Matrix metalloproteinases and tissue inhibitors of metalloproteinases in coxsackievirus-induced myocarditis

Abstract

Background: Coxsackievirus B3 (CVB3) is the major causative agent of myocarditis in humans. In the mouse model, the inflammatory phase of myocarditis results in extensive damage to the heart and triggers profound extracellular matrix (ECM) remodeling, which may ultimately lead to dilated cardiomyopathy. Matrix metalloproteinases (MMPs) are regulators of the ECM and can degrade all the components in the matrix.

Methods: Adolescent male mice were infected with cardiovirulent CVB3 and sacrificed at 3, 9, and 30 days post infection (pi). Transcription of MMP-2, MMP-9, and MMP-12 was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). Protein expression of these enzymes was examined using immunohistochemistry, and the activation status of MMP-2 and MMP-9 was assessed using gelatin zymography. Tissue inhibitors of metalloproteinases (TIMPs) were analyzed using immunoblotting assays. Myocarditic hearts were also stained with picrosirius red and viewed under polarizing light to examine the collagen network.

Results: MMP-2, MMP-9, and MMP-12 transcription was increased at 9 days pi, as determined by RT-PCR. Immunohistochemistry confirmed an increase in translation of these MMP species, and zymographic analysis further showed elevated activation of MMP-2 and MMP-9 following CVB3 infection. TIMP-3 and TIMP-4 expression was down-regulated, while TIMP-1 and TIMP-2 remained constant throughout the infection. Mouse hearts stained with picrosirius red showed an increase in total amount of collagen during the acute phase of infection and disrupted fibrils at later timepoints.

Conclusion: After CVB3 infection, ECM remodeling is triggered, and this response may involve increased expression and activation of MMPs.