Role of Nrf2 Signaling in the Regulation of Vascular BK Channel β1 Subunit Expression and BK Channel Function in High-Fat Diet–Induced Diabetic Mice
Article Figures & Tables
Figures
- Figure 1
Downregulation of BK-β1 and Nrf2 protein expressions and upregulation of MuRF1 protein expressions in vascular SMCs are associated with coronary BK channel dysregulation and coronary arterial dysfunction in HFD mice. A: Inside-out single BK currents were recorded from freshly isolated coronary SMCs of LFD and HFD mice at baseline and after exposure to 100 nmol/L DHS-1 (a specific BK-β1 activator). The total channel nPo was significantly lower in HFD mice at baseline compared with LFD mice. DHS-1 robustly enhanced the channel activities, but the DHS-1 effects in HFD mice were diminished compared with those in LFD mice (n = 23 cells). Dashed lines indicate the closed state (C) of channels. B: BK-β1 protein expression was markedly downregulated and was accompanied by reduced Nrf2 expression and increased MuRF1 expression in the arterial vessels of HFD mice (n = 6 mice for each group). C: No difference in BK-β1 mRNA levels in coronary arteries from LFD and HFD mice (n = 6 mice for each group). D: Coronary vasodilation to NS-1619 (a BK channel activator) was impaired in HFD mice (n = 4 mice). *P < 0.05 vs. controls.
- Figure 2
Regulation of HO-1, MuRF1, and BK-β1 expressions in cultured coronary SMCs by Nrf2. A: Ad-Nrf2 transduction (50 MOI) produced a 6.8-fold increase in Nrf2 expression and a 2.1-fold increase in HO-1 expression in human coronary SMCs. These changes were associated with a 66.2% reduction in MuRF1 expression and a 2.3-fold increase in BK-β1 expression (n = 9 culture plates). B: Transduction with Ad-Nrf2 shRNA (50 MOI) in human coronary arterial SMCs resulted in a 58.9% knockdown in the expression of Nrf2, a 41.2% reduction in that of HO-1, a threefold upregulation in that of MuRF1 and a 68.9% downregulation in that of BK-β1. Transduction with Ad-GFP or Ad-scramble RNA served as controls (n = 9 culture plates). C: Adenoviral delivery of Nrf2 (50 MOI) enhanced the BK-β1 mRNA levels by 2.6-fold and 1.6-fold in cultured human and mouse coronary SMCs, respectively (n = 9 culture plates). *P < 0.05 vs. controls. Ctrl, control.
- Figure 3
Regulation of NF-κB protein expressions by HFD and Nrf2 in primary human coronary arterial SMCs. A: Protein expressions of NF-κB/p50 and NF-κB/p65 were increased by 0.3-fold and 2.4-fold, respectively, in the arteries of HFD mice compared with those of LFD mice (n = 6 mice for each group). B: A 48-h transduction with Ad-Nrf2 suppressed the protein expressions of NF-κB/p50, NF-κB/p65, and phospho-NF-κB/p65(S267) in cultured human coronary arterial SMCs by 64.5%, 61.2%, and 58.0%, respectively (n = 7 to 8 culture plates). *P < 0.05 vs. controls. p-NF-κB, phosphorylated NF-κB.
- Figure 4
Effects of DMF on the protein expressions of Nrf2, HO-1, NK-κB, MuRF1, and BK-β1 in primary human coronary arterial SMCs in vitro. A: A 12-h incubation with 10 μmol/L DMF augmented the protein expressions of Nrf2 and HO-1 by 1.9-fold and 1.6-fold, respectively, accompanied by 43.9% and 63.8% reductions, respectively, in those of NF-κB/p65 and MuRF1 in cultured human coronary SMCs. These changes were associated with a threefold increase in BK-β1 protein levels in DMF-treated cells, compared with cells with vehicle treatment (n = 6 culture plates). B: A significant increase in BK-β1 mRNA expression by 15.3% in human coronary SMCs treated with DMF for 12 h compared with cells treated with vehicle (n = 9 culture plates for each group). *P < 0.05 vs. controls. Ctrl, control.
