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Abstract
The structure of coxsackievirus and adenovirus receptor's CAR is similar to adhesion molecules. In the adult heart, the majority of CAR localizes at the intercalated disc. Germ line CAR deletion induces embryonic lethality at E11.5 with evidence of a cardiac abnormality. The CAR role as a viral receptor is well known; however, its precise function in the heart for enterovirus infection is not clear. To understand the role of CAR in the cardiac myocyte, we generated cardiac-specific CAR knockout mice using a CAR floxed allele and alpha-MHC-Mer CRE Mer mice. Western blot analysis and immunofluorescent stain of ventricles at 6 weeks after 2 weeks tamoxifen administration, CAR expression was significantly decreased in CARf/f MCM mice but not in CARf/f mice heart. Enterovirus was intraperitoneally infected into CARf/f MCM and CARf/f mice (n=10 each). CAR disruption was dramatically reduced virus infection and replication in the heart but not different in liver, spleen, and pancreas. Cardiac myocyte damage was significantly reduced in the CARf/f MCM mutant mice by evans blue dye stain. In addition, the CARf/f MCM mutant mice heart inflammation and fibrosis were decreased in H&E and trichrome stain compare to CARf/f control mice. CAR expression was required for normal ventricular function, but it is the cause of enterovirus infection. In the adult mice heart, CAR deletion was significantly reduced viral infection, proliferation, and myocarditis. These results suggested that CAR deletion could be useful therapeutic strategy to prevent viral myocarditis.
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Figure 1.
Generation of Mice with Cardiac-Specific Deletion of CAR. After 2 weeks tamoxifen administration CAR expression was observed. CAR expression in CARf/f (control) and CARf/f MCM (Mutant) mice were measured by western blot analysis (A), and immunofluorescent staining (B) using an antibody specific for CAR (Green). The typical intercalated disc staining for CAR was observed in the CARf/f mice, but not in the CARf/f MCM mice.
Figure 2.
Enterovirus infection in cardiac-specific CAR deletion mice. (A) Scheme of animal study for CAR deletion and enterovirus infection. Enterovirus was infected after 2 weeks tamoxifen administration. (B) Tissue virus titer was measured by PFU assay. In the CARf/f MCM mutant mice (Mut) heart virus titer was significantly reduced compare to CARf/f control mice (WT) heart 4 days following enterovirus infection. (Mean ± S.E.M, **p < 0.01, each data point is represented on graphs).
Figure 3.
Decrease cardiac myocyte damage. (A) Evans blue dye (EBD) uptake was significantly decreased in the heart of CARf/f MCM mutant mice 4 days following enterovirus infection compare to CARf/f control mice. (B) EBD uptake was measured in CARf/f mice (1.4 ± 0.5%) and CARf/f MCM mice (0.2 ± 0.1%). (Mean ± S.E.M, ***p < 0.001, each data point is represented on graphs).
Figure 4.
Decrease inflammation and fibrosis. CAR deletion was effectively blocked enterovirus infection and proliferation in the heart. Inflammation and fibrosis were significantly reduced in CARf/f MCM mutant mice heart compare to CARf/f mice heart 10 days following enterovirus infection. Without CAR expression enterovirus was not able to infect into cardiac myocyte.
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