Collagen XV preserves heart function and protects from pathological remodelling after myocardial infarction

Abstract

Increasing knowledge of the components involved in left ventricle (LV) remodelling and fibrotic processes after a myocardial infarction is crucial to understanding heart pathology. We have here analysed collagen XV (ColXV) expression in human myocardial infarct samples and assessed how its deficiency affects cardiac responses, such as fibrogenesis and tissue stiffness, after acute myocardial infarction (AMI) in mice. We first observed high ColXV expression in human infarction scars. After ligating the left anterior descending artery in mice, cardiac function and remodelling were monitored by echocardiography, elasticity assessment, immunohistochemical analysis and ultrastructural assessments. After AMI, Col15a1^-/- mice showed significantly increased tissue stiffness and upregulation of fibrosis-related genes in the remote myocardium. Striking differences were observed between the genotypes in the scar ultrastructure, protein compositions, cardiomyocyte morphology and intracellular architecture. Furthermore, the proportion of immature collagen fibres in the infarct border zone increased in Col15a1^-/- mice, suggesting fragility and poor scar resistance to mechanical stress. Structural parameters indicated more substantial LV remodelling in the knockout mice, leading to a more dilated ventricle. Functionally, the ejection fraction and fractional shortening decreased significantly in Col15a1^-/- mice, indicating impaired heart contractile capacity. The results show that in the event of an AMI, ColXV plays an essential role in sustaining cardiac structure and function. In the absence of ColXV, dysregulated remodelling results in disrupted scar and infarct border zone, and stiffer left ventricle. These changes lead to a more severe cardiac phenotype and may affect long-term survival after AMI.

Keywords: collagen XV; extracellular matrix remodelling; fibrosis; infarct scar; tissue stiffness.

Fig. 1

Expression and localisation of collagen XV in normal and infarcted human myocardia. Representative immunohistochemical figures of (A) a normal human myocardia from control individuals (n = 5) and (B) an infarcted myocardia (n = 20 individuals) showing ColXV upregulation in an infarcted myocardia. ColXV localises to the BM zones of the vascular structures (black arrowheads), adipocytes (white arrowheads) and cardiomyocytes (arrows), and is upregulated in the interstitial matrix and the areas of perivascular fibrosis (stars) in the infarcted samples. Original magnifications were 4× and 10×.

Fig. 2

Expression and localisation of collagen XV in fractionated cardiac cells and in the acute myocardial infarction (AMI) scar, and representation of the infarct model and heart morphology in mice. Quantitative PCR analysis of the expression of Col1a1, Myh6, Tek (Tie2) and Col15a1 mRNA in fractionated mouse cardiac cells (WT hearts, n = 5; Col15a1 ^−/− hearts, n = 5) showed that (A) the collagen XV was mainly expressed in endothelial cells and fibroblasts, showing a lower relative expression level in cardiomyocytes. Cells isolated from Col15a1 ^−/− mice did not express Col15a1. (B) The expression levels of markers for endothelial cells, fibroblasts and cardiomyocytes [e.g. Tek (Tie2), Col1a1 and Myh6, respectively] in different cell types indicated that the isolated cell fractions contained specific cell types (duplicate analyses, n = 5 mice/group). (C) Schematic representation of the left anterior descending (LAD) coronary artery ligation to induce in vivo ischemia in mice. Sham‐control operation was done without the ligation of LAD. (D) Schematic figure of the heart, showing the different areas analysed: scar, border zone and remote myocardium. Ligation site is marked with a thick black cross. (E) Representative image shows that immunofluorescence ColXV signal in the WT mouse (n = 7) scar area after AMI localises to the BM zones of the capillaries (arrowheads), the cardiomyocytes (arrows) and the interstitial matrix (stars). Cd31 was used as a marker for endothelial cells. (F) qPCR analysis for Col151a in the scar area (the apex of the heart) 5 weeks after AMI (duplicate analyses, n = 6 mice/group). (G) Representative images of the mid‐left ventricles of the sham‐operated (WT hearts, n = 5; Col15a1 ^−/− hearts, n = 6) and AMI mice (n = 7 mice/group) and the corresponding Masson trichrome‐stained tissue sections, showing the fibrotic scar area (blue). Error bars represent the standard deviation (SD). Original magnifications: E, 20×; G, (Masson staining) 4×. AMI, acute myocardial infarction; KO, knock out (Col15a1 ^−/− ); LAD, left anterior descending coronary artery; LV, left ventricle; RV, right ventricle; WT, wild‐type; *P < 0.05; (Student's t‐test). Schematic figures were created in BioRender; Karppinen, S‐M (2025).

