NOX4 stimulates ANF secretion via activation of the Sirt1/Nrf2/ATF3/4 axis in hypoxic beating rat atria

Abstract

Silent information regulator factor 2-related enzyme 1 (Sirt1) is involved in the regulation of cell senescence, gene transcription, energy balance and oxidative stress. However, the effect of Sirt1 on atrial natriuretic factor (AN F) secretion, especially under hypoxic conditions is unclear. The present study aimed to investigate the effect of Sirt1, regulated by NAD PH oxidase 4 (NO X4), on AN F secretion in isolated beating rat atria during hypoxia. AN F secretion was analyzed using radioimmunoassays and protein expression levels were determined by western blotting and immunofluorescence staining. Intra-atrial pressure was recorded using a physiograph. Hypoxia significantly upregulated Sirt1 and nuclear factor erythroid-2-related factor 2 (Nrf2) protein expression levels, together with significantly increased ANF secretion. Hypoxia-induced protein expression of Sirt1 was significantly blocked by a NO X4 inhibitor, GLX351322, and Nrf2 protein expression levels were significantly abolished using the Sirt1 inhibitor, EX527. Hypoxia also significantly elevated the protein expression levels of phosphorylated-Akt and sequestosome 1 and significantly downregulated Kelch-like EC H-associated protein 1 protein expression levels. These effects were significantly blocked by EX527, preventing hypoxia-induced Nrf2 expression. An Nrf2 inhibitor, ML385, significantly abolished the hypoxia-induced upregulation of activating transcription factor (ATF)3, ATF4, T cell factor (TCF)3 and TCF4/lymphoid enhancer factor 1 (LE F1) protein expression levels, and significantly attenuated hypoxia-induced AN F secretion. These results indicated that Sirt1 and Nrf2, regulated by NO X4, can potentially stimulate TCF3 and TCF4/LE F1 signaling via ATF3 and ATF4 activation, thereby potentially participating in the regulation of AN F secretion in beating rat atria during hypoxia. In conclusion, intervening with the Sirt1/Nrf2/ATF signaling pathway may be an effective strategy for resisting oxidative stress damage in the heart during hypoxia.