The Experimental Study On The Role Of Sema4D In Myocardial Infarction And Its Clinical Significance

Objective:Myocardial infarction is one of the severe acute clinical events commonly causedby the rupture of coronary atherosclerotic plaques and is one of the major factors thatcause the death of patients. Although increasing effort has been make in its earlydiagnosis, monitoring and treatment, the mortality rate still remains high and has been adifficult task to the majority of clinical and basic research workers. Our previous studyhas shown that Sema4D is involved in platelet activation and thrombus formationduring the development of atherosclerosis, suggesting that Sema4D may also beinvolved in the pathophysiology of myocardial infarction. Therefore, using the in vivoand in vitro experimental methods, we measured the plasma soluble Sema4D level ofthe patients with myocardial infarction and explored the mechanisms by which Sema4Dplays a role in the process of myocardial infarction in order to provide a theoreticalbasis to the pathogenesis of myocardial infarction and to develop a novel approachforthe early diagnosis of myocardial infarction.Method:We purified the recombinant protein of Sema4D extracellular fragment. Usingantibodies (#3and#4) against Sema4D extracellular domain, we have established asandwich ELISA method and detected the plasma soluble Sema4D level of myocardialinfarction patients.To study potential mechanism by which Sema4D participates thedevelopment of myocardial infarction, we established a modified mouse model ofmyocardial ischemia and reperfusion injury. Randomly grouped8-week-old maleSema4D-/-mice and wild-type mice with the similar size were operated for the leftanterior coronary artery ligation for45min. After reperfusion, mice were re-anesthetizedand perfused with0.9%saline through the abdominal aorta followed by injection ofTTC (2,3,5-triphenyltetrazolium chloride)(Sigma) for over7min to stain infarct area. To delineate the area of risk (AAR), the coronary ligature was retied and Evans bluewas injected. The atrium was removed and the heart was frozen and sectioned into7equal slices. The slices of heart were weighed and fixed in10%formalin. The viablemyocardium was stained red and the infarcts remained a pale white. The sections oneach side of slice were digitally photographed.The size of the infarct and AAR wasdetermined by weight and the infarct size was expressed as a percentage of the AAR.To examine whether deletion of Sema4D affect cardiac function, echocardiographywere performed for the two groups of mice before surgery and at day28ofpostoperation. Immunohistochemistry was performed to detect the microvascularthrombosis in the two groups of mice with ischemia for45minutes and reperfusion for2,15,30, and60minutes.Blood were detected by flow cytometry for the interaction between platelets andleukocytes and Western blot was used to examine platelet Syk phosphorylation andreal-Time PCR was performed to detect the expression of Sema4D and it’s receptors inmouse myocardium.Finally, ROS generation, apoptosis, and Ca2+content in myocardiumaftermyocardial ischemia and reperfusion were examined.Result:1. Using ELISA assay, we detected the content of plasma soluble Sema4D in healthdonor and patients with myocardial infarction. Results showed that the plasmasoluble Sema4D level in myocardial infarction patients were significantly higherthan health donors (10.980±6.150ng/ml，n=87vs.5.183±3.301ng/ml，n=53，P<0.0001), implying that Sema4D may be involved in the process of myocardialinfarction.2. A modified mouse model of myocardial ischemia and reperfusion injury wassuccessfully established for the mechanism study.3. Infarct size after myocardial ischemia and reperfusion injury in two groups wereperformed and the results showed that Sema4D knockout significantly reduced theinfarct size (10.7%，n=7vs.30.2%，n=9，P<0.01).4. Echocardiographic examination before and at the day28post-operation showed thatSema4D knockout improves mouce cardiac function after myocardial ischemia andreperfusion injury (LVID,s，P<0.05；LVID,d，P<0.05；LVEF，P<0.05；LVFS%， P<0.05).5. Immunohistochemistry showed that Sema4D knockout effectively inhibits mousecardiac microvascular thrombosis after myocardial ischemia-reperfusion injury.6. Flow cytometry showed that Sema4D knockout can reduce the interaction betweenplatelets and leukocytes after myocardial ischemia-reperfusion (P<0.01).7. Sema4D and its receptors, plexin B1and plexin B2are expressed in heart asconfirmed by Real-Time PCR.8. Sema4D knockout reduces ROS generation in myocardial ischemia and reperfusioninjury (P<0.01).9. Sema4D knockout increases the intramyocardial Ca2+content compared towild-type mice (P<0.05).Our study showed that Sema4D is involved in the pathophysiology of myocardialinfarction. The mechanism by which Sema4D participates myocardial infarction mayinvolve two pathways. One of the pathways is the non-myocardium-dependent pathwaythat involves the decreased thrombus formation, the decreased Syk phosphorylation,and the attenuated interaction of platelets and leucocytes in sema4D knockout mice. Theother potential mechanism may be the myocardium–dependent signaling pathway as weshowed that Sema4D and its receptors are expressed in heart and deletion of Sema4Dgene reduced apoptosis during myocardial ischemia reperfusion injury. Our study on therole of Sema4D in myocardial infarction has provided an additional mechanism for thedevelopment of myocardial ischemia and further study will facilitate our understandingof the pathophysiology of myocardial infarction.