Mitochondrial function of cardiomyocytes during CVB3 infection. (A–C) Primary cardiomyocytes from WT and ISG15^−/− mice were infected with CVB3 at an MOI 1.0 for 6–8 h. (B–C) Seahorse analysis of mitochondrial oxygen consumption rate (representative of n = 5 independent experiments). (B) Oligomycin inhibits the respiratory chain ATP synthase thus reducing oxygen usage, while FCCP decouples the other complexes, resulting in maximal activity and, hence oxygen consumption. Rotenone and antimycin A inhibit complex I and III of the respiratory chain, respectively, thereby halting cellular oxygen consumption by oxidative phosphorylation. (C) Basal respiration, ATP production, and maximal respiration are depicted and were calculated relative to wild-type untreated control cells. Significances were determined using two-way ANOVA with Sidak’s multiple comparisons test. (D–E) MitoTracker Deep Red^TM (MTDR) live cell staining in cardiomyocytes to analyze mitochondrial integrity. FCCP pre-treatment for 4 h served as a control. Cells were analyzed by confocal microscopy. Maximum intensity Z-projection is displayed with staining of MTDR in red and DAPI in blue. Scale bar: 10 µm. Quantitative comparison of intensity distributions of MTDR fluorescence (n = 24) (D). Flow cytometry analysis of MTDR mean fluorescence intensity, set relative to WT control (E). Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. (F, G) Mitochondrial function of heart tissue during CVB3 infection. Heart tissue biopsies obtained from wild-type (WT), and ISG15^−/− mice at T0, T1, and T2 were analyzed by Seahorse measurement, determining oxygen consumption rates that were normalized to uninfected controls. (F) Basal and (G) maximal respiration are displayed. P-values were determined by Student’s t-test. (H) To distinguish between effects of free ISG15 and ISGylation, Ube1L^−/− mice were infected and cardiac biopsies were analyzed as described above.