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Myostatin negatively regulates satellite cell activation and self-renewal.

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R - J. Cell Biol. (2003)

Bottom Line: BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type.Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells.Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Animal Genomics, AgResearch, Hamilton 2015, New Zealand.

ABSTRACT
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

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Immunocytochemistry of isolated satellite cells. Adult myoblasts were cultured from the hind leg muscles of either wild-type or Mstn−/−mice, fixed, and immunostained for M-cadherin (A), MyoD (B), Desmin (C), and CD34 (D). DAPI staining of the nuclei in the corresponding fields is also shown; panel E shows the background immunofluorescence when anti–mouse secondary antibody was used in the absence of primary antibody. Similar background was observed for all other secondary antibody negative controls. Greater than 95% of the isolated cultured cells were myogenic.
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fig2: Immunocytochemistry of isolated satellite cells. Adult myoblasts were cultured from the hind leg muscles of either wild-type or Mstn−/−mice, fixed, and immunostained for M-cadherin (A), MyoD (B), Desmin (C), and CD34 (D). DAPI staining of the nuclei in the corresponding fields is also shown; panel E shows the background immunofluorescence when anti–mouse secondary antibody was used in the absence of primary antibody. Similar background was observed for all other secondary antibody negative controls. Greater than 95% of the isolated cultured cells were myogenic.

Mentions: To gain insight into the role of Myostatin in satellite cell activation and proliferation, low passage number primary cultures were isolated from hind limb muscles of 8-wk-old myostatin- and wild-type mice to generate highly purified satellite cell–derived cultures and preclude the inclusion of neonatal myoblasts (see Materials and methods). Previous work has demonstrated that proliferating primary adult myoblasts derived from satellite cells express myoblast markers such as MyoD, Desmin, and adhesion molecules, but not terminal differentiation markers such as Myogenin or Myosin heavy chain. Therefore, to characterize the cultures used in this experiment, we immunostained the adult myoblast cultures with MyoD- and M-cadherin–specific antibodies. Over 95% of proliferating primary cells derived from the hind limb muscles of wild-type and myostatin knockout mice expressed M-cadherin and MyoD, indicating that the isolation procedure generated highly purified cultures of myogenic cells from both wild-type and myostatin knockout mice (Fig. 2, A and B). Proliferating myogenic precursor cells in vivo, and myoblasts in vitro, express the intermediate filament Desmin, whereas, satellite cells do not express Desmin (George-Weinstein et al., 1993). Hence, primary cultures were immunostained with antibodies reactive with Desmin to assess their developmental status. As shown in Fig. 2 C, uniform expression of Desmin is detected, indicating that the primary cultures are all activated myogenic precursor cells. It has been recently demonstrated that expression of CD34 protein is associated with skeletal muscle precursors but not with the differentiated fibers. To rule out any myogenic differentiation during the isolation procedure of primary cultures, the cells were immunostained with anti-CD34 antibodies. As shown in Fig. 2 D, almost all of the cultured cells were immunopositive for CD34, indicating that under the culturing conditions, the myogenic precursor cells are in active proliferation status (Beauchamp et al., 2000). The negative control did not show any nonspecific background (Fig. 2 E).


Myostatin negatively regulates satellite cell activation and self-renewal.

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R - J. Cell Biol. (2003)

Immunocytochemistry of isolated satellite cells. Adult myoblasts were cultured from the hind leg muscles of either wild-type or Mstn−/−mice, fixed, and immunostained for M-cadherin (A), MyoD (B), Desmin (C), and CD34 (D). DAPI staining of the nuclei in the corresponding fields is also shown; panel E shows the background immunofluorescence when anti–mouse secondary antibody was used in the absence of primary antibody. Similar background was observed for all other secondary antibody negative controls. Greater than 95% of the isolated cultured cells were myogenic.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172861&req=5

fig2: Immunocytochemistry of isolated satellite cells. Adult myoblasts were cultured from the hind leg muscles of either wild-type or Mstn−/−mice, fixed, and immunostained for M-cadherin (A), MyoD (B), Desmin (C), and CD34 (D). DAPI staining of the nuclei in the corresponding fields is also shown; panel E shows the background immunofluorescence when anti–mouse secondary antibody was used in the absence of primary antibody. Similar background was observed for all other secondary antibody negative controls. Greater than 95% of the isolated cultured cells were myogenic.
Mentions: To gain insight into the role of Myostatin in satellite cell activation and proliferation, low passage number primary cultures were isolated from hind limb muscles of 8-wk-old myostatin- and wild-type mice to generate highly purified satellite cell–derived cultures and preclude the inclusion of neonatal myoblasts (see Materials and methods). Previous work has demonstrated that proliferating primary adult myoblasts derived from satellite cells express myoblast markers such as MyoD, Desmin, and adhesion molecules, but not terminal differentiation markers such as Myogenin or Myosin heavy chain. Therefore, to characterize the cultures used in this experiment, we immunostained the adult myoblast cultures with MyoD- and M-cadherin–specific antibodies. Over 95% of proliferating primary cells derived from the hind limb muscles of wild-type and myostatin knockout mice expressed M-cadherin and MyoD, indicating that the isolation procedure generated highly purified cultures of myogenic cells from both wild-type and myostatin knockout mice (Fig. 2, A and B). Proliferating myogenic precursor cells in vivo, and myoblasts in vitro, express the intermediate filament Desmin, whereas, satellite cells do not express Desmin (George-Weinstein et al., 1993). Hence, primary cultures were immunostained with antibodies reactive with Desmin to assess their developmental status. As shown in Fig. 2 C, uniform expression of Desmin is detected, indicating that the primary cultures are all activated myogenic precursor cells. It has been recently demonstrated that expression of CD34 protein is associated with skeletal muscle precursors but not with the differentiated fibers. To rule out any myogenic differentiation during the isolation procedure of primary cultures, the cells were immunostained with anti-CD34 antibodies. As shown in Fig. 2 D, almost all of the cultured cells were immunopositive for CD34, indicating that under the culturing conditions, the myogenic precursor cells are in active proliferation status (Beauchamp et al., 2000). The negative control did not show any nonspecific background (Fig. 2 E).

Bottom Line: BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type.Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells.Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Animal Genomics, AgResearch, Hamilton 2015, New Zealand.

ABSTRACT
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

Show MeSH
Related in: MedlinePlus