Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

Abstract

Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of methionine in proteins involving the enzyme peptide methionine sulfoxide reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

Fig. 1.

Hypoxia increases ROS production and promotes cell death. (A) Hypoxia enhances ROS production in PC12 cells. The slope of the DHR123 signal in the first 4 min of the treatment was used to estimate the ROS level. The results obtained from the cells treated with blank control adenovirus particles, bMSRA adenovirus particles, TEMPOL (5 mM), and bMSRA particles and TEMPOL (5 mM) together are shown. The cells were treated with the virus particles 24 h before the measurements and then subjected to normoxia (open bar), hyperoxia (shaded bar), and hypoxia (filled bar) (10 μM DHR123; n = 4–10). (B) ROS production during reoxygenation is inhibited by MSRA overexpression. The cells were treated with hypoxia, and the DHR123 signals were measured as in A (20 μM DHR123; n = 3). (C) MSRA overexpression protects cells from hypoxia-induced cell death. Data regarding nonviable fractions in the groups treated with no virus, bMSRA adenovirus particles, control EGFP adenovirus particles, and TEMPOL (5 mM) are shown. The cells were challenged with normoxia (open bar), hyperoxia (shaded bar), or hypoxia (filled bar) for 5 min. The cell viability assay with trypan blue was performed 24 h later. The effect of ethanol (1%) on cell viability was indistinguishable from that of control (data not shown).

Fig. 2.

MSRA overexpression preferentially prevents apoptosis. (A) Flow cytometric analysis of PC12 cells double-stained with Annexin-V-FLOUS and PI. Cells were treated with normoxia, hyperoxia, or hypoxia for 10 min, and the measurements were made 24 h later. In each of the six plots, the bottom left quadrangle indicates viable cells that are negative for both Annexin–V binding and PI uptake; the bottom right quadrangle includes apoptotic cells positive for Annexin–V binding but negative for PI uptake; and the top right quadrangle primarily includes necrotic cells positive for both Annexin–V binding and PI uptake. (Upper) Representative dot plots from the control cells treated with normoxia (Left), hyperoxia (Center), and hypoxia (Right). (Lower) Representative dot plots from the cells overexpressing bMSRA. (B) The fractional increases in the number of apoptotic Annexin-V-positive cells. The results from the control and MSRA overexpressing cells are shown. In each cell group, the results after normoxia (open bar), hyperoxia (shaded bar), and hypoxia (filled bar) treatments are shown (n = 3). The measurements were made as in A, and the average percentage of cells undergoing apoptosis without any treatment (20.8 ± 3.7%) has been subtracted. (C) The fractional increases in the number of necrotic PI-positive cells. The average percentage of necrotic cells (1.7 ± 0.1%) was subtracted (n = 3). (D) Flow cytometric cell death analysis of PC12 cells after 24-hr treatments. Cells were treated with normoxia or hypoxia and immediately assayed for cell death. (E) MSRA overexpression partially inhibits the increase in the number of apoptotic cells after 24-hr hypoxia treatment. The measurements are made as in A and B. The average percentage of apoptotic cells in the group treated with normoxia (8.9%) was subtracted (n = 3). (F) MSRA overexpression does not alter the number of necrotic cells. The measurements are made as in A and B. The average percentage of necrotic cells in the group treated with normoxia (2.7%) was subtracted (n = 3).

Fig. 3.

Membrane potentials of intact mitochondria in PC12 cells estimated by JC-1. (A) Control PC12 cells treated with blank control virus particles (Left) and the cells treated with bMSRA adenovirus particles (Right) were challenged with normoxia (Top), hyperoxia (Middle), and hypoxia (Bottom). The images were obtained ≈5 min after the start of the treatment. The JC-1 red/green ratio signals are represented with the color scale shown. The full scale represents the JC-1 red/green ratios of 0 and 2. Greater JC-1 red/green ratio values indicated by brighter colors reflect more polarized ΔΨ_m. The size scale bar represents 20 μm. (B) Linescan analysis of the JC-1 results. The JC-1 red/green ratio results from the mitochondria in two groups are shown. The control group received blank control virus particles, and the MSRA group received bMSRA particles (n = 19–25 in each condition). (C) JC-1 signals are attenuated by the mitochondrial uncoupler FCCP. FCCP (0.2 μM) was applied for 20 min, and the cells were imaged.

Fig. 4.

Membrane potentials of isolated rat liver mitochondria measured by JC-1. (A) JC-1 red/green ratio after the onset of normoxia, hyperoxia, and hypoxia. Isolated mitochondria were placed in a cuvette and challenged with normoxia, hyperoxia, or hypoxia starting at time 0. It took ≈15 s to load the cuvettes into the spectrofluorometer, and the recording could not be performed during this period (line width indicates SEM; n = 3–4 in each condition). The values shown are slightly different from those in the Fig. 3B, because the measurements here were made by using isolated mitochondria. Similar results were obtained when the emission results at 535 nm only were compared. (B) Mean JC-1 red/green ratio slope values in the normoxia, hyperoxia, and hypoxia conditions. The slopes were calculated by differentiating the JC-1 fluorescence ratio traces as shown in A.