Abstract W P240: S1177-eNOS Phosphorylation Protects Against Stroke Injury in Akt1 Deficient Mice
Background: Phosphorylation of eNOS at serine 1177 (S1177) by Akt1 increases NO production and vascular reactivity. The goal of this study is to test the hypothesis that Akt1 deficiency increases susceptibility to stroke because of insufficient eNOS phosphorylation. Thus, constitutively phosphorylated eNOS will overcome Akt1 deficiency and protect against stroke. We generated knock-in mice (S1177D) carrying phosphomimetic mutation (serine to aspartate substitutions) in the eNOS gene. We bred S1177D mice with Akt1 knockout (Akt1KO) mice to test whether constitutively active eNOS could improve stroke outcome in Akt1KO mice.
Methods: Adult male mice were anesthetized by 30% oxygen, 70% nitrous oxide, and 1.5% isoflurane. Body temperature was maintained at 36-37°C. The common carotid artery was catheterized to measure systemic blood pressure using a blood pressure transducer. For stroke studies we used the middle cerebral artery occlusion (MCAO) by filament with subsequent reperfusion with Laser Doppler cortical blood flow monitoring. Mice were examined for neurologic deficits by 5 point scale 24 hours after stroke. Infarct sizes were determined by the indirect method by 2,3,5-triphenyltetrazolium chloride staining.
Results: Mean blood pressure was higher in Akt1KO (95±7 mmHg, Mean±SD, n=9) and WT mice (93±3 mmHg, n=8) than in S1177D/Akt1KO mice (80±11 mmHg, n=6, P<0.05). One hour of MCAO with 23 hours of reperfusion produced increased cerebral infarct volume in Akt1KO mice (106±21 mm3, n=9) as compared with WT (70±33, n=9, p< 0.05) mice. S1177D phosphomimetic mutation decreased cerebral infarct volume in Akt1KO mice (64±20 mm3, n=9, P<0.05). This was associated with a functional impairment in the neurologic deficit, as quantities by neurologic scoring (2.7 point deficit for Akt1KO; 1.9 for WT and 1.3 for S1177D/Akt1KO mice).
Conclusion: Stroke injury was more pronounced in Akt1KO mice than in WT mice. Introducing S1177D mutation in Akt1KO mice resulted in decreased infarct volume, neurological deficit and blood pressure. These results demonstrated that S1177D eNOS-derived NO is able to protect against stroke in the absence of Akt1.
Author Disclosures: D.N. Atochin: None. P.L. Huang: None.
This research has received full or partial funding support from the American Heart Association, Founders - Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont.
- © 2015 by American Heart Association, Inc.