Figure 3.

Blocking MK reduces cardiac fibrosis and preserves cardiac function during the chronic phase of myocarditis. (a) Representative cross sections of cardiac tissue of sham-treated control mice, vehicle-treated EAM mice, and EAM mice after anti-N-MK Ab treatment on day 63 using Masson’s trichrome staining. Fibrotic tissue appears blue. Bars, 1 mm (overview); 100 µm (magnification). (b) Degree of fibrosis in the cardiac tissue using a semiquantitative score. Single data points represent individual mice (sham: mean fibrosis score, 0 ± 0; EAM + vehicle: mean fibrosis score, 1.68 ± 0.32; EAM + anti-N-MK: mean fibrosis score, 0.80 ± 0.22). n = 10 for sham mice; n = 25 for EAM groups. (c) Echocardiographic images on day 63 after immunization. Representative M-mode short axis view images depicting diastolic and systolic diameters of vehicle-treated animals (EAM + vehicle) as well as anti-N-MK Ab–treated mice (EAM + anti-N-MK). (d and e) Parameters describing systolic function using (d) fractional shortening (sham, 32.1 ± 1.0 [%]; EAM + vehicle, 24.4 ± 1.1 [%]; EAM + anti-N-MK, 27.9 ± 1.0 [%]) and (e) left ventricular ejection fraction (sham, 60.8 ± 1.4 [%]; EAM + vehicle, 48.6 ± 1.9 [%]; EAM + anti-N-MK, 54.2 ± 1.5 [%]). n = 10 for sham mice; n = 25 for the EAM groups. (f–i) Intravital microscopy of cremaster muscle postcapillary venules of WT mice treated with anti–N-MK blocking Ab (anti-N-MK) or the appropriate isotype Ab (isotype ctrl) as control 2 h after intrascrotal application of TNFα. n = 15 venules from four mice (isotype ctrl); n = 17 venules from four mice (anti-N-MK). (f) Leukocyte rolling flux fraction (isotype control, 0.047 ± 0.013 [%]; anti-N-MK, 0.048 ± 0.019 [%]). (g) Leukocyte rolling velocity including 35 cells from four control mice (isotype ctrl) and 41 cells from four mice after blocking MK (anti-N-MK; isotype ctrl, 2.47 ± 0.18 µm/s; anti-N-MK, 3.32 ± 0.25 µm/s). (h) Number of adherent leukocytes per mm² (isotype control, 490.6 ± 48.8 cells/mm²; anti-N-MK, 259.8 ± 51.2 cells/mm²). (i) Hemodynamic parameters of investigated vessels. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for b, d, and e, and an unpaired Student's t test was used for f–h. For all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Data are presented as mean ± SEM or mean ± individual data points.