The Protecting Effects Of Sphingosine 1-phosphate On The Myocardium And It's Electrophysiological Mechanisms During The Acute Myocardial Infarction

S1P is a immediated metobolites which produced from SM by sphingomyelinase. In addition, platelate is an important site where SIP synthesize and releases. S1P receptors is high homologous families of G protein coupling receptors. SIP1-SIP5 were subgrouped. S1P could show multiple bio-effects by introduction of specific receptors and coresponding signal transduction pathway. These bio-effects include inhibiton of cellular apoptosis, induction of growth and transmigration vascular endothelial cell, regulation of blood vessles development, regulation of immune response, promotion of skeletal system growth, and also concerns with growth and transmigration of tumor. It has been paid attention by people gradully for its involement of some physiolocal and pathologcal progress.Activation of phospholipase becomes more stronger at the time infarction. The amount of S1P becomes larger after degradation of membrane phopholipid. At the mean while, since the activation of agglutination platelet locally, the S1P would be realease more form platelet. It would lead to the concentration of SIP increasing at plasma and storing at myocardium locally. While S1P receptor distributed at the myocardium could bind with SIP tightly. Thus SIP is supposed to bind to it's own receptor then affect the process of infarction. However, less report about S1P function at the time of acute infarction has been published. Especially, the function and mechanism of S1P affect cardiocyte electriphysiologcial features during the period of acute infarction. Electriphysiological technique, surface electrocardiogram were applied. Parameters of cardiocyte action potential and membrane current of ion channels were determined as some indexes. The function and mechanism of S1P would be stuied on electriphysiological features of cardiocyte at the time of acute infarction. In order to reveal the effects of this substance on progress of myocardium infarction.I The effects of S1P on arrhythmogenesis of acute myocardium infarction. Surface electrocardiogram of rat was used to be studied. Proceed with administration by injection from tongue vein on the groups of control, S1P and S1P plus suramin 5min, acute myocardium infarction model were prepared. The dose to each group is 50μl respectively. The results has been shown that S1P could delay the VP(p﹤0.05) of the model of acute myocardium infarction and shorten the lasting time of VT(p﹤0.01)compared to controls. While the group of SIP plus suramin show no significant difference compared with control group(p﹥0.05). It has been suggested that SIP reduce the ratio of ventricular arrhythmia on the progress of acute infarction. The function is protective and it's function may couple with G-protein.II The protective function of S1P on ischamia reperfusion arrhythmia in rat. Surface electrocardiogram of rat was used to be studied. Proceed with administration by injection from tongue vein on the groups of control, S1P and S1P plus suramin 5min, acute myocardium infarction model were prepared. The dose to each group is 50μl respectively. The results has been shown that S1P could delay the VP(p﹤0.05) of the model of acute myocardium infarction and shorten the lasting time of VT(p﹤0.01)compared to controls. While the group of SIP plus suramin show no significant difference compared with control group(p﹥0.05). It has been suggested that SIP reduce the ratio of ventricular arrhythmia on the progress of acute infarction. The function is protective and it's function may couple with G-protein.III The effects of S1P on action potential of ventricular myocyte in guinea pig The action potential columna papillares was recorded by means of standard capillary glass microelectrode. Action potential was decreased from 74.84±5.55mV to 49.78±9.4mV(p﹤0.01 n=6)after treating with the concentration of S1P(2.2μmol/L). Action potential was decreased from 72.01±6.80mV to 59.11±5.38mV(p﹤0.01 n=6)after treating with the concentration of S1P(1.1μmol/L). While action potential showed no