IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice.

Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn-/-) mice with mice overexpressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes of male mice; wild type (Mstn+/+ ), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn+/+ , 33% over Mstn+/- and 49% over Mstn-/- (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1 Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1+ independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn-/-mice, while phosphorylation of AKTS473 was increased in Igf1+mice (Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.

Brief-reports: elevated myostatin levels in patients with liver disease: a potential contributor to skeletal muscle wasting.

Loss of skeletal muscle mass is a poorly understood complication of end-stage liver disease (ESLD). Based on recent stem cell literature, we hypothesized that the potent negative regulator of muscle mass, myostatin, could play a role in the muscle loss associated with ESLD. In this preliminary investigation, we measured myostatin levels in patients undergoing liver transplant evaluation, using a novel enzyme-linked immunosensitivity assay. Myostatin levels were significantly elevated in patients with ESLD compared with healthy controls. These data suggest that myostatin deserves further investigation as a target for therapies designed to preserve muscle mass in patients with ESLD.

Antagonism of myostatin enhances muscle regeneration during sarcopenia.

A reduction in muscle mass and strength is often observed with aging, and this phenomenon is known as sarcopenia. This age-related atrophy frequently correlates with insufficient levels of muscle regeneration resulting from impairment of satellite cell involvement and myogenesis brought about by the aged environment. Using myostatin-null mice, we recently showed that negative regulators of muscle mass such as myostatin play an active role in the regulation of myogenesis during aging. The present study specifically tests the therapeutic value of a myostatin antagonist in sarcopenia. We report here that a short-term blockade of myostatin, through stage-specific administration of a myostatin antagonist, significantly enhanced muscle regeneration in aged mice after injury and during sarcopenia. Antagonism of myostatin led to satellite cell activation, increased Pax7 and MyoD protein levels, and greater myoblast and macrophage cell migration, resulting in enhanced muscle regeneration after notexin injury in aged mice. In addition, the antagonist demonstrated a high degree of efficacy, as only minimal doses during the critical period of regeneration after injury were sufficient to restore the myogenic and inflammatory responses in the aged environment. Thus, we propose that the antagonism of myostatin has significant therapeutic potential in the alleviation of sarcopenia.

Myostatin negatively regulates the expression of the steroid receptor co-factor ARA70.

Myostatin is a transforming growth factor-beta (TGF-beta) superfamily member and a key negative regulator of embryonic and postnatal muscle growth. In order to identify downstream target genes regulated by Myostatin, we performed suppressive subtraction hybridization (SSH) on cDNA generated from the biceps femoris muscle of wild-type and myostatin-null mice. Sequence analysis identified several known and unknown genes as Myostatin downstream target genes. Here, we have investigated the regulation of gene expression of an androgen receptor (AR) binding co-factor, androgen receptor associated protein-70 (ARA70), by Myostatin. We show that in mouse there are two isoforms of ARA70 with high homology (79%) to human ARA70; an alpha-isoform which is a canonical ARA70 and a beta-isoform which has a 9 consecutive amino acid deletion and 6 amino acid substitutions in the carboxyl-terminal portion. Reverse Northern analysis on the differentially expressed cDNA library indicated that there is increased expression of ARA70 in the muscles of myostatin-null mice. In addition, Northern blot, together with semi-quantitative PCR analysis, confirmed that there is increased expression of ARA70 in myostatin-null biceps femoris muscle when compared to wild-type muscle. In corroboration of these results, addition of exogenous Myostatin results in down-regulation of ARA70 expression confirming that Myostatin is a negative regulator of ARA70 gene expression. Expression analysis further confirmed that ARA70 is up-regulated during myogenesis and that peak expression of ARA70 is observed following the peak expression of MyoD in differentiating myoblasts. Given that lack of Myostatin and increased expression of AR leads to hypertrophy, we propose that absence of Myostatin, at least in part, induces the hypertrophy phenotype by increasing the activity of AR by up-regulating the expression of ARA70, a known stimulating co-factor of AR.

Proteolytic processing of myostatin is auto-regulated during myogenesis.

Myostatin, a potent negative regulator of myogenesis, is proteolytically processed by furin proteases into active mature myostatin before secretion from myoblasts. Here, we show that mature myostatin auto-regulates its processing during myogenesis. In a cell culture model of myogenesis, Northern blot analysis revealed no appreciable change in myostatin mRNA levels between proliferating myoblasts and differentiated myotubes. However, Western blot analysis confirmed a relative reduction in myostatin processing and secretion by differentiated myotubes as compared to proliferating myoblasts. Furthermore, in vivo results demonstrate a lower level of myostatin processing during fetal muscle development when compared to postnatal adult muscle. Consequently, high levels of circulatory mature myostatin were detected in postnatal serum, while fetal circulatory myostatin levels were undetectable. Since Furin proteases are important for proteolytically processing members of the TGF-beta superfamily, we therefore investigated the ability of myostatin to control the transcription of furin and auto-regulate the extent of its processing. Transfection experiments indicate that mature myostatin indeed regulates furin protease promoter activity. Based on these results, we propose a mechanism whereby myostatin negatively regulates its proteolytic processing during fetal development, ultimately facilitating the differentiation of myoblasts by controlling both furin protease gene expression and subsequent active concentrations of mature myostatin peptide.

