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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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myostatin- adult myoblasts proliferate faster than the wild-type myoblasts. Adult myoblasts were isolated from hind limbs of C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) and seeded at a low number. Proliferation rate determined for the myoblasts isolated from E17 (A) and 4-wk-old muscle (B). In addition, the proliferation rate determined for m. tibialis anterior (C) and m. soleus (D) muscle fiber–specific adult myoblasts is also shown. Experiments were done in triplicate. Data shown are an average of three animals. *, P < 0.05; **, P < 0.01 (as compared with wild type). To determine the direct effect of Myostatin on satellite cell proliferation, adult myoblasts were isolated from wild-type and myostatin- mice. Exogenous Myostatin was added to the proliferating myostatin- myoblast cultures. As the concentration increased, the enhanced proliferative potential of the myostatin- myoblast decreased to that of the wild type (E).
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fig5: myostatin- adult myoblasts proliferate faster than the wild-type myoblasts. Adult myoblasts were isolated from hind limbs of C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) and seeded at a low number. Proliferation rate determined for the myoblasts isolated from E17 (A) and 4-wk-old muscle (B). In addition, the proliferation rate determined for m. tibialis anterior (C) and m. soleus (D) muscle fiber–specific adult myoblasts is also shown. Experiments were done in triplicate. Data shown are an average of three animals. *, P < 0.05; **, P < 0.01 (as compared with wild type). To determine the direct effect of Myostatin on satellite cell proliferation, adult myoblasts were isolated from wild-type and myostatin- mice. Exogenous Myostatin was added to the proliferating myostatin- myoblast cultures. As the concentration increased, the enhanced proliferative potential of the myostatin- myoblast decreased to that of the wild type (E).

Mentions: Previously, we have shown that high concentrations of Myostatin inhibit the proliferation of myoblasts by controlling the G1 to S transition during cell cycle (Thomas et al., 2000). Thus, here, we determined the proliferation rate and the doubling time of adult myoblasts isolated from both wild-type and myostatin knockout mice. For this purpose, we plated secondary myoblasts cultured from embryonic day (E) 17 and adult myoblasts from postnatal muscle and determined their proliferation. Primary myoblasts isolated from wild-type and myostatin knockout mice underwent a lag period of 3 d before they became proliferative (unpublished data). Cultured secondary myoblasts, isolated from E17 embryos from myostatin knockout mice, proliferated significantly faster, resulting in an increase in the number of Mstn−/− myoblasts as compared with the wild-type myoblasts (Fig. 5 A). The increased proliferation rate of Mstn−/− myoblasts is also reflected by three times higher cumulative population doublings attained by the myostatin- myoblasts, compared with the wild-type myoblasts (unpublished data). In addition, the proliferation rate of adult myoblasts derived from hind limbs of 4-wk-old myostatin- and wild-type mice were also assayed using the methylene blue cell proliferation assay. As seen in Fig. 5 B, myostatin- myoblasts proliferate significantly faster than wild-type myoblasts after 3 d in culture. The proliferation experiments described above were performed on myoblasts derived from satellite cells that were isolated from mixed muscles that contain both fast and slow twitch fibers. A recent report, however, suggests that the satellite cells isolated from fast and slow fibers are inherently different (Rosenblatt et al., 1996). Thus, to address the role of Myostatin in the proliferation of myoblasts from fast or slow fibers, we cultured adult myoblasts from m. tibialis anterior (fast twitch) or m. soleus (slow twitch) of both normal and myostatin knockout mice. The proliferation assay again indicates that both fast (Fig. 5 C) and slow (Fig. 5 D) fiber–specific adult myoblasts isolated from myostatin knockout mice proliferate much faster as compared with the wild-type adult myoblasts. Thus, regardless of the origin of satellite cells, lack of myostatin appears to increase the proliferation rate of both fast and slow fiber–specific satellite cells.


Myostatin negatively regulates satellite cell activation and self-renewal.

