Oxidative stress-induced mitochondrial fragmentation and movement in skeletal muscle myoblasts

Abstract

Mitochondria are dynamic organelles, capable of altering their morphology and function. However, the mechanisms governing these changes have not been fully elucidated, particularly in muscle cells. We demonstrated that oxidative stress with H2O2 resulted in a 41% increase in fragmentation of the mitochondrial reticulum in myoblasts within 3 h of exposure, an effect that was preceded by a reduction in membrane potential. Using live cell imaging, we monitored mitochondrial motility and found that oxidative stress resulted in a 30% reduction in the average velocity of mitochondria. This was accompanied by parallel reductions in both organelle fission and fusion. The attenuation in mitochondrial movement was abolished by the addition of N-acetylcysteine. To investigate whether H2O2-induced fragmentation was mediated by dynamin-related protein 1, we incubated cells with mDivi1, an inhibitor of dynamin-related protein 1 translocation to mitochondria. mDivi1 attenuated oxidative stress-induced mitochondrial fragmentation by 27%. Moreover, we demonstrated that exposure to H2O2 upregulated endoplasmic reticulum-unfolded protein response markers before the initiation of mitophagy signaling and the mitochondrial-unfolded protein response. These findings indicate that oxidative stress is a vital signaling mechanism in the regulation of mitochondrial morphology and motility.

Keywords: mitochondria; mitochondrial morphology; mitochondrial movement; oxidative stress.

Fig. 1.

Oxidative stress halts mitochondrial movement in C₂C₁₂ myoblasts. A: representative myoblast co-transfected with mito-DsRED2. B: the first frame (top) in a live cell imaging series with representative kymographs generated from the videos of untreated [control (CON)] and 300 μM H₂O₂-treated myoblasts (bottom). Time (0–5 min) progresses from top to bottom in the kymograph, whereas the x-axis represents mitochondrial position. Vertical white lines correspond to stationary mitochondria, and horizontal deviations (vibrations from the vertical) depict moving mitochondria. C: the percentage of time that mitochondria spent in motion with and without the addition of 300 μM H₂O₂ was determined by monitoring the position of the organelle throughout the 5-min capturing time in 10 separate experiments. D: quantification of the total path length (top) and average speed (bottom) traveled by mitochondria assessed in control and 300 μM H₂O₂-treated myoblasts with or without the presence of N-acetylcysteine (NAC; 60-min pretreatment). The movement of mitochondria was determined and averaged from n = 18–80 mitochondria from 6–10 separate cells. All values are expressed as averages ± SE. *P < 0.05 vs. CON.

Fig. 2.

Oxidative stress results in changes to mitochondrial dynamics. A, middle: the first frame from a live cell imaging series of a representative myoblast with time-lapse images of myoblasts expressing mito-DsRed2. Left, representative images of a 6-s time-lapse series of a myoblast undergoing mitochondrial fission. The arrows indicate sites of mitochondrial division. Right, sequential events of mitochondrial fusion monitored over 19 s. The arrow denotes the extension of a mitochondrial filament, resulting in a fusion event. B: numbers of mitochondrial fission and fusion events were determined in a 7 × 7-μm area over the time course of 5 min from 6–10 separate cells. Numbers of fission and fusion events were determined in control and H₂O₂ (300 μM)-treated myoblasts with or without NAC. Values are expressed as averages ± SE. *P < 0.05 vs. CON (without H₂O₂ and NAC).

Fig. 3.

