Type V collagen from macrophages regulates initial collagen assembly and alignment in post-infarcted hearts

Fig. 1. Fibrillar collagen deposition in regenerative and fibrotic mouse hearts at 4-days post-injury. Confocal imaging of an immunofluorescent-stained cross-section from P1 or P7 hearts at day 4 post-MI (4dpi) showing endomucin (EMCN)-stained vessels (red) and Collagen I (Col I, white; a, a’ and d, d’). Collagen III (Col III, white; b, b’ and e, e’) and Collagen V (Col V, white; c, c’ and f, f’). a’-f’ are magnified views of the inset boxes in (a–f). LV: left ventricle. Epi: epicardium. Representative images of n = 3, scale bar: 100 μm. Transmission electron microscopy (TEM) of the scar region of a P1 heart at 4dpi showing the injured area and cardiomyocytes (g). A fibroblast and surrounding collagen fibrils (white arrows) in the same P1 heart at 4dpi (h). Magnified collagen fibrils (i). Scar area of a P7 heart 4dpi showing dying cardiomyocytes, fibroblasts and collagen (j). A fibroblast and collagen fibrils (k). Magnified collagen fibrils in the P7 heart 4dpi (l). Scale bar as indicated. M: cardiomyocyte. Fb: fibroblast; Nu: neutrophil; ER: endoplasmic reticulum. Cl: collagen.

Fig. 2. Fibrillar collagen deposition in regenerative and fibrotic mouse hearts at 7 days post-injury. Confocal imaging of an immunofluorescent-stained cross-section from P1 or P7 hearts at day 7 post-MI (7dpi) showing endomucin (EMCN)-stained vessels (red) and Collagen I (Col I, white; a, a’, d, d’:). Collagen III (Col III, white; b, b’, e, e’) and Collagen V (Col V, white; c, c’, f, f’). a’-f’ are magnified views of the inset boxes in (a–f). LV: left ventricle. Epi: epicardium. Representative images of n = 3, scale bar: 100 μm. TEM of three different area in the scar region of a P1 heart at 7dpi showing the low electron density vacuoles and cardiomyocytes (g–i). TEM of a section of the scar area of a P7 heart at 7dpi showing damaged myocardium and dying cardiomyocytes (j). A magnified view from the inset box in (j) showing a fibroblast and surrounding collagen fibrils (k). A magnified view from the inset box in (k) showing collagen fibril bundles (l). Scale bar as indicated. CM: cardiomyocyte. Cp: capillary. Fb: fibroblast. Cl: collagen.

Fig. 3. Fibrillar collagen deposition in regenerative and fibrotic mouse hearts at 21 days post-injury. Confocal imaging of an immunofluorescent-stained cross-section from P1 or P7 hearts at day 21 post-MI (21dpi) showing endomucin (EMCN)-stained vessels (red) and Collagen I (Col I, white; a, a’, d, d’:). Collagen III (Col III, white; b, b’, e, e’) and Collagen V (Col V, white; c, c’, f, f’). a’-f’ are magnified views of the inset boxes in (a–f). LV: left ventricle. Epi: epicardium. Representative images of n = 3, scale bar: 100 μm. TEM of myocardium proximal to suture of a P1 heart at 21dpi showing cardiomyocytes and collagen fibrils (g). Magnified view of the inset box in (g) showing the cardiomyocyte and the collagen fibrils (h). Magnified view of collagen fibrils in the same section (i). TEM of the scar area of a P7 heart at 21dpi showing cardiomyocytes, fibroblasts and collagen fibrils (j). Magnified view from the inset box of (j) showing abundant collagen fibrils (k). Magnified view of collagen fibrils in the same section (l). Scale bar as indicated. CM: cardiomyocyte. Cl: collagen. FB: fibroblast.

