The microRNA 221/222 cluster controls CVB3-induced myocarditis: could tiny microRNAs explain adverse inflammation in the heart?

Abstract

Background: Viral myocarditis is an important cause of heart failure and sudden cardiac death in young adults. Our previous studies have shown that many microRNAs are differentially expressed in the heart during viral myocarditis, including microRNA-221 and -222. Therefore we aimed at studying the biological role of these miRs during viral myocarditis in mice, induced by human Coxsackie B3 virus. Materials and methods: We measured cardiac microRNA levels during VM with microarrays and qRT-PCR. Wild type C3H mice were intraperitoneally infected with CVB3. Mice were sacrificed at day 4 and day 7 post infection. The hearts, and other organs, were weighted and collected for histological analyses, immunoblotting and qRT-PCR. In vitro experiments were performed in neonatal rat cardiomyocytes (nRCMs). Results: Levels of miR-221 and miR-222 were significantly elevated during acute viral myocarditis in mice, and depressed in biopsies from viral myocarditis patients. In situ hybridization and in vitro experiments showed that both miRs are expressed by cardiomyocytes and cardiac endothelial cells, and are up regulated in neonatal rat cardiomyocytes (nRCMs) in response to enteroviral infection. In vivo inhibition of miR-221 and miRNA-222 by systemically delivered antagomirs (day +1, +3 and +6) aggravated cardiac injury and inflammation at day 7 post-infection, related to a threefold increase of viral loads at day +7 as compared to scrambled antagomiRs. Likewise, inhibition of miR-221 and -222 significantly increased the number of viral particles in nRCMs, whereas overexpression of miR-221 and -222 in nRCMs inhibited enteroviral replication by 30 percent at 48h post-infection. We identified and confirmed a set of conserved miR-221/-222 targets, including ETS1 and ETS2, the chemokine CXCL12, the type I interferon inhibitor IRF2, the T cell development factor TOX and the anti-apoptotic proteins BCL2L11 and BMF. Furthermore, the Coxsackie and adenovirus receptor (CXADR), up regulated in biopsies of patients with dilated cardiomyopathy, is also targeted by miR-221/-222 in mice and rats. De-repression of CXADR upon inhibition of miR-221/-222 may explain the higher viral load observed in vivo and in vitro. Higher CXADR levels contribute to increased viral entry in the cardiomyocyte. Conclusions: Systemic inhibition of miR-221 and miR-222 increases initial cardiac viral loads and resulting inflammation and necrosis during VM in mice. Both targets involved in increased viral replication and enhanced inflammation with inhibition of the miR-221/-222 family are involved. This miR cluster thus represents a master switch in VM.