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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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Lack of Myostatin increases the activation of satellite cells on myofibers. (A, i) In vivo quantification of activated satellite cells in the muscle of wild-type and myostatin- mice. Activated satellite cells were labeled with BrdU in wild-type or myostatin- (Mstn −/−) mice of 4 wk, 8 wk, or 6 mo and were isolated using Percoll gradient. 5,000–10,000 satellite cells were immunostained for BrdU, and percentages of nuclei that were positive for BrdU labeling are shown. **, P < 0.01 (as compared with wild type). At least a total of 1,000 cells were counted in each of three replicates. The data provided are an average of three animals each. (A, ii) Myostatin inhibits the migration of satellite cells from fibers. Single muscle fibers (n = 32) were isolated from the muscle and incubated in media conducive to the migration of myogenic precursor cells. The addition of Myostatin in increasing concentrations decreases the percentage of fibers with migrated satellite cells (**, P < 0.01). (B, i) The number of satellite cells (CD34 positive) per 100 myonuclei in wild-type and myostatin- (Mstn−/−) myofibers is shown. **, P < 0.01 (as compared with wild type). More than 1,000 nuclei were counted in each of three replicates. The data presented are an average of three animals each. (B, ii) Micrograph showing typical CD34-immunostained satellite cell and (iii) myonuclei were visualized by counterstaining with DAPI. (C, i) An increased number of satellite cells migrate from myostatin- fibers. Quantitative analysis demonstrates that an increased number of myogenic progenitor cells migrates from single myofibers isolated from myostatin- mice as compared with wild-type myofibers (*, P < 0.05). (C, ii) An example of satellite cells surrounding an isolated myofiber after 72 h.
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fig4: Lack of Myostatin increases the activation of satellite cells on myofibers. (A, i) In vivo quantification of activated satellite cells in the muscle of wild-type and myostatin- mice. Activated satellite cells were labeled with BrdU in wild-type or myostatin- (Mstn −/−) mice of 4 wk, 8 wk, or 6 mo and were isolated using Percoll gradient. 5,000–10,000 satellite cells were immunostained for BrdU, and percentages of nuclei that were positive for BrdU labeling are shown. **, P < 0.01 (as compared with wild type). At least a total of 1,000 cells were counted in each of three replicates. The data provided are an average of three animals each. (A, ii) Myostatin inhibits the migration of satellite cells from fibers. Single muscle fibers (n = 32) were isolated from the muscle and incubated in media conducive to the migration of myogenic precursor cells. The addition of Myostatin in increasing concentrations decreases the percentage of fibers with migrated satellite cells (**, P < 0.01). (B, i) The number of satellite cells (CD34 positive) per 100 myonuclei in wild-type and myostatin- (Mstn−/−) myofibers is shown. **, P < 0.01 (as compared with wild type). More than 1,000 nuclei were counted in each of three replicates. The data presented are an average of three animals each. (B, ii) Micrograph showing typical CD34-immunostained satellite cell and (iii) myonuclei were visualized by counterstaining with DAPI. (C, i) An increased number of satellite cells migrate from myostatin- fibers. Quantitative analysis demonstrates that an increased number of myogenic progenitor cells migrates from single myofibers isolated from myostatin- mice as compared with wild-type myofibers (*, P < 0.05). (C, ii) An example of satellite cells surrounding an isolated myofiber after 72 h.

Mentions: Using single-cell PCR analysis, Cornelison and Wold (1997) have previously speculated that Myostatin is an important regulator of the quiescent state of satellite cells. Thus, to determine the involvement of Myostatin in satellite cell quiescence, the cell cycle phase of the satellite cells attached to myofibers in vivo in wild-type and myostatin knockout mice was assessed. Both wild-type and myostatin knockout mice were pulsed with BrdU, and satellite cells from m. tibialis anterior muscle were isolated by Percoll gradient. Characterization of satellite cells with M-cadherin and CD34 immunostaining revealed that the cultures were >95% pure and that there is no difference in the yield or purity between wild-type and myostatin knockout mice (unpublished data). BrdU-positive satellite cells were identified by immunostaining. As shown in Fig. 4 A (i), ∼18% of satellite cells isolated from 4-wk-old wild-type mice were labeled with BrdU. However, in myostatin knockout mice, a significantly higher number (33%) of satellite cells was activated to enter S phase. In 8-wk-old wild-type muscle, 8% of the satellite cells isolated were BrdU positive, whereas in the myostatin- muscle, 15% of the satellite cells were BrdU positive. When the same experiment was repeated on older mice (6 mo old), only 3.2% of the wild-type satellite cells had incorporated BrdU. A threefold higher number, ∼10% of Myostatin-deficient satellite cells, had incorporated BrdU (Fig. 4 A i), indicating that irrespective of the age, lack of functional Myostatin results in an increased number of activated satellite cells in uninjured muscle. To investigate the role of Myostatin in inhibiting satellite cell activation ex vivo, recombinant Myostatin was added to single isolated fibers in varying concentrations, and satellite cells were left to detach from the fiber and migrate to the well. Fig. 4 A (ii) graphically demonstrates that on average, 80% of the wells had identifiable satellite cells, which were subsequently differentiated in low serum to show they were myogenic. In comparison, in media containing 1 μg/ml of Myostatin, only 37% of fibers had migrated myogenic precursor cells in the well, while media with increased amounts of Myostatin, 2 and 5 μg/ml, had 12 and 0% migrated myogenic cells, respectively. These results confirm that a lack of myostatin expression leads to increased activation of satellite cells in vivo, and overexpression of myostatin inhibits activation.


Myostatin negatively regulates satellite cell activation and self-renewal.

