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First published online 19 November 2003
doi: 10.1242/jcs.00828

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Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation

Centro de Regulación Celular y Patología, Facultad de Ciencias Biológicas, MIFAB, P. Universidad Católica de Chile, Santiago, Chile

View larger version (68K): [in a new window]   Fig. 1. Time course of skeletal muscle regeneration after barium chloride-induced injury. (A) Skeletal muscle regeneration is completed 10-15 days after the injection of 60 µl of 1.2% w/v BaCl2 into mice TA muscle. Degenerating myofibers together with abundant mononuclear cells, mostly inflammatory and muscle precursor cells, are observed the first days after the injection. Regenerated fibers, with nuclei in a central position, appear by the fourth or fifth day after the injection. Haematoxylin and Eosin stained cross sections from control and regenerating muscles at different times after barium chloride injection are presented in the first column, and the double-detection of -dystroglycan by immunofluorescence and AChRs by labeling with rhodamine-conjugated -bungarotoxin is presented in the second and third column, respectively. -Dystroglycan staining surrounds normal and regenerating muscle fibers and is associated with the remnants of basement membranes 3 days after the injection. Clusters of AChR can be detected throughout the whole process, associated with necrotic fibers or regenerating myotubes, but their number is reduced after barium chloride induced damage. Scale bars: 50 µm. (B) Transient protein expression of myogenin and embryonic myosin during skeletal muscle regeneration. Aliquots of extracts containing equivalent amounts of protein, obtained from control TA on different days after injury were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and analyzed by western blot as described in the Materials and Methods.

View larger version (48K): [in a new window]   Fig. 2. Heparan sulfate proteoglycans increase in regenerating skeletal muscle fibers after barium chloride-induced damage. Transverse sections from adult TA in control conditions (A,E-F), and 4 (B), 5 (C) and 15 (D) days after barium chloride injection. Staining with anti--heparan sufate antibody 3G10 (A-D, red) in cross sections pre-treated with the enzyme heparitinase, suggests that heparan sulfate proteoglycans are associated with regenerating skeletal muscle fibers. As a specificity control, anti--heparan sulfate antibody staining of a section not pre-treated with heparitinase is shown (E). Hoechst 33258 staining of the same section is shown in (F). Scale bar: 50 µm. (G) On western blots with anti--heparan sulfate antibody, several bands appear to increase during skeletal muscle regeneration, and according to their electrophoretic migration pattern are probably perlecan (per), syndecan-3 (syn-3), glypican (gly) and syndecan-4 (syn-4, which appears with longer exposure times, lower inset), as is indicated at the right of the figure. Western blot detection of decorin (dcn), a proteoglycan that shows only minor variations during skeletal muscle regeneration (Casar et al., manuscript in preparation), in aliquots of the same proteoglycan-enriched samples, treated with chondroitinase ABC, is shown in the lowest inset as a loading control. (H) Electrophoretic migration pattern of the core proteins of heparan sulfate proteoglycan is similar to that observed in C2C12 myoblasts extracts with the anti--heparan sulfate antibody. Molecular mass standards are shown at the left (kDa).

View larger version (99K): [in a new window]   Fig. 3. Expression of syndecan-3, glypican and perlecan by skeletal muscle fibers during TA regeneration. Heparan sulfate proteoglycans syndecan-3 (A-D), glypican-1 (E-H) and perlecan (I-L) were detected by immunofluorescence with the corresponding specific polyclonal antibodies in cross sections from control TA (A,E,I), and TA 4 (B,F,J), 5 (C,G,K) and 15 (D,H,L) days after barium chloride injection. Heparan sulfate proteoglycans are localized at the surface or basal lamina of myofibers, and increase in newly regenerated myotubes. Scale bar: 50 µm.

