Fig 1.
Effect of hyperglycemia (HG) and hypoxia/reperfusion (H/R) on intracellular Gln, cell viability, and apoptosis.
H9C2 cells were treated with HG (33 mM), H/R, or HG + HR. (A) Intracellular Gln level in each group. (B) Cell viability of each group was established using an MTT assay. (C) HG and H/R-induced cell apoptosis was examined by DAPI staining and TUNEL assay. Blue spots represent cell nuclei and green spots represent apoptotic bodies. (D) Bars represent the percentage of TUNEL-positive cells based on the total number of cells stained by DAPI. The results are expressed as mean ± S.D. of 3 independent experiments. * p < 0.05 compare with the control group; # p < 0.05 compare with the HG and H/R group; a.u. = arbitrary units.
Fig 2.
Effect of Gln on cell viability and apoptosis.
H9C2 cells were exposed to HG + H/R with different concentrations of Gln (0.5, 1, 2, 4, 8, 16, and 32 mM). (A) Cell viability of each group was determined by MTT assay. (B) Results of the TUNEL assay and DAPI staining of each group. Green spots represented apoptotic bodies and blue spots represent cell nuclei. (C) Bars represent the percentage of TUNEL-positive cells based on the total number of cells stained by DAPI. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM and 4 mM Gln groups. Data are expressed as mean ± S.D. of 3 independent experiments; a.u. = arbitrary units.
Fig 3.
Effect of Gln on cytosolic GSH, GSSG and intracellular ROS.
H9C2 cells were exposed to HG+H/R with different concentrations of Gln (1, 4, or 16 mM). (A) Cytosolic GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were detected. (D) Representative images of DCFH flow cytometry of each group. (E) Mean DCFH fluorescence intensities are presented as percentage relative to the control value. (F) Representative images of DHE flow cytometry of each group. (G) Mean DHE fluorescence intensities are presented as percentage relative to the control value. These data are expressed as the mean ± S.D. of 3 independent experiments. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups, a.u. = arbitrary units.
Fig 4.
Effect of Gln on mitochondrial GSH, GSSG, S-glutathionylation, and complex I activity.
H9C2 cells were treated as described in Fig 3. (A) Mitochondrial GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were analyzed. (D and E) S-glutathionylated Complex I protein expression (51—and 75—kDa subunits) from each group were detected by immunoprecipitation and western blot analysis. The up panel shows the anti-complex I 75 and 51 kDa subunits positive-IP sample followed by identification by an anti-GSH antibody. As a control, mitochondrial protein (20 μg) was also immunoblotted with complex I 75 kDa and 51 kDa subunit antibody. Bar graphs summarize the protein band intensities of S- glutathionylated Complex I. (F) Complex I activity (200 μg mitochondrial protein) of each group are detected. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. The data are expressed as the mean ±S.D. of 3 independent experiments, a.u. = arbitrary units.
Fig 5.
Effect of Gln on mitochondrial ROS formation.
H9C2 cells were treated as described in Fig 3. (A) Representative imaging of Mito Sox confocal image of each group. (B) Mean Mito Sox fluorescence intensities are presented as percentage relative to the control value. The data are expressed as the means ±S.D. of 3 independent experiments. * P < 0.05 compared with the control group; # P < 0.05 compare with the 1 mM and 4 mM Gln groups. a.u. = arbitrary units.
Fig 6.
Effects of Gln on mitochondrial membrane potential stability and ATP level.
H9C2 cells were treated as described in Fig 3. (A). Representative images of JC-1 confocal images of each group; the red image detected in the left panel represents JC-1 aggregates, and the green image represents JC-1 monomer; the blue image represents DAPI, right panel shows the merged image. (B) Mean ± S.D. of the changes in radiometric JC-1 fluorescence for each group as indicated. (C) Cellular ATP levels of each group were detected. * P < 0.05 compare with the control group; # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. Data are expressed as the mean ±S.D. of 3 independent experiments. a.u. = arbitrary units.
Fig 7.
Influence of Gln on mitochondrial apoptosis-related protein.
H9C2 cells were treated as described in Fig 3. The expression of cytosolic (A) and mitochondrial (B) cytochrome c (cyto-c) as well as cleaved caspase-3 and pro caspase-3 (C) in each group were measured by western blot. Beta-actin and VADC1 were used as internal protein loading controls. Bar graphs summarize the protein band intensities of cyto-c and cleaved caspase 3. * P < 0.05 compare with the control group; # P <0.05 compare with the 1 mM and 4 mM Gln groups.
Fig 8.
Possible mechanism of the protective effect of Gln on HG and H/R-treated H9C2 cells.
Abbreviations: HG = high glucose; H/R = hypoxia-reoxygenation; GSH = glutathione; GSSG = oxidized glutathione; ROS = reactive oxygen species; ATP = adenosine triphosphate.