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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02482

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Improved muscle healing through enhanced regeneration and reduced fibrosis in myostatin-null mice

Seumas McCroskery¹, Mark Thomas¹, Leanne Platt², Alex Hennebry¹, Takanori Nishimura¹, Lance McLeay², Mridula Sharma^1,* and Ravi Kambadur^1,*,

¹ Animal Genomics, AgResearch, Private Bag 3123, East Street, Hamilton, New Zealand
² School of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand

View larger version (31K): [in a new window]   Fig. 1. Mstn inhibits SC proliferation. (A) Isolated single muscle fibers (n=30) were treated with increasing amounts of Mstn. After 48 hours, the fibers were fixed for immunofluorescence to detect BrdU-positive SCs. Increasing Mstn levels reduced the number of BrdU-positive SCs. (B) In the rescue experiment, single muscle fiber cultures were maintained in FM with 1 µg/ml Mstn either throughout the experiment (FM + 1 µg/ml Mstn) or for the first 24 hours, and then in FM without Mstn (Rescue) for a further 24 hours. Control muscle fibers were maintained in FM media throughout the experiment. The activated SCs were identified by BrdU incorporation and immunofluorescence. Whereas addition of Mstn inhibits the activation of SCs, removal of Mstn from the media rescues the SCs from the inhibitory effects of Mstn (***P<0.001). (C) Single muscle fibers (n=30) were isolated from muscle and incubated in media conducive to the migration of satellite cells in the presence of increasing concentrations of Mstn (***P<0.001). The average number of migrated satellite cells at varying Mstn concentrations is shown. (D, i) Example of a typical myofiber with BrdU-positive nuclei. (D, ii) The same myofiber with DAPI-stained nuclei.

View larger version (69K): [in a new window]   Fig. 2. Lack of Mstn facilitates muscle regeneration. Tibialis anterior muscle from wild-type and Mstn-/- mice were injured using notexin, and frozen muscle samples were sectioned. (A) Hematoxylin and eosin staining of control uninjured muscle sections from wild-type and Mstn-/- mice are shown. (B) One day (D1) after the notexin treatment there was extensive muscle necrosis in both wild-type and Mstn-/- mice (low power view shown in B). (C) Myofibers contained eosinophilic (e) cytoplasm and some cells showed fine intracellular vacuolation (v). There was an increase in the intracellular spaces with marked myofiber disruption visible (arrows). (D) In day 2 Mstn-/- muscle sections (D2), increased numbers of nuclei within the injured area were seen (arrows). Arrow heads denote the myonuclei along the margins of the necrotic myofibers. (E) Three days after notexin treatment (D3), both muscle sections have infiltrating mononucleated cells, but higher numbers were present in the Mstn-/- sections. (F) In day 5 sections (D5), increased numbers of nuclei were seen within the injured area of Mstn-/- muscle sections. (A,C-F) Bar, 10 µm. (B) Bar, 100 µm.

View larger version (50K): [in a new window]   Fig. 3. Lack of Mstn increases the accretion of myoblasts and macrophages. In a unit regenerating area (20 mm2), Myod1+ or Mac-1+ nuclei were counted and expressed as percentage of the total nuclei identified by DAPI incorporation. The results were an average of three independent sections and the counting of the nuclei was carried out in triplicate on individual sections. (A) Twice the number of Myod1+ myogenic precursor cells were present in the regenerating area of Mstn-/- sections at all times as compared with wild type, indicating a greater migration and/or increased proliferation of myoblasts in the regenerating muscle. (B) Anti-Myod1 immunostaining of regenerating muscle (day 2) from wild-type and myostatin-null (Mstn-null) mice. A DAPI-counterstained section is displayed within the inserts. Examples of positive nuclei and cells are indicated with an asterisk (*). Bar, 10 µm. (C) Anti-Mac-1 antibodies were used to identify infiltrating macrophages. An enhanced inflammatory response in the Mstn-/- muscle is observed at day 2 compared with the wild-type muscle. The number of macrophages in the wild-type muscle was maximum on day 3 and macrophages were still present on day 5 in the wild-type muscle. However, macrophages were not detectable on day 5 in the Mstn-/- muscle section. (**P>0.001; *P>0.01). (D) Anti-Mac-1 immunostaining of regenerating muscle (day 2) from wild-type and myostatin-null (Mstn-null) mice. A DAPI-counterstained section is displayed within the inserts. Examples of positive nuclei and cells are indicated with an asterisk (*). Bar, 10 µm. (E) Expression profiles of genes in control uninjured muscle and regenerating wild-type and Mstn-/- muscle are shown. Quantitative RT-PCR was performed for Myod1 and Myog. The amplicons were detected by Southern blot hybridization. GAPDH was used to show equal amount of RNA used.

