L6-GLUT4myc cells, a L6 skeletal muscle cell line (Kerafast, Inc., Boston, MA, USA), were harvested in α-MEM supplemented with a 10% FBS and 1% penicillin-streptomycin solution according to a previous study [12]. As prolonged exposure to low concentrations of EtBr inhibits the replication of mtDNA selectively, a L6-GLUT4myc cell line depleted of mtDNA was prepared by incubating the cells with EtBr (0.2 µg/ml) and uridine (50 µg/ml) for 3 weeks. The reversal of mtDNA depletion was achieved within 7 days by the removal of EtBr and uridine. The control cells were cultured for the same time period under normal harvesting conditions.

Total cellular DNA was extracted by using the DNeasy Tissue kit (Qiagen, Valencia, CA, USA), and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The reverse transcription reaction was performed using M-MLV reverse transcriptase (Promega, Madison, WI, USA). The primers and PCR conditions used in this study are shown in Table 1. The quantitative real-time RT-PCR (qRT-PCR) amplification was carried out using SYBR green master mix in a LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA).

The myoblasts were treated with the active mitochondrial-specific fluorescent dye, MitoTracker (Molecular Probes, Eugene, OR, USA), for 15 min at 37℃ and washed three times with cold PBS. The fluorescent intensity, visualized, and photographed in a fluorescence microscope (Leica, Germany), correlates with the integrity of mitochondrial function [21].

The intracellular ROS levels were analyzed using 2′,7′-dichlorofluorescin diacetate (DCFH-DA), a cell-permeable oxidationsensitive fluorescent probe, based on the oxidation of DCFH-DA to fluorescent dichlorofluorescein (DCF). Briefly, the cells were treated with 10 µM of DCFH-DA at 37℃ for 30 min and observed by fluorescence microscopy (Carl Zeiss, Göttingen, Germany) because the fluorescence intensity corresponds to the amount of ROS generated in the cells. The fluorescence was determined immediately with excitation and emission wavelengths set at 485 and 530 nm, respectively. The level of mitochondrial ROS was also determined by MitoSOX Red (Life Technology, Carlsbad, CA, USA) dye. Briefly, myoblasts were loaded with 5 µM MitoSOX Red at 37℃ for 30 min. The fluorescence intensity was determined with excitation and emission wavelengths set at 510 nM and 580 nM, respectively. To determine the cellular ATP contents, the cells were pelleted in an ice-cold lysis buffer, and the supernatants were measured by using an ATP Bioluminescence Detection Kit (Promega).

The myoblasts were homogenized with a lysate buffer containing 0.1% Triton X-100 and centrifuged to remove the cell debris and insoluble materials. The activities of the various antioxidant enzymes in the cell lysates were analyzed using a commercially available enzyme assay kit according to the manufacturer's instructions. The GPx, Catalase, GR, and MnSOD enzyme activities were determined quantitatively using enzyme assay kits from BioVision (Mountain View, CA, USA). The kits for measuring the glutathione S-transferase (GST) and glucose-6 phosphate dehydrogenase (G6PD) activities were obtained from Sigma-Aldrich. The GR activity was analyzed using Bioxytech GR-340 from OxisResearch (Portland, OR, USA).

GSH level was measured by using a Glutathione Assay Kit (BioVision). GSH generates a yellow colored complex, 2-nitro-5-thiobenzoic acid, and the absorbance was measured at 405 nm. GSSG level was measured using an OxiSelect Glutathione Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's protocol.

To analyze the H₂O₂-induced cytotoxicity in myoblasts, the L6-GLUT4myc cells were incubated with the designated concentration (0–500 µM) of H₂O₂ for 24 h. The cell viability based on the metabolic activity was determined using an MTT Cell Proliferation Assay Kit (BioVision). The release of LDH resulting from a disruption of the cell membrane integrity was determined by assaying the activity of LDH released into the culture medium using a Cytotoxicity Detection Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol.