View larger version (49K): [in a new window]   Fig. 4. Early up-regulation of syndecan-3 mRNA during skeletal muscle regeneration. (A) Double-labeling with anti-embryonic myosin F1.652 antibody (EM; detected with fluorescein-conjugated secondary antibodies, green) and specific antibodies (detected with rhodamine-conjugated secondary antibodies, red) against laminin (Lam), syndecan-3 (Syn3) and perlecan (Per) was performed in TA sections from muscles injected with barium chloride 4 days before. The arrowhead indicates elongated cells beneath the basal lamina of intact myofibers that express embryonic myosin, probably nascent myotubes, being formed by fusion of activated satellite cells. Syndecan-3 is detected on the surface of these cells expressing embryonic myosin. Perlecan, on the contrary, has a pattern similar to laminin staining. Nuclei were stained with Hoechst 33258 (blue) to facilitate morphological identification. The bar indicates 50 µm. (B) A significant and early up-regulation of syndecan-3 mRNA during skeletal muscle regeneration was detected by northern blot, whereas only a small increase on the fifth day was observed for glypican-1 mRNA content.

View larger version (59K): [in a new window]   Fig. 5. Proliferation capacity in damaged muscle is not impaired in AN-10i as compared to C2C12i myoblasts 24 hours after transplantation. Representative microphotographs of skeletal muscle transverse sections 1 day after grafting with C2C12i (A,B) and AN-10i (C-D) myoblasts, showing X-gal staining of ß-galactosidase (A,C) and immunofluorescent detection of BrdU labeling in the same sections (B,D). Arrowheads indicate nuclei positive for both BrdU and ß-galactosidase. Scale bar: 50 µm. (E) No difference was found between C2C12i and AN-10i myoblasts when BrdU labeling indexes were quantified in ß-galactosidase-positive cells. (F) C2C12i and AN-10i proliferate at similar rates in normal growing conditions in cell culture.

View larger version (68K): [in a new window]   Fig. 6. AN-10i myoblast differentiation is impaired in regenerating skeletal muscle. (A) Representative photomicrographs of skeletal muscle transverse sections 7 days after grafting with C2C12i and AN-10i myoblasts. ß-Galactosidase was detected with X-Gal staining and skeletal muscle fibers were detected by immunohistochemistry against -dystroglycan. ß-Galactosidase-positive nuclei can be found incorporated into myofibers (C2C12i and AN-10i, upper panels) or in mononuclear cells in the interstitial tissue (C2C12i and AN-10i, lower panels). Scale bar: 25 µm. (B-D) Quantification of the ratio of ß-galactosidase-positive cells expressing differentiation markers (embryonic myosin or -dystroglycan) among total ß-galactosidase-positive cells in transplantation experiments shows that only a minority of the grafted cells from both groups had differentiated 3 days after transplantation, and that on the fifth and seventh day fewer AN-10i myoblasts were incorporated into myofibers than wild-type C2C12i myoblasts (*, P<0.01, unpaired Student's t-test).

View larger version (61K): [in a new window]   Fig. 7. Two subclones of C2C12 cells with inhibited syndecan-3 expression show a similar impairment in their differentiation capacity when transplanted into regenerating skeletal muscle. (A) Comparison of the quantification of the ratio of ß-galactosidase positive cells that are also positive for -dystroglycan (-DG) on sections taken day 5 after transplantation shows that syndecan-3 antisense clones, AN-10i and AN-8i, have similarly defective expression of this differentiation marker in contrast to C2C12i (*P<0.01, unpaired Student's t-test). Owing to its great variability, the total number of ß-galactosidase-positive cells was not significantly different among the groups, but, interestingly, the two antisense clones showed opposite tendencies: a higher number of AN-8i cells and a lower number of AN-10i cells were observed compared to the mean number of C2C12i cells detected per section [for C2C12i, 206±224 cells/section (n=41) were observed vs. 294±180 cells/section (n=38) for AN-8i and 109±143 cells/section for AN-10i (n=39); mean±s.d.]. (B) A similar result is obtained when quantification of ß-galactosidase-positive myotubes is performed with embryonic myosin (EMHC), a second differentiation marker (*P<0.01; **P<0.05, unpaired Student's t-test). (C) Representative transverse sections of ß-galactosidase-positive cells in skeletal muscle 5 days after grafting, stained using anti-embryonic myosin histochemistry, for C2C12i, AN-10i and AN-8i myoblasts. Scale bar: 25 µm.

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