| OBJECTIVE:The risk of ventricular arrhythmia significantly increased post skeletal myoblast (SKMs) intramyocardial transplantation in spite of improving cardiac function.The exact mechanism may be related to the fact that mutual skeletal muscle cells are electrically isolated after differentiation.However, un-differentiated SKMs and cardiomyocytes are coupled by functional gap junctions in vitro. Studies have shown differentiation and proliferation of graft cells were affected by the ventricular remodeling after MI. Hence, we hypothesized that the arrhythmogenic effect of intracardiac SKMs transplantation may be related to the differentiation state of SKMs. Improves ventricular remodeling can improve the microenvironment of cells after transplantation,which can result in reduction of arrhythmias post SKMs transplantation. MicroRNA-181(miR-181) could downregulate the homeobox protein All (Hox-All, a repressor of the differentiation process) and affect the terminal differentiation of myoblasts. We tested the hypothesis that miRNA-181 knockdown could reduce the arrhythmias by suppressing differentiation of SKMs post SKM transplantation into ischemic myocardium of rats. Further studies on the myocardial microenvironment on myoblast differentiation and cardiac arrhythmia post myoblast transplantationMETHODS:1.We comparied two different SKM culture methods in order to construct effective SKM culture System.2.Construct recombined lentivirus against miRNA-181. Lentivirus against luciferase (Lenti-siLUC) and equal volume saline were employed as Negative control (Nc) and control group (Cn). The total RNA and whole cell protein were collected after 1 day,3 days, and 5 days and 7days respectively, miRNA-181 level was examined.3. MTT and Flow Cytometry were used to explore the the cell proliferate ability. And the-cx43 levels of SKM transduced with Lenti-siR-miR-181 were assayed.4.Rat myocardial infarction model were produced by left coronary artery (LCA) ligation. One week after LCA ligation, the animals were randomly assigned to three groups and 5×106 SKMs (MI-SKM group) or 5×106 SKM with miRNA-181 knockout (MI-KO group)in 100μL PBS or 100μL PBS (MI-Cn) were injected into peri-infarct area through rethoracotomy.14 days and 1 month after cell injection the cardiac systolic function were detected by 2-dimensional mode echocardiography, serum BNP levels were evaluated by ELASA, and ventricular arrhythmia induceability were invested in vivo by programmed stimuli.5.Valsartan (20mg/Kg.D-') were administrated after SKM transplantation to improve cardiac remodeling. We do electrophysiological examination again to study the incidence of arrhythmia 2 weeks and 1 month later.1-type collagen expression of infract myocaedio marginal zone and vascular density were observed by immunohistochemistry, Cx43 expression were detected by Western Blot. The aim of those study were to investigate the effect of myocardial microenvironment after transplantation of SKM on the incidence of arrhythmia.RESULTS:1. The purity (91.5±4.1% vs85.3±5.3%, P= 0.062) and the cell survival rate(90.7±2.9% vs.94±6.1%, P= 0.058) was not statistically significant in two methods, However, cell purity in tissue culture method is relatively low.2. Lenti-siR-miR-181 transfected SKMs efficiently (peak efficiency was 85.2%). At days 3, days 5 and days 7 post Lentivirus transfection, miRNA-181 level in Tr groups was about 60±6.5%,35±3.2% and 30±2.4% of that in Cn groups (P<0.05 respectively). Hox-All protein level in Cn group decreased significantly compared with that of dayl (Day3:0.42019±0.0231 vs.0.691±0.0712, P＜0.001), (Day5: 0.2157±0.0107v.s.0.6911±0.0712,P＜0.001), (Day7:0.0880±0.0211 vs.0.6911±0.0712, P<0.001), Hox-All protein level in Tr group were significantly higher than those in Cn group at day5 and day7.(Day 5:0.3807±0.0317 vs. 0.1803±0.050, P＜0.001); (Day 7:0.2981±0.0178 vs.0.2010±0.0319,P<0.001). Hox-All mRNA level had no significant differences among the three groups at any indicated time point (P=0.071).3.Knockdown of miRNA-181 significantly promotes SKM's growth in vtro. (MTT OD:Day7:0.91968±0.063 vs.0.8206±0.072 vs.0.6302±0.059, P<0.05). miRNA-181 knockdown could partly ablate the CX43 mRNA downregulation in the differentiation process of SKMs (Tr vs.Cn 0.94±0.07 vs.0.63±0.09,P<0.05)).4 LVEF was significantly improved in MI-SKM or MI-SKM+V group at 2 weeks post cell transplantation (MI-KO vs. Ml-Cn:42.05±3.16%vs.34.17±1.99%, P<0.05;MI-SKMvs MI-Cn 41.98±3.7% vs.34.17±2.09%, P<0.05). Self-terminating VT could be evoked in 71% of SKMs-injected rat (MI-SKM), MI-KO engraftment caused a marked decrease (34%,P=0.006) in the incidence of VT and was similar to values of the MI-PBS group (27.7% P=0.588).However, there was no significant difference about the incidence of VT 1 month post SKM transplantation. 5. LVEF in MI-KO, MI-KO+V, MI-SKM, MI-SKM+V was significantly higher than that in Ml-Cn (14 days post transplantation, MI-KO vs. MI-Cn:41.05±3.1% vs.33.9±2.0%, P<0.05; MI-KO+V vs.MI-Cn:42.5±2.4% vs.33.9±2.0%; MI-SKM vs.MI-Cn:41.91±2.7% vs.33.9±2.0%, P＜0.05; MI-SKM+V vs. MI-Cn: 42.6±2.0% vs.33.9±2.0%, P<0.05.1 month MI-KO vs. MI-Cn:40.35±2.3% vs.34.5±2.5%, P<0.05; MI-KO+V vs.MI-Cn:44.1±3.0% vs.34.5±2.5%; MI-SKM vs.MI-Cn:41.6±2.7% vs.34.5±2.5%, P<0.05; MI-SKM+V vs.MI-Cn: 43.9±2.2% vs.34.5±2.5%, P<0.05). Valsartan treatment can further improve the cardiac function 1 month post transplantation (MI-KO vs. MI-KO+V:40.35±2.3% vs.44.1±3.0%; MI-SKM vs. MI-SKM+V:41.6±2.7% vs.43.9±2.2%, both P <0.05). There was no statistically differences about LVEF 14 days after transplantation. There was no significant difference about the incidence.of VT between valsartan treatment group and corresponding untreated control group. The incidence of VT in valsartan treatment group(MI-KO+V 29%) were significantly lower than those in untreated group (MI-KO,46%) or control group (MI-Cn,64%). Immunohistochemistry showed that infarct border zone collagen I in valsartan treatment group were significantly lower than that in the control group,and vascular density were higher (MI-KO+V vs. MI-KO:148.52±34.57 vs 102.31±30.75/mm2, Pb0.05), Cx43 protein levels higher than in control group notablely.CONCLUSIONS:1. miRNA-181 knockout can promote myoblast proliferation in vitro, inhibition of cultured myoblast differentiation. Prevent myoblast differentiation induced reduction of Cx43.2. The progress of SKMs with miRNA-181 knockout in cardiac differentiation become slow, but still further differentiation and maturation. The incidence of arrhythmia were significantly reduced early after transplantation. 3. Valsartan can improve cardiac remodeling and improve the proliferation of SKM, reduce the incidence of arrhythmia after transplantation without significant effect on SKM differentiation... |
PDF Full Text Request