Oxidative stress induces mitochondrial fragmentation as well as reductions in membrane potential and O₂ consumption. Myoblasts were treated with 300 μM H₂O₂ for 5 h, transfected with mito-DsRed2 for visualization of mitochondria, and imaged immediately or after 1, 3, or 5 h of incubation. Mitochondrial morphology was assessed as the percentage of the cell occupied by either fragmented or network mitochondria. A: representative images for each time point (top) with magnified images of the mitochondrial network condition (bottom). H₂O₂ altered the morphology of mitochondria, such that more fragmented organelles were observed beginning at 3 h posttreatment. B: percentages (means ± SE) of total cell area displaying fragmented mitochondria from 6 separate experiments. C: membrane potential was analyzed by assessing the fluorescence of Tetramethylrhodamine ethyl ester (TMRE). The mean fluorescence intensity of each condition was expressed as a percentage of the untreated CON. Mean percentages ± SE of 13–18 cells are shown. D: O₂ consumption was determined in C₂C₁₂ cells treated with 300 μM H₂O₂ for a total time of 5 h. n = 4. *Significantly different from 0 h (P < 0.05).

Fig. 4.

Oxidative stress-induced mitochondrial fragmentation is mediated by dynamin-related protein 1 (Drp1). Mitochondrial morphology for myoblasts expressing mito-DsRed2 after 100 μM H₂O₂ exposure is shown. A: percentages of fragmented mitochondria were expressed relative to the total area of the cells with or without the presence of mDivi1 (25 μM, 60 min). B: C₂C₁₂ myoblasts were treated with and without 300 μM H₂O₂ for 5 h. Cells were cotransfected with mito-DsRed2 (red) and pEYFP-C1-Drp1 (green) and imaged at ×150 magnification. Visualization of CON myoblasts displayed long, reticular mitochondria with diffuse Drp1. Cells treated with H₂O showed small, fragmented mitochondria and an accumulation of Drp1-positive puncta concentrated by mitochondria, in a time-dependent manner. The hatched boxes are magnified on the insert (bottom). Scale bar = 10 μm. C: Drp1 dephosphorylation compared with total Drp1 levels with 100 μM H₂O₂ exposure. Expression of phosphorylated (p-)Drp1 (Ser⁶³⁷) and total Drp1 was assessed on the same gel with and without 25μM mDivi1 (60-min preincubation). The line on the blot indicates that this image was taken from two separate blots to best illustrate the difference observed in the presence and absence of mDivi1, representative of 6 independent experiments. n = 6 for each condition. *Significantly different from 0 h (P < 0.05); †significantly different from untreated conditions (P < 0.05).

Fig. 5.

Acute oxidative stress specifically induces the endoplasmic reticulum (ER)-unfolded protein response (UPR) without affecting the mitochondrial (mt)-UPR or mitophagy proteins. A: effect of oxidative stress on mitophagy. Representative Western blots of the mitophagy protein markers light chain 3 (LC3) and p62 were assessed with acute (5 h) and long exposure (24 h) with 300 μM H₂O₂ in mitochondrial-enriched fractions from C₂C₁₂ myoblasts. Porin was used as the loading marker. B: protein expression of markers for the mt-UPR. Levels of mitochondrial heat shock protein 60 (mtHSP60), mitochondrial heat shock protein 70 (mtHSP70), and chaperonin 10 (Cpn10) protein were determined in whole cell lysates after 300 μM H₂O₂ exposure. Tubulin was used as the loading control. C and D: representative Western blots of the ER-UPR markers C/EBP homology protein (CHOP), p-eukaryotic translation initiation factor 2α (p-eIF2α), and eIF2α (top) as well as graphical quantification (bottom). Tubulin was unchanged between the conditions and was used as a loading control for CHOP. Values are expressed as averages ± SE; n = 6. *P < 0.05 vs. CON.

Fig. 6.

Summary and working hypothesis on the effects of excess ROS on cellular responses based on the temporal changes described by our results. Exposure of the cell to increased levels of ROS resulted in reductions in mitochondrial membrane potential (ΔΨ; left). Subsequently, the ROS-induced decreases in membrane potential lead to reductions in respiration as well as enhanced mitochondrial fragmentation mediated by Drp1. Previous work has shown that inhibition of respiratory components results in enhanced organelle fission. These changes in mitochondrial morphology lead to increases in the ER-UPR, which then activates mitophagy along with an upregulation of the mt-UPR.