The Impact Of Sphingosine Phosphate On L-type Calcium Current In Hydrogen Peroxide （H₂O₂） Injury Myocardial Cell

Even though the medical technology is highly developed now,cardiovascular disease（CVD）remains endanger human life and health with its high morbidity and mortality.Myocardial infarction（MI）caused by coronary artery occlusion is the most common cardiovascular disease,it is a fatal and acute disease of the cardiovascular system.Early reperfusion of the ischemic myocardium is the most effective intervention for restoring cardiac function in the event of acutemyocardial infarction（AMI）,however,it is invariably accompanied by reperfusion induced cellular damage and mechanical/electrical impairment of ventricular function.It is called myocardial ischemia/reperfusion（I/R）injury.I/R injury is a complex process involving numerous mechanisms,including release of reactive oxygen species（ROS）,cytosolic and mitochondrial Ca2+ overload,acute inflammatory response,and shift in substrate use.Overproduction of reactive oxygen species may cause oxidative damage and lead to cell apoptosis.Calcium homeostasis has an important effect on maintaining normal activities of myocardial cell.The changes of Ca2+ distribution and concentration will directly affect electrophysiological properties of cardiomyocytes.L-type calcium channel（LTCC）on the cell membrane plays an important role in mediating calcium influx.Sphingosine-1-phosphate（S1P）is a bioactive sphingolipid metabolite that circulates flow in the body.In recent years,S1 P was found to play a protective role in cardiovascular disease,acting as an endogenous metabolites.A large number of experimental studies have found that exogenous S1 P can reduce the infarct size of ischemia/reperfusion model and protect the heart.Our laboratory pre-study found that S1 P can enhance the viability of damaged cells and reduce the calcium concentration on the cellular level.In order to further explore the protection of S1 P against injury of myocardial cells and the role of L-type calcium channel in reducing calcium ion concentration,we subject in primary cultured neonatal rat ventricular myocytes which were treated withhydrogen peroxide,discussion the injuryed myocardial cell’s morphology,ROS generation,apoptotic rate,I_Ca-Land channel dynamic influence affected by S1 P.The results are as follows:1、Using the inverted microscope,the result showed that,compared with the control group,the group of H₂O₂-injured myocytes gradually shrinking,and away from the surface of the cell culture plate,floating in pieces,cell death and cell morphology was significantly damaged.After treated with 1μmol/L 、 5μmol/L 、10μmol/L of S1 P,cell morphology is significantly improved,5μmol/L S1 P group is the best.2、Using DCFH-DA incubate cell to detect the influence of S1 P on the ROS generation of H₂O₂-injured myocytes.The flow cytometry analysis results showed,compared with the control group（mean fluorescent intensity,MFI=10.5%）,the generation of ROS in H₂O₂-injured myocytes was significantly increased（MFI=33.7%,P<0.01）.Treated with 1μmol/L（MFI=24.2%,P<0.05）、5μmol/L（MFI=17.6%,P<0.01）、10μmol/L（MFI=22.0%,P<0.01）of S1 P can significantly reduced intracellular ROS generation.The most significant decrease of ROS was observed in 5μmol/L S1 P group,tthe result of fluorescence microscopy is same with it.3、Using flow cytometry to detect cell apoptosis rate.The result showed that,compared with the control group（apoptotic rate,AR=10.2%）,the cell apoptosis rate of H₂O₂-injured myocytes group is significantly increased（AR=43%,P<0.01）.Treated with 1μmol/L（AR=27.3%,P<0.01）、5μmol/L（AR=17.1%,P<0.01）、10μmol/L（AR=24.1%,P<0.01）of S1 P,cell apoptosis rate decreased to varying degrees,5μmol/L S1 P group is the most significant reduction in cell apoptosis.4、Using whole cell patch clamp technique to detect the effect of 5μmol/L S1 P on I_Ca-L of oxidative injured ventricular myocytes.The result showed that,compared with the control group,I_Ca-L was no significant change in group of H₂O₂-injured myocytes（P>0.05）,treated with S1 P can reduce the I_Ca-L（P<0.01）..5、Using patch clamp technique to detect the effect of 5μmol/L S1 P on L-type calcium single-channel.The result showed that,compared with the control group,there is no significant change in open time 、 closed time and open probability of single-channel in group of H₂O₂-injured myocytes.treated with S1 P can extension the closed time（P<0.05）,shorten open time（P<0.05）and decrease open probability（P<0.01）.In conclusion,S1 P can improve the H₂O₂-injured myocytes morphology,decrease intracellular R0 S generation,decrease cell apoptosis rate,plays a protective role on H₂O₂-injured myocytes.In the meantime,S1 P can decrease the H₂O₂-injured myocytes I_Ca-L by extension the closed time,shorten open time and decrease open probability of L-type calcium single-channel.