significant difference after treating with the concentration of S1P(0.1μmol/L)compared to the controls. ADD50 and APD90 were prolonged from 226.2±16.98 mS and 267.75±16.08 mS respectively to 261.33±24.46 mS and 301.25±25.94 mS respectively after treating with the concentration of S1P(2.2μmol/L). ADD50 and APD90 were prolonged from 214.55±19.83 mS and 239.33±16.95 mS respectively to 242.03±20.43 mS and 264.18±20.52 mS(p﹤0.01 n=6)respectively after treating with the concentration of S1P(2.2μmol/L). While action potential showed no significant difference(p﹥0.05 n=6) after treating with the concentration of S1P(0.1μmol/L)compared to the controls.APA was (69.75±2.92)mV before administration in the group of S1P plus suramin. And was (70.37±3.01)mV(P>0.05 n=6)after administration. APD90 was (179.36±2.86) ms before administration in the group of S1P plus suramin. And was (179.33±2.40)ms (P>0.05 n=6)after administration. No significant difference has been noted (P>0.05 n=6). It has been suggested that S1P reduce action potential APA and prolong phase of action potential. In addition, the effects showed dose-dependence and it may concern with coupling with G-protein.IV Effects of S1P on delayed rectifier potassium current of ventricular myocyte in guinea pigWhole cell patch clamp technique was applied to study the delayed rectifier potassium current of ventricular myocyte in guinea pig. The holding potential was set at -40mV. 10mV was stage jump. From -40mV depolarizing to +50mV, a group of IK value was obtained. Making diagram of current versus tested voltage, I-V curve of IK was obtained. It has been shown that the change of IK has significant difference at the groups of the concentration of S1P(1.1μmol/L) and 2.2μmol/L compared to control group. Under the appointed voltage of 50mV, IK of S1P(1.1μmol/L)decreased from (1.2±0.26)nA to (0.95±0.23)nA. While IK of S1P(2.2μmol/L)decreased from (1.43±0.31)nA to (1.02±0.28)nA. There is significant difference compared to control group( P < 0.01 n=6). The IK peak value was decreased from(1.29±0.26)nA to(1.26±0.37)nA at the group of S1P (1.1μmol/L)plus suramin (200μmol/L) and show no significant difference compared to control group. The above results suggested that S1P inhibit delayed rectifier potassium current of ventricular myocyte in guinea pig remarkably.It's mechanism may concern coupling of G-protein and S1P receptor. And correlation with those of effects on action potential.V Effects of S1P on inward rectifier potassium current(IK1) of ventricul- ar myocyte in guinea pigWhole cell patch clamp technique was applied to study the inward rectifier potassium current of ventricular myocyte in guinea pig. The holding potential was set at -40mV. From -100mV depolarizing to +30mV,10mV was stage jump and last 300ms appointed voltage. a group of IK 1 value was obtained without change along the time. The IK1 showed features of inward rectifier. It has been shown that the change of IK has insignificant difference at the groups of the concentration of S1P(1.1μmol/L) and 2.2μmol/L compared to control group. IK1 of S1P(1.1μmol/L)decreased from (-8.94±2.01)nA to (-8.81±1.55)nA. While IK of S1P(2.2μmol/L)decreased from (-8.86±1.59)nA to (-8.55±1.39) nA. There is no significant difference compared to control group( P >0.05,n=6). The above results suggested that S1P show no effects on delayed rectifier potassium current of ventricular myocyte in guinea pigVI Effects of S1P on ATP- sensitive potassium channel current(IKATP) of ventricular myocyte in guinea pigWhole cell patch clamp technique was applied to study the ATP- sensitive potassium channel(IKATP)of ventricular myocyte in guinea pig. The holding potential was keep at -40mV to inactivate sodium channel. Testing potential was from -100mV to +100mV. The order of stage jump was set at 10mV and phase at 300ms. After 30min of mimic of ischemia perfusion, IKATP current was recorded. The results show that the current peak value increased from (5.47±0.68)nA and (5.18±0.32)nA to (5.51±0.53)nA and (5.24±0.45)nA respectively at the concentration of S1P(1.1μmol/L)and S1P(2.2μmol/L). There is no significant difference compared to