Basal and reovirus-induced beta interferon (IFN-beta) and IFN-beta-stimulated gene expression are cell type specific in the cardiac protective response.

Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-beta) and IFN-beta-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-beta response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-beta response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-beta, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-beta), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-beta, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-beta in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-beta resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-beta response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-beta, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-beta and ISG expression in myocytes may play an important protective role.

PKR's protective role in viral myocarditis.

Reovirus-induced murine myocarditis provides an excellent model for the human disease. Previously, we showed that reovirus induction of and sensitivity to interferon-beta (IFN-beta) are important determinants of protection against cardiac damage. IFN-beta induces a number of genes with antiviral activities, including the dsRNA-activated protein kinase, PKR. Once bound to viral dsRNA, PKR becomes activated and phosphorylates eukaryotic initiation factor-2 alpha (eIF2 alpha) leading to the cessation of host cell translation. Additionally, activated PKR can exert its antiviral effects by inducing phosphorylation of I kappa B, leading to the activation of the transcription factor NF kappa B and subsequent induction of IFN-beta. Thus, activated PKR can both induce and be induced by IFN-beta. Recently, numerous reports have shown PKR to be dispensable for both induction of IFN as well as protection against disease. However, both PKR's role in the heart in response to viral infection and its ability to prevent cardiac damage have gone largely unexplored. Here, we demonstrate PKR to be critical for viral induction of IFN-beta in primary cardiac myocyte cultures. Additionally, we show that loss of PKR leads to an increase in virulence for both myocarditic and nonmyocarditic reoviruses. Finally, we demonstrate PKR to be critical for protection against reovirus-induced viral myocarditis.

Interferon regulatory factor-1, interferon-beta, and reovirus-induced myocarditis.

Viral myocarditis is an important human disease, and reovirus-induced myocarditis in mice provides an excellent model to study direct viral damage to the heart. Previously, we showed that reovirus induction of and sensitivity to interferon-beta (IFN-beta) is an important determinant of viral pathogenicity in the heart and that the transcription factor interferon regulatory factor-3 (IRF-3) is required for reovirus induction of IFN-beta in primary cardiac myocyte cultures. Given several lines of evidence suggesting a possible distinctive environment for IRFs in the heart, we have now focused on IRF-1. Previous studies demonstrated that viruses, double-stranded-RNA (dsRNA), and IFN-alpha/beta can each induce IRF-1 and that IRF-1 plays a role in dsRNA, but perhaps not viral, induction of IFN-alpha/beta. Importantly, none of these studies used a virus with a dsRNA genome (such as reovirus), none of them used a highly differentiated nonlymphoid cell type, and none of them addressed whether viral induction of IRF-1 is direct or is mediated through viral induction of IFN-beta. Indeed, as recently as this year it has been assumed that viral induction of IRF-1 is direct. Here, we found that reovirus induced IRF-1 in primary cardiac myocyte cultures, but that IRF-1 was not required for reovirus induction of IFN-beta. Surprisingly, we found that reovirus failed to induce IRF-1 in the absence of the IFN-alpha/beta response. This provides the first evidence that viruses may not induce IRF-1 directly. Finally, nonmyocarditic reovirus strains induced more cardiac lesions in mice deficient for IRF-1 than they did in wildtype mice, directly demonstrating a protective role for IRF-1. Together, the results indicate that while IRF-1 is downstream of the IFN-beta response, it plays an important protective role against viral myocarditis.