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Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells.

Konigsberg M, Pérez VI, Ríos C, Liu Y, Lee S, Shi Y, Van Remmen H - Redox Biol (2013)

Bottom Line: We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals.Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2.H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs.

View Article: PubMed Central - PubMed

Affiliation: Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center, San Antonio, TX 78229, USA ; Universidad Autonoma Metropolitana-Iztapalpa, Mexico City, Mexico.

ABSTRACT
In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2), despite the fact that physiological O2 at the tissue level in vivo is much lower (~1-5% O2). We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation) or oxidative damage to proteins (protein disulfides, carbonyls and aggregates) in myoblast precursor cells (MPCs) isolated from young (3-4 m) and old (29-30 m) C57BL/6 mice. MPCs were grown under physiological (3%) or ambient (21%) O2 for two weeks prior to exposure to an acute oxidative insult (H2O2). Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

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No differences in protein disulfides content in both age nor oxygen conditions. Protein disulfides were measured in the cytosolic fraction isolated from MPCs with and without oxidative treatment (100 μM H2O2 for 19 h). Young MPCs are represented by black bars for 3% O2 and hatched bars for 21% O2. Old MPCs are represented by white bars for 3% O2 and gray bars for 21% O2. Each point represents the mean±SE of three independent experiments.
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f0015: No differences in protein disulfides content in both age nor oxygen conditions. Protein disulfides were measured in the cytosolic fraction isolated from MPCs with and without oxidative treatment (100 μM H2O2 for 19 h). Young MPCs are represented by black bars for 3% O2 and hatched bars for 21% O2. Old MPCs are represented by white bars for 3% O2 and gray bars for 21% O2. Each point represents the mean±SE of three independent experiments.

Mentions: In order to determine whether oxygen tension has an effect on the level of protein oxidation (protein disulfides and protein carbonyls) and protein aggregates, we measured these parameters in the cytosolic fraction isolated from MPCs grown at 3 or 21% O2, with or without oxidative treatment (100 μM H2O2 for 19 h). As shown in Fig. 3, we found no difference in the levels of protein disulfides between young and old MPCs at the two O2 levels. In addition, even though there was a mild increment in the protein disulfides in the H2O2-treated samples, the difference was not statistically significant. With respect to protein carbonyls (Fig. 4), we found no difference in carbonyl levels in proteins isolated from young MPCs at 21 and 3% O2 and no effect from H2O2 treatment in young MPCs. In contrast, there was a 30 to 40% increase in carbonyls in the proteins isolated from H2O2-treated old MPCs compared to untreated old MPCs, concurring with the results in Fig. 2 that show a decrease in mitochondrial respiration after H2O2 treatment and suggesting oxidative damage. In addition, old treated MPCs grown at 21% O2 had significantly higher carbonyl levels (~40%) than old treated MPCs grown at 3%. Protein aggregates increased in response to oxidative treatment in all cell types (Fig. 5), but the effect of O2 was only evident in the old MPCs, whereas cells cultured at 21% O2 showed more protein aggregates. These findings suggest that old cells are in general more sensitive to oxidative treatment.


Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells.

Konigsberg M, Pérez VI, Ríos C, Liu Y, Lee S, Shi Y, Van Remmen H - Redox Biol (2013)

No differences in protein disulfides content in both age nor oxygen conditions. Protein disulfides were measured in the cytosolic fraction isolated from MPCs with and without oxidative treatment (100 μM H2O2 for 19 h). Young MPCs are represented by black bars for 3% O2 and hatched bars for 21% O2. Old MPCs are represented by white bars for 3% O2 and gray bars for 21% O2. Each point represents the mean±SE of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814963&req=5

f0015: No differences in protein disulfides content in both age nor oxygen conditions. Protein disulfides were measured in the cytosolic fraction isolated from MPCs with and without oxidative treatment (100 μM H2O2 for 19 h). Young MPCs are represented by black bars for 3% O2 and hatched bars for 21% O2. Old MPCs are represented by white bars for 3% O2 and gray bars for 21% O2. Each point represents the mean±SE of three independent experiments.
Mentions: In order to determine whether oxygen tension has an effect on the level of protein oxidation (protein disulfides and protein carbonyls) and protein aggregates, we measured these parameters in the cytosolic fraction isolated from MPCs grown at 3 or 21% O2, with or without oxidative treatment (100 μM H2O2 for 19 h). As shown in Fig. 3, we found no difference in the levels of protein disulfides between young and old MPCs at the two O2 levels. In addition, even though there was a mild increment in the protein disulfides in the H2O2-treated samples, the difference was not statistically significant. With respect to protein carbonyls (Fig. 4), we found no difference in carbonyl levels in proteins isolated from young MPCs at 21 and 3% O2 and no effect from H2O2 treatment in young MPCs. In contrast, there was a 30 to 40% increase in carbonyls in the proteins isolated from H2O2-treated old MPCs compared to untreated old MPCs, concurring with the results in Fig. 2 that show a decrease in mitochondrial respiration after H2O2 treatment and suggesting oxidative damage. In addition, old treated MPCs grown at 21% O2 had significantly higher carbonyl levels (~40%) than old treated MPCs grown at 3%. Protein aggregates increased in response to oxidative treatment in all cell types (Fig. 5), but the effect of O2 was only evident in the old MPCs, whereas cells cultured at 21% O2 showed more protein aggregates. These findings suggest that old cells are in general more sensitive to oxidative treatment.

Bottom Line: We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals.Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2.H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs.

View Article: PubMed Central - PubMed

Affiliation: Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center, San Antonio, TX 78229, USA ; Universidad Autonoma Metropolitana-Iztapalpa, Mexico City, Mexico.

ABSTRACT
In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2), despite the fact that physiological O2 at the tissue level in vivo is much lower (~1-5% O2). We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation) or oxidative damage to proteins (protein disulfides, carbonyls and aggregates) in myoblast precursor cells (MPCs) isolated from young (3-4 m) and old (29-30 m) C57BL/6 mice. MPCs were grown under physiological (3%) or ambient (21%) O2 for two weeks prior to exposure to an acute oxidative insult (H2O2). Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

Show MeSH
Related in: MedlinePlus