Pathophysiological Mechanisms of Cardiac Dysfunction in Transgenic Mice with Viral Myocarditis

Figure 1. (a) Schematic illustration of the CVBΔVP0 expression system which was used to generate a transgenic mouse line. (b) Exemplary cardiac magnetic-resonance-imaging (CMR) of a wt mouse (left) and a CVB3ΔVP0 transgenic mouse (right). (c) Statistical analysis of measured cardiac output, ejection fraction and stroke volume (* indicates n < 0.05; ** indicates n < 0.01; *** indicates n < 0.001). (d) Volume–time filling curves of the left ventricle. (e) Statistical analysis of the early filling rate (E), atrial ejection (A) and EA ratio of CVB3 mice and control mice. (f) Analysis of tissue Doppler imaging (TDI) of transgenic CVB3 mice and control mice.

Figure 2. (a) Electrocardiogram (ECG) recordings of transgenic CVB3 mice and control mice under basal conditions and under β-adrenergic stimulation with isoprenaline. (b) Analysis of ECG recordings of CVB3 mice and control mice considering P-wave, T-wave and arrhythmic event emergence (* indicates n < 0.05; *** indicates n < 0.001). (c) Analysis of QT, QRS and JT (JT = QT − QRS duration) of transgenic CVB3 mice and control mice under basal conditions and after isoprenaline application.

Figure 3. (a) Isolated mouse cardiomyocytes were optically measured under control conditions and under CVB3 expression. The cell length was quantified (n = 11; * indicates n < 0.05;) (b) Isolated mouse cardiomyocytes were optically measured under control conditions and under CVB3 expression. The cell area was quantified (n = 11; * indicates n < 0.05;) (c) Two exemplary induced action potentials of two different cardiomyocytes during patch clamp recordings and statistics on occurrence of delayed action potentials as relative events after stimulation at 0.5 to 7 Hz. (a) Example of a normal and a train of delayed depolarizations (DAD). (d) Cardiomyocytes isolated from transgenic CVB3ΔVP0 mice show a higher number of DAD events than the control group. The mean values ± SEM and the statistically relevant differences after a Mann–Whitney test (non-parametric) were plotted (n = 10–12 each group).

Figure 4. (a) Time to peak of intracellular Ca2+ (Ca2+I) transients and of sarcomere shortening, (b) Ca2+-transient (ΔCa2+i) and sarcomere shortening amplitudes, (c) time constant τ of a single exponential decay function fitted to the decay phase of Ca2+-transients and to sarcomere relaxation phase. * indicates p < 0.05 vs. WT controls. All parameters recorded under acute stimulation with isoproterenol (ISO) were significantly altered vs. basal (p < 0.05, not indicated in the figure for clarity). (box: 25th–75th percentile, whiskers: 10th–90th percentile, horizontal line: median, square: mean). (d) decay of Ca2+ and time to peak were simultaneously measured and analyzed. Ca2+ decay time was significantly altered between control and CVB3-expressing cardiomyocytes under basal and stimulated conditions. Time to peak was only altered under basal conditions indicating a calcium-contraction uncoupling due to CVB3 expression (* indicates p < 0.05).

Figure 5. Electron microscopic image of sarcomeric structures with co-localized mitochondria of mouse cardiomyocytes from wt mice (left) and CVB3ΔVP0-expressing mice (right). Scale bar top right—100 nm, scale bar bottom right—30 nm. Statistical analysis of the mitochondrial area in mouse cardiomyocytes from wt mice and CVB3ΔVP0 expressing mice shows mitochondrial degradation due to CVB3 expression (*** indicates p < 0.001; n = 5).

Figure 6. (a) Gene expression pattern of control and CVB3-expressing mice illustrated as a heatmap. Gene expression levels were averaged and normalized to the expression of cardiac ACTC1, which were not altered under CVB3 expression. (b) Gene array analysis and qPCR validation of regulated key genes in age-matched wt and CVB3-transgen mice. GO analyses of biological processes and cellular components are shown. (c) RT-PCR verified downregulation of five key genes (* indicates p < 0.05).