The myoblasts were lysed in ice-cold buffer containing 10 mM Tris, 5 mM EDTA, and 1% Triton X-100, and the supernatants after centrifugation were solubilized using a Laemmli solution, subjected to SDS-PAGE, and analyzed for the indicated proteins by immunoblotting, as described elsewhere [22]. Protein expression was determined using ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The immunoblot intensities were quantified by densitometry using an analytical scanning system (Alpha Innotech, San Leandro, CA, USA).

The antibodies against catalase (ab16731), G6PD (ab993), GR (ab16801), and MnSOD (ab13533) were purchased from Abcam (Cambridge, UK), and the antibody against GPx (sc-22145) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin was supplied by Sigma. All other unspecified reagents were obtained from Sigma.

The values are expressed as the mean ± standard error of the mean (SEM) from at least four independent experiments. Where applicable, the significance of the differences was determined using a Student's t-test for unpaired data.

As the prolonged incubation of cells with low dose EtBr is a useful model for examining the cellular processes affected by the mtDNA content, the myoblasts containing varying quantities of mtDNA were prepared by long-term incubation with a low dose of EtBr (0.2 µg/ml) as described in the Methods. As shown in Fig. 1, COX-I and COX-II, which are mtDNA-encoded genes, were barely detected by PCR of the genomic DNA from the cells cultured with EtBr, whereas COX-IV, a nuclear DNA-encoded gene, was not changed in the control, mtDNA-depleted, and -reverted myoblasts. Under these experimental conditions, the mtDNA content was reduced to < 10% of the control by incubation with EtBr, whereas the removal of EtBr in the medium reversed the change in mtDNA content dramatically to the control level within 7 days (Fig. 1A). This shows that prolonged exposure to low EtBr concentration can modulate the cellular mtDNA content without affecting the replication of nuclear DNA in myoblasts. The levels of COX-I, -II, and -IV transcription were also determined in the control, mtDNA-depleted, and -reverted cells by qRT-PCR (Fig. 1B). The mtDNA-depleted cells exhibited very low levels of COX-I and -II mRNAs, which are mtDNA-encoded genes. In contrast, the reverted cells included similar amounts of COX-I and -II mRNA to those of the control cells. In addition, the mRNA of COX-IV, which is encoded by nuclear DNA, was not changed in the mtDNA-depleted or -reverted myoblasts, suggesting that the transcription levels of the genes encoded by mtDNA are in accordance with the cellular mtDNA content in myoblasts. This study further examined whether the depletion of mtDNA provoked mitochondrial dysfunction, which was assessed by the mitochondrial transmembrane potential (ΔΨm) and ATP content, in the cells. Mitotracker, which stains the mitochondria in membrane potential (ΔΨm)-dependent manner [21], was used to evaluate the integrity of the mitochondrial function. As shown in Fig. 1C, dust-like fluorescent granules scattered in the cytoplasm were observed clearly in the control myoblasts. In contrast, in the mtDNA-depleted cells, the intensity of fluorescent granules was dark and almost indistinct, whereas the reverted cells exhibited similar fluorescence intensity to the control cells. This suggests that the depletion of mtDNA disrupted ΔΨm in the myoblasts. As anticipated from the impairment of ΔΨm by mtDNA depletion, the total cellular ATP level was significantly lower in the depleted cells than in the control cells (Fig. 1D). Following mtDNA repletion, however, the total cellular ATP content was similar to that of the control cells in the reverted cells (Fig. 1D).

As the expression of many genes is affected by the retrograde stress signaling from the mitochondria [1123], the differentially expressed genes were evaluated using Affymetrix GeneChip microarrays. The levels of certain antioxidant enzymes were found to be affected by mtDNA depletion (data not shown). Therefore, this study next verified the effect of the cellular mtDNA content on the expression of antioxidant enzymes, such as GR, GPx, GST, G6PD, catalase, and MnSOD, in myoblasts. As shown in Fig. 2A, the depletion of mtDNA increased the mRNA levels of GPx and catalase drastically compared to the control and mtDNA-reverted cells. On the other hand, the transcription of GR, GST, G6PD, and MnSOD were not changed by the depletion or restoration of the mtDNA content. qRT-PCR analysis confirmed the significant upregulation of GPx (10.3-fold) and catalase (8.4-fold) in the mtDNA-depleted myoblasts compared to the control (Fig. 2B). The increased transcription of GPx and catalase in the mtDNA-depleted cells was restored to the control level in the mtDNA-reverted cells, clearly showing that the transcription of GPx and catalase was inversely correlated with the mtDNA content in myoblasts.