Fig. 4. Col V is expressed in early infarcted myocardium. Confocal imaging of an immunofluorescent-stained 4-chamber cross-section from a P1 heart at 1dpi showing injured area (green), endomucin (red) and Col V deposition (white; a–e). Immunofluorescent staining of Col I (f) and Col III (g) of serial sections in the same region as in (b). LV: left ventricle. RV: right ventricle. Scale bar: 100 μm. TEM showing cells with macrophages features in a P1 heart at 4dpi and proximal collagen fibrils (h, i). A magnified view of the inset box in (h) showing transverse section of collagen fibrils (j). TEM of a P7 heart at 4 dpi showing cells with macrophage features proximal to collagen fibrils (k, l). Magnified view from (k) showing close association of collagen fibrils and macrophages (m). Scale bar as indicated. M: macrophage. Cl: collagen. Ly: lysosome. Rb: rudiment body.

Fig. 5. Colocalization of macrophages and Col V after injury. Confocal imaging of Immunofluorescent-stained cross-sections from P1 (a), P7 (b) and adult (c) hearts at 1dpi respectively, showing localization of CD68+ macrophages (red), Col V (white) and nuclei (DAPI, blue). Epi: epicardium. LV: left ventricle. Confocal imaging of immunofluorescent-stained cross-sections of P1 (d), P7 (e), and adult (f) hearts at 7dpi showing localization of CD68+ macrophages (red), Col V (white) and nuclei (DAPI, blue). Epi: epicardium. LV: left ventricle. Scale bar: 100 μm.

Fig. 6. Col V deletion from macrophages results in altered scarring and compromised collagen alignment. Schematic showing the Col5a1 deletion strategy in CD68+ macrophage lineages. Col5a1fl/fl, hCD68-CreERT2/+ males were crossed with Col5a1fl/fl, tdTomatoAi14/tdTomatoAi14 females. Progenies were administered 200 mg tamoxifen for 5 days and then subjected to LAD ligation surgery and analysed at the following timepoints (a). Masson-Trichrome staining of serial sections from three controls and three mKO samples showing the dilated and thinned left ventricles (b). Scale bars: 5 mm. Magnified Masson-Trichrome staining of representative sections from a control (CON) and an mKO heart (c, upper panel). Polarized laser microscopy on picrosirius red staining (lower panel) of serial sections from the same control and mKO hearts showing the collagen fibrils (red) in the left ventricle myocardial wall (c, lower panel). Scale bars 10 μm. Degree of anisotropy analysed with Fibriltool based on polarized laser microscopy on picrosirius red staining of serial sections from the control (CON) hearts and mKO hearts (d) N = 3 per group; p = 0.0082. Ejection fraction (EF, e), Fractional area change (FAC, f)…

Fig. 7. Ultrastructure defects in the extracellular matrix (ECM) of macrophage-Col V depleted mice. TEM showing the morphology of cells and associated collagen fibrils in infarcted hearts of Col5a1fl/fl (CON) and Col5a1fl/fl, CD68-CreERT2+ (mKO) mice at 4dpi (a–l), 7dpi (M-S) and 21dpi (T-Z). Lower magnification views of serial sections of the injured area within the myocardium of a CON (a) and mKO heart at 4dpi (d). Collagen fibril bundles in the myocardium of CON (b, c) and mKO hearts (e, f). Macrophages in close proximity to collagen fibrils in CON (g) and mKO hearts (j) at lower magnified views highlighting differences in collagen fibril bundle formation, deposition and alignment (h, i, k, l). Lower magnification views of serial sections of the injured area within the myocardium of a CON (m) and mKO heart at 7dpi (q) with higher magnification insets (n) and (r). Higher magnification to show differences in collagen fibril bundles and fibres in the myocardium of CON (o, p) and mKO hearts (s). Macrophages proximal to collagen fibrils in CON (N) and mKO hearts (R). Lower magnification views of serial sections of the injured area within the myocardium of a CON (t) and mKO heart at 21dpi…