Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy

Abstract

Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies.

Keywords: Collagen VI; Ullrich congenital muscular dystrophy (UCMD); metalloproteinases; pericellular matrix; tendon.

Figure 1

(A) Ultrastructural analysis of normal peroneal tendon showing a tenocyte with cellular processes (p, upper panel) which define extracellular matrix compartments containing collagen fibrils. Collagen fibrils are closely associated with the cell membrane (c, lower panel). Scale bar, 1 μm. (B) Ullrich congenital muscular dystrophy (UCMD) tenocytes display reduced and irregular cellular processes (p). A necrotic cell with hypercondensed heterochromatin (n) and vacuoles (v) is shown (upper lane, right panel). Note the presence of abnormal microfibrillar material accumulated in the pericellular matrix (PCM) of UCMD fibroblasts. Scale bar, 1 μm. (C) Transmission electron microscopy of cross-sectioned normal and UCMD peroneal tendon. Normal tendon displays fibrils with regular profile. In contrast, UCMD tendon displays smaller diameter fibrils; several aberrant fibrils with irregular profile are also observed (arrows). Scale bar, 200 nm. (D) Fibril diameter distribution in normal and UCMD tendon. The fibrils diameter distribution was shifted toward smaller diameters in UCMD tendon compared to normal tendon, with a significant difference in mean fibril diameter (± SEM *p < 0.001).

Figure 2

(A) Immunoelectron microscopy analysis of collagen VI (COLVI) on tissue sections of normal (upper row) and UCMD (lower row) peroneal tendon sections. In normal tendon, COLVI, identified by 15 nm colloidal gold particles, is detected among collagen fibrils (c). In contrast, in UCMD tendon, COLVI appears concentrated in the pericellular matrix of tenocytes (PCM) and scarcely associated with the collagen fibrils (c). Scale bar, 200 μm. (B) Immunoelectron microscopy of collagen I (COLI) (left panels) and fibronectin (FBN; right panels) in normal (upper panels) and UCMD (lower panels), showing a marked reduction of COLI and FBN in the pericellular matrix (PCM) of UCMD tenocytes compared to normal control (c, collagen fibrils). Scale bar, 200 nm. (C) Quantitative analysis of the density of colloidal gold particles (cgp) in the pericellular matrix (PCM), and in ECM displaced from the cells (central region, CR) of the UCMD and normal control tendon labeled with anti-COLVI, COLI and FBN antibodies. (± SEM *p < 0.001).

Figure 3

(A) Immunofluorescence microscopy of COLVI antibody in normal (CTRL, left panels) and UCMD patient (UCMD, right panels) tenocyte cultures treated with ascorbic acid for 24 h. COLVI is early secreted in normal tenocyte culture and associates with the cell surface (arrowheads). In UCMD tenocyte culture, COLVI is severely reduced in the matrix and form anomalous aggregates (arrows). The association of COLVI with the cell surface is also impaired. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (B) Immunofluorescence microscopy of COLVI in normal (CTRL, left panel) and UCMD (right panel) long term tenocyte cultures showing the abnormalities of COLVI organization in patient sample compared to complex network developed in normal culture. Nuclei were stained with DAPI (blue). Scale bar, 20 μm.

Figure 4

(A) Confocal microscopy of (COLVI) and FBN in normal (upper row) and UCMD patient (lower row) tenocyte cultures; 3D surface shaded reconstruction of an enlargement of the area defined by the white box is shown on the right. In normal tendon fibroblast culture, COLVI network co-localizes at discrete site with FBN, as visualized in merge and 3D reconstruction images. In UCMD sample, COLVI forms anomalous aggregates, which also include FBN staining (arrows). Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (B) Confocal microscopy of (COLVI) and COLI in normal (upper row) and UCMD patient (lower row) tendon fibroblast cultures; 3D surface shaded reconstruction of an enlargement of the area defined by the white box are shown on the right. In normal tendon fibroblast culture, COLI and COLVI form distinct interconnected networks, as indicated by the partial association visualized in 3D reconstruction. In UCMD tendon culture, COLI forms aggregates that match with COLVI abnormal structures. 3D reconstruction of a particular of the merge image clearly shows that collage I associates with COLVI aggregates. Nuclei were stained with DAPI (blue). Scale bar, 50 μm.

Figure 5

(A) Western blot analysis of cell lysate (Cell Layer) and conditioned medium (Medium) of normal (CTRL) and UCMD patient (UCMD) cultured tenocytes of FBN and COLI, and of the relative densitometric quantification; actin was used as a loading control of cell lysates. For cell lysate quantification, protein levels were calculated as relative intensity with respect to actin. The respective protein levels in UCMD patient cells and media were compared to the control (showed by the dark line, set as 1) ± SD (*p < 0.06 vs. control). (B) Gelatin zymography of conditioned medium from normal (CTRL) and UCMD patient showing an increased gelatinolitic activity of MMP2 in the culture medium of patient cells. (C) Western blot analysis with anti-MMP2 antibody, recognizing both the active (63 kDa) and non-active pro-MMP2 (72 kDa) form in normal (CTRL) and UCMD patient (UCMD) cultured tenocytes (Cell Layer) and conditioned medium (Medium), and the relative densitometric quantification; actin was used as a loading control of cell lysates, while tenomodulin (TNMD), a tenocyte marker, was used to assess the cell phenotype. For cell lysates, protein levels were calculated as relative intensity with respect to actin. The respective protein levels in UCMD patient cells and media were compared to the control (showed by the dark line, set as 1). Error bars indicate SD. (*p < 0.001 vs. control).