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Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species.

Kolamunne RT, Dias IH, Vernallis AB, Grant MM, Griffiths HR - Redox Biol (2013)

Bottom Line: However, L-NAME only afforded protection during H.Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R.These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK ; Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.

ABSTRACT
Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 (-•) scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 (-•).To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4 h hypoxia with and without 2 h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage.

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Nrf2 is a protective factor for cell survival during hypoxia/reperfusion and sustained hypoxia but does not contribute to the salvage of glutathione in H9C2 cells. H9C2 cells grown to 50–70% confluence were transiently transfected with Nrf2-siRNA or scramble (scr)-siRNA using Lipofectamine RNAimax and incubated for 16–24 h. Transfected cells were then incubated in hypoxic or normoxic medium for 4 h followed by further incubation for 2 h either at 2% or 21% O2. (A) Metabolic activity (MTT reducing activity) of cells was measured over last 2 h incubation in each experiment. (B) Cellular glutathione (GSH) was determined by the DTNB recycling assay after H/R or sustained hypoxia and is expressed as µmol/mg protein. Data are the mean±S.E.M of three independent experiments conducted in triplicate. Compared to controls in the absence of inhibitors, ⁎ represents P<0.05, ⁎⁎ represents P<0.01 and ⁎⁎⁎ represents P<0.001 (one-way ANOVA), Tukey′s post-hoc test.
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f0030: Nrf2 is a protective factor for cell survival during hypoxia/reperfusion and sustained hypoxia but does not contribute to the salvage of glutathione in H9C2 cells. H9C2 cells grown to 50–70% confluence were transiently transfected with Nrf2-siRNA or scramble (scr)-siRNA using Lipofectamine RNAimax and incubated for 16–24 h. Transfected cells were then incubated in hypoxic or normoxic medium for 4 h followed by further incubation for 2 h either at 2% or 21% O2. (A) Metabolic activity (MTT reducing activity) of cells was measured over last 2 h incubation in each experiment. (B) Cellular glutathione (GSH) was determined by the DTNB recycling assay after H/R or sustained hypoxia and is expressed as µmol/mg protein. Data are the mean±S.E.M of three independent experiments conducted in triplicate. Compared to controls in the absence of inhibitors, ⁎ represents P<0.05, ⁎⁎ represents P<0.01 and ⁎⁎⁎ represents P<0.001 (one-way ANOVA), Tukey′s post-hoc test.

Mentions: Further investigation of the role for Nrf2 during H and H/R in cardiomyoblasts was undertaken following Nrf2 knockdown (KD). siRNA-treated Nrf2 cells exhibited a significant decrease (>40%) in metabolic activity during H/R and S-H in comparison to non-transfected controls and cells that had been transfected with scrambled si-RNA implying an important role for Nrf2 in survival during H and H/R (Fig. 6A). Nrf2-KD cells grown under normoxia showed little cytotoxicity after knockdown beyond 6 h (data not shown). Analysis of cellular glutathione confirmed a significant loss of glutathione from cells during H with or without reoxygenation which was not exacerbated by Nrf2 knockdown (Fig. 6B).


Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species.

Kolamunne RT, Dias IH, Vernallis AB, Grant MM, Griffiths HR - Redox Biol (2013)

Nrf2 is a protective factor for cell survival during hypoxia/reperfusion and sustained hypoxia but does not contribute to the salvage of glutathione in H9C2 cells. H9C2 cells grown to 50–70% confluence were transiently transfected with Nrf2-siRNA or scramble (scr)-siRNA using Lipofectamine RNAimax and incubated for 16–24 h. Transfected cells were then incubated in hypoxic or normoxic medium for 4 h followed by further incubation for 2 h either at 2% or 21% O2. (A) Metabolic activity (MTT reducing activity) of cells was measured over last 2 h incubation in each experiment. (B) Cellular glutathione (GSH) was determined by the DTNB recycling assay after H/R or sustained hypoxia and is expressed as µmol/mg protein. Data are the mean±S.E.M of three independent experiments conducted in triplicate. Compared to controls in the absence of inhibitors, ⁎ represents P<0.05, ⁎⁎ represents P<0.01 and ⁎⁎⁎ represents P<0.001 (one-way ANOVA), Tukey′s post-hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814985&req=5

f0030: Nrf2 is a protective factor for cell survival during hypoxia/reperfusion and sustained hypoxia but does not contribute to the salvage of glutathione in H9C2 cells. H9C2 cells grown to 50–70% confluence were transiently transfected with Nrf2-siRNA or scramble (scr)-siRNA using Lipofectamine RNAimax and incubated for 16–24 h. Transfected cells were then incubated in hypoxic or normoxic medium for 4 h followed by further incubation for 2 h either at 2% or 21% O2. (A) Metabolic activity (MTT reducing activity) of cells was measured over last 2 h incubation in each experiment. (B) Cellular glutathione (GSH) was determined by the DTNB recycling assay after H/R or sustained hypoxia and is expressed as µmol/mg protein. Data are the mean±S.E.M of three independent experiments conducted in triplicate. Compared to controls in the absence of inhibitors, ⁎ represents P<0.05, ⁎⁎ represents P<0.01 and ⁎⁎⁎ represents P<0.001 (one-way ANOVA), Tukey′s post-hoc test.
Mentions: Further investigation of the role for Nrf2 during H and H/R in cardiomyoblasts was undertaken following Nrf2 knockdown (KD). siRNA-treated Nrf2 cells exhibited a significant decrease (>40%) in metabolic activity during H/R and S-H in comparison to non-transfected controls and cells that had been transfected with scrambled si-RNA implying an important role for Nrf2 in survival during H and H/R (Fig. 6A). Nrf2-KD cells grown under normoxia showed little cytotoxicity after knockdown beyond 6 h (data not shown). Analysis of cellular glutathione confirmed a significant loss of glutathione from cells during H with or without reoxygenation which was not exacerbated by Nrf2 knockdown (Fig. 6B).

Bottom Line: However, L-NAME only afforded protection during H.Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R.These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK ; Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.

ABSTRACT
Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 (-•) scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 (-•).To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4 h hypoxia with and without 2 h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage.

Show MeSH
Related in: MedlinePlus