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

Lack of Myostatin increases the activation of satellite cells on myofibers. (A, i) In vivo quantification of activated satellite cells in the muscle of wild-type and myostatin- mice. Activated satellite cells were labeled with BrdU in wild-type or myostatin- (Mstn −/−) mice of 4 wk, 8 wk, or 6 mo and were isolated using Percoll gradient. 5,000–10,000 satellite cells were immunostained for BrdU, and percentages of nuclei that were positive for BrdU labeling are shown. **, P < 0.01 (as compared with wild type). At least a total of 1,000 cells were counted in each of three replicates. The data provided are an average of three animals each. (A, ii) Myostatin inhibits the migration of satellite cells from fibers. Single muscle fibers (n = 32) were isolated from the muscle and incubated in media conducive to the migration of myogenic precursor cells. The addition of Myostatin in increasing concentrations decreases the percentage of fibers with migrated satellite cells (**, P < 0.01). (B, i) The number of satellite cells (CD34 positive) per 100 myonuclei in wild-type and myostatin- (Mstn−/−) myofibers is shown. **, P < 0.01 (as compared with wild type). More than 1,000 nuclei were counted in each of three replicates. The data presented are an average of three animals each. (B, ii) Micrograph showing typical CD34-immunostained satellite cell and (iii) myonuclei were visualized by counterstaining with DAPI. (C, i) An increased number of satellite cells migrate from myostatin- fibers. Quantitative analysis demonstrates that an increased number of myogenic progenitor cells migrates from single myofibers isolated from myostatin- mice as compared with wild-type myofibers (*, P < 0.05). (C, ii) An example of satellite cells surrounding an isolated myofiber after 72 h.
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Related In: Results  -  Collection

Show All Figures
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fig4: Lack of Myostatin increases the activation of satellite cells on myofibers. (A, i) In vivo quantification of activated satellite cells in the muscle of wild-type and myostatin- mice. Activated satellite cells were labeled with BrdU in wild-type or myostatin- (Mstn −/−) mice of 4 wk, 8 wk, or 6 mo and were isolated using Percoll gradient. 5,000–10,000 satellite cells were immunostained for BrdU, and percentages of nuclei that were positive for BrdU labeling are shown. **, P < 0.01 (as compared with wild type). At least a total of 1,000 cells were counted in each of three replicates. The data provided are an average of three animals each. (A, ii) Myostatin inhibits the migration of satellite cells from fibers. Single muscle fibers (n = 32) were isolated from the muscle and incubated in media conducive to the migration of myogenic precursor cells. The addition of Myostatin in increasing concentrations decreases the percentage of fibers with migrated satellite cells (**, P < 0.01). (B, i) The number of satellite cells (CD34 positive) per 100 myonuclei in wild-type and myostatin- (Mstn−/−) myofibers is shown. **, P < 0.01 (as compared with wild type). More than 1,000 nuclei were counted in each of three replicates. The data presented are an average of three animals each. (B, ii) Micrograph showing typical CD34-immunostained satellite cell and (iii) myonuclei were visualized by counterstaining with DAPI. (C, i) An increased number of satellite cells migrate from myostatin- fibers. Quantitative analysis demonstrates that an increased number of myogenic progenitor cells migrates from single myofibers isolated from myostatin- mice as compared with wild-type myofibers (*, P < 0.05). (C, ii) An example of satellite cells surrounding an isolated myofiber after 72 h.
Mentions: Using single-cell PCR analysis, Cornelison and Wold (1997) have previously speculated that Myostatin is an important regulator of the quiescent state of satellite cells. Thus, to determine the involvement of Myostatin in satellite cell quiescence, the cell cycle phase of the satellite cells attached to myofibers in vivo in wild-type and myostatin knockout mice was assessed. Both wild-type and myostatin knockout mice were pulsed with BrdU, and satellite cells from m. tibialis anterior muscle were isolated by Percoll gradient. Characterization of satellite cells with M-cadherin and CD34 immunostaining revealed that the cultures were >95% pure and that there is no difference in the yield or purity between wild-type and myostatin knockout mice (unpublished data). BrdU-positive satellite cells were identified by immunostaining. As shown in Fig. 4 A (i), ∼18% of satellite cells isolated from 4-wk-old wild-type mice were labeled with BrdU. However, in myostatin knockout mice, a significantly higher number (33%) of satellite cells was activated to enter S phase. In 8-wk-old wild-type muscle, 8% of the satellite cells isolated were BrdU positive, whereas in the myostatin- muscle, 15% of the satellite cells were BrdU positive. When the same experiment was repeated on older mice (6 mo old), only 3.2% of the wild-type satellite cells had incorporated BrdU. A threefold higher number, ∼10% of Myostatin-deficient satellite cells, had incorporated BrdU (Fig. 4 A i), indicating that irrespective of the age, lack of functional Myostatin results in an increased number of activated satellite cells in uninjured muscle. To investigate the role of Myostatin in inhibiting satellite cell activation ex vivo, recombinant Myostatin was added to single isolated fibers in varying concentrations, and satellite cells were left to detach from the fiber and migrate to the well. Fig. 4 A (ii) graphically demonstrates that on average, 80% of the wells had identifiable satellite cells, which were subsequently differentiated in low serum to show they were myogenic. In comparison, in media containing 1 μg/ml of Myostatin, only 37% of fibers had migrated myogenic precursor cells in the well, while media with increased amounts of Myostatin, 2 and 5 μg/ml, had 12 and 0% migrated myogenic cells, respectively. These results confirm that a lack of myostatin expression leads to increased activation of satellite cells in vivo, and overexpression of myostatin inhibits activation.

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