Effect of postdevelopmental myostatin depletion on myofibrillar protein metabolism

It is unclear whether the muscle hypertrophy induced by loss of myostatin signaling in mature muscles is maintained only by increased protein synthesis or whether reduced proteolysis contributes. To address this issue, we depleted myostatin by activating Cre recombinase for 2 wk in mature mice in which Mstn exon 3 was flanked by loxP sequences. The rate of phenylalanine tracer incorporation into myofibrillar proteins was determined 2, 5, and 24 wk after Cre activation ended. At all of these time points, myostatin-deficient mice had increased gastrocnemius and quadriceps muscle mass (≥27%) and increased myofibrillar synthesis rate per gastrocnemius muscle (≥19%) but normal myofibrillar synthesis rates per myofibrillar mass or RNA mass. Mean fractional myofibrillar degradation rates (estimated from the difference between rate of synthesis and rate of change in myofibrillar mass) and muscle concentrations of free 3-methylhistidine (from actin and myosin degradation) were unaffected by myostatin knockout. Overnight food deprivation reduced myofibrillar synthesis and ribosomal protein S6 phosphorylation and increased concentrations of 3-methylhistidine, muscle RING finger-1 mRNA, and atrogin-1 mRNA. Myostatin depletion did not affect these responses to food deprivation. These data indicate that maintenance of the muscle hypertrophy caused by loss of myostatin is mediated by increased protein synthesis per muscle fiber rather than suppression of proteolysis.     

Muscle growth after postdevelopmental myostatin gene knockout

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass approximately 25% over the following 3 mo in both male and female mice (P<0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass.     

Plasticity of mandibular biomineralization in myostatin-deficient mice

Compared with the normal or wild-type condition, knockout mice lacking myostatin (Mstn), a negative regulator of skeletal muscle growth, develop significant increases in relative masticatory muscle mass as well as the ability to generate higher maximal muscle forces. Wild-type and myostatin-deficient mice were compared to assess the postweaning influence of elevated masticatory loads because of increased jaw-adductor muscle and bite forces on the biomineralization of mandibular cortical bone and dental tissues. Microcomputed tomography (microCT) was used to quantify bone density at a series of equidistant external and internal sites in coronal sections for two symphysis and two corpus locations. Discriminant function analyses and nonparametric ANOVAs were used to characterize variation in biomineralization within and between loading cohorts. Multivariate analyses indicated that 95% of the myostatin-deficient mice and 95% of the normal mice could be distinguished based on biomineralization values at both symphysis and corpus sections. At the corpus, ANOVAs suggest that between-group differences are due to the tendency for cortical bone mineralization to be higher in myostatin-deficient mice, coupled with higher levels of dental biomineralization in normal mice. At the symphysis, ANOVAs indicate that between-group differences are related to significantly elevated bone-density levels along the articular surface and external cortical bone in the knockout mice. Both patterns, especially those for the symphysis, appear because of the postweaning effects of increased masticatory stresses in the knockout mice versus normal mice. The greater number of symphyseal differences suggest that bone along this jaw joint may be characterized by elevated plasticity. Significant differences in bone-density levels between normal and myostatin-deficient mice, coupled with the multivariate differences in patterns of plasticity between the corpus and symphysis, underscore the need for a comprehensive analysis of the plasticity of masticatory tissues vis-à-vis altered mechanical loads.     

Effect of myostatin depletion on weight gain, hyperglycemia, and hepatic steatosis during five months of high-fat feeding in mice

