Undernutrition regulates the expression of a novel splice variant of myostatin and insulin-like growth factor 1 in ovine skeletal muscle.

Undernutrition suppresses the growth of skeletal muscles and alters the expression of insulin-like growth factor 1 (IGF1), a key mitogen, and myostatin, a potent inhibitor of myogenesis. These changes can explain, at least in part, the reduced growth of skeletal muscles in underfed lambs. We have recently identified a myostatin splice variant (MSV) that binds to and antagonizes the canonical signaling of myostatin. In the present study, we hypothesized that the expression of MSV would be reduced in conjunction with myostatin and IGF1 in response to underfeeding in skeletal muscles of sheep. Young growing ewes were fed either ad libitum or an energy-restricted diet (30% of maintenance requirements) for 28 d. This regime of underfeeding resulted in a 24% reduction in body mass (P < 0.001) and a 36% reduction in the mass of the semitendinosus muscles relative to controls (P < 0.001) by day 28. The concentrations of MSV and IGF1 messenger RNA (mRNA) were reduced (both P < 0.001), but myostatin mRNA was not altered in semitendinosus muscles. Unlike the reduced expression of mRNA, the abundance of MSV protein was increased (P < 0.05) and there was no change in the abundance of myostatin protein. Our results suggest that undernutrition for 28 d decreases the signaling of myostatin by increasing the abundance of MSV protein. Although this action may reduce the growth inhibitory activity of myostatin, it cannot prevent the loss of growth of skeletal muscles during undernutrition.

Prolonged underfeeding of sheep increases myostatin and myogenic regulatory factor Myf-5 in skeletal muscle while IGF-I and myogenin are repressed.

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.

Genomic organization and neonatal expression of the bovine myostatin gene.

Myostatin belongs to the Transforming Growth Factor-beta (TGF-beta) superfamily and is expressed in developing and mature skeletal muscle. Biologically, the role of myostatin seems to be extremely well conserved during evolution since inactivating mutations in myostatin gene cause similar phenotype of heavy muscling in both mice and cattle. In this report we have analysed the genomic structure and neonatal expression of the bovine myostatin gene. The molecular analysis shows that the bovine myostatin gene consists of three exons and two introns. The sizes of the first and second exons are 506 and 374 base pairs (bp) respectively. The size of the third exon was found to be variable in length (1701 or 1812 or 1887 nucleotides), whereas the size of the two introns is 1840 and 2033 bps. In the first exon of bovine myostatin, a single transcription initiation site is found at 133 bps from the translation start codon ATG. Sequencing the 3' untranslated region indicated that there are multiple polyadenylation signals at 1301, 1401 and 1477 bp downstream from the translation stop codon (TGA). Furthermore, 3' RACE analysis confirmed that all three polyadenylation sites are used in vivo. Using quantitative RT-PCR we have analysed neonatal expression of myostatin gene. In both the M. biceps femoris and M. semitendinosus, the highest level of myostatin expression was observed on day 1 postnatally, then gradually reduced on days 8 and 14 postnatally. In contrast, in the M. gastrocnemius, myostatin expression was highest on day 14 and lowest on day 8. These results indicate that myostatin gene structure and function is well conserved during evolution and that neonatal expression of myostatin in a number of predominantly fast twitch muscles is differentially regulated.

Molecular expression of myostatin and MyoD is greater in double-muscled than normal-muscled cattle fetuses.

Excessive muscling in double-muscled cattle arises from mutations in the myostatin gene, but the role of myostatin in normal muscle development is unclear. The aim of this study was to measure the temporal relationship of myostatin and myogenic regulatory factors during muscle development in normal (NM)- and double-muscled (DM) cattle to determine the timing and possible targets of myostatin action in vivo. Myostatin mRNA peaked at the onset of secondary fiber formation (P < 0.001) and was greater in DM (P < 0.001) than in NM. MyoD expression was also elevated throughout primary and secondary fiber formation (P < 0.001) and greater in DM (P < 0.05). Expression of myogenin peaked later than MyoD (P < 0.05); however, it did not differ between NM and DM. These data show that myostatin and MyoD increase coincidentally during formation of muscle fibers, indicating a coordinated role in the terminal differentiation and/or fusion of myoblasts. Myostatin mRNA is also consistently higher in DM than NM, suggesting that a feedback loop of regulation is also disrupted in the myostatin-deficient condition.