| Transplantation of bone marrow mesenchymal stem cells (BMSCs) to repair myocardial tissue after acute myocardial infarction is currently the focus of medical research. Animal experiments and clinical studies have found that BMSCs transplantation can inhibit ventricular remodeling and improve cardiac function after acute myocardial infarction. However, Some problems, at present,such as the survival of transplanted BMSCs in vivo ,the degree of BMSCs differentiation into myocardial cells, the connection between new cardiomyocytes and myocardial tissue and BMSCs transplantation in the treatment of ischemic heart failure by transplanting cells into myocardial cells or cell paracrine effects, are not entirely clear. Ryanodine receptor (RyR2) are the major calcium release channel in the sarcoplasmic reticulum. It has an important role in the excitement - contraction coupling .The present study suggests that FKBP12.6 is critical in the regulation of RyR2. combined with FKBP12.6, RyR2 are in the Stable closed state. When dissociation of FKBP12.6 and RyR2, RyR2 are in the open state , Therefore, calcium in the sarcoplasmic reticulum release into the cytoplasm. In addition, FKBP12.6 is also involved in the regulation of adjacent RyR2 synchronous opening and closing. Study found that RyR2 and FKBP12.6 expression decreased in heart failure. Abnormal structure and function of RyR2 increased calcium leakage. Sarcoplasmic reticulum stored and released calcium reduce. So that myocardial contractility decreased and cardiac function reduced. In addition, reduction in the number of RyR2 led to further reduction of sarcoplasmic reticulum calcium release in systole and further deterioration of heart failure. Real-time quantitative PCR is currently the new molecular biology methods. It's higher detection accuracy than the semi-quantitative PCR.Therefore, In our experiments, transplantation of marrow stromal cells repaired myocardium after acute myocardial infarction, RyR2 and FKBP12.6 mRNA were detected by Real-time quantitative PCR, to Study the mechanisms of BMSCs transplantation in the treatment of ischemic heart failure.Objective:BMSCs were isolated by density gradient centrifugation and adherence method.to observe the changes of RyR2 and FKBP12.6 mRNA expression in rabbits after autologous BMSCs transplantation after acute myocardial infarction and the ultrastructure of BMSCs induced by 5-azacytidine and cultured in vitro for 4 weeks.To study the mechanisms of BMSCs transplantation in the treatment of ischemic heart failure.Methods:Thirty New Zealand rabbits were randomly divided into three groups:control group(only thoracotomy),Acute myocardial infarction group(thoracotomy and ligation of left anterior descending coronary artery)and BMSCs group(ligation of left anterior descending coronary artery and BMSCs transplantation).The models of acute myocardial infarction were produced by ligation of left anterior descending coronary artery of the rabbits.Rabbit's BMSCs were isolated by density gradient centrifugation and adherence method. Before implantation,the BMSCs were induced by 5-azacytidine for 24 hours and labeled with DAPI.10 days after the rabbit models of AMI were produced,the 1.0×107 BMSCs were injected directly into 4 locations of the ischemic myocardial area in BMSCs group with a micro syringe. The same 600μl volume of cultural medium was injected into those in AMI group.4 weeks after BMSCs transplantation, RyR2 and FKBP12.6 mRNA in the infarct border zone of myocardial were determined by real time quantitative RT-PCR. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography. The morphology of BMSCs was Observed by inverted microscope.The labeled BMSCs were observed by fluorescence microscope. The ultrastructure of the BMSCs induced by 5-azacytidine and cultured for 4 weeks in vitro was observed by transmission electron microscopy.Data were analyzed by SPSS 13.0 statistical software. All values were expressed as mean±standard deviation, The data differences between three groups were evaluated by analysis of one-way ANOVA with LSD test,The correlation was tested by pearson correlation analysis. Values of p<0.05 were considered statistically significant.Results : The BMSCs which were isolated by density gradient centrifugation and adherence method showed morphological homogeneity and were high purity. After BMSCs were induced by 5-azacytidine ,their form were longer, their volume were increased, their reproduction reduced. About 14 days, The multinucleated cells could be seen. The characteristics of BMSCs and cardiac myocytes could be observed from the BMSCs which were induced by 5-azacytidine and cultured for 4 weeks in vitro by transmission electron microscopy. The structure of myofilament could be observed, However, sarcomere structure was not found. Before the AMI, the left ventricular ejection fraction (LVEF) of rabbits between the three groups were no significant difference(F=0.403,P=0.673). 7 days after the AMI, the LVEF of the rabbits in the control group was 67.88±3.91% , the LVEF of the rabbits in the AMI group was 53.63±3.38% and the LVEF of the rabbits in the BMSCs group was 50±3.51%.The differences of the LVEF of the rabbits between the three groups were statistically significant(F=48.836 P=0.0001), The difference of the LVEF of the rabbits between in the AMI group and in the BMSCs group was not statistically significant. 4 weeks after BMSCs transplantation, The LVEF of the rabbits in the BMSCs group improved significantly;The labeled BMSCs could be observed in the myocardium of the rabbits in the BMSCs group by fluorescence microscopy;The real-time fluorescence quantitative PCR amplification and melting curves showed that there were GAPDH, RyR2 and FKBP12.6 mRNA in myocardial tissue;RyR2 mRNA in the infarct border zone of myocardial in the AMI group decreased 1.97 times Compared with in the control group;FKBP12.6 mRNA in the infarct border zone of myocardial in the AMI group decreased 4.41 times Compared with in the control group;RyR2 mRNA in the infarct border zone of myocardial in the BMSCs group increased 1.81 times Compared with in the AMI group; FKBP12.6 mRNA in the infarct border zone of myocardial in the BMSCs group increased 2.75 times Compared with in the AMI group.LVEF was positively correlated with RyR2 and FKBP12.6 mRNA, Their coefficient of determination (R2) were 0.679 and 0.606.Conclusion:The BMSCs who were induced by 5-azacytidine and cultured in vitro for 4 weeks had the characteristics of differentiation toward cardiomyocytes. Cardiac function was positively correlated with the expression of FKBP12.6 and RyR2. The Downregulation of FKBP12.6 and RyR2 was one of the factors that affect the heart function to reduce. The expression of RyR2 and FKBP12.6 increased to improve the heart function, by paracrine role,was one of the mechanisms of BMSCs transplantation in the treatment of ischemic heart failure .
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