Empirical Study Of Rapamycin Inhibiting Fibrosis Of Viral Myocarditis By Blocking MTOR Signal Pathway

Objective: mTOR (mammalian target of rapamycin, mTOR) signal pathway plays a central role in the cell survival, growth and proliferation. This study employed CVB3 to infect primary cultured myocardial fibroblasts and cardiocytes of neonatal rats to construct the experimental cell model in vitro, and which were treated with rapamycin, to evaluate the possible mechanism of mTOR signal pathway in the development of myocardial fibrosis induced by viral myocarditis.Methods: To culture primary myocardial fibroblasts and cardiocytes of neonatal rats with an improved method and the cells were identified with the cell morphology and immunocytochemical stain. The experimental cell models were established by infecting the cells with CVB3. We employed these methods such as RT-PCR, Western Blot or immunocytochemical stain to detect the expression of mTOR, eIF-4E, collagen typeⅠand Smad3 of the cell models treated by Rapamycin which is an inhibitor of mTOR signal pathway. SPSS 11.5 software was used for the statistical analysis. P＜0.05 was considered to be statistically significant.Results:(1) After digestion with pancreatin and collagenaseⅡwith the technique of differential adhesion twice in a short period, which can increase the survival rate and contraction rate of primary cardiocytes, and which can purify cardiocytes and myocardial fibroblasts from each other. The myocardial fibroblasts stained with monoclonal antibodies against vimentin were positive, stained with monoclonal antibodies againstα-sarcomeric actin were negative. For cardiocytes, stained with monoclonal antibodies againstα-sarcomeric actin were positive, stained with monoclonal antibodies against vimentin were negative.(2) CVB3 can cause structure change in primary cultured myocardial fibroblasts. Cell morphology change and viral particles in cytoplasm can be found with transmission electron microscope. CVB3 mRNA was persistent expressed in myocardial fibroblasts infected by CVB3. And CVB3 cause cytopathic effect of myocardial fibroblasts when the multiplicity of infection (MOI) was 1.0 PFU/cell. The same changes were detected in infected cardiocytes with transmission electron microscope. CK-MB and LDH were increased in the supematant of the cells infected by CVB3.(3) The primary cultured rat myocardial fibroblasts were treated for 48 hours with Rapamycin in different concentration. The gray scale values of mTOR/β-actin in control group (0 nM), 1 nM, 10 nM and 100 nm groups were 1.45±0.04, 0.38±0.02, 0.28±0.01, 0.26±0.01 respectively checked with RT-PCR, and both the 10 nM and 100 nM groups were lower than that in In M group (all P＜0.05). There was no significant difference between 10 nM group and 100 nM group (P＞0.05). The gray scale values of mTOR/β-actin in 0 h (control), 24 h, 48 h, and 72 h groups were 0.34±0.02, 0.20±0.02, 0.16±0.02, 0.14±0.01 respectively with RT-PCR. The longer the cells treated with Rapamycin, the lower the mTOR expression (all P＜0.05). The primary cultured rat myocardial fibroblasts were infected by CVB3 with different tite (MOI=0, 0.2, 0.5 and 1.0PFU/cell) for 24 hours and the OD values of the groups were 0.36±0.04, 0.35±0.05, 0.66±0.07 and 0.32±0.07 respectively with MTT. The OD value of 0.5PFU/cell group was higher than that in control group (all P＜0.05). There was no significant difference between control group and 0.2 PFU/cell group or 1.0 PFU/cell group (both P＞0.05). The OD values in control, CVB3, Rap and CVB3+Rap groups were 0.55±0.09, 0.66±0.09, 0.42±0.09 and 0.36±0.12 respectively With MTT when the myocardial fibroblasts were treated with 10nM Rapamycin. These results suggest that the OD value in CVB3 group was higher than that in control group, and Rap group was lower than that in control group, CVB3+Rap group was lower than that in CVB3 group (all P＜0.05).(4) The gray scale values of mTOR/β-actin of myocardial fibroblasts in control group were 0.36±0.03, 0.39±0.02 and 0.37±0.04 respectively with RT-PCR at dl, d2 and d3, there were no significant differences each other (all P＞0.05). The gray scale values of mTOR/β-acfin of myocardial fibroblasts in CVB3 group were 0.86±0.06, 0.97±0.06 and 0.93±0.06 respectively with RT-PCR at d1, d2 and d3, there were no significant differences each other (all P＞0.05). The gray scale values of the mTOR/β-actin of myocardial fibroblasts in CVB3+Rap group were 0.26±0.03, 0.18±0.02 and 0.10±0.02 respectively with RT-PCR at d1, d2 and d3 and the gray scale values were decreased significantly at d3 than that at d1 and d2(all P＜0.05). The gray scale values of CVB3 group were increased at d1, d2 and d3, compared with control groups at same time point, the differences were significant (all P＜0.05). The gray scale values in CVB3+Rap group were lower than that in CVB3 group at same time point, the differences were significant (all P＜0.05). The gray scale values of the mTOR/β-actin of myocardial fibroblasts in control, CVB3, Rap and CVB3+Rap groups were 0.22±0.09, 0.92±0.13, 0.19±0.03 and 0.20±0.05 respectively with Western Blotting. The gray scale value in CVB3 group was higher than that in control group at same time point, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point (all P＜0.05).