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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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Hypoxia and hypoxia/reoxygenation induces cell death and a decrease in metabolic activity but not protein concentration. H9C2 cells at 70–80% confluence were incubated in 2% 10% or 21% O2 equilibrated, HEPES-buffered, phenol red-free DMEM and exposed to (4 h) hypoxia. (A); The ratio of propidium iodide (PI)-positive stained cells to total Hoechst stained cells was measured as an index of necrosis. ((B) and (C)); Procaspase 3 activation measured as cleavage relative to tubulin expression and determined by western blotting. (D); Metabolic activity determined by MTT assay. (E); After 4 h hypoxia (2% O2), the medium was replaced with reoxygenated medium (2 h; 21% O2) for control and H/R experiments, and with hypoxic medium (2 h; 2% O2) for sustained hypoxia experiments and MTT activity was determined. Data represent the mean ±S.E.M of three independent experiments conducted in triplicate. ⁎ represents P<0.05 and ⁎⁎ represents P<0.001 (one-way ANOVA) compared to controls, Tukey′s post-hoc test.
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f0010: Hypoxia and hypoxia/reoxygenation induces cell death and a decrease in metabolic activity but not protein concentration. H9C2 cells at 70–80% confluence were incubated in 2% 10% or 21% O2 equilibrated, HEPES-buffered, phenol red-free DMEM and exposed to (4 h) hypoxia. (A); The ratio of propidium iodide (PI)-positive stained cells to total Hoechst stained cells was measured as an index of necrosis. ((B) and (C)); Procaspase 3 activation measured as cleavage relative to tubulin expression and determined by western blotting. (D); Metabolic activity determined by MTT assay. (E); After 4 h hypoxia (2% O2), the medium was replaced with reoxygenated medium (2 h; 21% O2) for control and H/R experiments, and with hypoxic medium (2 h; 2% O2) for sustained hypoxia experiments and MTT activity was determined. Data represent the mean ±S.E.M of three independent experiments conducted in triplicate. ⁎ represents P<0.05 and ⁎⁎ represents P<0.001 (one-way ANOVA) compared to controls, Tukey′s post-hoc test.

Mentions: Analysis of necrotic death was undertaken by co-staining with Hoechst and PI. PI uptake was significant during 4 h H (2% O2P<0.05) but not at 10% O2 compared to normoxia (Fig. 2A). After incubation at 2%, 10% and 21%O2 for 4 h, total cell lysates (20 µg) were resolved by SDS-PAGE and transferred to PVDF membranes for immunoblotting for procaspase 3 and cleaved caspase 3. The larger cleaved fragment (18 kDa) of procaspase-3 was detected in cardiomyoblasts exposed to 2% and 10% O2 for 4 h (Fig. 2B and C). However, no cleavage band was detected under normoxia confirming that apoptosis is increased by H only.


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)

Hypoxia and hypoxia/reoxygenation induces cell death and a decrease in metabolic activity but not protein concentration. H9C2 cells at 70–80% confluence were incubated in 2% 10% or 21% O2 equilibrated, HEPES-buffered, phenol red-free DMEM and exposed to (4 h) hypoxia. (A); The ratio of propidium iodide (PI)-positive stained cells to total Hoechst stained cells was measured as an index of necrosis. ((B) and (C)); Procaspase 3 activation measured as cleavage relative to tubulin expression and determined by western blotting. (D); Metabolic activity determined by MTT assay. (E); After 4 h hypoxia (2% O2), the medium was replaced with reoxygenated medium (2 h; 21% O2) for control and H/R experiments, and with hypoxic medium (2 h; 2% O2) for sustained hypoxia experiments and MTT activity was determined. Data represent the mean ±S.E.M of three independent experiments conducted in triplicate. ⁎ represents P<0.05 and ⁎⁎ represents P<0.001 (one-way ANOVA) compared to controls, Tukey′s post-hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0010: Hypoxia and hypoxia/reoxygenation induces cell death and a decrease in metabolic activity but not protein concentration. H9C2 cells at 70–80% confluence were incubated in 2% 10% or 21% O2 equilibrated, HEPES-buffered, phenol red-free DMEM and exposed to (4 h) hypoxia. (A); The ratio of propidium iodide (PI)-positive stained cells to total Hoechst stained cells was measured as an index of necrosis. ((B) and (C)); Procaspase 3 activation measured as cleavage relative to tubulin expression and determined by western blotting. (D); Metabolic activity determined by MTT assay. (E); After 4 h hypoxia (2% O2), the medium was replaced with reoxygenated medium (2 h; 21% O2) for control and H/R experiments, and with hypoxic medium (2 h; 2% O2) for sustained hypoxia experiments and MTT activity was determined. Data represent the mean ±S.E.M of three independent experiments conducted in triplicate. ⁎ represents P<0.05 and ⁎⁎ represents P<0.001 (one-way ANOVA) compared to controls, Tukey′s post-hoc test.
Mentions: Analysis of necrotic death was undertaken by co-staining with Hoechst and PI. PI uptake was significant during 4 h H (2% O2P<0.05) but not at 10% O2 compared to normoxia (Fig. 2A). After incubation at 2%, 10% and 21%O2 for 4 h, total cell lysates (20 µg) were resolved by SDS-PAGE and transferred to PVDF membranes for immunoblotting for procaspase 3 and cleaved caspase 3. The larger cleaved fragment (18 kDa) of procaspase-3 was detected in cardiomyoblasts exposed to 2% and 10% O2 for 4 h (Fig. 2B and C). However, no cleavage band was detected under normoxia confirming that apoptosis is increased by H only.

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