Selective Nrf2 Gene Deletion in the RVLM Increases Blood Pressure and Evokes Sympatho‐Excitation in Mice
Authors: Gao Lie , Zucker Irving H 
Published: November 2023

Nrf2 is a key transcription factor that regulates redox status by up‐regulating antioxidant genes. Previous data from our laboratory demonstrated that increased ROS in the RVLM contributed to the sympatho‐excitation in rabbits with chronic heart failure. Accordingly, we hypothesized that deletion of Nrf2 in the RVLM of mice will increase sympathetic drive due to oxidative stress by down‐regulating antioxidant protein expression. Twenty seven Nrf2 Floxed mice were randomized into two groups: control mice received Lenti‐GFP microinjection into bilateral RVLM, while Nrf2‐deleted mice received Lenti‐Cre‐GFP. Real‐time RT‐PCR, Western blot analysis, immunofluorescence, DHE staining, and ELISA were used to evaluate mRNA level, protein expression, ROS production, and NE concentration. Blood pressure in the conscious state was measured by the radiotelemetry. Renal sympathetic nerve activity (RSNA) was recorded under anesthesia. We found that (1) Nrf2 mRNA and protein levels in the RVLM of Cre‐mice were significantly lower than GFP‐mice (mRNA: 0.3 ± 0.1 vs 1.0 ± 0.1; protein: 0.07 ± 0.02 vs 0.32 ± 0.03. **P < 0.01, n = 6). The down‐regulated Nrf2 protein in the RVLM of Cre‐mice was further confirmed by immunofluorescence staining, where the Lenti‐Cre‐GFP positive neurons exhibited a markedly decreased Nrf2 protein compared to the adjacent negative neurons. (2) The expression of principle antioxidant proteins targeted by Nrf2, including NQO1, HO1, SOD2, and Catalase, were significantly lower in the RVLM of Cre‐mice (HO1: 0.93 ± 0.10 vs 1.27 ± 0.12; NQO1: 0.57 ± 0.08 vs 0.93 ± 0.12; SOD2: 0.46 ± 0.10 vs 1.68 ± 0.14; Catalase: 0.34 ± 0.13 vs 1.11 ± 0.20; *P < 0.05, n = 6). Consistent with the change in antioxidant protein expression, the superoxide level in the Lenti‐Cre‐GFP positive neurons was significantly increased (arbitrary DHE signal: 71.8 ± 2.95 vs 39.2 ± 0.67; *P < 0.05, n = 10 and 12). (3) In the conscious state, Cre‐mice displayed a significant increase in blood pressure compared to GFP‐mice (24h averaged MAP: 123.7 ± 3.8 vs 100.2 ± 2.2 mm Hg, *p < 0.05, n = 6). In addition, Cre‐mice had significantly elevated urinary NE concentration (456.4 ± 16.9 vs 356.5 ± 19.9 ng/mL, *P < 0.05, n = 13–14) and 24‐hour NE excretion (676.5 ± 64.1 vs 416.5 ± 28.1 ng, *P < 0.05, n = 13–14), and decreased spontaneous baroreflex gain (Up‐sequence: 1.66 ± 0.17 vs 3.61 ± 0.22 ms/mm Hg, *P < 0.05, n = 6; Down‐sequence: 1.89 ± 0.12 vs 2.98 ± 0.19 ms/mmHg, *P < 0.05, n = 6) as compared to GFP‐mice. (4) Under anesthesia, Cre‐mice displayed elevated baseline RSNA (63.75 ± 8.1 vs 24.21 ± 4.3 %/Max, *P < 0.05, n = 5) and impaired inducible baroreflex function (Ave Slope: 0.08 ± 0.01 vs 0.13 ± 0.02 %mmHg; Gain Max: 1.64 ± 0.2 vs 3.46 ± 0.2 %mmHg; Minimum RSNA: 8.98 ± 1.21 vs 46.32 ± 5.51 %/Max; PB50: 122.34 ± 8.9 vs 90.13 ± 11.5 mmHg; Range of RSNA Response: 79.9 ± 9.8 vs 107.4 ± 12.4 %/Max, *P < 0.05, n = 5–6). Based on the above data, we conclude that selective Nrf2 gene deletion in the RVLM elevates blood pressure, increases sympathetic outflow, and impairs baroreflex function potentially by the oxidative stress due to the impaired antioxidant mechanisms.

Supported by NIH grant P01‐HL62222