Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations

Abstract

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Keywords: Bethlem myopathy; NG2 proteoglycan; Ullrich congenital muscular dystrophy; cell polarization; cell-extracellular matrix interactions; collagen VI; extracellular matrix remodeling; metalloproteinase 2; pericellular matrix.

Figure 1

Collagen VI expression in tendon primary cultures of Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD) patients. (A) Immunofluorescence analysis of collagen VI (green) in proliferating (left panels) and confluent (right panels) tendon cultured fibroblasts from control (CTRL), BM, and UCMD patients. A clear cell membrane association of collagen VI is detectable in normal proliferating cells, while confluent cultures display a filamentous arrangement (inset in right panel). Note the presence of collagen VI aggregates (arrows) both in proliferating and confluent cultures of BM and UCMD cultures. Nuclear staining, DAPI. Scale bar, 10 μm. (B) Transmission electron microscopy visualization of rotary shadowed replicas from controls (CTRL; upper panels), BM (middle panels), and UCMD (right panels) showing well extended webs (arrows) associated with the cell membrane in normal control; disorganized webs (asterisks) are evident in BM and UCMD samples. Scale bar, 1 μm.

Figure 2

Collagen VI-NG2 axis is disrupted in tendon fibroblasts from BM and UCMD patients. (A) Confocal microscopy analysis of tendon fibroblasts double-labeled with anti-collagen VI (COL6, green fluorescence) and NG2 (NG2, red fluorescence) on proliferating samples from control (upper panels), BM (middle panels), and UCMD (lower panels). Collagen VI co-localizes with NG2 at the cell membrane of control cells. Note the moderate reduction of NG2 in BM cells, which correlates with the altered collagen VI signal. NG2 signal is reduced in UCMD cells, while collagen VI staining is dispersed among the cells. Nuclear staining, DAPI. Scale bar, 40 μm. (B) Co-localization analysis of the representative perinuclear area in collagen VI-NG2 double-labeled samples, from control (upper panel), BM (middle panel), and UCMD (right panel). White signal identifies areas of high co-localization. Nuclear staining, DAPI. Scale bar, 10 μm. (C) Western blot analysis of NG2 in cell cultures of control (CTRL), BM, and UCMD cell lysates shows reduced NG2 expression in the UCMD cells. In BM cells, NG2 is expressed at levels similar to that of the control cell lysate. Tenomodulin (TNMD), used as a marker of tendon cells, and actin, used as a loading control, do not show differences between control and patient cell lysates. (D) Densitometric analysis of NG2 and TNMD. Protein levels were calculated as relative intensity with respect to actin. The respective protein levels in BM and UCMD cells were compared to the control (CTRL; showed by the dark line, set as 1) ± SE. *** p < 0.001. (E) CSPG4 mRNA analysis in UCMD and BM cells showing no obvious differences with respect to control (CTRL) cells. Data represent mean ± SE of three independent experiments; ns, non-significant.

Figure 3

Disruption of collagen VI-NG2 axis affects BM and UCMD cell polarization during migration. (A) Immunofluorescence microscopy of collagen VI (COL6) and NG2 double-labeled cells subjected to scratch wound assay. Collagen VI microfilaments and NG2 co-localize at the rear of control (CTRL) cells. In BM and UCMD migrating cells, collagen VI and NG2 staining appears reduced, with a spot-like pattern. Nuclear staining, DAPI. Scale bar, 50 μm. (B) Immunofluorescence microscopy of Golgin-97, a marker of the Golgi apparatus, in tendon fibroblasts subjected to the scratch wound assay. In migrating cells of the control (CTRL), the Golgi apparatus was located between the cell’s leading edge and the nucleus. In BM and UCMD samples, cells at the wound edge appear incorrectly oriented (arrows indicate the direction of the single cells). Scale bar, 20 μm. (C). The graph indicates the percentage of cells whose Golgi apparatus is not facing the wound. Data represent mean ± SE of three independent experiments. *** p < 0.0001. (D) Tracking of the individual cell movement during In vitro scratch wound assay. On the left, phase contrast images at T0 of scratched samples. Colored dots identify the position of the nucleus of monitored cells. On the right, graphical representation of the tracks of each cell monitored for 20 h (T20). Scale bar, 0.5 mm. (E) Graphs showing the mean velocity (upper) and the ratio of the accumulated distance (Ad) to the Euclidean distance (Eu) (lower) of control (CTRL), BM, and UCMD patient cells. Data represent mean ± SE of three independent experiments. * p < 0.05, ** p < 0.001.

Figure 4

(A) Immunofluorescence microscopy of fibronectin (FBN), collagen type I (COLI), collagen type XII (COLXII), and their merge with collagen VI (COL6, green) in control (CTRL), BM, and UCMD long-term cultures. Note the longitudinal pattern of fibronectin fibrils in BM and UCMD samples, when compared with the intertwined pattern in normal cells. In both BM and UCMD cultures, collagen I and collagen XII staining, and their merge with collagen VI, show the presence of protein aggregates (arrows). Nuclear staining, DAPI. Scale bar, 50 μm. (B) Western blot analysis of active MMP2 (MMP2) and pro-MMP2 (p-MMP2) in control (CTRL), BM, and UCMD cell layer and medium showing an increase of the MMP2 active form in the conditioned medium of both patient cultures. (C) Gelatin zymography of cell layer and conditioned medium of two controls (CTRL), BM, and UCMD patients. MMP2 was loaded at a concentration of 50 mg/mL as a reference to the mobility of the active form. Note the increase of both pro-MMP2 (p-MMP2) and MMP2 activity in the UCMD medium. No obvious differences are detectable in BM cell layer and medium when compared with two controls (CTRL). (D) MMP2 mRNA analysis in UCMD and BM cells showing no significant (ns) differences with respect to control (CTRL) cells. Data represent mean ± SE of three independent experiments.

Figure 5

Transmission electron microscopy analysis of tendon biopsies. (A) Ultrastructural analysis of normal piriformis and pedidium tendon showing long cellular processes. Scale bar, 1 µm. (B) BM and UCMD tenocytes displaying reduced and irregular cellular processes. Necrotic cell with hypercondensed heterochromatin and vacuoles is shown (upper and lower lane, right panels). Scale bar, 1 µm. (C) Transmission electron microscopy of cross-sectioned BM piriformis tendon showing altered fibrils with irregular profiles and a ragged appearance (inset). Scale bar, 1 µm. 60 nm. (D) UCMD pedidium tendon with altered fibrils (arrows) showing irregular profile. Scale bar, 1 µm. 60 nm.