significantly higher in H2O2-treated L6 SKMs (46.54% ± 3.99% vs. 1.26% ± 0.21%). This effect can be reduced by DZ pretreatment to the control level (Fig. 7D).
Gene expression involved in apoptosis on protein level has also been checked by both immunocytochemisty and Western blot. It has been suggested that mitochondria participate apoptosis by releasing cytochrome c and this effect can be prevented by antiapoptosis gene bcl-2 but induced by bax (Otani, 2008). The integrity of mitochondrial membrane was assessed by immunostaining of cytochrome c. In normal control group, positive brown particles were located in mitochondria. In H2O2-treated cells, the immunoreactivity of cytochrome c was found significantly increased and diffused in cytoplasm, which indicated cytochrome c transferred from mitochondria to cytoplasm. DZ treatment caused more localized cytochrome c staining, which was similar to those observed in control cells (Fig. 7A–C).
In this study, we found that compared with control groups, H2O2 treatment significantly increased the gene expression of bax, while decreased the expression of bcl-2 on protein level. And significantly decreased bax gene expression and increased bcl-2 expression were all observed when cells were pretreated with DZ, indicating DZ may protect L6 SKMs from the apoptosis induced by H2O2 (Fig. 8).
To reduce the damage of transplanted cells in host situation is widely studied in recent years. Under some ischemic cardiac pathologic situations, persisted oxidative stress and increased local reactive oxygen concentration have all been suggested to participate in cell damage, which also including transplanted cells, contributing to reduce transplantation efficiency (Djordjevic et al., 2008).
H2O2 is one of ROS and has been widely used in the experiment to mimic situation with oxidative stress. However, the concentration of H2O2 used in experiments differs widely in different cell types. In experiments conducted in myoblast, very low concentration of H2O2 was found to enhance growth, while moderate and high concentration (Caporossi et al., 2003; Djordjevic et al., 2008) was detrimental. Different type of cells showed different response to oxidative stress induced by H2O2 (Djordjevic et al., 2008). In this study, we are first to evaluate the effect of H2O2 on L6 SKMs. We have found that L6 SKM damage induced by H2O2 was almost time- and dose-dependent. Also, we have chosen the relative lower concentration of H2O2, which may cause almost 50% cell loss for the further experiment. Similar idea has also been described by Niagara et al. (Niagara et al., 2007; Salucci et al., 2010; Choi, 2011).
DZ is a mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel opener, which may increase membrane permeability to potassium ions. This switches off voltage-gated calcium ion channels causing intracellular calcium overload. It has been found that DZ may protect cells against hypoxia. For example, Choi et al. (2011) have fount that, DZ may restore mitochondrial membrane potential dissipation, ATP loss, and intracellular calcium elevation and prevented cell death in osteoblastic MC3T3-E1 induced by antimycin A. Similar result has also been found by Robin et al. (2010) in rats with cereal ischemia-reperfusion models. However, the concentration of DZ used in the experiments also varies from 30 to 500 μmol/L (Minners et al., 2001, 2007; Niagara et al., 2007). In this study, we have checked several different concentration of DZ, we have found that, its protective effect was also dose dependent, and the maximal effect was already achieved at 200 μmol/L. Therefore, for L6 SKM damage induced by H2O2, 200 μmol/L DZ might be the minimal dose that may achieve the maximal effect.
Apoptosis, which is also termed as programmed cell death, participates in pathology of myocardial infarction and heart failure (Qu et al., 1998; Toma et al., 2002; Pagani et al., 2003). Mitochondria is one of the most important organelles where energy and ROS generation, calcium homeostasis are taking place. Therefore, improve mitochondria tolerance to stress by some treatments, such as “ischemic preconditioning,” may protect cell from apoptosis by reducing some stress factors (Abraham and Gerstenblith, 2007). Apoptosis regulator bcl-2 family may govern mitochondrial outer membrane permeabilization and can be either proapoptotic (bax) or antiapoptotic (bcl-2). Bax induces the release of cytochrome c into the cytosol, which may consequently activate caspase-9 and caspase-3, leading to apoptosis, and this effect can be inhibited by bcl-2 (Scorrano and Korsmeyer, 2003; Huang et al., 2007). Bcl-2-modified rat L6 myoblast and MSCs were significant resistant to apoptotic stimuli in the infracted myocardium, and cardiac function was improved significantly (Li et al., 2007; Kitabayashi et al., 2010; Siltanen et al., 2011).
DZ may protect cells from apoptosis but mechanism underlying remains contentious (Abraham and Gerstenblith, 2007). Consistent with the studies performed previously, DZ may protect cells against damage induced by ischemia (Niagara et al., 2007). Except that, we also found H2O2 may cause cell damage and cell death, which was characterized by both TEM and SEM findings, increased apoptotic cell numbers and increased bax gene expression. In this experiment, we are first to find that DZ pretreatment may inhibit cell damage by reducing apoptosis cell number and decrease proapoptotic gene bax and increase antiapoptosis bcl-2 gene expression on protein levels. The protective effects of DZ might be achieved by modulating bcl-2/bax gene expression. This result seems controversial with previous study performed by Kicinska et al. (2003) in ventricular cardiomyocytes isolated from neonatal rat, which showed that DZ has no affection on bcl-2 expression. We assume that different type of cells in the experiment might respond stress and damage through different pathways, and other signal pathways might be also involved in DZ effects. In addition, we also found that DZ may maintain the integrity of cell membrane, especially of the mitochondria. ROS stress may cause increased permeability of cell membrane, leading to increased LDH activity and diffusion of cytochrome c in cytoplasm. In this study, we have found that these pathological alterations can be relieved by DZ pretreatment, which may also partly contribute to reduce cell apoptosis.
Interestingly, in this study, we also found firstly that DZ also may promote SKM proliferation, which was identified by increased PCNA expression and increased PI measured by FCM. It has been suggested that mitoK(ATP) channel is involved in cell proliferation in some cell types (Zhao et al., 2009; Fogal et al., 2010). It has been found that DZ may induce proliferation of glioma cell (Huang et al., 2009) and HPASMC (Wang et al., 2007), but not cancer cells (Ding et al., 2009). Increased NF-kB levels might be involved in this procedure (Afzal et al., 2010). No proliferation effect was found in H2O2 treatment group, we think that it might because the concentration of H2O2 used in this study was higher, since lower concentration of H2O2 may induce cell proliferation, while high concentration only can induce cell damage.
In summary, DZ may protect L6 SKMs from damage induced by H2O2 and this effect might be achieved by maintaining integrity of cell membrane, reducing apoptosis and increasing proliferation in vitro. This may provide a promising strategy to improve the efficacy of L6 SKM transplantation. Further in vivo study still needs to be done to check its clinical feasibility.
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