control group(p>0.05 n=6). The above results suggested that S1P show no effects on ATP- sensitive potassium channel current of ventricular myocyte in guinea pigVI Effects of S1P on the rapidly activating component of delayed rectifier potassium current(IKr) of ventricular myocyte in guinea pigWhole cell patch clamp technique was applied to study the rapidly activating component of delayed rectifier potassium current(IKr) of ventricular myocyte in guinea pig. The holding potential was keep at -80mV. Depolarization pulse was put from -100mV to +100mV and phase 225 mS to record Ikr current firstly. I(kr.tail) current was recorded by depolarization pulse from 0mV to -20mV during the process of repolarization. Ikr and I(kr.tail) were inhibit significantly compared to control group after adding S1P( 1.1μmol/L). These two index are decreased from (0.85±0.53)nA and (0.65±0.40)nA to (0.63±0.37)nA and(0.56±0.29)nA(p<0.05 n=6)respectively. While Ikr and I(kr.tail) were inhibit no significantly compared to control group after adding S1P ( 1.1μmol/L)plus Suramin(200μmol/L). These two index are decreased from (0.87±0.37)nA and (0.68±0.40)nA to (0.85±0.41)nA and(0.71±0.43)nA(p<0.05 n=6)respectively. The above results has been suggested that S1P inhibit significantly the rapidly activating component of delayed rectifier K+ current(IKr) of ventricular myocyte in guinea pig. The mechanism may concern with coupling G-protein receptor.VIII Effects of S1P on the slowly activating component of delayed rectifier potassium current(IKs) of ventricular myocyte in guinea pigWhole cell patch clamp technique was applied to study the slowly activating component of delayed rectifier potassium current(IKs) of ventricular myocyte in guinea pig. Chlorpheniramine (1.0μmol/L)was applied to block rapid activating component of delayed rectifier K+ current (Ikr). The holding potential was keep at -50mV. Appointed potential was from -50mV to +60mV. The order of stage jump was set at 10mV to record IKs and tail current.The results show that IKs and tail current value decreased from (1.53±0.61)nA and (0.82±0.34)nA to (1.47±0.46)nA and (0.79±0.41)nA respectively at the concentration of S1P(2.2μmol/L). There is no significant difference compared to control group(p>0.05 n=6). The above results suggested that S1P show no effects on the slowly activating component of delayed rectifier K+ current(IKs) of ventricular myocyte in guinea pigIX The effects S1P on endogenous NO generating system of myocardium after ischemia reperfusionIn vivo rat model on ischemia reperfusion was applied. The rats were subdivided into 3 groups. And they were control group, S1P group (1.1μmol/L)and SIP( 1.1μmol/L) plus Suramin(200μmol/L)group. After 20 min of ischemia reperfusion on myocardium, the myocardium tissue were stored at -70. Nitrate reductase method was applied to determine the contents of NO. The results show that the contents of NO at the concentration of S1P( 1.1μmol/L)increased to(17.94±2.28)μmol/g.p ro ( P < 0.01n=8) significantly compared to compare control ((13.68±3.21)μmol/g.p ro.) While the S1P plus suramin group ((12.73±2.83)μmol/g.p ro )show no significant compared to control group. The above results suggested that S1P promote NO synthesis on myocardium significantly and show protective function to myocardium.The above results show that S1P increase ratio of ventricular arrhythmia at the time of myocardium infarction and this is protective to myocardium. It's mechanism are the following: 1. S1P could prolong the phase of action potential and decrease the degree of action potential of musculi papillares in guinea pig and result in low heart rate in model of ischemia myocardium, decreased ratio of arrhythmia caused by ischemia reperfusion. 2. S1P could decrease the value of IK of ventricular cardiocyte and result in decreased ratio of arrhythmia caused by ischemia reperfusion. 3. S1P could decrease the value of Ikr which is the target site for Sotalol,E-4031,Dofetilide belong to the III subclass of the anti arrhythmia. 4. S1P could increase NO contents of ischemia myocardium and lead to decreased ratio of arrhythmia caused by ischemia reperfusion.