Fig. 3

Analyses of cardiomyocytes and heart elasticity in wild‐type and Col15a1 ^−/− mice. (A) Representative phase‐contrast images and dystrophin/phalloidin staining of adult mouse primary cardiomyocytes cultured in laminin‐coated wells for 6 h. (B) The lengths and (C) widths of the cardiomyocytes (CM) were measured from the 10× microscopic fields (3 mice/genotype, 5 fields/mouse in (A–D) and (C) the length‐to‐width aspect ratio was counted. (D) The histogram shows the relative frequency distribution of the aspect ratios of the WT and Col15a1 ^−/− cardiomyocytes. (E) The numbers of attached cardiomyocytes were counted from the 10× microscopic fields (four mice/genotype, five fields/mouse) of laminin‐coated wells and following treatment with recombinant collagen XV (COLXV). (F) Cross‐sectional HE sections and (G) longitudinal laminin‐stained sections were used to define cardiomyocyte characteristics. (H) The mean diameter (black line) of the cardiomyocytes (black arrows) were measured in the Col15a1 ^−/− and WT hearts. (I) Abnormally shaped wavy myofibres (white arrows in B) as percentages in the microscopic fields were analysed after AMI. Ten separate 40× microscopic fields from three mice per genotype were analysed in (F–I). (J) The elastic modulus was calculated for the sham‐operated and AMI hearts (n = 4 mice per group, WT hearts, n = 3; Col15a1 ^−/− hearts, n = 4 samples/group, and three measurements of each sample were performed). Error bars represent SD. Original magnifications: (A), 10×, 20× and 60×; (F, G) 40×. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (nested t‐test, nested one‐way ANOVA followed by Tukey's HSD (alpha = 0.05), or chi‐squared and Fisher's exact test); AMI, acute myocardial infarction; CM, cardiomyocyte; COLXV, collagen XV; HE, haematoxylin–eosin; n.s., not significant; WT, wild‐type.

Fig. 4

Quantification of scar size and peripheral fibrosis, and co‐localisation of collagen XV with α‐SMA and Cd31 in WT and Col15a1 ^−/− hearts. The amount of fibrosis was quantified from the Masson trichrome‐stained sections (A) in the scar area and (B) in the remote myocardium after AMI in the WT and Col15a1 ^−/− mice (n = 7/genotype). Representative triple‐label IF staining of 20× microscopic fields (3 mice/genotype, 3 fields/mouse) with antibodies against Cd31 (magenta), collagen XV (green) and α‐SMA (purple) shows that (C) α‐SMA and Cd31 co‐localise in Col15a1 ^−/− (C) sham and (D) AMI hearts in larger vascular structures (arrows) but not in the spindle shaped cells (stars) or in smaller vascular structures (arrow heads). Collagen XV shows negative staining in Col15a1 ^−/− hearts. (E–F) Collagen XV co‐localises with some α‐SMA ‐positive cells in the WT scar area (stars), and additionally with CD31 in vascular structures (arrows) and with both α‐SMA and Cd31 in larger vascular structures (arrow head). DAPI (blue) was used for nuclear staining. Error bars represent SD. Original magnifications 20×. *P < 0.05 (Student's t‐test); α‐Sma, alpha‐smooth muscle Actin; AMI, acute myocardial infarction; ColXV, collagen XV; ns., not significant; WT, wild‐type.

Fig. 5

Proliferative cell counts from cryosections of WT and Col15a1 ^−/− mice. (A) Proliferating myofibroblasts (arrows) shown in the 20× microscopic fields of the α‐SMA/Ki67‐double‐stained cryosections. (B) The proportions of proliferating myofibroblasts of all proliferating cells were counted from α‐SMA/Ki67 double‐stained cryosections, and proliferating (C–D) immune cells and (E–F) endothelial cells (arrows) of all proliferating cells from CD45/Ki67‐ and CD31/Ki67‐double‐stained cryosections, respectively. (G–H) The proportions of proliferating cells (Ki‐67 positive cells, arrows) of all cells (DAPI) were counted from DAPI/Ki67‐stained cryosections from the remote myocardium of the infarcted WT and Col15a1 ^−/− hearts. Ki67 was used as a marker for proliferating cells. 20× microscopic fields (four mice/genotype, three fields/mouse) were analysed at each point. Error bars represent SD. **P < 0.01 (nested t‐test); α‐SMA, alpha‐smooth muscle Actin; AMI, acute myocardial infarction; ColXV, collagen XV; ns., not significant; WT, wild‐type.