Milk L-lactate concentration is increased during mastitis.

A study was undertaken in cattle to evaluate changes in milk L-lactate in relation to mastitis. A healthy, rear quarter of the udder of each of ten cows in mid-lactation was infused with 1000 colony-forming units (cfu) of Streptococcus uberis following an afternoon milking. Foremilk samples were taken at each milking from control and treated quarters and antibiotic treatment was applied following the onset of clinical mastitis or after 72 h. One cow did not become infected. Six quarters showed clinical symptoms of mastitis within 24-40 h and this was associated with a more than 30-fold increase in milk L-lactate (to 3.3 mM) and an increase in somatic cell count (SCC) from 4.5 x 10(3) to 1 x 10(7) cells/ml. Three cows were subclinical, with cell counts ranging from 1.5 x 10(6) to 1 x 10(7) cells/ml. In these animals, milk lactate ranged from 0.7 to 1.5 mM in the infected quarters up to 40 h post-infection, compared with less than 0.1 mM in control quarters. Milk was examined from 137 cows in mid-lactation which were known to have mastitis. Foremilk samples were taken aseptically from control and infected quarters of cows on commercial farms. Mean milk L-lactate concentrations and SCC were 0.14 +/- 0.02 mM and 1.85 +/- 0.3 x 10(5) cells/ml, respectively, in control (bacteriologically negative) samples. However, L-lactate concentrations exceeded 2.5 mM in the presence of some types of infection, the level of the lactate response being closely related to the impact of the infection on SCC. L-Lactate concentrations were relatively elevated in milk samples taken post partum, declining from 0.8 to 0.14 mM oyer the first few days of lactation. In conclusion, milk L-lactate has potential as an indicator of clinical and subclinical mastitis in dairy cows.

Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis.

Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development.

Effects of stage of lactation and time of year on plasmin-derived proteolytic activity in bovine milk in New Zealand.

The objective of this study was to determine the effects of stage of lactation (SOL) and time of year on plasmin-derived proteolytic activity in the milk of pasture-fed dairy cows in New Zealand. Four herds of 20 Friesian cows were used, one herd calving in each of January, April, July and October. Cows grazed ryegrass/white clover pasture only, except during June (winter) when all cows received supplementary pasture silage. Milk samples were collected on four occasions during the year (spring, summer, autumn and winter) from each cow in milk, to give a total of three samples per cow (early, mid and late lactation; c. 30, 120 and 220 days after calving, respectively). Milk samples were analysed for plasmin-derived proteolytic activity. There was no effect of either SOL or time of year on plasmin activity and therefore yields of plasmin followed patterns in milk yield (highest in early lactation and in summer). There were effects of both SOL and time of year on plasminogen-derived and total plasmin plus plasminogen-derived activity, both of which were highest in late lactation and in spring. Changes in plasminogen-derived activity and total plasmin plus plasminogen-derived activity due to SOL were not only due to the decrease in milk yield associated with advancing lactation, because enzyme yields were also increased with advancing lactation. Similarly, effects of time of year on plasminogen-derived activity and total plasmin plus plasminogen-derived activity could not be attributed solely to concomitant changes in milk yield, and may be influenced by the variation in the quality and quantity of feed during the year inherent in a pasture-based dairy system. Effects of SOL on proteolytic activity were greater than, and independent of, effects of time of year.

Titin-cap associates with, and regulates secretion of, Myostatin.

Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth. To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins. Using a yeast two-hybrid screen, we identified Titin-cap (T-cap) protein as interacting with Myostatin. T-cap is a sarcomeric protein that binds to the N-terminal domain of Titin and is a substrate of the titin kinase. Mammalian two-hybrid studies, in vitro binding assays and protein truncations in the yeast two-hybrid system verified the specific interaction between processed mature Myostatin and full-length T-cap. Analysis of protein-protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T-cap with a KD of 40 nM. When T-cap was stably overexpressed in C(2)C(12) myoblasts, the rate of cell proliferation was significantly increased. Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T-cap, but an increase in the retention of mature Myostatin indicated that T-cap may block Myostatin secretion. Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T-cap contained lower levels of Myostatin. Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T-cap could thus be due to reduced Myostatin secretion. These results suggest that T-cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin.