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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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Related in: MedlinePlus

Schematic plan of experimental hypoxia and reoxygenation experiment. Media was pre-equilibrated at desired oxygen tension for 24 h prior to each experiment. At the start of each experiment, pre-equilibrated media was added to near confluent cells and the incubator was flushed with appropriate oxygen tension at a high flow rate for 3 min, then flow rate was returned to 25 ml/min for the remainder of the study period. After 4 h, for H/R experiments, 2% oxygen media was removed and pre-equilibrated 21% oxygen media added with 21% oxygen flushed into the incubator for the remaining 2 h. Cells that were to be retained in the same oxygen tension (either sustained hypoxia or normoxia) also underwent a change in media at the same time points but oxygen tension remained unchanged.
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f0005: Schematic plan of experimental hypoxia and reoxygenation experiment. Media was pre-equilibrated at desired oxygen tension for 24 h prior to each experiment. At the start of each experiment, pre-equilibrated media was added to near confluent cells and the incubator was flushed with appropriate oxygen tension at a high flow rate for 3 min, then flow rate was returned to 25 ml/min for the remainder of the study period. After 4 h, for H/R experiments, 2% oxygen media was removed and pre-equilibrated 21% oxygen media added with 21% oxygen flushed into the incubator for the remaining 2 h. Cells that were to be retained in the same oxygen tension (either sustained hypoxia or normoxia) also underwent a change in media at the same time points but oxygen tension remained unchanged.

Mentions: Cells were maintained in preincubated hypoxic or normoxic phenol red-free DMEM which was supplemented with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), with penicillin/streptomycin as above (complete media). Estimates of O2 tension in tissue vary between 10 and 14% O2 and will fluctuate during physiological stress and pathophysiological conditions which reduce O2 delivery to tissues [25]. In normal myocardial perfusion in rat hearts, mean PO2 values were reported as 240 ±140 Torr and during local ischemia, the mean PO2 changed to 50±90 Torr which is equivalent to 6.7% oxygen [26]. For hypoxia or normoxia experiments, the medium was preincubated for 24 h in a hypoxic environment (2% O2+98% N2) and 10% O2+90% N2 and 21% O2+79% N2 for control experiments (BOC, UK). pH stability was assessed as described previously [27,28]. The experimental design is shown in Fig. 1.


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)

Schematic plan of experimental hypoxia and reoxygenation experiment. Media was pre-equilibrated at desired oxygen tension for 24 h prior to each experiment. At the start of each experiment, pre-equilibrated media was added to near confluent cells and the incubator was flushed with appropriate oxygen tension at a high flow rate for 3 min, then flow rate was returned to 25 ml/min for the remainder of the study period. After 4 h, for H/R experiments, 2% oxygen media was removed and pre-equilibrated 21% oxygen media added with 21% oxygen flushed into the incubator for the remaining 2 h. Cells that were to be retained in the same oxygen tension (either sustained hypoxia or normoxia) also underwent a change in media at the same time points but oxygen tension remained unchanged.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0005: Schematic plan of experimental hypoxia and reoxygenation experiment. Media was pre-equilibrated at desired oxygen tension for 24 h prior to each experiment. At the start of each experiment, pre-equilibrated media was added to near confluent cells and the incubator was flushed with appropriate oxygen tension at a high flow rate for 3 min, then flow rate was returned to 25 ml/min for the remainder of the study period. After 4 h, for H/R experiments, 2% oxygen media was removed and pre-equilibrated 21% oxygen media added with 21% oxygen flushed into the incubator for the remaining 2 h. Cells that were to be retained in the same oxygen tension (either sustained hypoxia or normoxia) also underwent a change in media at the same time points but oxygen tension remained unchanged.
Mentions: Cells were maintained in preincubated hypoxic or normoxic phenol red-free DMEM which was supplemented with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), with penicillin/streptomycin as above (complete media). Estimates of O2 tension in tissue vary between 10 and 14% O2 and will fluctuate during physiological stress and pathophysiological conditions which reduce O2 delivery to tissues [25]. In normal myocardial perfusion in rat hearts, mean PO2 values were reported as 240 ±140 Torr and during local ischemia, the mean PO2 changed to 50±90 Torr which is equivalent to 6.7% oxygen [26]. For hypoxia or normoxia experiments, the medium was preincubated for 24 h in a hypoxic environment (2% O2+98% N2) and 10% O2+90% N2 and 21% O2+79% N2 for control experiments (BOC, UK). pH stability was assessed as described previously [27,28]. The experimental design is shown in Fig. 1.

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