The Novel Role And Mechanism Of Sphingosine-1-Phosphate In Myocardial Remodeling After Myocardial Infarction

Heart failure(HF)is a common cause of hospitalization and death of patients with cardiovascular diseases.HF is also a "fortress" that is difficult to overcome in the cardiovascular field.Myocardial infarction(MI)is the most common cause of HF.Myocardial remodeling occurs early after acute MI and can lead to HF.Sphingosine kinase(Sph K)/ sphingosine-1-phosphate(S1P)/ S1 P receptor(S1PR)signal pathway participates in a variety of signal transduction pathways and regulates a variety of cell functions.In recent years,studies have shown cardioprotective effects of the Sph K/S1P/S1 PK signaling pathway.Autophagy plays an important role in maintaining intracellular homeostasis in response to intracellular stress.Previous studies have shown that the regulation of autophagy through the relevant signal pathways improves myocardial remodeling.However,the effect of early administration of S1 P on myocardial remodeling after MI remains unclear.Little is known about whether the effect of S1 P on myocardial remodeling is mediated by cardiomyocyte autophagy and its underlying signal pathway.Accordingly,in the present study we investigated whether S1 P improves myocardial remodeling by mediating cardiomyocyte autophagy and the underlying signaling pathway.First,we determined the time course of changes in plasma S1 P and myocardial Sph K and S1 PR after MI in rats;Second,we examined whether S1 P administration improves myocardial remodeling and the alterations of autophagy early after MI in rats;Finally,we explored the role of S1 P in autophagy and the signaling pathway in H9C2 rat cardiomyocyte under mimic ischemia conditions.Objective:To examine the time course of changes in plasma S1 P and myocardial Sph K and S1 PR expression after MI;To investigate whether S1 P improves myocardial remodeling and the changes of autophagy after MI.To determine the role of S1 P in autophagy and the underlying signal pathway in cultured cardiomyocytes under mimic ischemia condition.Methods:The rat model of MI was used in this study.In the first phase,the rats were divided into 4 groups: 1 day after MI(MI-1d group);3 days after MI(MI-3d group);7 days after MI(MI-7d group);7 days after sham operation(Sham group).Cardiac structure and function were measured by echocardiography at 1,3 and 7 days after surgery.Plasma S1 P was measured by enzyme-linked immunosorbent assay,and myocardial Sph K1,Sph K2,S1PR1,S1PR2 and S1PR3 m RNA expression was measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction.In the second phase,the rats were divided into 4 groups: Sham + Placebo group;Sham + S1 P group;MI + Placebo group;MI + S1 P group.Sham and MI rats were randomized to receive S1P(50μg / kg / day,intraperitoneal injection)or placebo for 7 days.Cardiac structure and function were measured by echocardiography.Hemodynamics was measured by the multi-channel physiological recorder.Electronic analytical balance and vernier caliper were used to measure organ weight and tibial length.Infarct size(the ratio of infarct area to whole left ventricle)and myocardial fibrosis in non-infarcted areas were assessed by Masson trichrome staining.Cardiomyocyte size was measured by hematoxylin eosin staining.The expression of microtubule associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ)was evaluated by immunohistochemical staining.Autophagy and apoptosis were measured by Western blot.The H9C2 rat cardiomyocytes were cultured in serum-and glucose-deficient DMEM to mimic ischemia.The H9C2 cells were divided into 4 groups: Control group,S1 P group,Ischemia group(IS group),IS S1 P group.Control group: H9C2 cardiomyocytes were cultured in DMEM medium in the incubator for 3 hours.S1 P group:H9C2 cardiomyocytes were transferred into DMEM medium,S1P(5 m M)was added and cultured in the incubator for 3 hours.IS group: H9C2 cardiomyocytes were transferred into serum-and glucose-free medium,and cultured in the incubator for 3hours.IS S1 P group: H9C2 cardiomyocytes were transferred into serum-and glucose-free medium,S1P(5 m M)was added,and then cultured in incubator for 3 hours.Autophagy and apoptosis were measured by Western blot.Results:1.Compared with the sham operation group,there were no marked changes in