The marked hypermuscularity in mice with constitutive myostatin deficiency reduces fat accumulation and hyperglycemia induced by high-fat feeding, but it is unclear whether the smaller increase in muscle mass caused by postdevelopmental loss of myostatin activity has beneficial metabolic effects during high-fat feeding. We therefore examined how postdevelopmental myostatin knockout influenced effects of high-fat feeding. Male mice with ubiquitous expression of tamoxifen-inducible Cre recombinase were fed tamoxifen for 2 weeks at 4 months of age. This depleted myostatin in mice with floxed myostatin genes, but not in control mice with normal myostatin genes. Some mice were fed a high-fat diet (60% of energy) for 22 weeks, starting 2 weeks after cessation of tamoxifen feeding. Myostatin depletion increased skeletal muscle mass ～30%. Hypermuscular mice had ～50% less weight gain than control mice over the first 8 weeks of high-fat feeding. During the subsequent 3 months of high-fat feeding, additional weight gain was similar in control and myostatin-deficient mice. After 5 months of high-fat feeding, the mass of epididymal and retroperitoneal fat pads was similar in control and myostatin-deficient mice even though myostatin depletion reduced the weight gain attributable to the high-fat diet (mean weight with high-fat diet minus mean weight with low-fat diet: 19.9 g in control mice, 14.1 g in myostatin-deficient mice). Myostatin depletion did not alter fasting blood glucose levels after 3 or 5 months of high-fat feeding, but reduced glucose levels measured 90 min after intraperitoneal glucose injection. Myostatin depletion also attenuated hepatic steatosis and accumulation of fat in muscle tissue. We conclude that blocking myostatin signaling after maturity can attenuate some of the adverse effects of a high-fat diet.     

Is functional hypertrophy and specific force coupled with the addition of myonuclei at the single muscle fiber level?

Muscle force is typically proportional to muscle size, resulting in constant force normalized to muscle fiber cross-sectional area (specific force). Mice overexpressing insulin-like growth factor-1 (IGF-1) exhibit a proportional gain in muscle force and size, but not the myostatin-deficient mice. In an attempt to explore the role of the cytoplasmic volume supported by individual myonuclei [myonuclear domain (MND) size] on functional capacity of skeletal muscle, we have investigated specific force in relation to MND and the content of the molecular motor protein, myosin, at the single muscle fiber level from myostatin-knockout (Mstn(-/-)) and IGF-1-overexpressing (mIgf1(+/+)) mice. We hypothesize that the addition of extra myonuclei is a prerequisite for maintenance of specific force during muscle hypertrophy. A novel algorithm was used to measure individual MNDs in 3 dimensions along the length of single muscle fibers from the fast-twitch extensor digitorum longus and the slow-twitch soleus muscle. A significant effect of the size of individual MNDs in hypertrophic muscle fibers on both specific force and myosin content was observed. This effect was muscle cell type specific and suggested there is a critical volume individual myonuclei can support efficiently. The large MNDs found in fast muscles of Mstn(-/-) mice were correlated with the decrement in specific force and myosin content in Mstn(-/-) muscles. Thus, myostatin inhibition may not be able to maintain the appropriate MND for optimal function.     

Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls (n = 6/group). Wild-type mice lost 8% of body mass and approximately 13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass (P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.     

The myostatin null mutation and clenbuterol administration elicit additive effects in mice

In mice, the myostatin (Mstn) null mutation and treatment with clenbuterol both increase muscle growth and decrease fat mass. Our objective was to determine whether mechanistic overlap exists by administering clenbuterol to Mstn null mice. Male Mstn null and wild-type mice of similar genetic backgrounds received either 0 (control) or 20 p.p.m. clenbuterol in tap water free choice for 14 days. Several traits were measured to estimate muscle and fat growth. The Mstn null mutation resulted in increased body and empty carcass weight, increased muscle weights and decreased fat pad weights. Fat content was reduced and protein content was increased in the empty carcasses of Mstn null mice. Similarly, treatment with clenbuterol resulted in increased body and empty carcass weight, increased muscle weights and reduced fat pad weights. Fat content of empty carcasses and viscera was reduced and protein content of empty carcasses was increased with clenbuterol treatment. A significant interaction of genotype and clenbuterol treatment would indicate an altered responsiveness of Mstn null mice to clenbuterol. However, only the weight of gastrocnemius muscles exhibited a significant (P = 0.01) interaction of genotype and clenbuterol treatment, indicating that Mstn null mice were less responsive to clenbuterol compared with wild-type mice. Thus, for all other traits, the impact of Mstn null mutation and clenbuterol treatment was completely additive. These data suggest that disruption of Mstn function does not alter the response of mice to β-adrenergic agonists.     

