The Myocardial Injury And Abnormal Expression Of Fas/FasL In Murine Acute Myocarditis Caused By Coxsackievirus B3

Objectives: The mechanisms responsible for myocardial injury and cell death in myocarditis are still unclear. Necrosis and apoptosis were proved exist in virus myocarditis (VM). Fas/FasL belong to the tumor necrosis factor receptor/ligand superfamily of costimulatory molecules and are known to play a critical role in the induction of apoptosis, as well as in the cytotoxicty mediated by T-cells and natural killer cells. In this study, we examined that myocardial cell death occurs in murine acute myocarditis caused by Coxsackie virus B3 (CVB3) and whether the Fas/Fas ligand (FasL) system plays a role in myocardial damage(especially in the myocytes apoptosis) and the development of VM .Methods: One hundred and thirty BALB/c mice were included in the experiment. Fifteen mice in experimental group inoculated with CVB3 and five mice in control group injected with Eagle's minimum essential medium (MEM) were sacrificed 5, 7, 10, 14, 21, 28 days post-inoculation (p.i.). Light microscopy, electronic microscopy and flow cytometry (FCM) were used to detect the inflammation, necrosis and apoptosis in myocardium. Serum creatine phosphokinase (CK) measurements were performed as marker enzymes of the mice myocytes lesion progression induced by CVB3 infection. The expressions of Fas and FasL protein in myocardium were determined by immunohistochemistry. Fas mRNA and FasL mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR).Results: (1) The typical myocardial lesions including myocardial necrosis and lymphocyte infiltration at the 5th day p.i. were observed, much more obviously at the 7th to 14th day, and recovered gradually from the 21th to 28th day p.i. The mice in control group did not show any sign of inflammation. The level of serum CK increased at the 5th day p.i., much more obviously at the 7th to 14th day p.i., and recovered gradually from the 21th to 28th day p.i. But the level of serum CK in mice of VM did not increase as it changed in acute myocardial infarction. (2) Apoptosis myocytes were seen in seven of twelve myocarditis mice by electronic microscopy. Apoptosis cells including myocytes (around the nidus) and infiltrating lymphocytes were detected in the myocardium of myocarditis mice by electronic microscopy.But apoptosis cells were not observed in the mice of control group. (3) The detection rate apoptosis cells in myocardium was much higher in mice with VM than the micein control group . The percent of apoptosis cells increased significantly after the infection 7 to 14 days than 5 p.i.and 21 to 28 days p.i. (4) In experimental group, Fas protein expressed mainly in myocytes and FasL protein expressed mainly in infiltrating lymphocytes and increased remarkably from 7 to 14 days compared with control group(P<0.001) . The dynamic changes of FasL protein expression showed a significantly positive correlation with the changes of myocardial histopathologic scores (r= 0.9082, P<0.001) . Fas mRNA in experimental group increased remarkably from 7 to 14 days p.i. compared with control group(P<0.00l).Conclusion: These findings suggest that cell death via necrosis and apoptosis existed in murine acute myocarditis caused by CVBs at the same time, apoptosis of cardiomyocytes and lymphocytes is one of the mechanisms of myocardial injury in VM, and that the cytotoxic T lymphocyte mediatied myocardium damage through Fas/FasL pathway might play an important role in the development of VM.