Alteration Of Cx43, Cx45 And Cardiac Electrophysiology In Rats With Acute Myocardial Infarction After Transplantation Of Mesenchymal Stem Cells

Background Acute myocardial infarction (AMI) is a common disease that severely threatens people's lift. Although medical ways nowadays such as coronary artery bypass surgery can establish reperfusion, necrotic myocardiums can not regenerate, which can lead to heart failure and even death. Mesenchymal stem cells (MSCs) have a potential of multi-differentiation and characters of easy-gain and remarkable amplification, which make them be the hot spot of research on the treatment of myocardial infarction.Gap junction (GJ), which is composed of connexins (Cx), is a specialized membrane structures consisting of arrays of intercellular channels that directly connect adjacent cells by providing chemical and electrical communication. Cx43 is the major gap junction protein in the adult mammalian ventricle, whereas Cx45 is exprssed at special areas in ventricle such as conducting system. In myocardial infarction, the total amount of Cx43 was reduced and the distribution of Cx43 gap junctions was markly disturbed. These changes form the anatomical basis for conduction block, anisotropy and reentry arrhythmias. Recent researches found that Cx45 may regulate the change of Cx43 in ischemic heart disease.Although MSCs can exprss myocardial sepecific proteins and Cx in vitro, they emerge many questions after the transplantation: whether they can survive in vivo and improve the function of the host? Whether they are arrhythmogenesis? Whether they can express Cx and form functional GJ with the host in vivo? What are the influences of MSCs on Cx43 and Cx45 after they are transplanted into the host? These problems need more basic researches.Our study emploied the way of transplanting MSCs, which were induced and labeled in vitro, into ischemic heart under an open-chest operation to observe the surviving time and the influences on the host myocardium, and the effect of MSCs on the alteration of cardiac electrophysiology after myocardial infarction. And, the study also observe the influences on Cx43 and Cx45 remodeling after MSCs transplantation. The study was divided into four parts.PartⅠThe purification, culture, induction and identification of allogenic MSCs in vitroObjectives To purify, culture, induce and indentify MSCs in vitro in order to investigate their potential of differentiating into myocardium.Methods The MSCs were isolated from bone marrow aspiration of neonate Wistar rats by using density gradient centrifugation with 1.073g/ml percoll cell separating medium. All cells were cultured at 37°C, under 5% CO2 atmosphere, with saturated humidity in L-DMEM supplemented with penicilin and streptomycin. MSCs were induced by 10μmol/L 5-aza and then were detected aboutα-actin, cTnT and Cx43 by immunohistochemistry and the myofilament by transmission electron microscope.Results MSCs derived from bone marrows of neonate Wistar rats by density gradient centrifugation growed well in L-DMEM. The adherent MSCs growed fast after 7 to 10 days of culture and fully spreaded after 10 to 12 days. MSCs appeared many forms such as polygon, triangle and fusiform. After 10μmol/L 5-aza induction, MSCs could differentiate into cardiac muscle cell-like cells which were capable of pulsing spontaneously, expressingα-actin, cTnT and Cx43 and forming myofilament in vitro.Conclusions MSCs is easy to purify and culture. They have the plasticity of differentiating into cardiac muscle cell-like cells which can pulse spontaneously, expressα-actin, cTnT and Cx43 and form myofilament in vitro. MSCs are ideal for the treatment of ischemic heart diseases.PartⅡResearch on the transplantation of mesenchymal stem cells for the treatment of rats with acute myocardial infarctionObjectives To establish a stable model of myocardial infarction. And to observe the alterations on the left ventricular function and pathology after transplantation of MSCs labeled in vitro.Methods Wistar rats were randomly divided into Normal group, Sham gorup, MI group and MSCs group. MSCs were induced into cardiac muscle cell-like cells by 5-aza after purification and culture and were labeled by DAPI before transplantation. MI group and MSCs group were underwent a ligation of the left anterior descending (LAD) coronary artery through a left thoracotomy. Seven days after MI, 2×106/100μl MSCs (MSCs group) were transplanted into the border and the center area of myocardial infarction. After 4, 8 and 12 weeks of transplantation, heart function of each group was measured by echocardiography. Histopathologic examination (HE stain) was used for the detection of infarcted areas. Fresh left ventricular samples were uesed to detect the migration and the distribution of MSCs.Results (1)Ligation of the left anterior descending (LAD) coronary artery was an effective way to establish an MI model. The total survival rate of MI group and MSCs group 7 days after LAD ligation was 87.8%. According to the second operation, the survival rates of MI group and MSCs group were 68.9% and 80.0% respectively. DAPI labeling rate was 100%. (2) Echocardiography showed that, LVID and LVIDs increased and EF and FS