Increased energy expenditure and leptin sensitivity account for low fat mass in myostatin-deficient mice

Myostatin deficiency causes dramatically increased skeletal muscle mass and reduced fat mass. Previously, myostatin-deficient mice were reported to have unexpectedly low total energy expenditure (EE) after normalizing to body mass, and thus, a metabolic cause for low fat mass was discounted. To clarify how myostatin deficiency affects the control of body fat mass and energy balance, we compared rates of oxygen consumption, body composition, and food intake in young myostatin-deficient mice relative to wild-type (WT) and heterozygous (HET) controls. We report that after adjusting for total body mass using regression analysis, young myostatin-deficient mice display significantly increased EE relative to both WT (+0.81 ± 0.28 kcal/day, P = 0.004) and HET controls (+0.92 ± 0.31 kcal/day, P = 0.005). Since food intake was not different between groups, increased EE likely accounts for the reduced body fat mass (KO: 8.8 ± 1.1% vs. WT: 14.5 ± 1.3%, P = 0.003) and circulating leptin levels (KO: 0.7 ± 0.2 ng/ml vs. WT: 1.9 ± 0.3 ng/ml, P = 0.008). Interestingly, the observed increase in adjusted EE in myostatin-deficient mice occurred despite dramatically reduced ambulatory activity levels (-50% vs. WT, P < 0.05). The absence of hyperphagia together with increased EE in myostatin-deficient mice suggests that increased leptin sensitivity may contribute to their lean phenotype. Indeed, leptin-induced anorexia (KO: -17 ± 1.2% vs. WT: -5 ± 0.3%) and weight loss (KO: -2.2 ± 0.2 g vs. WT: -1.6 ± 0.1, P < 0.05) were increased in myostatin-deficient mice compared with WT controls. We conclude that increased EE, together with increased leptin sensitivity, contributes to low fat mass in mice lacking myostatin.     

Negative Auto-Regulation of Myostatin Expression is Mediated by Smad3 and MicroRNA-27

Growth factors, such as myostatin (Mstn), play an important role in regulating post-natal myogenesis. In fact, loss of Mstn has been shown to result in increased post-natal muscle growth through enhanced satellite cell functionality; while elevated levels of Mstn result in dramatic skeletal muscle wasting through a mechanism involving reduced protein synthesis and increased ubiquitin-mediated protein degradation. Here we show that miR-27a/b plays an important role in feed back auto-regulation of Mstn and thus regulation of post-natal myogenesis. Sequence analysis of Mstn 3′ UTR showed a single highly conserved miR-27a/b binding site and increased expression of miR-27a/b was correlated with decreased expression of Mstn and vice versa both in vitro and in mice in vivo. Moreover, we also show that Mstn gene expression was regulated by miR-27a/b. Treatment with miR-27a/b-specific AntagomiRs resulted in increased Mstn expression, reduced myoblast proliferation, impaired satellite cell activation and induction of skeletal muscle atrophy that was rescued upon either blockade of, or complete absence of, Mstn. Consistent with this, miR-27a over expression resulted in reduced Mstn expression, skeletal muscle hypertrophy and an increase in the number of activated satellite cells, all features consistent with impaired Mstn function. Loss of Smad3 was associated with increased levels of Mstn, concomitant with decreased miR-27a/b expression, which is consistent with impaired satellite cell function and muscular atrophy previously reported in Smad3-null mice. Interestingly, treatment with Mstn resulted in increased miR-27a/b expression, which was shown to be dependent on the activity of Smad3. These data highlight a novel auto-regulatory mechanism in which Mstn, via Smad3 signaling, regulates miR-27a/b and in turn its own expression. In support, Mstn-mediated inhibition of Mstn 3′ UTR reporter activity was reversed upon miR-27a/b-specific AntagomiR transfection. Therefore, miR-27a/b, through negatively regulating Mstn, plays a role in promoting satellite cell activation, myoblast proliferation and preventing muscle wasting.     