- Figure 5
Effects of DMF on the protein expressions of Nrf2, NK-κB, MuRF1, and BK-β1 in HFD mouse arteries in vivo. A 10-day course of treatment with DMF by oral administration (25 mg/kg/daily) enhanced the protein expressions of Nrf2 and BK-β1 in the arteries of HFD mice by 1.5-fold and 1.8-fold, whereas it downregulated the protein expressions of NF-κB/p50, NF-κB/p65, and MuRF1 by 56.8%, 75.5%, and 66.3%, respectively, compared with HFD mice treated with placebo (n = 5 mice for each group). *P < 0.05 vs. placebo. Ctrl, control.
- Figure 6
Protective effects of DMF administration on coronary BK channel current density, BK channel–mediated coronary vasodilation, and BK-β1–dependent activation in HFD mice. A: Representative tracings of whole-cell BK channel recordings and current voltage curves obtained from freshly isolated coronary SMCs of HFD mice after a 10-day course of treatment with DMF by gavage. Treatment with DMF restored the BK current density to a level comparable to that in LFD mice (dashed line) (n = 8 cells for each group). B: Treatment with DMF preserved NS-1619–mediated coronary vasodilation in HFD mice to a level similar to that in LFD mice (dashed line) (n = 8 mice for each group). C: Representative tracings of single-BK channel recordings in inside-out patches from freshly isolated coronary SMCs of HFD mice, elicited at 60 mV before and after exposure to DHS-1 (n = 13–14 cells). Animals were treated with DMF or placebo for 10 days, with DMF treatment preserving BK channel activity at baseline and restoring the channel response to DHS-1 in HFD mice to a level similar to that in LFD mice (open bar graphs). *P < 0.05 vs. baseline; †P < 0.05 vs. placebo. Ctrl, control.
- Figure 7
Illustration showing the role of Nrf2 signaling in the regulation of BK-β1 expression in arterial myocytes. Under normal conditions, Nrf2 is bound to Keap1, which promotes the degradation of Nrf2 through the UPS. Upon activation by DMF, Nrf2 dissociates from Keap1 and translocates into the nuclei, where the transcription of downstream effectors such as BK-β1 and HO-1 is facilitated. The metabolites of HO-1, such as CO, bilirubin, and Fe2+, inhibit NF-κB activity. With increased oxidative stress in diabetic vessels, Nrf2 is downregulated, resulting in a reduction of BK-β1 and HO-1 expressions. In addition, increased ROS generation directly phosphorylates NF-κB/p65 at serine residues, which in turn promotes NF-κB/p65 nuclear translocation, facilitates MuRF1-dependent BK-β1 protein degradation, and suppresses Nrf2 transcriptional activity on BK-β1.
Tables
- Table 1
Metabolic characterization and blood pressure of mice (strain: C57BL/6J) 6 months after the consumption of a 10% fat diet (LFD) or a 60% fat diet (HFD)
Animal (n) Body weight (g) Glucose (mg/dL) Insulin (ng/mL) Systolic/diastolic pressure (mmHg) Mean arterial pressure (mmHg) LFD (n = 8–10) 30.4 ± 0.9 146.5 ± 12.6 0.97 ± 0.06 110.4 ± 4.4/82.6 ± 5.2 91.9 ± 4.8 HFD (n = 8–12) 48.1 ± 1.8 230.1 ± 9.7 6.64 ± 0.45 126.0 ± 3.5/94.8 ± 3.8 105.2 ± 3.7 P value <0.05 <0.05 <0.05 <0.05 <0.05 There were significant increases in body weight, serum glucose level, serum insulin level, and mean blood pressure in HFD mice compared with LFD mice. Data are presented as mean ± SEM.
In this Issue
