| Viral myocarditis is a common disease in humans. Interferon-beta (IFN-beta) has been identified as critical for protection against viral myocarditis in mouse models, and IFN-alpha or -beta treatment is beneficial in the treatment of human viral myocarditis. IFN-beta expression and its antiviral effects are cell-type specific in murine cardiac myocytes and fibroblasts. However, expression and function of individual IFN-alpha subtypes in cardiac cells has not previously been investigated. Therefore, IFN-alpha subtype expression and antiviral effects were studied in reovirus-infected murine primary cardiac myocyte and cardiac fibroblast cultures. In order to quantify the thirteen highly conserved IFN-alpha subtypes, a quantitative Real-Time PCR assay was developed. Results demonstrated that IFN-alpha induction by reovirus T3D in cardiac cells is both subtype- and cell type-specific, and that some individual IFN-alpha subtypes are likely important in the antiviral cardiac response. In brief, reovirus T3D induced five IFN-alpha subtypes in primary cultures of cardiac myocytes and fibroblasts: IFN-alpha1, -alpha2, -alpha4, -alpha5, and -alpha8/6. The levels of IFN-alpha expression were both higher and spanned a greater range in cardiac myocytes than in fibroblasts. Viral induction of IFN-alpha1, -alpha2, -alpha5, and -alpha8/6 required IFN-alpha/beta signaling in both cell types, while induction of IFN-beta and -alpha4 was more dependent on IFN signaling in myocytes than fibroblasts. Murine IFN-alpha1, -alpha2, -alpha4, or -alpha5 treatment induced IRF7 and ISG56 in both cardiac cell types, however induction was always greater in cardiac fibroblasts than in cardiac myocytes. Finally, each IFN-alpha subtype inhibited reovirus T3D replication in both cell types, but protection was subtype-specific.;To discover novel proteins or protein post-translational modifications involved in the IFN pathway or displaying antiviral effects against viral myocarditis, a proteomics tool, two-dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix absorption laser desorption ionization-time of flight-time of flight (MALDI-TOF-TOF) mass spectrometry, was used to investigate the reovirus-induced proteome changes in murine primary cardiac myocyte cultures. Results demonstrated that the 2D-DIGE technique is quantitative and reproducible. Whole proteome changes based on differentially expressed proteins were clustered according to viral pathogenic phenotypes and induction of IFN. One hundred and twenty-four differentially expressed proteins were identified, including those involved in calcium signaling, ERK/MAPK signaling, protein ubiquitination, mitochondrial dysfunction, oxidative stress, amino acid metabolism, and other pathways. Interestingly, 2D-DIGE results and additional studies demonstrated that heat shock protein Hsp25 is modulated differentially by myocarditic and non-myocarditic reoviruses, and suggested that it may play a role in the cardiac antiviral response. This is the eighth virus family found to modulate Hsp25 or its human homolog, Hsp27, suggesting that Hsp25/27 participation in the antiviral response may be widespread. However, results here provide the first evidence for a virus-induced decrease in Hsp25/27, and suggest that viruses may have evolved a mechanism to subvert this protective response, as they have for IFN. |
