Korean J Physiol Pharmacol.  2019 Nov;23(6):519-528. 10.4196/kjpp.2019.23.6.519.

Alteration of mitochondrial DNA content modulates antioxidant enzyme expressions and oxidative stress in myoblasts

• Min KH
• Lee W

• KMID: 2461045
• DOI: http://doi.org/10.4196/kjpp.2019.23.6.519

Abstract

Mitochondrial dysfunction is closely associated with reactive oxygen species (ROS) generation and oxidative stress in cells. On the other hand, modulation of the cellular antioxidant defense system by changes in the mitochondrial DNA (mtDNA) content is largely unknown. To determine the relationship between the cellular mtDNA content and defense system against oxidative stress, this study examined a set of myoblasts containing a depleted or reverted mtDNA content. A change in the cellular mtDNA content modulated the expression of antioxidant enzymes in myoblasts. In particular, the expression and activity of glutathione peroxidase (GPx) and catalase were inversely correlated with the mtDNA content in myoblasts. The depletion of mtDNA decreased both the reduced glutathione (GSH) and oxidized glutathione (GSSG) slightly, whereas the cellular redox status, as assessed by the GSH/GSSG ratio, was similar to that of the control. Interestingly, the steady-state level of the intracellular ROS, which depends on the reciprocal actions between ROS generation and detoxification, was reduced significantly and the lethality induced by Hâ‚‚Oâ‚‚ was alleviated by mtDNA depletion in myoblasts. Therefore, these results suggest that the ROS homeostasis and antioxidant enzymes are modulated by the cellular mtDNA content and that the increased expression and activity of GPx and catalase through the depletion of mtDNA are closely associated with an alleviation of the oxidative stress in myoblasts.

Keyword

Antioxidant; Catalase; Glutathione peroxidase; Mitochondrial DNA; Myoblasts; Reactive oxygen species

MeSH Terms

Catalase
DNA, Mitochondrial*
Glutathione
Glutathione Disulfide
Glutathione Peroxidase
Hand
Homeostasis
Myoblasts*
Oxidation-Reduction
Oxidative Stress*
Reactive Oxygen Species
Catalase
DNA, Mitochondrial
Glutathione
Glutathione Disulfide
Glutathione Peroxidase
Reactive Oxygen Species

Figure

• 

  Fig. 1 Alteration of mitochondrial DNA (mtDNA) contents, functional mitochondria, and ATP levels by ethidium bromide (EtBr). (A) The genomic DNA was isolated from the control (C), mtDNA-depleted (D) and -reverted (R) myoblasts, and mtDNA-encoded genes, such as cytochrome c oxidase subunit I (COX-I), subunit II (COX-II), and nuclear DNA-encoded genes, such as cytochrome c oxidase subunit IV (COX-IV), were amplified by PCR. (B) The total RNA was extracted from the myoblasts, and the mRNA levels were quantified by qRT-PCR. The densities were normalized to the β-actin signals, and the relative intensities are expressed in arbitrary units, where the intensity of the control was set to one hundred. ***p < 0.001. (C) To analyze the functional mitochondria, control, mtDNA-depleted and -reverted L6-GLUT4myc myoblasts were stained with MitoTracker. The magnification is ~×400. (D) The total cellular ATP levels were measured by the luciferin-luciferase assay. All results represent the mean ± SEM from five independent experiments. ***p < 0.001 vs. control.

• 

  Fig. 2 Effect of mitochondrial DNA (mtDNA) depletion on the transcripts of the antioxidant defense enzymes. The total RNA from the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts was prepared. The transcript level of the antioxidant defense enzymes was quantified by RT-PCR (A) and qRT-PCR (B). β-Actin was used as the control. All results represent the mean ± SEM from five independent experiments. GR, glutathione reductase; GPx, glutathione peroxidase; GST, glutathione S-transferase; SOD, superoxide dismutase. ***p < 0.001 vs. control.

• 

  Fig. 3 Effect of mitochondrial DNA (mtDNA) depletion on the expression and activity of glutathione peroxidase (GPx) and catalase. The total cell lysates were prepared in the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts. (A–C) The expression levels of GPx and catalase were analyzed by immunoblotting. The densities were normalized to the β-actin signals, and the relative intensities are expressed in arbitrary units, where the intensity of the control was set to one. (D) The total GPx activity was measured using the coupled enzyme procedure with glutathione reductase. The specific activity was calculated using the extinction coefficient obtained from the NADPH standard. (E) The total catalase activity was measured by monitoring the decomposition of 10 mM H2O2 at 240 nm in a medium. One unit of catalase decomposes 1 mM of H2O2 per min. The values are expressed as the mean ± SEM from four independent experiments. GR, glutathione reductase; SOD, superoxide dismutase. ***p < 0.001 vs. control.

• 

  Fig. 4 Effect of mitochondrial DNA (mtDNA) depletion on the reduced glutathione (GSH) or oxidized glutathione (GSSG) contents and glutathione S-transferase (GST) activity. (A, B) The cellular GSH and GSSG contents were measured in the control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts. (C) The cellular redox status is presented as the GSH/GSSG ratio. (D) The GST activity was assayed in the supernatant of the cell lysates and was calculated using a known extinction coefficient. The values are expressed as the mean ± SEM from three independent experiments. **p < 0.01 vs. control.

• 

  Fig. 5 Effects of mitochondrial DNA (mtDNA) depletion on the cellular reactive oxygen species (ROS) content and H2O2-induced cell death. (A) Control, mtDNA-depleted (Depleted) and -reverted (Reverted) myoblasts were analyzed for the cellular ROS content using a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe. The fluorescent images were observed by confocal microscopy (excitation, 485 nm; emission, 530 nm). The relative fluorescence intensities obtained from 50 cells are expressed in arbitrary units where the intensity of the control was set to one. (B) The level of mitochondrial ROS (mt ROS) was also determined by MitoSOX. The fluorescence intensity was determined with excitation (510 nM) and emission (580 nM) wavelengths. (C, D) Control (circle), mtDNA-depleted (triangle) and -reverted (rectangle) myoblasts were incubated with 0, 25, 250, or 500 mM H2O2 for 24 h. The effects of H2O2-induced cell death were evaluated by MTT reduction assay (C) and LDH release (D) as described in the Methods. The results of MTT reduction are expressed as the percentage change where the absorbance of myoblasts without H2O2 was set to one hundred. LDH release was determined by assaying the activity of LDH released into the culture medium. All results represent the mean ± SEM from four independent experiments. ***p < 0.001; **p < 0.01 vs. control.

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