View larger version (16K): [in a new window]   Fig. 4. Mstn inhibits the migration of SCs and macrophages. (A) Graph showing the chemo-inhibitory effect of Mstn on myoblasts. The addition of a chemoattractant to the bottom well of a blind-well chamber increases the migration of myoblasts across the filter by 200%. When Mstn was added with the chemoattractant (5 µg/ml), the migration is reduced by 50%. Mstn alone does not inhibit chemokinesis (**P<0.01; Student's t-test). (B) Graph showing the chemo-inhibitory effect of Mstn on macrophages using blind-well chambers. The addition of a chemoattractant to the bottom well increases the migration of macrophages across the filter by over 200%. When Mstn was added with the chemoattractant (5 µg/ml), the migration is reduced by 50%. Mstn alone does not inhibit chemokinesis. (**P<0.01; Student's t-test).

View larger version (50K): [in a new window]   Fig. 5. Reduced fibrosis in the Mstn-/- mice. (A) Low-power micrograph (i), and high-power micrograph (ii) of hematoxylin and eosin staining (H&E) and Van Geisen (iii) staining of day 28 (D28) wild-type and Mstn-/- muscle sections; bar, 100 µm. Bar, 10 µm. Thick connective tissue (arrows) is only seen in wild-type muscle sections with H&E staining (ii). Similarly, considerably more collagen (red) staining was seen in the wild-type muscle sections (iii; Van Geisen), indicating over-expression of extracellular membrane proteins, leading to fibrosis. Further evidence of increased fibrosis was observed in the scanning electron micrographs of wild-type muscle after 24 days of regeneration (iv); bar, 120 µm. (B) Increased expression of decorin mRNA in the Mstn-/- regenerated muscle. (i) Quantitative RT-PCR was performed on total RNA isolated from day 28 wild-type and Mstn-/- muscle and the amplicons were detected by southern hybridization. (ii) Densitometry analysis and normalization of results to GAPDH expression indicates that there is increased expression of decorin mRNA in the regenerating Mstn-/- muscle.

View larger version (28K): [in a new window]   Fig. 6. HGF inhibits Mstn expression. (A) Quantitative RT-PCR was performed for Mstn and Hgf on regenerating and wild-type muscle. The amplicons were detected by Southern blot hybridization. GAPDH was used to show equal amount of RNA used. (B) C2C12 cells stably transfected with the 1.6 kb Mstn promoter luciferase reporter construct were treated with either 20 ng of HGF or with vehicle, and the luciferase assay was performed on cell lysates. Luciferase activity is normalized to ß-galactosidase activity. HGF treatment significantly downregulates Mstn promoter activity. (***P<0.001; Student's t-test).

View larger version (28K): [in a new window]   Fig. 7. A model for the role of Mstn in skeletal muscle healing. Myotrauma activates SCs and the inflammatory response. As a result, macrophages and SCs migrate to the site of injury. Mstn negatively regulates SC activation and inhibits migration of macrophages and SCs. Activated SCs proliferate at the site of injury and resulting myoblasts (MB) either fuse with the damaged myofiber or form a new myotube.

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