end-diastolic dimension(EDD)and end-systolic dimension(ESD)at 1 and 3 days after MI.At 7 days after MI,EDD tended to increase,ESD was markedly increased,and ejection fraction(EF)and fractional shortening(FS)were significantly decreased.In the early stage after MI,the level of plasma S1 P was decreased,and the levels of S1PR1,S1PR2,S1PR3,Sph K1 and Sph K2 m RNA expression in myocardium tended to decrease,especially,the m RNA levels of S1PR1 and Sph K2 were markedly decreased.These data indicate the downregulation of Sph K / S1 P / S1 PR signal pathways in the early stage after MI.2.Compared with the sham operation group,EDD and ESD were increased and FS,EF,the maximal rate of LV pressure rise(+dp/dt)and the maximal rate of LV pressure decline and(-dp/dt)were decreased at 7 days after MI.Administration of S1 P attenuated the increases in EDD and ESD and the decreases in FS,EF,+dp/dt and-dp/dt after MI.In the MI placebo group,the ratio of whole heart weight to tibial length and the lung wet to dry weight ratio were increased,and the increases were reduced by the treatment of S1 P after MI.The results suggest that S1 P administration attenuates myocardial remodeling and cardiac dysfunction in the early stage after MI.3.Immunohistochemistry and Western blotting showed that myocardial expression of LC3 II,a marker of autophagy,was increased after MI,and S1 P treatment further increased the expression of LC3 Ⅱ after MI.The ratio of LC3 II/I,indicative of autophagy activity,was increased after MI,and increased further by S1 P treatment.In addition,S1 P administration markedly increased the expression of autophagy-related proteins Atg4 b and Atg5 after MI.The results suggest that cardiomyocyte autophagy is increased early after MI,and S1 P treatment further promotes cardiomyocyte autophagy.4.The ratio of p-AMPK / AMPK was increased early after MI,and the ratio of p-m TOR / m TOR had no change;S1P treatment further increased the p-AMPK / AMPK ratio and reduced the p-m TOR / m TOR ratio.The results suggest that S1 P induces AMPK activation and m TOR inhibition early after MI.5.Compared with the sham operation group,myocyte cross-sectional area,myocardial caspase 3 activity and myocardial fibrosis were increased in the non-infarcted myocardium after MI.The increases were attenuated by the treatment of S1 P.The results suggest that S1 P reduces cardiomyocyte hypertrophy,cardiomyocyte apoptosis and myocardial fibrosis in the non-infarcted myocardium in early after MI.6.In H9C2 cardiomyocyte in culture,under mimic ischemia conditions(serum-and glucose-free medium),compared with the control group,the ratio of LC3 Ⅱ/I,an index of autophagy,was increased,and S1 P treatment further increased the LC3 II/I ratio.S1 P treatment had no effect on P62,but induced an increase in Atg5 protein expression under mimic ischemia.The results indicate that cardiomyocyte autophagy is increased during mimic ischemia,and S1 P further increases cardiomyocyte autophagy.7.S1 P treatment further increased the p-AMPK / AMPK ratio and further decreased the p-m TOR / m TOR ratio in H9C2 cardiomyocytes under mimic ischemia conditions.The results suggest that S1 P promotes autophagy by inducing AMPK activation and m TOR inhibition in cardiomyocytes during mimic ischemia.8.Compared with the control group,the expression of cleaved caspase 3 protein,a marker of apoptosis,was increased in H9C2 cardiomyocytes under mimic ischemia conditions,and S1 P treatment alleviated the increase of cleaved caspase 3.S1 P treatment markedly increased the expression of anti-apoptotic protein Bcl-2 and reduced the expression of pro-apoptotic protein Bax.The results indicate that cardiomyocyte apoptosis is increased during mimic ischemia,and S1 P reduced cardiomyocyte apoptosis by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.Conclusion:In the present study,we have demonstrated for the first time the novel role and mechanism of S1 P in myocardial remodeling early after MI in rats and in cultured cardiomyocytes.S1 P increases myocyte autophagy through activation of AMPK and inhibition of m TOR,leading to attenuation of myocardial remodeling and improvement of cardiac function after MI.The role of S1 P on myocyte autophagy may provide a novel theoretical basis for the prevention and treatment of myocardial remodeling after MI.