Gina Nicholas

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 C2C12 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. J. Cell. Physiol. 193: 120–131, 2002.

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.

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.

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

Myostatin is a transforming growth factor‐β (TGF‐β) 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 α‐isoform which is a canonical ARA70 and a β‐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.

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.

Lack of association between allelic status and myostatin content in lambs with the myostatin g+6723G > A allele

Lambs with the myostatin (MSTN) g+6723G>A mutation have a greater muscle mass, which is believed to be associated with reduced myostatin protein abundance. This experiment was designed to determine if differences in allelic frequency of the MSTN g+6723G>A mutation affected abundance of myostatin protein from birth to 24 wk of age. A Poll Dorset cross White Suffolk ram (MSTN A/G) was mated to 35 White Suffolk cross Border Leicester cross Merino ewes (MSTN A/G, n=21, and MSTN G/G, n=14). The progeny of these matings delivered 44 lambs with MSTN A/A (n=9), MSTN A/G (n=21), and MSTN G/G (n=14) genotypes. At approximately 1, 4, and 12 wk of age, a biopsy sample was collected and a blood sample was taken to measure the abundance of myostatin protein in muscle and plasma. At approximately 24 wk of age, the wether lambs were slaughtered to determine carcass characteristics and muscle samples were taken from the bicep femoris. The abundance of mature myostatin protein in muscle from 1 wk old lambs was less (P=0.05) in MSTN A/A and MSTN A/G compared with MSTN G/G lambs. However, at 4 and 24 wk the MSTN A/A lambs had a greater (P=0.04) abundance of myostatin protein compared with the MSTN A/G and MSTN G/G lambs. The abundance of mature myostatin did not differ between genotypes in plasma but the myostatin protein did increase as the lambs aged. At slaughter the MSTN A/A wether lambs had greater dressing percentages (P=0.04), shortloin (P=0.01), topside (P<0.001), and round (P=0.01) weights but did not differ in final BW or HCW (P>0.05). The MSTN A/A lambs had more muscle fibers (P=0.02) in the cross-section of LM between the 12th and 13th rib. The MSTN A/A lambs also had greater lean (P=0.002), less fat (P=0.009), and reduced organ (heart, liver, spleen, and kidneys) mass as determined by computed tomography scanning than MSTN G/G lambs. The results of this study demonstrated that lambs homozygous for the MSTN g+6723G>A mutation have changes in carcass characteristics (dressing and total lean), organ weights, and muscle fiber number. This may be due to reduced myostatin protein early in utero, but after 4 wk of age there was no difference in the abundance of mature myostatin protein in muscle or plasma among MSTN A/A, MSTN A/G, and MSTN G/G genotypes.

Identification of amino acids associated with skeletal muscle growth in late gestation and at weaning in lambs of well-nourished sheep.

The objective of this study was to determine the association between intracellular free AA (FAA) profiles in skeletal muscle with muscle growth in twin and singleton fetuses in late pregnancy and at weaning, under an ad libitum feeding regime of the dam. Plasma from singleton- (n = 9) and twin-bearing (n = 10) ewes at d 140 of pregnancy and FAA in the semitendinosus muscle (STM) from the corresponding fetuses were studied. At weaning, intracellular STM FAA concentrations were compared between twins at the same age as singletons (Twin(age); n = 17) and at the same weight as singletons (Twin(wt); n = 17) to that of singletons (n = 20). Twin fetuses were 15% lighter (P = 0.03) with a 20% lighter STM (P = 0.02) compared to singletons. Maternal plasma FAA were similar (P ≥ 0.17) between singleton- and twin-bearing ewes. Twin fetuses had greater (P < 0.05) plasma concentrations of glutamine, histidine, and methionine and lower (P < 0.05) concentrations of aspartate, citrulline, glutamate, and ornithine compared with singletons. In fetal STM, twins had lower (P < 0.05) concentrations of aspartate and valine and greater (P < 0.01) concentration of methionine. Correlations were found between fetal STM weight and intracellular concentrations of arginine (r = 0.66, P < 0.01) and glutamine (r = 0.49, P < 0.01). Compared to singletons at weaning, Twin(age) were 16% lighter (P < 0.01) and the STM weight was proportionately 16% lighter (P < 0.01). For Twin(wt), the magnitude of the difference for STM weight was reduced to 8% lighter (P = 0.02). Compared to singletons, Twin(age) lambs had greater (P < 0.05) intracellular concentrations of glutamine, histidine, threonine, asparagine, alanine, serine, and glutamate but reduced taurine. The differences in FAA concentrations were less between Twin(wt) and singletons than between Twin(age) and singletons. Positive correlations were found between leucine, lysine, methionine, phenylalanine, proline, threonine, and tyrosine muscle concentration and STM weight at weaning. Males differed from females in intracellular FAA both in late pregnancy and at weaning. Twins had reduced RNA content during pregnancy and at weaning, suggesting a lower capacity for protein accretion. These data suggest that specific FAA concentrations are associated with differences in muscle growth during late pregnancy, notably arginine and glutamine, and reduced protein synthesis capacity. However, the relevance of specific FAA varies according to stage of development and sex of the lamb.

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.