| Myocardial infarction is the leading reason of cardiovascular death in patientswith acute coronary by sustained hypoxia caused by ischemia, leading to myocardialhypoxia, insufficient supply of nutrients, cellular energy metabolism, eventuallyleading to myocardial ischemia and necrosis. Nowadays, the most common way isreperfusion therapy such as thrombolytic therapy in patients with early onset restoreblood supply to the heart, reduce infarct size, thereby reducing mortality rate. Duringthe process of restore blood flow, some patients could occur arrhythmias, heart failureand even death. The injury due to ischemic does not improve after the recovery ofischemic cardiac dysfunction but leads to further exacerbation is myocardial ischemia/reperfusion (I/R) injury. Therefore, the study of pathogenesis and prevention ofcardiovascular disease is more and more important. Nowadays the pathogenesis of I/R injury is not fully understood and a large number of experiments show thatoxidative stress, calcium dyshomeostasis etc. are contribute to I/R injury. Theoutbreak of oxygen free radicals is one of the important reason. Reactive oxygenspecies (ROS) play an important role in cardiac hypertrophy, myocardial infarctionand myocardial ischemia-reperfusion injury and a series of cardiac dysfunctiondisease. H2O2is a kind of ROS which can freely get through the cell membrane intothe cell, so we use H2O2treated myocardial cell to simulate oxidative damage.H2O2can enter into cells and severely damaged membrane structures of the cellorganelles, releasing large amounts of calcium, leading to intracellular calciumoverload and apoptosis. Until now, calcineurin phosphatase (CaN) is the only proteinphosphatase regulated by Ca2+that is found in cells. It mainly expressed in the heart,and can regulate a variety of physiological activity. We measured CaN activity ofevery group cells, the results show that compared with normal myocardial cells, thelevel of CaN activity of H2O2damage significantly increased, S1P can reduce thelevel of H2O2damaged myocardial cells CaN activity, while S1P do not affect thelevel of ventricular myocytes CaN activity. CaN can mediate downstream signaling ofnuclear factor of activated T cells (NFAT) to regulate the physiological activity of myocardial cells. A large number of experimental studies have shown,Ca2+—CaN—NFAT signaling pathway involved in the regulation of a variety ofcardiac diseases such as cardiac hypertrophy, heart failure. L-type calcium channels(Cav1.2) is the main channel that regulate the intracellular calcium ion concentration.Sphingosine phosphate (sphingosine-1-phosphate S1P) is a bioactive sphingomyelinwhich has important physiological functions. Many studies have shown that S1P canactivate a variety of signaling pathways within cells and it has cardioprotective effect,but the protection mechanism is still not clear enough.According to early study results, we use200μmol/L H2O2to treat myocardialcells for4h to simulate the oxidative damage, we use neonatal rat ventricularmyocytes1、 In the experiment of methyl thiazolyl tetrazolium (MTT) cell viability test, wetreat normal myocardial cell and H2O2damaged myocardial cell with differentconcentration of S1P and treating time. We find S1P can improve normalventricular myocytes and H2O2injury myocardial cell viability. Calcium behavesas a second messenger cells, is very important for the normal function ofmyocardial cells. So we use Fluo3-AM to load cells and then detect thefluorescence intensity by Flow Cytometry, that is, the concentration ofintracellular calcium. We use different concentrations of S1P to treat normalventricular myocytes and H2O2damage ventricular myocytes, and found thatH2O2can significantly elevated Ca2+concentration of myocardial cells. S1P canreduce Ca2+concentration of H2O2damage ventricular cells and also reduce Ca2+concentration of normal ventricular myocytes. It has a certainconcentration-dependent manner. According to the results of our experiments incell viability and the result of flow cytometry, we selected5umol/L S1P,4h asdosing concentration and treatment time in the following study of S1P myocardialprotective effects.2、 In immunofluorescence experiments, we observed H2O2damaged myocardialcells shrinkage and compared with normal cells the size of cells is smaller that isthe typical apoptotic morphology. The calcium ion concentration of H2O2 damaged myocardial cells increased significantly, S1P treated H2O2damagedmyocardial cells’ calcium ion concentration increased slightly compared with thenormal group and the cells has normal cell morphology.3、 We used immunofluorescence method to detect the impact of S1P on theNFAT expression and translocation to the nucleus in normal cardiac cells andH2O2damaged myocardial cells. The results show that in normal ventricularmyocytes NFAT mainly exists in the cytoplasm. The NFAT expression is increasedin H2O2damaged myocardial cells, a lot of NFAT translocate into the nucleus, S1Pcan reduce the expression and translocation of NFAT of H2O2damagedmyocardial cells, but it do not affect the expression and translocation of NFAT innormal ventricular myocytes. Therefore, we believe that S1P could play acardioprotective effect of H2O2injury by inhibiting Ca2+—CaN—NFAT signalingpathway.4、 Real-time Polymerase Chain Reaction (RT-PCR)，Western Blot test resultsshowed that, H2O2can reduce expression of Cav1.2mRNA and protein, S1P canalso reduce the expression of Cav1.2mRNA and protein of normal myocardialcells and H2O2damaged myocardial cells. S1P may be regulated the number ofL-type calcium channel and thus regulate the intracellular Ca2+concentration.Our study shows that S1P can regulate the expression of L-type calcium channelprotein, decrease Ca2+concentration, then inhibit the Ca2+—CaN—NFAT signalingpathway of H2O2damaged myocardial cells. S1P plays a protective role in myocardialcells and provide new ideas for the treatment of I/R injury. |
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