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

• Cardiomyoblast toxicity during hypoxia is dependent on O2− and NO
• .
• Nrf2 activation is important for cardiomyoblast survival during hypoxia or hypoxia/reoxygenation, but, restoration of GSH is not required.
• NOS activity is essential for the adaptation of cardiomyoblasts to hypoxia/reoxygenation but survival may be independent of Nrf2.

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.

ROS and RNS in cell survival and death during hypoxia and reoxygenation; the role of Nrf2. There is evidence of superoxide anion radical and nitric oxide production in both hypoxia and H/R. While MnTBap inhibited the toxicity of both stressors, L-NAME only protected against the toxicity of hypoxia implying that nitric oxide was required for survival during H/R. Both superoxide anion radical and nitric oxide elicited Nrf2 activation during H/R and to a lesser extent during hypoxia. Nrf2 knockdown prevented GSH induction and also reduced survival during H/R by 50% but only by 30% during hypoxia. RNS are essential for Nrf2 activation but may also exert cytoprotective effects during H/R by other means.Figure optionsDownload as PowerPoint slide