(5) The gray scale values of the eIF-4E/β-actin of myocardial fibroblasts in control, CVB3, Rap and CVB3+Rap groups were 0.73±0.07, 0.87±0.03, 0.32±0.03 and 0.56±0.04 respectively with RT-PCR, and were 0.79±0.09, 1.35±0.12, 0.55±0.04 and 0.62±0.07 respectively with Western Blotting. The gray scale value in the CVB3 group was higher than that in control group, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point (all P＜0.05).(6) The gray scale Values of the collagen typeⅠ/β-actin of myocardiol fibroblasts in control, CVB3, Rap and CVB3+Rap groups were 1.13±0.06, 1.30±0.01, 0.73±0.03 and 0.82±0.03 respectively with RT-PCR. The gray scale value in CVB3 group was higher than that in control group at same time point, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point (all P＜0.05).The staining in myocardiol fibroblasts with monoclonal antibodies against collagen type I was positive: endochylema was stained brownish while nucelus was stained to be blue. The endochylema became yellowish-brown when the cells were infected by CVB3 for 48 h, while the color of the endochylema became fight when treated by Rapamycin.(7) The gray scale values of Smad3/β-actin of myocardiol fibroblasts in control, CVB3, Rap and CVB3+Rap groups were 0.63±0.06, 1.18±0.03, 0.35±0.05 and 0.77±0.08 respectively with RT-PCR, and were 0.89±0.07, 2.27±0.13, 0.36±0.02 and 0.13±0.01 respectively with Western Blotting. The gray scale value in CVB3 group was higher than that in control group, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point (all P＜0.05).(8) The gray scale values of mTOR/β-actin of cardiocytes in control, CVB3, Rap and CVB3+Rap groups were 0.32±0.08, 0.93±0.06, 0.15±0.05 and 0.32±0.08 respectively with RT-PCR, and were 0.79±0.11, 2.97±0.23, 0.45±0.08 and 1.23±0.19 respectively with Western Blotting. The gray scale value in CVB3 group was higher than that in control group, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point(all P＜0.05).(9) The gray scale values of eIF-4E/β-actin of cardiocytes in control, CVB3, Rap and CVB3+Rap groups were 0.80±0.03, 1.21±0.09, 0.45±0.04 and 0.44±0.06 respectively with RT-PCR, and were 1.22±0.09, 2.52±0.31, 0.66±0.03 and 0.99±0.11 respectively with Western Blotting. The gray scale value in CVB3 group was higher than that in control group, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point(all P＜0.05).(10) The gray scale values of collagen typeⅠ/β-actin of cardiocytes in control, CVB3, Rap and CVB3+Rap groups were 0, 0.18±0.06, 0 and 0.06±0.02 respectively with RT-PCR. The gray scale value in CVB3 group was higher than that in control group, and CVB3+Rap group was lower than that in CVB3 group (all P＜0.05).(11) The gray scale values of Smad3 /β-actin of cardiocytes in control, CVB3, Rap and CVB3+Rap groups were 0.55±0.04, 1.14±0.05, 0.41±0.10 and 0.35±0.07 respectively with RT-PCR ,and were 0.11±0.01, 0.35±0.09, 0.06±0.01 and 0.08±0.02 respectively with Western Blotting. The gray scale value in CVB3 group was higher than that in control group, Rap group was lower than that in control group and CVB3+Rap group was lower than that in CVB3 group at same time point(all P＜0.05).Conclusion:(1)When cardiocytes were isolated and cultured, the low concentration pancreatin and collagenasⅡwith the technique of differential adhesion should be employed twice, which can increase the survival rate, and can purity of primarycardiocytes from other cells, and can improve the cells contraction.(2)Myocardial fibroblasts could be an experimental cell model of CVB3 infection.(3)Rapamycin can down-regulate the mTOR expression of myocardial fibroblasts in dose-dependent way and time-dependent way in definite dosage. MTOR was considered as a target of rapamycin.(4) CVB3 can cause the proliferation of myocardial fibroblasts. The mechanism of rapamycin inhibit the proliferation may be associated with mTOR/eIF-4E signal pathway.(5)CVB3 can up-regulate the expression of collagen typeⅠand Smad3 in rat myocardial fibroblasts model and Rapamycin can inhibit their expression in these cells. These results suggested that mTOR/eIF-4E signal pathway combining with Smad signal pathway in the rat myocardial fibroblasts may play an important role in the mechanism of myocardial fibrosis caused by CVB3 infection.(6)CVB3 can up-regulate the expression of mTOR, eIF-4E, collagen typeⅠand Smad3 in rat cardiocytes and Rapamycin can inhibit their expression in these cells. These results suggested that the cell phenotype of cardiocytes may be changed when infected by CVB3 and these cells may be involved in myocardial fibrosis caused by CVB3. The mechanism may be also associated with the mTOR/eIF-4E signal pathway and Smad signal pathway.