ABSTRACT
OBJECTIVE@#To observe the effeet of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), on mTOR and eukaryotic initiation factor-4E(eIF-4E)expression in coxsac-kievirus B3 (CVB3)-induced rat myocardial cells and to investigate the role of mTOR/eIF-4E signal pathway in viral myocarditis.@*METHODS@#To construct a cell model of viral myocarditis with primary cultured myocardial cells. Myocardial cells infected by CVB3 were treated with 10 nmol/L rapamycin according to the cell toxicity test. The mTOR and eIF-4E expressions of cells were determined by RT-PCR and Western Blot.@*RESULTS@#Rapamycin inhibited the degeneration of CVB3-induced myocardial cells. Expressions of mTOR and eIF-4E mRNA or protein in CVB3-induced myocardial cells were significantly upregulated compared with the control group (P < 0.05), and rapamycin (10 nmol/L) inhibited the upregulation (P < 0.05).@*CONCLUSION@#Rapamycin can downregulate the expressions of mTOR and eIF-4E in CVB3-induced myocardial cells, suggesting that mTOR/eIF-4E signal transduction may play an important role in viral myocarditis.
Subject(s)
Animals , Rats , Animals, Newborn , Coxsackievirus Infections , Enterovirus B, Human , Eukaryotic Initiation Factor-4E , Myocarditis , Metabolism , Virology , Myocytes, Cardiac , Metabolism , Protein Kinases , Rats, Sprague-Dawley , Signal Transduction , Sirolimus , Pharmacology , TOR Serine-Threonine KinasesABSTRACT
<p><b>OBJECTIVE</b>Mammalian target of rapamycin (mTOR) plays a central role in controlling cell proliferation, survival and growth. We investigated the role of mTOR signal transduction on viral myocarditis by observing the effect of mTOR inhibitor rapamycin on Smad 3 and collagen type I expression in rat myocardial fibroblasts infected with coxsackievirus B 3 (CVB 3).</p><p><b>METHODS</b>Primary cultured myocardial fibroblasts of SD rats infected with CVB 3 were treated with or without rapamycin. The Smad 3 and collagen type I expression of the cells were determined by RT-PCR and Western blot.</p><p><b>RESULTS</b>(1) mTOR/beta-actin ratio was dose-dependently reduced (1 nmol/L, 0.381 +/- 0.022; 10 nmol/L, 0.282 +/- 0.014; 100 nmol/L, 0.263 +/- 0.012 vs. control 1.45 +/- 0.04, all P < 0.05 vs. control) after 48 hours rapamycin treatments and time-dependently reduced after 10 nmol/L rapamycin treatment (24 h, 0.203 +/- 0.021; 48 h, 0.163 +/- 0.022; 72 h, 0.144 +/- 0.013 vs. 0 h, 0.341 +/- 0.022, all P < 0.05 vs.0 h) in CVB 3 infected myocardial fibroblasts. (2) Smad 3/beta-actin ratio of myocardial fibroblasts was significantly increased in CVB 3 infected cardial fibroblasts and this increase could be significantly attenuated by rapamycin (control, 0.63 +/- 0.06; CVB 3, 1.18 +/- 0.03; CVB 3 + Rapamycin, 0.77 +/- 0.08 by RT-PCR and 0.89 +/- 0.07, 2.27 +/- 0.13 and 0.131 +/- 0.013 by Western blot). Collagen type I/beta-actin ratio was also significantly increased by CVB 3 and this increase could be reversed by rapamycin (1.13 +/- 0.06, 1.303 +/- 0.012, 0.82 +/- 0.03 by RT-PCR).</p><p><b>CONCLUSION</b>Rapamycin can inhibit the Smad 3 and collagen type I expressions in CVB 3 infected myocardial fibroblasts suggesting that the mTOR signal pathway may play an important role in the pathogenesis of CVB 3 induced myocardial fibrosis.</p>
Subject(s)
Animals , Female , Male , Rats , Cells, Cultured , Collagen Type I , Metabolism , Coxsackievirus Infections , Metabolism , Enterovirus , Fibroblasts , Metabolism , Myoblasts, Cardiac , Metabolism , Virology , Rats, Sprague-Dawley , Signal Transduction , Sirolimus , Pharmacology , Smad3 Protein , MetabolismABSTRACT
<p><b>OBJECTIVE</b>This study examined the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), on eukaryotic initiation factor (eIF- 4E) expression in rat myocardial fibroblasts infected by Coxsackievirus B3 (CVB3) in order to identify the drug target for treatment of viral myocarditis.</p><p><b>METHODS</b>Primary cultured rat myocardial fibroblasts were treated with CVB3 with multiplicity of infection (MOI=0.5 PFU/cell). The experiment consisted of four groups in which the cultured rat fibroblasts cells were treated with CVB3, rapamycin (10 nM) and CVB3 + rapamycin or placebo (control). Experimental model of CVB3-infected myocardial fibroblasts was confirmed by detection of CVB3 mRNA expression with RT-PCR and observation of morphological changes of the infected cells with microscopy. eIF-4E expression was determined by both RT-PCR and Western Blot methods.</p><p><b>RESULTS</b>Morphological changes were found in the fibroblasts treated with MOI 0.5 PFU/cell of CVB3 by transmission electron microscope and the viral particles were found in the cytoplasm. CVB3 mRNA was expressed in CVB3-infected fibroblasts after 1, 2, and 3 days after infection and 2 days after passage. The gray scale values of the eIF- 4E /beta -actin in the control, the CVB3, the rapamycin and the CVB3+rapamycin groups were 0.73 +/- 0.07, 0.87 +/- 0.03, 0.32 +/- 0.03 and 0.56 +/- 0.04 respectively detected by RT-PCR, and were 0.79 +/- 0.09, 1.35 +/- 0.12, 0.55 +/- 0.04, and 0.62 +/- 0.07 respectively detected by Western blot. EIF- 4E expression in the CVB3 group was higher than that in the control group. Both the rapamycin and the CVB3+rapamycin groups had lower eIF- 4E expression than the control and the CVB3 groups.</p><p><b>CONCLUSIONS</b>CVB3 can infect myocardial fibroblasts and up-regulate the eIF- 4E expression in rat myocardial fibroblasts. Rapamycin can inhibit eIF- 4E expression and may be a potential medicine for treatment of viral myocarditis. It was suspected that mTOR/eIF- 4E signal pathway in rat myocardial fibroblasts might play an important role in the pathogenesis of viral myocarditis.</p>