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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 results in deregulated S phase entry of adult myoblasts. (A) Freshly isolated adult myoblasts were stained with propidium iodide and analyzed on a flow cytometer. 10,000 cells of each genotype were analyzed on FACS® and were distributed into the phases of the cell cycle based on the DNA content. Percentage of myostatin- (Mstn−/−) and wild-type (Mstn+/+) myoblasts in S phase are shown in panel A. (B) Isolated myoblasts were synchronized to G1 phase, and once released into S phase, they were BrdU labeled and the percentage of BrdU-positive cells was counted at various time points. myostatin- cells progress into the S phase more rapidly as compared with wild-type myoblasts. **, P < 0.01. At least a total of 1,000 cells were counted in each of three replicates. Data presented are an average of three animals. (C) Western blot showing the levels of p21 and Cdk2 protein in primary myoblasts cultured with (+) or without (−) Myostatin for 24 h. p21 protein was detected using anti-p21 antibodies, and Cdk2 protein was detected using Cdk2 antibody. Tubulin protein levels are included to show equal loading.
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fig6: Lack of Myostatin results in deregulated S phase entry of adult myoblasts. (A) Freshly isolated adult myoblasts were stained with propidium iodide and analyzed on a flow cytometer. 10,000 cells of each genotype were analyzed on FACS® and were distributed into the phases of the cell cycle based on the DNA content. Percentage of myostatin- (Mstn−/−) and wild-type (Mstn+/+) myoblasts in S phase are shown in panel A. (B) Isolated myoblasts were synchronized to G1 phase, and once released into S phase, they were BrdU labeled and the percentage of BrdU-positive cells was counted at various time points. myostatin- cells progress into the S phase more rapidly as compared with wild-type myoblasts. **, P < 0.01. At least a total of 1,000 cells were counted in each of three replicates. Data presented are an average of three animals. (C) Western blot showing the levels of p21 and Cdk2 protein in primary myoblasts cultured with (+) or without (−) Myostatin for 24 h. p21 protein was detected using anti-p21 antibodies, and Cdk2 protein was detected using Cdk2 antibody. Tubulin protein levels are included to show equal loading.

Mentions: Proliferation assays clearly demonstrate that adult myoblasts that lack Myostatin proliferate much faster than the wild-type myoblasts. Furthermore, our previous results with C2C12 cells indicate that addition of exogenous Myostatin to the growth medium (GM) arrests the proliferation of C2C12 cells in G1 phase of the cell cycle (Thomas et al., 2000). Thus, we next asked if the increased proliferation observed in the myostatin knockout myoblasts is due to deregulated G1 to S phase transition. FACS® analysis of the cell cycle distribution of asynchronous populations of wild-type and myostatin knockout adult myoblasts in proliferation media revealed that myostatin knockout myoblasts had a twofold increase in S phase cell population (Fig. 6 A). To confirm these results, we then synchronized the adult myoblasts in G0 phase by growing them in methionine-free media and initiated S phase entry by serum stimulation. To monitor the number of cells in S phase, the cells were pulsed with BrdU, and the percent of labeled cells was determined by immunostaining the fixed cells at the end of the assay. The results indicate that within the first hour after serum stimulation, 7% of Mstn−/− myoblasts entered S phase. In contrast, only 2% of wild-type myoblasts were found to be in S phase. Similarly, an enhanced number of BrdU-positive cells was observed up to 8 h after serum stimulation in Mstn−/− myoblasts (Fig. 6 B). These results indicate that lack of Myostatin increases the proliferation of myoblasts by deregulating the S phase entry.


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 results in deregulated S phase entry of adult myoblasts. (A) Freshly isolated adult myoblasts were stained with propidium iodide and analyzed on a flow cytometer. 10,000 cells of each genotype were analyzed on FACS® and were distributed into the phases of the cell cycle based on the DNA content. Percentage of myostatin- (Mstn−/−) and wild-type (Mstn+/+) myoblasts in S phase are shown in panel A. (B) Isolated myoblasts were synchronized to G1 phase, and once released into S phase, they were BrdU labeled and the percentage of BrdU-positive cells was counted at various time points. myostatin- cells progress into the S phase more rapidly as compared with wild-type myoblasts. **, P < 0.01. At least a total of 1,000 cells were counted in each of three replicates. Data presented are an average of three animals. (C) Western blot showing the levels of p21 and Cdk2 protein in primary myoblasts cultured with (+) or without (−) Myostatin for 24 h. p21 protein was detected using anti-p21 antibodies, and Cdk2 protein was detected using Cdk2 antibody. Tubulin protein levels are included to show equal loading.
© Copyright Policy
Related In: Results  -  Collection

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fig6: Lack of Myostatin results in deregulated S phase entry of adult myoblasts. (A) Freshly isolated adult myoblasts were stained with propidium iodide and analyzed on a flow cytometer. 10,000 cells of each genotype were analyzed on FACS® and were distributed into the phases of the cell cycle based on the DNA content. Percentage of myostatin- (Mstn−/−) and wild-type (Mstn+/+) myoblasts in S phase are shown in panel A. (B) Isolated myoblasts were synchronized to G1 phase, and once released into S phase, they were BrdU labeled and the percentage of BrdU-positive cells was counted at various time points. myostatin- cells progress into the S phase more rapidly as compared with wild-type myoblasts. **, P < 0.01. At least a total of 1,000 cells were counted in each of three replicates. Data presented are an average of three animals. (C) Western blot showing the levels of p21 and Cdk2 protein in primary myoblasts cultured with (+) or without (−) Myostatin for 24 h. p21 protein was detected using anti-p21 antibodies, and Cdk2 protein was detected using Cdk2 antibody. Tubulin protein levels are included to show equal loading.
Mentions: Proliferation assays clearly demonstrate that adult myoblasts that lack Myostatin proliferate much faster than the wild-type myoblasts. Furthermore, our previous results with C2C12 cells indicate that addition of exogenous Myostatin to the growth medium (GM) arrests the proliferation of C2C12 cells in G1 phase of the cell cycle (Thomas et al., 2000). Thus, we next asked if the increased proliferation observed in the myostatin knockout myoblasts is due to deregulated G1 to S phase transition. FACS® analysis of the cell cycle distribution of asynchronous populations of wild-type and myostatin knockout adult myoblasts in proliferation media revealed that myostatin knockout myoblasts had a twofold increase in S phase cell population (Fig. 6 A). To confirm these results, we then synchronized the adult myoblasts in G0 phase by growing them in methionine-free media and initiated S phase entry by serum stimulation. To monitor the number of cells in S phase, the cells were pulsed with BrdU, and the percent of labeled cells was determined by immunostaining the fixed cells at the end of the assay. The results indicate that within the first hour after serum stimulation, 7% of Mstn−/− myoblasts entered S phase. In contrast, only 2% of wild-type myoblasts were found to be in S phase. Similarly, an enhanced number of BrdU-positive cells was observed up to 8 h after serum stimulation in Mstn−/− myoblasts (Fig. 6 B). These results indicate that lack of Myostatin increases the proliferation of myoblasts by deregulating the S phase entry.

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