Fig. 6

Isolated, cultured fibroblasts and cell counts after AMI in WT and Col15a1 ^−/− hearts. (A) Adult mouse primary fibroblasts were photographed under phase‐contrast microscopy after 6 h (Day 0) and 24 h (Day 1) in representative culture plates. Dividing fibroblasts are indicated by arrows. (B) The fibroblast numbers were counted 24 h after culturing from five 20× microscopic fields from four mice per genotype. Statistical differences were not observed in the quantities of (C) leukocytes, (D) oedema, (E) focal necrosis and (F) apoptotic cells, nor in (G) the average capillary counts between genotypes. Ten separate 40× microscopic fields from three mice per genotype were analysed for C–G. Error bars represent SD. Original magnifications 20×. **P < 0.01; ****P < 0.0001 [nested t‐test or nested one‐way ANOVA followed by Tukey's HSD (alpha = 0.05)]. AMI, acute myocardial infarction; WT, wild‐type.

Fig. 7

Histological and ultrastructural analyses of collagen fibres and sarcomere structure. (A) Picrosirius red was used to stain the thin (green, arrowheads) and thick (red, arrows) collagen fibres in the scar, scar border zone and remote myocardium. (B) Representative transmission electron microscopy (TEM) images show different proportions of poorly visible areas of cross‐sectional collagen fibres (squared area in the WT sample) and areas where the fibres were readily visualised (squared area in the Col15a1 ^−/− sample) between the genotypes (P < 0.0001, nested t‐test). (C) The diameters of the fibrils were measured from the TEM images. (D) Longitudinal sections of collagen fibres revealed disorganised areas of the collagenous matrix in Col15a1 ^−/− scars (arrows). (E) The distances between the fibrils were measured from the TEM images, and (F–G) the distance between the Z‐lines (indicated by stars and white lines) was analysed. Ten separate microscopic fields for light microscopy (A) and five fields for TEM (B–G) from three mice per genotype were analysed. Original magnifications: A 20×; B and D 11000×; F 7400×. Error bars represent SD. ****P < 0.0001 (nested t‐test); AMI, acute myocardial infarction; WT, wild‐type.

Fig. 8

Immunofluorescence analyses after AMI in mouse hearts. (A) The fibrillin‐1 immunosignal was highly reduced and focally aggregated (arrows) in the Col15a1 ^−/− infarction scars compared with the stronger and fibrillar‐like signal in the WT scars. (B) Quantification of Fibrillin‐1 immunofluorescence signal. (C) The signal for decorin was slightly but not significantly decreased in the Col15a1 ^−/− scars. (D–E) The signal for gelsolin was highly downregulated in the Col15a1 ^−/− remote area. Perivascular areas often had high gelsolin signals (arrowheads). (F) The alpha‐actinin signal was unevenly distributed in the Col15a1 ^−/− remote area, showing both low‐ (stars) and high‐ (arrows) intensity areas. Equal signals for (G) desmin, (H) dystrophin, (I) connexin 43 and (J) desmoplakin were observed between WT and Col15a1 ^−/− remote area. Ten separate 20× microscopic fields from three mice per genotype were analysed for each staining. Error bars represent SD. ***P < 0.001 (nested t‐test); AMI, acute myocardial infarction; WT, wild‐type.

Fig. 9

Representative transthoracic M‐mode SAX images showing the measurement of echocardiography parameters. Images of wild‐type sham (noninfarcted) (uppermost figure), wild‐type AMI (middle figure) and Col15a1 ^−/− AMI (lowest figure) mice 5 weeks after AMI to show the difference in contractile functions. IVS;d, interventricular septum in diastole; IVS;s, interventricular septum in systole; LVID;d, left ventricular internal diameter in diastole; LVID;s, left ventricular internal diameter in systole; LVPW;d, left ventricular posterior wall thickness in diastole; LVPW;s, left ventricular posterior wall thickness in systole. Vevo Lab 5.8.1 ultrasound analysis software was used to view and measure the acquired data.

Fig. 10

Echocardiography results in mice. The boxplot (left) shows (A) the ejection fraction, (B) fractional shortening, (C) LV end‐diastolic diameter, (D) LV end‐diastolic area (ENDO area; d), (E) left ventricular diastolic volume (LV volume; d) and (F) isovolumic relaxation time at different time points visualised as the median (black line within each box), 25^th and 75^th percentiles, with whiskers indicating maximal and minimal values (i.e. the variability outside the upper and lower quartiles), and dots denoting outliers. The scatter plot (right) shows individual datapoints. The normal distribution was assessed using the Kolmogorov–Smirnov test. Statistical analysis was performed to compare the two groups using unpaired, two‐tailed Student's t‐tests. n = 5–7 mice per genotype. *P < 0.05; AMI, acute myocardial infarction; d, diastolic; LV, left ventricle; WT, wild‐type.