NNOS Is Involved In Ischemic Postconditioning By Improving Myocardial Sarcoplasmic Reticulum Function

Ischemic heart disease is one of the diseases that have highest morbidity and fatality in the world. Nowadays, reperfusion therapy has become the main treatment for acute myocardial ischemic heart disease. However, the reperfusion after ischemia may lead to further tissue damage and dysfunction, resulting in myocardial infarction, systolic dysfunction, arrhythmias and other complications, which is called ischemia-reperfusion (I/R) injury. Myocardial ischemic preconditioning (IPC), which was firstly proposed by Murry in 1986, has been proved a cardiac protective effect against I/R injury. But the unpredictability of myocardial ischemia makes the clinical application of IPC be very restricted. In the year 2003, Zhao reported that the postconditioning can significantly reduce the infarct size in dog’s heart after ischemic repufusion. This is the first time confirmed the protective effect and the concept of ischemic postconditioning (IPostC). Thus, a new strategy against myocardial I/R injury-" ischemic postconditoning" comes into people’s vision. Experimental results have shown that IPostC has a similar protective effect to IPC.Ca2+, an important intracellular second messenger, is the key for cardiac excitation-contraction coupling. Under physiological conditions, intracellular Ca2+ homeostasis is in a balance. If the balance is broken, body will tend to die. So the Ca2+ signal is also considered as the survival and death signals. In myocardium, Ca2+ uptake ability of sarcoplasmic reticulum (SR) plays an essential role in myocardial contraction and relaxation.Studies have shown that NOS been involved in the regulation of intracellular Ca2+ homeostasis. nNOS is considered to be connected with the ryanodine receptor (RyR) in SR. The intracellular distribution suggests that nNOS may be a regulator of cardiac systolic and diastolic function. Under physiological conditions, the distribution of nNOS in SR relates closely to RyR, SERCA2a (Ca2+-ATPase of sarcoendoplasmic reticulum) and PLB (phospholamban). This will impact the myocardial systolic and diastolic function, and will also directly affect the SR’s "reservoir of calcium" capability. Some literatures indicate the presence of nNOS translocation after I/R. However, the protective mechanism of IPostC about whether or not nNOS is involved, or how it plays a role, has not been reported. In this paper, we want to explore some new clues of the protective mechanisms of IPostC against myocardial I/R injury. We established myocardial I/R model and cellular hypoxia and reoxygenation (H/R) model to explore how nNOS affect IPostC, especially the role of nNOS on SR function.Aims:To study whether or not nNOS is involved in the protection of IPostC against myocardial I/R injury, via improving the SR function; To test whether or not the distribution of nNOS is involved in the protection of IPostC against myocardial I/R injury.Methods:Mouse heart I/R injury model and cardiomyocytes H/R model were established. Cardiac function such as left ventricular developed pressure (LVDP) and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by TTC staining. Annexin V-FITC assay was used for quantification of apoptosis rate. Western blot and RT-PCR were used to measure the expression of SERCA2a, PLB and p-PLBSer16. Intracellular concentration of Ca2+([Ca2+]i) was determined by Fluo-3/AM as a fluorescent signals using confocal microscopy. Immunogold labeling was used to express the distribution of nNOS.Results:IPostC protected hearts against I/R injury by elevating the recovery of LVDP, decreasing LVEDP, and reducing the infarct size. HPostC decreased the cell apoptosis compared with H/R group. Postconditoning could raise the gene expression of SERCA2a and phosphorylation of PLBSer16, and thus increased the SERCA2a activity, reduced overloaded of intracellular calcium and improved the ability of SR Ca2+ transients. Further more, IPostC could reverse the nNOStranslocation caused by I/R injury. Administration of nNOS inhibitor--L-VNIO, could cancel the protective effects of IPostC. Conclusion:These results suggest that mediated by nNOS, IPostC improved cardiac SR function, reduced damage and apoptosis of myocardial cells, protected myocardium against I/R injury.