Nox4 regulates Nrf2 and glutathione redox in cardiomyocytes in vivo

Abstract

NADPH oxidase-4 (Nox4) is an important modulator of redox signaling that is inducible at the level of transcriptional expression in multiple cell types. By contrast to other Nox enzymes, Nox4 is continuously active without requiring stimulation. We reported recently that expression of Nox4 is induced in the adult heart as an adaptive stress response to pathophysiological insult. To elucidate the potential downstream target(s) regulated by Nox4, we performed a microarray screen to assess the transcriptomes of transgenic (tg) mouse hearts in which Nox4 was overexpressed. The screen revealed a significant increase in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in tg compared to wild-type (wt) mouse hearts, and this finding was subsequently confirmed by Q-PCR. Expression of glutathione biosynthetic and recycling enzymes was increased in tg hearts and associated with higher levels of both GSH and the ratio of reduced:oxidised GSH, compared to wt hearts. The increases in expression of the antioxidant genes and the changes in glutathione redox effected by Nox4 were ablated in an Nrf2-null genetic background. These data therefore demonstrate that Nox4 can activate the Nrf2-regulated pathway, and suggest a potential role for Nox4 in the regulation of GSH redox in cardiomyocytes.

Fig. 1

Expression of Nox4 in wt and tg hearts. (A) Q-PCR analyses of relative expression of endogenous Nox4 mRNA, in postnatal hearts at birth (day 0), 1 week, and 2 weeks. (B) Q-PCR analyses of Nox4 transgene expression in Day 0, 1 week, and 2 week postnatal hearts. Hearts samples from 3 littermate animals were analysed in all cases. Values are shown normalised to β-actin, and are plotted as arbitary units (AU). (C) Nox4 protein levels in 15 day wt and tg embryonic hearts (amounts of protein loaded as inducated); (D) Nox4 protein levels in tg hearts, at 2 days and 2 weeks after birth, as indicated (15 μg protein loaded in each case). (E) Analyses of hyperoxidised Prdx in wt and tg littermate controls at 2 weeks after birth, as indicated (15 μg protein loaded in each case). ** P < 0.01.

Fig. 2

Expression of Nrf2-regulated genes in Nox4 tg mouse hearts. (A–G) Q-PCR analyses of Nrf2-regulated genes, and Nrf2 itself in 2 week postnatal tg and wt mouse hearts as indicated. Triplicate tg and wt littermate controls were analysed, and relative expression is shown normalised to β-actin in all cases, plotted as arbitary units (AU). (H) Analyses of binding activity of Nrf2 in wt and tg littermate mouse hearts. In both cases binding to the immobilised Nrf2 oligonucleotide is ablated by self-competition (+ wt Nrf2 oligo). ** P < 0.01, * P < 0.05.

Fig. 3

Expression of KEAP 1 and Nrf2 in Nox4 tg mouse hearts. Immunocytochemistry showing stronger cytoplasmic expression of KEAP 1 in wt (A) compared to Nox4 tg (B) hearts (brown stain). Increased nuclear localisation of Nrf2 expression is seen in tg (D) compared to wt (C) hearts (brown stain). Arrows depict strong nuclear expression of Nrf2 that is not apparent in wt sections. Nucleii are stained with hematoxylin (violet) in all panels. Scale bar: 10 μm.

Fig. 4

Glutathione levels and GSH/GS-SG redox couple in Nox4 tg hearts. Levels of (A) total (GSH + GS-SG), (B) reduced (GSH), and (C) oxidised (GS-SG) glutathione in 12 day postnatal Nox4 tg and littermate wt hearts (males and females). (D) Ratio of GSH/GS-SG in these hearts. Cohorts of 3 tg and 3 wt littermate controls were used for this study. (E) GSH levels in hearts of tg and wt, males and females, from 2 pooled 12 day litters. Cohorts of 3 animals were assessed in each case. (F) GSH levels in 3 tg and 3 wt littermate control hearts of 2 week postnatal mice of a second Nox4 tg line. (G) Time course of endogenous GSH levels in hearts of wt mice. Three littermate samples were assessed at birth (Day 0), 1 week, and 2 weeks. Results are plotted on an arbitrary scale of relative light units (RLU) ** P < 0.01, * P < 0.05.

Fig. 5

Upregulation of Nrf2-regulated genes and GSH levels is ablated in an nrf2-null genetic background. (A–D) Q-PCR analyses of Nrf2-regulated genes in 2 week postnatal Nox4 tg and wt hearts in a normal (black bars) or Nrf2-null genetic background (gray bars), as indicated. Cohorts of 3 age-matched mice (males and females) were used for this study, from pooled litters, and relative expression is shown normalised to β-actin in all cases, plotted as arbitary units (AU). (E) GSH levels in the same cohorts of mice assessed in A–D, plotted on an arbitrary scale of relative light units (RLU). ** P < 0.01, * P < 0.05 in all cases.

Fig. 6

Nox2 overexpression does not activate Nrf2. (A, B) Q-PCR analyses of Nrf2-regulated genes in Nox2 tg, Nox4 tg, and wt mouse hearts as indicated. Triplicate heart samples were analysed from adult Nox2 tg and age-matched wt control animals, and 2 week Nox4 tg mice. Relative expression is shown normalised to β-actin in all cases, plotted as arbitary units (AU). ** P < 0.01. C; GSH levels in cohorts of 4 approx 6-week-old Nox2 tg mouse hearts and wt littermate controls (males and females) plotted on an arbitrary scale of relative light units (RLU).