title: Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species.
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.
authors.affiliation: Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK ; Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.
authors.forename: Rajitha T
authors.initials: RT
authors.lastname: Kolamunne
authors.forename: Irundika H K
authors.initials: IH
authors.lastname: Dias
authors.forename: Ann B
authors.initials: AB
authors.lastname: Vernallis
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Grant
authors.forename: Helen R
authors.initials: HR
authors.lastname: Griffiths
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D008665:Metalloporphyrins / Q000494:pharmacology*; D032386:Myoblasts, Cardiac / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology*; D051381:Rats; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D008665:Metalloporphyrins; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; D019331:NG-Nitroarginine Methyl Ester
keywords: Adaptive; CREB, cAMP-responsive element-binding protein; DAF-2-DA, 4,5-diaminofluorescein diacetate; DHE, dihydroethidium; Glutathione; HIF-1, hypoxia-inducible factor; KEAP1, Kelch-like ECH-associated protein 1; L-NAME; L-NAME, L-NG-nitroarginine methyl ester; MnTBap; MnTBap, manganese [III] tetrakis (4-benzoic acid) porphyrin; NFκB, nuclear factor kappa B; NO, nitric oxide; NOS, nitric oxide synthase; NOX, NADPH oxidase; Nrf2, nuclear factor erythroid 2-related factor 2; RNS; RNS, reactive nitrogen species; ROS; ROS, reactive oxygen species