Because mRNA of GPx and catalase were increased significantly in the mtDNA-depleted myoblasts, the protein expression and activity of those enzymes were next examined. As shown in Fig. 3, the protein expression and activity of GPx and catalase were increased significantly by mtDNA depletion, whereas the protein levels of GR, G6PD, GST, and MnSOD were unaffected by the depletion or restoration of mtDNA in myoblasts. Moreover, the drastic increase in GPx and catalase were returned to the control levels in the mtDNA-reverted myoblasts, indicating that the expression and activity of GPx and catalase were negatively correlated with the cellular mtDNA level.

Next, the soluble cellular antioxidants, such as GSH and GSSG, were analyzed in the myoblasts, because the expression of antioxidant enzymes was affected by the mtDNA content. As shown in Fig. 4A and B, mtDNA depletion decreased the cellular GSH and GSSG contents slightly but significantly compared to the control. Upon mtDNA repletion, however, the GSH and GSSG contents returned to near the control levels. Despite the lower levels of GSH and GSSG in the depleted cells, the GSH/GSSG ratio, which is considered a good index of the cellular redox status, were similar to the control (Fig. 4C). The cellular activity of GST, an antioxidant enzyme involved in GSH conjugation with reactive oxygen species, was unaffected by the mtDNA contents in the myoblasts (Fig. 4D). The decrease in the GSH and GSSG levels may be associated with the lower demand for the soluble GSH/GSSG pool due to increased GPx and catalase in the mtDNA-depleted myoblasts.

To further explore the interrelationship between the mtDNA content and oxidative stress response, the steady-state levels of intracellular ROS and mitochondrial ROS (mt ROS) were determined in myoblasts as describes in the Methods. The levels of cellular ROS (Fig. 5A) and mt ROS (Fig. 5B) were altered markedly by changes in the mtDNA content corresponding to the modulation of antioxidant enzymes. In the mtDNA-depleted cells, the contents of cellular ROS and mt ROS were drastically reduced as compared to the control level. Upon mtDNA restoration, both the cellular ROS and mt ROS contents returned to near the control levels, suggesting that the cellular mtDNA contents are positively correlated with the cellular and mitochondrial ROS levels in myoblasts. To determine the effects of mtDNA content changes on exogenous oxidative stress, L6-GLUT4myc myoblasts were treated with various amounts of H₂O₂ (125–500 µM), and the cell viability was measured by MTT reduction and LDH release. As shown in Fig. 5C, at concentrations ranging from 250 to 500 µM, treatment with H₂O₂ decreased the cell viability significantly in control. Upon exposure to 500 µM H₂O₂, more than 80% of the myoblasts detached and died. On the other hand, the depletion of mtDNA decreased the level of H₂O₂-induced cell death significantly compared to those of the control. In addition, when the mtDNA content was returned to near the control level, as shown in the reverted cells, the lethality induced by H₂O₂ in the myoblasts was similar to the control level. In addition, the release of LDH into the culture medium disrupted the cell membrane integrity, which is an indicator of cell death (Fig. 5D). Corresponding to the result from the MTT reduction assay, LDH release to the medium in H₂O₂-treated cells increased significantly in a dose-dependent manner. On the other hand, the depletion of mtDNA decreased the H₂O₂-induced release of LDH dramatically. Thus, these results indicate that mtDNA depletion in myoblasts mitigates oxidative stress-induced cell death as well as ROS accumulation.