decreased significantly at 4, 8 and 12 weeks of post-transplantation in MI group. Compared to MI group, LVIDs was smaller and EF and FS were higher in MSCs group at 4, 8 and 12 weeks of post-transplantation. (3) Histopathologic examination showed a significant decrease on the infarct sizes at 8 and 12 weeks of post-transplantation in MSCs group compared to MI group. There was no statistical difference between MSCs group and MI group at 4 weeks of post-transplantation. (4) MSCs labeled by DAPI could be detected at the ischemic zone by fluorescence microscope at 4, 8 and 12 weeks of post-transplantation, but the fluorescence quenched gradually.Conclusions LAD ligation is an effective way to establish an MI model. Allogenic MSCs can suvive in the host, improve left ventricular function and decrease infarcted size after transplantation.PartⅢInfluence of MSCs transplantation on the cardiac electrophysiology in rats with acute myocardial infarctionObjectives To evaluate the influence of MSCs transplantation on the cardiac electrophysiology after myocardial infarction, and to evaluate the relationship between MSCs transplantation and arrhythmia.Methods Wistar rats were randomly divided into Normal group, Sham gorup, MI group and MSCs group. Each group was further divided into 3 subgroups according to 4, 8 and 12 weeks of post-transplantation. All subgroup were examined on ECG, ventricular effective refractory period (VERP) and ventricular fibrillation threshold (VFT) by programmed electrical stimulation in vitro and in vivo using S1S2 scanning and S1S1 stimulation. The types and amounts of ventricular tachycardia (VT) were recorded.Results (1) Compared to Sham group, PR interval, QRS duration, QT interval, QTc interval and VERP increased and VFT decreased in MI group. Compared to MI group, PR interval, QRS duration and VERP decreased and VFT increased in MSCs group. There were no statistical differences on ECG parameters, VERP and VFT among 4, 8 and 12 weeks of post-transplantation. (2) There was no statistical difference on the VT induction rate between Normal group and Sham group (8.9% vs. 11.1%). The VT induction rate in MI group was higher than Sham group (37.8% vs. 11.1%, P<0.01), but there was no statistical difference with MSCs group (37.8% vs. 26.7%).Conclusions Allogenic MSCs transplantation can alter the abnormality of electrophysiology in MI rats.PartⅣInfluence of MSCs transplantation on the remodeling of Cx43 and Cx45 in rats with acute myocardial iInfarction Objectives To investigate the alterations of Cx43 and Cx45 expressions and their distributions in MI rats after transplantation of allogenic MSCs. And to approach the relationship between Cx43/Cx45 remodeling and arrhythmia.Methods Wistar rats were randomly divided into Normal group, Sham gorup, MI group and MSCs group. Each group was further divided into 3 subgroups according to 4, 8 and 12 weeks of post-transplantation. All subgroups were examined by examinations below: (1) Fluorescence PCR. Cx43mRNA and Cx45mRNA were extracted and detected by fluorescence PCR. (2) Immunohistochemistry. Cx43 and Cx45 were immuno-colorated by DAB immuno-system and AEC immuno-system respectively. Cx43 and Cx45 were semiquantified with Image Pro Plus 6.0 image analysis system. (3) Immunofluorescence. Cx43 and Cx45 were labeled by FITC and TR respectively and then were quantified by laser scanning confocal microscopy (LSCM) examination. MSCs labeled by DAPI were also detected by LSCM. (4) Electromicroscope. Fresh left ventricular samples were fixed by LR White and labeled by colloidal gold. Electromicroscope was then used to detect the remodeling of Cx43 and Cx45 and the changes of ultrastructural organelle.Results (1) LSCM examination found that DAPI-labeled MSCs distributed widely at the host ischemic zone and expressed Cx43. (2) Fluorescence PCR, Immunohistochemistry, Immunofluorescence and immuno-electron microscope studies showed that, Cx43mRNA and Cx43 protein reduced significantly at ischemic zone at 4, 8 and 12 weeks of post-transplantation in MI group. Compared to MI group, Cx43mRNA and Cx43 protein exprssions were statistically higher in MSCs group. There was no difference of Cx43 expression at infarcted zone between MI group and MSCs group. Cx45mRNA and Cx45 protein increased significantly at ischemic zone at 4, 8 and 12 weeks of post-transplantation in MI group. Compared to MI group, Cx45mRNA and Cx45 protein expressions were statistically lower in MSCs group. There were no differences of Cx45mRNA and Cx45 protein expressions at infarcted zone between MI group and MSCs group. (3) Coexpression zones of Cx43/Cx45 could be found in ischemic areas of MI groups at 8 and 12 weeks of post-transplantation. There was no any coexpression zones of Cx43/Cx45 in normal areas of MI groups and MSCs groups at 4, 8 and 12 weeks of post-transplantation.Conclusions (1) MSCs can survive at the host ischemic zone and express Cx43. (2) Decrease of Cx43, increase of Cx45 and regional coexpression of Cx43/Cx45 result in heterogeneity of electrical communication which is facilitative to arrhythmia after AMI. (3) MSCs transplantation can modulate the electrophysiological abnormality of AMI by altering Cx43 and Cx45 remodeling.