The Role Of Neuronal Nitric Oxide Synthase In Ischemic Postconditioning Protects Against Cardiac Ischemia-reperfusion Injury

As the development of the medical technology, people have known more about myocardial ischemia-reperfusion injury. Recently, the mechanism of ischemic preconditioning has been noticed in many studies, for its clear protective effect against ischemia-reperfusion injury. However, due to the unpredictability of myocardial ischemia, ischemic preconditioning is limited in clinical application. Studies have shown that ischemic postconditioning (IPostC) has the similar protect effect as ischemic preconditioning. Because IPostC is executed at the beginning of reperfusion, it is easier to be operated clinically.In recently years, scientists found that oxidative stress and Ca2+overload are the major triggers of ischemia-reperfusion injury, and IPostC can significantly reduce oxidative stress level and Ca2+overload in ischemia-reperfusion injured heart, but its molecular mechanism is still not clear.Based on its spatial confinement in cardiomyocytes, nitric oxide synthase (NOS)-derived NO plays different roles in cardiac function. Neuronal nitric oxide synthase (nNOS), located in the sarcoplasmic reticulum (SR) and mitochondria, regulates SR and mitochondrial function by maintaining Ca2+cycling and nitro-redox balance. In some pathological conditions, nNOS can reduce oxidative stress by decreasing generation of reactive oxygen species (ROS) and inhibiting xanthine oxidoreductase (XOR). Given that nNOS is closely associated with oxidative stress and Ca2+overload, we hypothesize that IPostC reduces oxidative stress and Ca2+overload in the early minutes of reperfusion via nNOS signaling pathway in cardiomyocytes.Part One:The role of nNOS in ischemic postconditioning protects against cardiac ischemia-reperfusion injuryAim:To test the role of nNOS in ischemic postconditioning protects against cardiac ischemia-reperfusion injury.Methods:Mouse heart ischemia/reperfusion injury model and cardiomyocytes hypoxia/reoxygenation model were established. Infarction size was measured by1%triphenyltetrazolium chloride staining. The histological changes of mouse myocardium were investigated by histopathology observation. Apoptotic cell death was measured by Annexin V-FITC staining with the flow-cytometer.Result:Compared with I/R group, IPostC protected hearts against I/R injury by reducing the infarct size and ultrastructural damage, which were abolished by the nNOS selective inhibitor L-VNIO. HPostC decreased the cell apoptosis compared with H/R group, which was abolished by L-VNIO. However, L-VNIO which was added into perfused buffer during the whole reperfusion time had similar protective effects of IPostC.Part Two:The molecular mechanism of nNOS in ischemic postconditioning protects against cardiac ischemia-reperfusion injuryAim:To explore the molecular mechanism of nNOS in ischemic postconditioning protects against cardiac ischemia-reperfusion injury.Methods:Western blot and isotope method were used to measure the expression of p-nNOSSer852and the activity of nNOS; Western blot was used to measure the production of nitrotyrosine, the peroxynitrite marker in cytosol and mitochondria; Lipid peroxidation (MDA) and reactive oxygen species (ROS) level were measured to evaluate the oxidative stress of myocardium; Western blot and RT-PCR were used to measure the expression of AMPK, p-AMPK (Thr172) and PGC-1α; Intracellular Ca2+([Ca2+]i) was determined with Fluo-3/AM as a fluorescent signals using confocal microscopy.Results:IPostC significantly decreased the activity of nNOS and reduced the production of ONOO-in mitochondria. IPostC increased the activity of nNOS in cytosol. IPostC reduced oxidative stress through nNOS/AMPK/PGC-la signaling pathway, on the other hand, IPostC increased the expression of p-PLBSer16, recovered the function of SR and reduced Ca2+overload. L-VNIO, a selective inhibitor of nNOS, abolished the cardioprotection of IPostC. L-VNIO administrated alone at the beginning of reperfusion inhibited the activity of nNOS in mitochondria, reduced the production of ONOO-, attenuated oxidative stress, and protected the ischemia-reperfusion injured heart.Conclusion:nNOS plays a deleterious role in myocardial I/R injury.ischemia reperfusion decreases the activity of nNOS in cytosol and increases the activity of nNOS in mitochondria, and then causes I/R injury. nNOS is involved in cardioprotection of IPostC against I/R injury. IPostC increases the activity of nNOS in cytosol and decreases the activity of nNOS in mitochondria, reduces oxidative stress and Ca2+overload to protect heart against ischemia-reperfusion injury.