Lack of Endothelial α1AMPK Reverses the Vascular Protective Effects of Exercise by Causing eNOS Uncoupling

Figures

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  Figure 1. Effects of exercise training on α1AMPK expression in the endothelium. Voluntary exercise training was performed on running wheels supplied in mouse cages, which were connected to a counter to measure running distance. Running distances were shown in km/d (A). Western blot experiments present the consequence of exercise training on α1AMPK protein expression in isolated mouse lung endothelial cells (MLEC) in α1AMPKfl/fl x TekCre+ and corresponding wild type controls (TekCre+). Representative western blot of 6 independent experiments is shown (B). Effects of voluntary exercise training on body and heart weight were shown for every group (C–F). Data are shown as mean ± SEM from 11–25 animals per group.

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  Figure 2. Mice with endothelial α1AMPK develop endothelial dysfunction in response to exercise training. Vascular relaxation in aortic rings was investigated in organ chamber experiments. Endothelial dependent relaxation with cumulative doses of Acetylcholine and endothelial independent relaxation with cumulative doses of Nitroglycerin are shown (A,B). Data are shown as mean ± SEM from 8–16 animals per group. Aortic NO production was assessed using EPR in repetitive measurements (C). Representative EPR traces are shown. Effects of exercise training on aortic endothelin-1 expression are presented with immunohistochemistry and the respective densitometry (D). Data are shown as mean ± SEM from 3–10 animals per group. * means p < 0.05 vs. TekCre+. # means p < 0.05 vs. α1AMPKfl/fl x TekCre+ + Exercise.

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  Figure 3. Deletion of endothelial α1AMPK diminishes eNOS expression. eNOS gene expression in response to exercise training was measured with real-time PCR in isolated mouse lung endothelial cells (A). Abrogated protein expression of eNOS in α1AMPKfl/fl x TekCre+ mice after exercise training in MLEC was determined in western blot experiments (B). Representative western blot is shown. Immunoblots of eNOS protein expression in the aortic tissue (C) phospho-eNOSSer1177/eNOS protein expression ratio (D) during exercise training are shown. Data represent as mean ± SEM. with pooled samples of 12-18 animals in each group.

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  Figure 4. Reactive oxygen species production in response to exercise training is aggravated in endothelial specific α1AMPK knockout mice. Effects of exercise training on 3-Nitrotyrosin formation in MLEC were determined by dot blot analysis (A). Immunohistochemistry staining using the 3-nitrotyrosin antibody was performed in sections of aortic tissue (B). Superoxide production in the vasculature was measured by in situ topographic DHE- staining in aortic cryosections (C). Representative pictures are shown. Production of hydrogen peroxide in the aortic tissue was determined with Amplex Red assay (D). Data are mean ± SEM from 5–11 mice per group.

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  Figure 5. Exercise training enhances ROS production and eNOS uncoupling in endothelial α1AMPK knockout mice. eNOS uncoupling in α1AMPKfl/fl x TekCre+ mice was shown in aortic cryosections using DHE staining and the NO-synthase inhibitor L-NAME. This results in exaggerated ROS production, aggravated in response to exercise in α1AMPKfl/fl x TekCre+ (A). NOX-2 mRNA expression in mouse lung endothelial cells (B) and aortic tissue (C) was determined with real-time PCR. Mitochondrial reactive oxygen species production were measured by using mitoSox staining in prepared cryosections of aortic tissue (D). Additionally we measured mitochondrial- specific reactive oxygen species production using mitoSox—HPLC based method, n = 3–9 (E). Representative chromatograms are presented for each group. Data are mean ± SEM from 3–20 mice per group or pooled samples from at least two mice.

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  Figure 6. Endothelial α1AMPK deletion results in diminished antioxidant capacity. Antioxidant capacity was determined with a DPPH assay in serum of mice performing exercise (A). Results display repetitive measurements of five independent experiments. Aortic gene expression of heme oxygenase 1 (B), Nrf-2 mRNA expression (F) and UCP-2 gene expression (C) were measured with quantitative real-time PCR. Representative western blot with densitometric analysis of Nrf-2 protein expression in isolated mouse lung endothelial cells of all experimental animal groups are shown (D). Each lane represents a pooled sample of at least three animals. Nrf-2 mRNA expression was additionally measured in mouse lung endothelial cells (E). Data are mean ± SEM from 4–18 mice per group.