Myostatin, a member of the transforming growth factor-␤ (TGF-␤) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C 2 C 12 myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G 1 -to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21Waf1, Cip1 , a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G 1 -phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation.

Myostatin, a member of the TGF-beta superfamily, is a key negative regulator of skeletal muscle growth. The role of myostatin during skeletal muscle regeneration has not previously been reported. In the present studies, normal Sprague-Dawley and growth hormone (GH)-deficient (dw/dw) rats were administered the myotoxin, notexin, in the right M. biceps femoris on day 0. The dw/dw rats then received either saline or human-N-methionyl GH (200microg/100g body weight/day) during the ensuing regeneration. Normal and dw/dw M. biceps femoris were dissected on days 1, 2, 3, 5, 9 and 13, formalin-fixed, then immunostained for myostatin protein. Immunostaining for myostatin revealed high levels of protein within necrotic fibres and connective tissue of normal and dw/dw damaged muscles. Regenerating myotubes contained no myostatin at the time of fusion (peak fusion on day 5), and only low levels of myostatin were observed during subsequent myotube enlargement. Fibres which survived assault by notexin (survivor fibres) contained moderate to high myostatin immunostaining initially. The levels in both normal and dw/dw rat survivor fibres decreased on days 2-3, then increased on days 9-13. In dw/dw rats, there was no observed effect of GH administration on the levels of myostatin protein in damaged muscle. The low level of myostatin observed in regenerating myotubes in these studies suggests a negative regulatory role for myostatin in muscle regeneration.

The IGF axis is nutritionally sensitive in vivo and IGFs stimulate myoblast proliferation and differentiation in vitro, while myostatin inhibits these processes in vitro. We hypothesised that underfeeding would reversibly inhibit the myogenic activity of satellite cells in vivo together with decreased IGF-I and increased myostatin in muscle. Satellite cell activity was measured indirectly from the expression of proliferating cell nuclear antigen (PCNA) and the myogenic regulatory factors (MRFs), MyoD, Myf-5 and myogenin. Young sheep were underfed (30% of maintenance) and some killed after 1, 4, 12, 17, 21 and 22 weeks. Remaining underfed animals were then re-fed a control ration of pellets and killed after 2 days, and 1, 6 and 30 weeks.Expression of PCNA and MRFs decreased during the first week of underfeeding. This coincided with reduced IGF-I and myostatin mRNA, and processed myostatin. Subsequently, Myf-5, MyoD, myostatin mRNA and processed myostatin increased, suggesting that satellite cells may have become progressively quiescent. Long-term underfeeding caused muscle necrosis in some animals and IGF-I and MRF expression was increased in these, indicating the activation of satellite cells for muscle repair. Re-feeding initiated rapid muscle growth and increased expression of PCNA, IGF-I and the MRFs concurrently with decreased myostatin proteins.In conclusion, these data indicate that IGF-I and myostatin may work in a coordinated manner to regulate the proliferation, differentiation and quiescence of satellite cells in vivo.

Myostatin inhibits skeletal muscle development. Therefore, we sought to determine whether larger body and muscle mass in male mice was associated with lower mRNA and protein expression of myostatin compared with females. Ten male and ten female mice of the C57 strain were killed at 16-18 wk of age, and their biceps femoris, gastrocnemius, and quadriceps femoris muscles were collected. Body and muscle masses were 40% heavier (P < 0.001) in males than in females. Northern analysis showed no difference in mRNA between males and females. In contrast, Western analysis showed that processed myostatin (26 kDa) was 40-60% lower (P < 0.001) in males compared with females. These data show first that decreased processed myostatin is a posttranscriptional and posttranslational event and, second, that decreased abundance of processed myostatin is associated with increased body mass and skeletal muscle mass in male compared with female mice.

GH enhances skeletal muscle growth, and IGF-II peptide is highly expressed during regeneration. We have therefore investigated the effect of GH administration on IGF-II binding and expression in regenerating rat skeletal muscle using the techniques of receptor autoradiography and in situ hybridisation. Notexin, a myotoxin, was injected into the right M. biceps femoris (day 0), causing affected fibres to undergo necrosis followed by rapid regeneration. Animals were administered either GH (200 micrograms/100 g body weight) or saline vehicle daily. Contralateral muscles were used as regeneration controls. GH administration during regeneration resulted in significant increases in body weight, and damaged and undamaged muscle weights (P < 0.001). IGF-II expression, which was examined in regenerating fibres, survivor fibres and undamaged fibres, varied according to tissue type (P < 0.001). Specifically, IGF-II expression in regenerating fibres was elevated relative to control and survivor fibres after day 3 (P < 0.05), with a peak on day 9 (P < 0.001). GH did not affect IGF-II message levels. 125I-IGF-II binding in regenerating muscle was examined in the same fibre types as well as in connective tissue. 125I-IGF-II binding in regenerating fibres was higher (P < 0.001) than in other tissue types on day 5. GH administration increased 125I-IGF-II binding in all damaged muscle tissues on day 5 (P < 0.001, regenerating fibres; P < 0.01, others). We believe that this shows for the first time an effect of GH on the Type 2 IGF receptor in regenerating skeletal muscle.