| 1. Research backgroundVentricular remodeling is a key event during chronic heart failure(HF) after acute myocardial infarction(AMI). Ventricular remodeling is not only due to the apoptosis and hypertrophy of the myocardial cells, but also due to the formation of fibrous tissue. The specific mechanisms regulating fibrosis development is not fully understood.Studies suggest a relationship between sphingosine-1-phosphate(S1P) and myocardial ischemia/reperfusion, coronary heart disease, and AMI. To date, three different types of S1P1 receptors-S1P1, S1P2 and S1P3, have been reported in cardiac tissue. The study has found that S1 P has a protective effect on myocardial cells under anoxic conditions, and vitro experiments have confirmed that this protective effect on myocardial cells can be achieved through S1P1.Current studies have demonstrated the role of the S1P1 signaling pathway in the regulation of myocardial inflammation reactions post myocardial infarction. In addition, in vivo and in vitro studies have confirmed that S1P2 and S1P3 signals are involved in cardiac fibrosis.After a literature survey, we found that S1 P plays a dual role in promoting cell survival and exacerbating cardiac myocardial fibrosis through its receptors. Thus, we conjectured that in the normal myocardial tissue, the expression of S1 P receptors is in a balanced state, which is involved in the maintenance of normal form and functioning of the heart. However, several questions in the field have not be adequately addressed and remain controversial. These include whether there is an imbalanced expression of S1P1, S1P2 and S1P3 in ventricular remodeling after AMI? Are the trends of their expression in the infarcted and non-infarcted areas of heart consistent? Can ventricular remodeling be restrained by regulating S1 P receptors? The change in the expression of these three receptors, especially in the infarcted area, has not been studied yet. The purpose of this study will be to address these three questions mentioned above and to provide a new idea for improving the prognosis of AMI patients through the intervention of the S1P1 and its downstream signals in vivo.2. Research purpose1) To investigate changes in the expression of S1P1, S1P2 and S1P3 in the infarcted and non-infarcted areas after AMI.2) To investigate the effects of intervening S1 P receptor after AMI on ventricular remodeling and cardiac function.3. Research methods(1) Experiment one: Select the male SD rats(220~250 g), divide them randomly into the sham operation group and myocardial infarction group. Model the heart failure after myocardial infarction by ligating the left coronary artery. After 8-weeks of operation, verify the reliability of this model by HE staining, Masson staining, Brain Natriuretic Peptide(BNP) detection, ECG and cardiac function measurement.(2) Experiment two: Divide the experimental animals into 4 groups: the random normal control group and 1, 4, and 8-weeks groups after myocardial infarction and model them according to Experiment 1. Detect the m RNA and protein expression of S1P1, S1P2 and S1P3 in the heart tissue from all the above mentioned groups using real-time fluorescence quantitative polymerase chain reaction(q RT-PCR) and enzyme-linked immuno sorbent assay(ELISA).(3) Experiment three: Divide the experimental animals into 4 groups: random sham operation group(Sham), myocardial infarction group(AMI), sham operation group+SEW2871(Sham+SEW2871), and myocardial infarction group+SEW2871(AMI+SEW2871). The Sham+SEW2871 group and AMI+SEW2871 group were fed S1P1 agonist SEW2871 until killed(dose: 7 mg/kg/ day). Eight weeks after operation, cardiac function was measured by echocardiography, myocardial microcirculation perfusion was detected by Laser Doppler, change in serum BNP(a sign of heart-failure) was detected by ELISA, and collagen volume fraction was evaluated by Masson staining.(4) Statistical methods: Data were represented as mean ± standard deviation(Mean + SD) and analyzed by Graph Pad Prism 6.02 software. T-test was performed as single factor analysis for the two groups and variance analysis for multi groups. The data was considered as statistical different when the P-value<0.05.4. Research results(1) Experiment 1(1) Model of the left coronary artery showed higher survival rate in rats, 75% and 72.5% in sham operated group and myocardial infarction groups, respectively.(2) Electrocardiogram in the MI group showed a rise in the J-point, up to 0.1 mv and ST segment to 0.2 mv in lead I, II, a VL after 5 minutes of the surgery.The sham group showed no changes.