Neuronal Nitric Oxide Synthase Is Involved In Ischemic Postconditioning Against Myocardial Ischemia-Reperfusion Injury By Improving Mitochondrial Function

Recently people have renewed the knowledge about myocardial ischemia/reperfusion injury that injury is initiated within the first minutes of reperfusion, which is known as "window of opportunity" for myocardial protection. In view of this, ischemic postconditioning (IPostC), a promising strategy for cardioprotection was issued. Oxidative stress and Ca2+ overload are major factors causing ischemia/reperfusion injury. Many studies have found IPostC can significantly reduce ischemic induced oxidative stress and Ca2+ overload, but the underlying molecular mechanism is not clear.Neuronal nitric oxide synthase (nNOS) has been found in the heart, and the role of nNOS in cardiac ischemia-reperfusion (IR) injury have been researched extensively. But the role of the subtype in the mitochondria nitric oxide synthase (mtNOS) in cardiac ischemia-reperfusion injury and ischemia postconditioning (IPostC) is still controversial.Objective To observe the role of nNOS in cardioprotective effect during IPostC and to explore the effect of nNOS on mitochondria structure and function.Methods C57/BL6 mice hearts were subjected to 30 minutes global ischemia followed by 120 minutes reperfusion (IR) in a Lagendorff apparatus. The hearts were randomly divided into 5 groups:control (sham); ischemia-reperfusion (IR); ischemic postconditioning treatment (IPostC), nNOS specific inhibitor L-VNIO treatment (IR +L-VNIO), and ischemia postconditioning treated with L-VNIO (IR+L-VNIO+ IPostC). Hearts were first balanced for 20 minutes and then:a) continued 150min reperfusion for sham; b) 30min global ischemia followed by 120 min reperfusion for IR group; c) 30min global ischemia followed by 6 cycles of 10s ischemia/10s reperfusion before 118 minutes reperfusion for IPostC group. L-VNIO was added into K-H reperfusion solution before reperfusion. Left ventricular developed pressure (LVDP) and heart rate (HR) were recorded. The cardiac injury (infarct size and lactate dehydrogenase) was assessed associated with the biomarker of lipid peroxides (malondialdehyde). Indicators of mitochondrial oxidative stress (GSH/GSSG) were determined. The values of GSH and GSSG were used to calculate the mitochondrial redox potential (Eh) using the Nernst Equation. Furthermore, the activities of succinate dehydrogenase (SDH, complex II) and cytochrome c oxidase (COX, complex IV) were also measured spectrophotometrically from LV tissue homogenates to determine wether mitochondrial integrity and function were preserved.Results a) IPostC improved cardiac systolic and diastolic function significantly and reduced infarct size. b) IPostC attenuated IR-induced cardiac injury was associated with reducing mitochondrial oxidative/nitrative stress. IPostC improved mitochondrial function and maintained the structural integrity of mitochondria, c) IR induced injury was partially attenuated by nNOS specific inhibitor L-VNIO. d) L-VNIO reversed the protective effect of IPostC.Conclusion nNOS mediated cardioprotective effect against ischemia-reperfusion injury in isolated heart of mice in certain degree and involved in postconditioning by maitaining the integrity of the mitochondria structure and preserving the mitochondria function.