Abstract 233: Knockout Of Vascular AMPK Leads To Increased Mitochondrial Stress And Decreased Vasodilator Sensitivity Towards Organic Nitrates.
Background: Chronic nitroglycerin (GTN) treatment causes nitrate tolerance associated with an increased production of mitochondrial derived reactive oxygen species (ROS) and formation of peroxynitrite. Since peroxynitrite is known to activate AMP-activated protein kinase (AMPK), we investigated whether knockout of vascular AMPK influences GTN-induced vasorelaxation, mitochondrial oxidative stress and the development of nitrate tolerance.
Methods: alpha1-AMPK knockout mice (AMPK −/−) and corresponding wildtype (WT) mice were treated with GTN (30 μg/kg/min) or solvent (ethanol) via osmotic minipump. After 4 days, we determined vascular relaxation by isometric tension studies and AMPK activity by phosphorylation of the enzyme and its downstream target ACC. Mitochondria were isolated from heart homogenates by differential centrifugation and mitochondrial ROS formation determined by addition of succinate and the luminescence dye L-012 (100 μM).
Results: In WT mice treated with GTN for 4 days we observed a twofold increase of AMPK activity. Animals with a knockout of the alpha1-AMPK isoform showed no detectable AMPK expression as detected by pan-alpha-AMPK antibody. In AMPK −/− mice, sensitivity to GTN was decreased, while chronic GTN treatment lead to a more pronounced decrease in GTN-induced relaxation. The degree of nitrate tolerance was not different between WT and AMPK −/− animals. Basal mitochondrial ROS production was increased in AMPK −/− compared to WT mice (WT 240033±8196 AMPK ko 403420±34480 counts/mg/min; p<0.05), while GTN treatment lead to a further increase in both groups (WT + GTN 335877±30028, AMPK ko + GTN 534990±47997 counts/mg/min; p<0.05). In parallel, the expression of the PPARgamma coactivator-1 (PGC-1alpha), a transcriptional coactivator required for mitochondrial biogenesis, was sharply decreased in AMPK −/− mice.
Conclusion: Knockout of vascular AMPK activity attenuates GTN-induced relaxation and increases mitochondrial oxidative stress. Since AMPK mediates mitochondrial biogenesis via PGC-1alpha, our results might be explained by a decrease in mitochondrial mass and GTN-biotransformation capacity.