(3) Cardiac ultrasound examination showed that 8 weeks after the operation, left ventricular fractional shortening(LVFS) of myocardial infarction group was 29.20%±1.75% and the sham group was 33.17%±1.32%, and the left ventricular ejection fraction(LVEF) of myocardial infarction group was 63.24%±1.88% and the sham group was 71.12%±0.79%. Both, the LVFS and LVEF in MI group were lower than those in sham operation group(P<0.05).(4) HE staining showed that the infarction ventricular wall became thinner, the muscle fibers were replaced by loose connective tissue, residual muscle cells and fibroblast proliferated and the vessels proliferated and congested significantly.(5) Masson staining showed the fibrous scar formation between the remnant myocardium and alternated each other.(6) The serum BNP levels were 648.44±9.37ng/m L and 541.65±33.34 ng/m L in the MI group and sham operation group respectively, after 8 weeks of surgery. It is significantly higher in the MI group than the sham group(P<0.05).(2) Experiment 2(1) The aim of the experiment was to detect m RNA and protein expression of S1P1 after 1, 4, and 8 weeks of surgery. As compared to the normal control group, we found that the expression level of S1P1 m RNA and protein in the infarct and non-infarct zones decreased after 1 and 4 weeks of surgery(P<0.05).(2) There was no significant difference in S1P2 m RNA expression between the control group and the MI group at different time points in the non-infarcted area. In contrast, its expression was significantly in the infarction area after 1 week of surgery and the trend continues up to 4 weeks(P<0.05). In non-infarct zone, the protein expression of S1P2 was decreased in 1- and 4-week groups while in 8 weeks group, it increased inversely. The difference were statistically significant between the two groups(P<0.05). There was no difference in S1P2 protein expression in infarcted area between different groups(P>0.05).(3) S1P3 m RNA expression in non-infarct region significantly increased after 1 week of the infarction and the tendency continued to the fourth week. 8 weeks after the operation, its expression was reduced to normal level. Similar trend was observed in the infarct area. The expression of S1P3 protein was up-regulated in both, the infarct area and the non-infarct area(P<0.05).(3) Experiment 3(1) Eight weeks after surgery, cardiac Doppler ultrasound measurement results showed that as compared with AMI group, AMI+SEW2871 group’s LVFS and LVEF both increased. The LVFS index of AMI+SEW2871 group was 32.12%±0.89% and that of AMI group was 29.65%±1.49%(P<0.05). The LVEF index of AMI+SEW2871 group was 66.32%±1.591% and AMI group was 62.32%±1.689%(P<0.05).(2) Eight weeks after surgery, heart surface blood flow of each rat was scanned by the Laser Doppler Flowmetry and the outcome showed that the red and yellow signals in AMI+SEW2871 group was significantly higher than AMI group but the blue signal was reduced. The perfusion units(PU) were 1015±96.17 in sham, 688.9± 70.19 in AMI, 985.4±46.47 in Sham+SEW2871 group and 823.2±110.8 in AMI+SEW2871 group.(3) Eight weeks after surgery, serum BNP level was 638.2±38.99 ng/m L in the AMI+SEW2871 group and 650.7±34.06 ng/m L in the AMI group, however this difference was not statistically significant(P>0.05).(4) Eight weeks after surgery, by Masson staining and image analysis, we calculated the collagen volume fraction(%). In AMI+SEW2871 group it was 34.33% ±4.43% in the infarcted zone and 16.83%±2.17% in the non-infarct zone. In the AMI group, the figure was 40.80%±6.92% in the infarcted zone and 20.61%±2.71% in the non-infarct zone. The collagen volume fraction in infarct area and non-infarct area of AMI+SEW2871 group were lower than that of AMI group, and in the infarct area there was significant difference between the two groups(P<0.05).5. Conclusions(1) This study is first to report the changes in the expression of three S1P receptors-S1P1, S1P2, and S1P3, in chronic heart failure after myocardial infarction.(2) We extended the study of S1 P receptor to the myocardial infarction area. The theory that the imbalanced expression of S1P1, S1P2, and S1P3 co-participated in the regulatory network of the ventricular remodeling stage after AMI was further(3) Through the intervention experiment, we found that ventricular remodeling and cardiac dysfunction after AMI in rats can be improved by up-regulating S1P1 expression. This study showed that S1 P receptor plays an important role in ventricular remodeling after AMI and it has potential therapeutic value to enhance the S1P1 signal. |
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