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

myostatin- adult myoblasts proliferate faster than the wild-type myoblasts. Adult myoblasts were isolated from hind limbs of C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) and seeded at a low number. Proliferation rate determined for the myoblasts isolated from E17 (A) and 4-wk-old muscle (B). In addition, the proliferation rate determined for m. tibialis anterior (C) and m. soleus (D) muscle fiber–specific adult myoblasts is also shown. Experiments were done in triplicate. Data shown are an average of three animals. *, P < 0.05; **, P < 0.01 (as compared with wild type). To determine the direct effect of Myostatin on satellite cell proliferation, adult myoblasts were isolated from wild-type and myostatin- mice. Exogenous Myostatin was added to the proliferating myostatin- myoblast cultures. As the concentration increased, the enhanced proliferative potential of the myostatin- myoblast decreased to that of the wild type (E).
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Related In: Results  -  Collection

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fig5: myostatin- adult myoblasts proliferate faster than the wild-type myoblasts. Adult myoblasts were isolated from hind limbs of C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) and seeded at a low number. Proliferation rate determined for the myoblasts isolated from E17 (A) and 4-wk-old muscle (B). In addition, the proliferation rate determined for m. tibialis anterior (C) and m. soleus (D) muscle fiber–specific adult myoblasts is also shown. Experiments were done in triplicate. Data shown are an average of three animals. *, P < 0.05; **, P < 0.01 (as compared with wild type). To determine the direct effect of Myostatin on satellite cell proliferation, adult myoblasts were isolated from wild-type and myostatin- mice. Exogenous Myostatin was added to the proliferating myostatin- myoblast cultures. As the concentration increased, the enhanced proliferative potential of the myostatin- myoblast decreased to that of the wild type (E).
Mentions: Previously, we have shown that high concentrations of Myostatin inhibit the proliferation of myoblasts by controlling the G1 to S transition during cell cycle (Thomas et al., 2000). Thus, here, we determined the proliferation rate and the doubling time of adult myoblasts isolated from both wild-type and myostatin knockout mice. For this purpose, we plated secondary myoblasts cultured from embryonic day (E) 17 and adult myoblasts from postnatal muscle and determined their proliferation. Primary myoblasts isolated from wild-type and myostatin knockout mice underwent a lag period of 3 d before they became proliferative (unpublished data). Cultured secondary myoblasts, isolated from E17 embryos from myostatin knockout mice, proliferated significantly faster, resulting in an increase in the number of Mstn−/− myoblasts as compared with the wild-type myoblasts (Fig. 5 A). The increased proliferation rate of Mstn−/− myoblasts is also reflected by three times higher cumulative population doublings attained by the myostatin- myoblasts, compared with the wild-type myoblasts (unpublished data). In addition, the proliferation rate of adult myoblasts derived from hind limbs of 4-wk-old myostatin- and wild-type mice were also assayed using the methylene blue cell proliferation assay. As seen in Fig. 5 B, myostatin- myoblasts proliferate significantly faster than wild-type myoblasts after 3 d in culture. The proliferation experiments described above were performed on myoblasts derived from satellite cells that were isolated from mixed muscles that contain both fast and slow twitch fibers. A recent report, however, suggests that the satellite cells isolated from fast and slow fibers are inherently different (Rosenblatt et al., 1996). Thus, to address the role of Myostatin in the proliferation of myoblasts from fast or slow fibers, we cultured adult myoblasts from m. tibialis anterior (fast twitch) or m. soleus (slow twitch) of both normal and myostatin knockout mice. The proliferation assay again indicates that both fast (Fig. 5 C) and slow (Fig. 5 D) fiber–specific adult myoblasts isolated from myostatin knockout mice proliferate much faster as compared with the wild-type adult myoblasts. Thus, regardless of the origin of satellite cells, lack of myostatin appears to increase the proliferation rate of both fast and slow fiber–specific satellite cells.

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