In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Figure 1. (a) Comparison of CVB3 load in different cell lines. CVB3 RNA copy numbers per ng RNA were obtained by qRT-PCR in CVB3-infected HeLa cells, HL-1 cells, H9c2 cells and iCell® Cardiomyocytes, eight hours pi. (b) Quantification of CVB3 RNA copy number per ng RNA by qRT-PCR in CVB3-infected iCell® Cardiomyocytes in the time course of infection.

Figure 2. RNA/RNA in situ hybridization for CVB3 RNA localization in different infected cell lines. Cells were infected with a multiplicity of infection (MOI) of 10 and analyzed after eight hours. The percentage of infected cells is indicated in the left lower corner, respectively.

Figure 3. Immunofluorescence analysis of VP1 protein localization in different CVB3-infected cell lines. Cells were infected with MOI 10 and analyzed after eight hours. The percentage of VP1 positively stained cells is indicated in the left lower corner, respectively.

Figure 4. (a) Course of CVB3 infection in different cell lines. Brightfield images of CVB3-infected HeLa cells, HL-1 cells, H9c2 cells and iCell® Cardiomyocytes. Cells were infected with MOI 10 and observed for 24 h. (b) Western blot analysis of caspase-1 (left panel, positive control: lysate of human heart tissue of a patient with myocarditis) and caspase-3 cleavage (right panel, positive control: iCell Cardiomyocytes treated with staurosporine) in different CVB3-infected cell lines eight hours pi (MOI 10).

Figure 5. (a) Electron microscopy images of iCell® Cardiomyocytes (A, non-infected, B–D infected) and murine heart tissue (E, non-infected; F, infected). iCell® Cardiomyocytes were analyzed eight hours post CVB3 infection (MOI 10) and compared with murine heart tissue of CVB3-infected mice obtained during acute infection (eight days pi). Yellow arrows indicate destruction of myofibrils; yellow triangles indicate structural changes in mitochondria; yellow asterisks indicate the evolvement of vesicular structures; black arrows (C, D) indicate the destruction of the cellular membrane, which was observed in > 90% of 100 analyzed cells. D illustrates end-stage infection with final cellular lysis. (b) LIVE/DEAD staining of iCell® Cardiomyocytes with an amine-reactive dye for analysis of cell membrane integrity. Cells were infected with MOI 10 and analyzed after eight hours. The percentage of positively stained cells is indicated in the left lower corner, respectively.

Figure 6. (a) Quantification of viral load in iCell® Cardiomyocytes after inhibition of RIPK3 kinase activity (GSK’872) and inhibition of RIPK1 kinase activity (necrostatin). (b) Western blot analysis of RIPK3 expression in CVB3-infected iCell® Cardiomyocytes eight hours pi (MOI 10) (left panel). Densitometric analysis is shown in the right panel. Data were normalized to GAPDH. (c) Western blot analysis of RPK1 expression in CVB3-infected iCell® Cardiomyocytes eight hours pi (MOI 10).

Figure 7. (a) Western blot analysis of RIPK1 expression in iCell® Cardiomyocytes transfected with either pIRES-EGFP-2A or pIRES-EGFP-3C (left panel). GFP expression is shown in the right panel. (b) Western blot analysis of RIPK1 expression in CVB3-infected iCell® Cardiomyocytes eight hours pi (MOI 10) after pre-incubation with the protease inhibitors E64d or pepstatin A. iCell® Cardiomyocytes were pre-incubated for one hour with the inhibitors before CVB3 infection. Densitometric analysis is shown in the right panel. Data were normalized to GAPDH.

Figure 8. (a) Western blot analysis of RIPK1 expression in CVB3-infected iCell® Cardiomyocytes eight hours pi (MOI 10) after pre-treatment with DMSO or Z-VAD-FMK (pan-caspase inhibitor) (left panel). Densitometric analysis is shown in the right panel. Data were normalized to GAPDH. (b) Quantification of viral load after DMSO treatment. iCell® Cardiomyocytes were pre-incubated with DMSO at different concentrations for one hour before CVB3 infection.