Quadrupling muscle mass in mice by targeting TGF-beta signaling pathways

Myostatin is a transforming growth factor-beta family member that normally acts to limit skeletal muscle growth. Mice genetically engineered to lack myostatin activity have about twice the amount of muscle mass throughout the body, and similar effects are seen in cattle, sheep, dogs, and a human with naturally occurring loss-of-function mutations in the myostatin gene. Hence, there is considerable interest in developing agents capable of inhibiting myostatin activity for both agricultural and human therapeutic applications. We previously showed that the myostatin binding protein, follistatin, can induce dramatic increases in muscle mass when overexpressed as a transgene in mice. In order to determine whether this effect of follistatin results solely from inhibition of myostatin activity, I analyzed the effect of this transgene in myostatin-null mice. Mstn(-/-) mice carrying a follistatin transgene had about four times the muscle mass of wild type mice, demonstrating the existence of other regulators of muscle mass with similar activity to myostatin. The greatest effect on muscle mass was observed in offspring of mothers homozygous for the Mstn mutation, raising the possibility that either myostatin itself or a downstream regulator may normally be transferred from the maternal to fetal circulations. These findings demonstrate that the capacity for increasing muscle growth by manipulating TGF-beta signaling pathways is much more extensive than previously appreciated and suggest that muscle mass may be controlled at least in part by a systemic mode of action of myostatin.     

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.     

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.     

Myostatin deficiency but not anti‐myostatin blockade induces marked proteomic changes in mouse skeletal muscle

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post‐developmental Mstn blockade. Using high‐resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn^?/?) and mice treated for 2‐weeks with REGN1033, an anti‐Mstn antibody. Relative to wild‐type animals, Mstn^?/? mice had a two‐fold greater muscle mass and a >1.5‐fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5‐fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn^?/? mice corroborates the mutiple physiological changes including slow‐to‐fast fiber type switch. Thus, the proteome‐wide protein expression differs between Mstn^?/? mice and mice subjected to specific Mstn blockade post‐developmentally, providing molecular‐level insights to inform mechanistic hypotheses to explain the observed functional differences.     

Myofibrillar protein degradation in the chicken. 3-Methylhistidine release in vivo and in vitro in normal and genetically muscular-dystrophic chickens

Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others.     

Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.     

The effect of chronic and acute dietary restriction on the growth and protein turnover of fast and slow types of rat skeletal muscle

Changes in the growth and protein turnover of the anterior tibialis and soleus muscles were studied in response to acute and chronic dietary restriction (50% of ad libitum intake) between 3 and 149 weeks post partum. The effect of long-term dietary restriction from weaning to senescence was to retard the growth and normal developmental of the two types of skeletal muscle. This was evident from measurements of various parameters of growth, i.e. total protein, RNA and DNA and protein/DNA-P, which were reduced by approximately 50% when compared with age-matched controls. These decreases, however, were not accompanied by a decline in the fractional rate of synthesis (%/day) or ribosomal activity (mg protein/day per mg RNAP). The slowing down of the age-related decline in muscle growth has been attributed to a reduction in RNA capacity (RNA/protein), with similar responses in the fast- and slow-twitch skeletal muscles. The initial effects of piecemeal feeding of this restricted diet on the two types of muscle were also monitored. Short term starvation effects, i.e. 24 hr after feeding a reduced ration, were measured on the protein content and RNA/protein of both the anterior tibialis and soleus muscles; both parameters were unchanged within 24 hr. In contrast, a rapid and significant decline in the ribosomal synthetic activity (mg/d per mg RNAP), and a corresponding fall in the fractional rate of synthesis, occurred within 24 hr of feeding.     

Turnover of muscle protein in the fowl (Gallus domesticus). Rates of protein synthesis in fast and slow skeletal, cardiac and smooth muscle of the adult fowl.

Rates of protein synthesis in skeletal, cardiac and smooth muscle of fully grown fowl (Gallus domesticus) were determined in vivo by means of the constant infusion method using [14C]proline. In the anterior latissimus dorsi muscle, containing predominantly slow fibres, the average synthesis rate of non-collagen muscle proteins was 17.0 +/- 3.1% per day, a value higher than that obtained for cardiac muscle (13.8 +/- 1.3% per day) and for smooth muscle of the gizzard (12.0 +/- 1.9% per day). In the posterior latissimus dorsi muscle, containing predominantly fast fibres, synthesis rates were much lower (6.9 +/- 1.8% per day). In each case these average rates for the non-collagen protein were similar to the average rate for the sarcoplasmic and myofibrillar protein fractions. The RNA concentration of these four muscles showed that relative rates of protein synthesis were determined mainly by the relative RNA concentrations. The rate of protein synthesis per unit of DNA (the DNA activity) was similar in the two skeletal muscles, but somewhat lower in cardiac muscle and gizzard, possibly reflecting the larger proportion of less active cell types in these two muscles. These quantitative aspects of protein turnover in the two skeletal muscles are discussed in terms of the determination of ultimate size of the DNA unit, and in relation to muscle ultrastructure.     

