Abstract WP292: Sleep Debt Induces Loss of S-Nitrosylation of Cysteine 93 Residue of Beta Chain Hemoglobin ( β Hb-Cys 93 ) to Impair Post-Stroke Brain Tissue Reoxygenation

Abstract

Introduction: Sleep debt attenuates nitric oxide synthase 3 (NOS3) activity and vasodilation which could diminish brain tissue oxygen (PbtO 2 ) after stroke. S-nitrosylated β Hb-Cys 93 promotes hypoxic vasodilation to enhance PbtO 2 . But the detection of s-nitrosylated β Hb-Cys 93 remain challenging and debated. We report a novel antibody and immunodetection of s-nitrosylated β Hb-Cys 93 and relate it to PbtO 2 after stroke. Hypothesis: Sleep debt in mice would attenuate erythrocyte-associated NOS3 ( ery NOS3) activity and β Hb-Cys 93 s-nitrosylation resulting in diminished PbtO 2 after stroke. Methods: Using click chemistry approach, a KLH-conjugated s-nitrosylated peptide, -SLSELH-C(NO)-DKLHVDPEN- (a conserved domain of β Hb in mouse and human carrying Cys 93 ), was synthesized. Rabbits were immunized using s-nitrosylated antigen, and polyclonal antibodies were affinity purified. Mice were subjected to progressive sleep deprivation (1-6h) for 3-wks to induce sleep debt. Blood samples were collected to detect s-nitrosylated β Hb-Cys 93 using western blot (WB) and FACS. Mice were subjected to transient middle cerebral artery occlusion (tMCAo; 45 minutes) followed by reperfusion. PbtO 2 at 2h post-stroke was recorded in vivo using an O 2 -sensor, followed by assessments of neurologic deficit at 24h and infarction volume at 48h (P<0.05). Results and Conclusion: Purities of antigens were confirmed with HPLC and mass spectrometry. Antibody against s-nitrosylated β Hb-Cys 93 exclusively detected it but not the non-nitrosylated β Hb-Cys 93 . WB and FACS results showed that sleep debt induces loss in β Hb-Cys 93 s-nitrosylation via reduced ery NOS3 activity. Post-reperfusion restoration of PbtO 2 in mice with sleep debt was significantly attenuated as compared to the non-sleep debt group. Sleep debt augmented the neurologic deficit and infarction volume as compared to the non-sleep debt controls subjected to stroke. In conclusion, immunodetection of β Hb-Cys 93 using this novel antibody could be translated as a biomarker to monitor the risk of sub-optimal brain tissue reoxygenation after stroke. Future studies are warranted to test therapies which could enhance the s-nitrosylated β Hb-Cys 93 to overcome the detrimental effects of sleep debt in stroke.