Selective Nrf2 Gene Deletion in the Rostral Ventrolateral Medulla Evokes Hypertension and Sympathoexcitation in Mice

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcriptional regulator of redox homeostasis that impacts antioxidant gene expression. Central oxidative stress and reduced antioxidant enzyme expression in the rostral ventrolateral medulla (RVLM) contributed to sympathoexcitation in chronic heart failure. In the current study, we hypothesized that deletion of Nrf2 in the RVLM would increase sympathetic drive and blood pressure. Experiments were performed in Nrf2-floxed mice treated with microinjection of lentiviral-Cre-GFP or lentiviral-GFP into the RVLM. Two weeks after viral administration, Nrf2 message, protein, oxidative stress, cardiovascular function, and sympathetic outflow were evaluated. We found that (1) Nrf2 mRNA and protein in the RVLM were significantly lower in Cre mice compared with control GFP mice. Nrf2-targeted antioxidant enzymes were downregulated, whereas reactive oxygen species were elevated. (2) Blood pressure measurements indicated that Cre mice displayed a significant increase in blood pressure (mean arterial pressure, 123.7±3.8 versus 100.2±2.2 mm Hg; P<0.05, n=6), elevated urinary norepinephrine (NE) concentration (456.4±16.9 versus 356.5±19.9 ng/mL; P<0.05, n=6), and decreased spontaneous baroreflex gain (up sequences, 1.66±0.17 versus 3.61±0.22 ms/mm Hg; P<0.05, n=6; down sequences, 1.89±0.12 versus 2.98±0.19 ms/mm Hg; P<0.05, n=6). (3) Cre mice displayed elevated baseline renal sympathetic nerve activity and impaired inducible baroreflex function. These data suggest that Nrf2 gene deletion in the RVLM elevates blood pressure, increases sympathetic outflow, and impairs baroreflex function potentially by impaired antioxidant enzyme expression.

Keywords: arterial baroreflex; blood pressure; brain stem; oxidative stress; sympathetic regulation.

Figure 1

Nrf2 mRNA (A) and Protein (B) expression in RVLM punches of Nrf2^flox/flox mice receiving Lentiviral-GFP (GFP) or Lentiviral-Cre-GFP (Cre). **P < 0.01 compared with GFP mice, n = 7 for each group. Panel C is immunofluorescence images of one brainstem section (5.3 mm ventral to the dorsal surface of the brain and 1.9 mm caudal to lambda) from a Nrf2^flox/flox mouse whose right RVLM received Lentiviral-Cre-GFP injection. Green is GFP signal indicating the infected region and neurons. Red is Nrf2 immuno-positive signal showing location of Nrf2 protein.

Figure 2

Expressions of Nrf2 downstream target proteins (NQO1, HO-1, SOD2, and Catalase) in the RVLM punches of Nrf2^flox/flox mice with bilateral microinjection of Lentiviral-GFP (GFP) or Lentiviral-Cre-GFP (Cre). *P < 0.05 and **P < 0.01 compared with GFP mice, n = 7 for each group.

Figure 3

Superoxide levels in the RVLM of mice following bilateral microinjection of Lentiviral-GFP (GFP) or Lentiviral-Cre-GFP (Cre). (A) left - DHE staining images of brainstem sections at 1.9 mm caudal to lambda; right- the quantified density of red fluorescence, *P < 0.05 vs GFP-mice, n = 6 for each group. (B) EPR spectra obtained from pooled RVLM punches from 8 mice, 4 GFP-mice and 4 Cre-mice. (a.u. = arbitrary units)

Figure 4

Blood pressure and heart rate (A), spontaneous arterial baroreflex sensitivity (B), and urinary norepinephrine excretion (C) in conscious mice with bilateral microinjection of Lentiviral-GFP (GFP) or Lentiviral-Cre-GFP (Cre). *P < 0.05 compared with GFP mice, n = 6 for each group.

Figure 5

Baseline renal sympathetic nerve activity (RSNA, A) and induced arterial baroreflex sensitivity (IBRS, B) in anesthetized mice with bilateral microinjection of Lentiviral-GFP (GFP) or Lentiviral-Cre-GFP (Cre). *P < 0.05 and **P < 0.01 compared with GFP mice, n = 6 for each group.