Mapping modifiers affecting muscularity of the myostatin mutant (Mstn(Cmpt-dl1Abc)) compact mouse

The hypermuscular Compact phenotype was first noted in a line of mice selected for high body weight and protein content. A new line, based on mice showing the Compact phenotype, was formed and selected for maximum expression of the Compact phenotype. Previously we mapped and identified a 12-bp deletion in the myostatin gene, denoted Mstn(Cmpt-dl1Abc), which can be considered as a major gene responsible for the hypermuscular phenotype. Genetic analysis revealed that full expression of the hypermuscular phenotype requires the action of modifier loci in addition to Mstn(Cmpt-dl1Abc). To map these modifier loci, an interspecific F(2) population was generated between Comp9, an inbred line homozygous for Mstn(Cmpt-dl1Abc), and CAST/Ei, an inbred line generated from Mus musculus castaneus. Selective DNA pooling and genotyping, separately by gender, was carried out within a subpopulation of the F(2) consisting of individuals homozygous for Mstn(Cmpt-dl1Abc). Significant association with hypermuscularity at a false discovery rate (FDR) of 0.05 was found for markers on chromosomes 3, 5, 7, 11, 16, and X. In all cases, the marker allele derived from the Comp9 parent showed a higher frequency in the hypermuscular group and the CAST/Ei allele in the normal group. The modifier loci apparently exerted their effects on muscularity only in the presence of Mstn(Cmpt-dl1Abc).     

Protein and RNA synthesis in the skeletal muscle of hereditary dystrophic mouse.

Protein and RNA contents in muscle of normal and hereditary dystrophic mice C57BL/6J-dy/dy were reexamined on the basis of DNA. It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dystrophic muscle had been found to increase. Rates of protein and RNA systhesis in the early stage of the disease were also determined with a concomitant check of the specific activities of free amino acids and free nucleotides. The rates of both protein and RNA synthesis (i.e., specific activities of protein and RNA) were higher in the dystrophic muscle, but when they were expressed on a DNA basis, the total protein synthesis per cell was the same as that of normal muscle and the total RNA synthesis per cell showed a smaller increase in dystrophic muscle. These apparent increases of protein and RNA synthesis were discussed in connection with the decreased protein and RNA contents in the cells of dystrophic muscle. The synthesized RNAs seemed to contain mRNA on the basis of sedimentation character and Millipore filter binding ability. However, no particular RNA was mainly synthesized in dystrophic muscle.     

Contractile properties of EDL and soleus muscles of myostatin-deficient mice.

Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (P(o)), but decrease the specific P(o) (sP(o)) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of extensor digitorum longus (EDL) and soleus muscles from wild-type (MSTN(+/+)), heterozygous-null (MSTN(+/-)), and homozygous-null (MSTN(-/-)) adult male mice were determined. For EDL muscles, the P(o) of both MSTN(+/-) and MSTN(-/-) mice were greater than the P(o) of MSTN(+/+) mice. For soleus muscles, the P(o) of MSTN(-/-) mice was greater than that of MSTN(+/+) mice. The sP(o) of EDL muscles of MSTN(-/-) mice was less than that of MSTN(+/+) mice. For soleus muscles, however, no difference in sP(o) was observed. Following two lengthening contractions, EDL muscles from MSTN(-/-) mice had a greater force deficit than that of MSTN(+/+) or MSTN(+/-) mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin-deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, activin type IIB. Compared with the soleus, the amount of activin type IIB receptor was approximately twofold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles but also in the contractility and the composition of the extracellular matrix of muscles.