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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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Low oxygen tension (3%) induces old MPCs to shift from glycolytic metabolism toward oxidative phosphorylation. Cellular bioenergetics in intact MPC was determined using the BOFA protocol in the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Fig. 2A shows a representative BOFA assay performed at 21% O2; Fig. 2B shows a representative BOFA assay performed at 3% O2. Young MPCs are represented by circles: black for 3% O2 and hatched for 21% O2. Old MPCs are represented by triangles: white for 3% O2 and gray for 21% O2. Fig. 2C shows the basal and maximal oxygen consumption rate (OCR) under different O2 tensions; Fig. 2D shows the extracellular acidification rate (ECAR) under different O2 tensions. Fig. 2E shows the OCR due to fatty acid oxidation (FAO). 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. The assays were performed with and without oxidative treatment (100 μM H2O2 for 19 h). Each point represents the mean±SE of four independent experiments. Statistical significance is marked as follows: &represents difference between different O2, i.e. OM 3% O2 vs. OM 21% O2; &p<0.05; &&p<0.005. ⁎represents difference between the same O2, i.e. YM vs. OM at 3% or YM vs. OM at 21%. ⁎p<0.05; ⁎⁎p<0.005. @represents difference between control and H2O2 treated, @p<0.05; @@p<0.005.
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f0010: Low oxygen tension (3%) induces old MPCs to shift from glycolytic metabolism toward oxidative phosphorylation. Cellular bioenergetics in intact MPC was determined using the BOFA protocol in the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Fig. 2A shows a representative BOFA assay performed at 21% O2; Fig. 2B shows a representative BOFA assay performed at 3% O2. Young MPCs are represented by circles: black for 3% O2 and hatched for 21% O2. Old MPCs are represented by triangles: white for 3% O2 and gray for 21% O2. Fig. 2C shows the basal and maximal oxygen consumption rate (OCR) under different O2 tensions; Fig. 2D shows the extracellular acidification rate (ECAR) under different O2 tensions. Fig. 2E shows the OCR due to fatty acid oxidation (FAO). 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. The assays were performed with and without oxidative treatment (100 μM H2O2 for 19 h). Each point represents the mean±SE of four independent experiments. Statistical significance is marked as follows: &represents difference between different O2, i.e. OM 3% O2 vs. OM 21% O2; &p<0.05; &&p<0.005. ⁎represents difference between the same O2, i.e. YM vs. OM at 3% or YM vs. OM at 21%. ⁎p<0.05; ⁎⁎p<0.005. @represents difference between control and H2O2 treated, @p<0.05; @@p<0.005.

Mentions: Shown in Fig. 2A (21% O2) and 2B (3% O2) are two complete representative BOFA experiments (total 97 min). Fig. 2C shows the quantification of both basal and maximal OCR under different oxygen tension conditions (arrows in Fig. 2A and B indicate the time points that were used to construct Fig. 2C). At 21% O2, old MPCs have lower basal and maximal OCR (64% and 72% respectively, in comparison to young MPCs). It is interesting to note that old MPCs cultured at 3% O2 respire at a rate similar to young MPCs (at both 21 and 3% O2) (white bars in Fig. 2C). When cells were treated with H2O2to induce a sublethal oxidative stress, there was an approximate 30% decrease in both basal and maximal OCR in young MPCs at 21% O2. In contrast, there was no decline in OCR in OM cells after oxidative insult, potentially because the OCR had already reached minimal threshold. These results concur with the proliferation data and suggest that old MPCs are more robust at physiological (3% O2) than at ambient air (21% O2), and although H2O2 treatment decreased basal (51%) and maximal (59%) OCR in OM cells at 3%, their performance was still significantly better than their homolog cells at 21% O2.


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)

Low oxygen tension (3%) induces old MPCs to shift from glycolytic metabolism toward oxidative phosphorylation. Cellular bioenergetics in intact MPC was determined using the BOFA protocol in the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Fig. 2A shows a representative BOFA assay performed at 21% O2; Fig. 2B shows a representative BOFA assay performed at 3% O2. Young MPCs are represented by circles: black for 3% O2 and hatched for 21% O2. Old MPCs are represented by triangles: white for 3% O2 and gray for 21% O2. Fig. 2C shows the basal and maximal oxygen consumption rate (OCR) under different O2 tensions; Fig. 2D shows the extracellular acidification rate (ECAR) under different O2 tensions. Fig. 2E shows the OCR due to fatty acid oxidation (FAO). 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. The assays were performed with and without oxidative treatment (100 μM H2O2 for 19 h). Each point represents the mean±SE of four independent experiments. Statistical significance is marked as follows: &represents difference between different O2, i.e. OM 3% O2 vs. OM 21% O2; &p<0.05; &&p<0.005. ⁎represents difference between the same O2, i.e. YM vs. OM at 3% or YM vs. OM at 21%. ⁎p<0.05; ⁎⁎p<0.005. @represents difference between control and H2O2 treated, @p<0.05; @@p<0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0010: Low oxygen tension (3%) induces old MPCs to shift from glycolytic metabolism toward oxidative phosphorylation. Cellular bioenergetics in intact MPC was determined using the BOFA protocol in the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Fig. 2A shows a representative BOFA assay performed at 21% O2; Fig. 2B shows a representative BOFA assay performed at 3% O2. Young MPCs are represented by circles: black for 3% O2 and hatched for 21% O2. Old MPCs are represented by triangles: white for 3% O2 and gray for 21% O2. Fig. 2C shows the basal and maximal oxygen consumption rate (OCR) under different O2 tensions; Fig. 2D shows the extracellular acidification rate (ECAR) under different O2 tensions. Fig. 2E shows the OCR due to fatty acid oxidation (FAO). 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. The assays were performed with and without oxidative treatment (100 μM H2O2 for 19 h). Each point represents the mean±SE of four independent experiments. Statistical significance is marked as follows: &represents difference between different O2, i.e. OM 3% O2 vs. OM 21% O2; &p<0.05; &&p<0.005. ⁎represents difference between the same O2, i.e. YM vs. OM at 3% or YM vs. OM at 21%. ⁎p<0.05; ⁎⁎p<0.005. @represents difference between control and H2O2 treated, @p<0.05; @@p<0.005.
Mentions: Shown in Fig. 2A (21% O2) and 2B (3% O2) are two complete representative BOFA experiments (total 97 min). Fig. 2C shows the quantification of both basal and maximal OCR under different oxygen tension conditions (arrows in Fig. 2A and B indicate the time points that were used to construct Fig. 2C). At 21% O2, old MPCs have lower basal and maximal OCR (64% and 72% respectively, in comparison to young MPCs). It is interesting to note that old MPCs cultured at 3% O2 respire at a rate similar to young MPCs (at both 21 and 3% O2) (white bars in Fig. 2C). When cells were treated with H2O2to induce a sublethal oxidative stress, there was an approximate 30% decrease in both basal and maximal OCR in young MPCs at 21% O2. In contrast, there was no decline in OCR in OM cells after oxidative insult, potentially because the OCR had already reached minimal threshold. These results concur with the proliferation data and suggest that old MPCs are more robust at physiological (3% O2) than at ambient air (21% O2), and although H2O2 treatment decreased basal (51%) and maximal (59%) OCR in OM cells at 3%, their performance was still significantly better than their homolog cells at 21% O2.

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