Myostatin Deficiency Research Articles

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.

Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice

There is no consensus about whether making muscles abnormally large by reducing myostatin activity affects force-generating capacity or the ability to perform activities requiring muscular endurance. We therefore examined grip force, contractile properties of extensor digitorum longus (EDL) muscles, and voluntary wheel running in mice in which myostatin was depleted after normal muscle development. Cre recombinase activity was induced to knock out exon 3 of the myostatin gene in 4-mo-old mice in which this exon was flanked by loxP sequences (Mstn[f/f]). Control mice with normal myostatin genes (Mstn[w/w]) received the same Cre-activating treatment. Myostatin depletion increased the mass of all muscles that were examined (gastrocnemius, quadriceps, tibialis anterior, EDL, soleus, triceps) by approximately 20-40%. Grip force, measured multiple times 2-22 wk after myostatin knockout, was not consistently greater in the myostatin-deficient mice. EDL contractile properties were determined 7-13 mo after myostatin knockout. Twitch force tended to be greater in myostatin-deficient muscles (+24%; P=0.09), whereas tetanic force was not consistently elevated (mean +11%; P=0.36), even though EDL mass was greater than normal in all myostatin-deficient mice (mean +36%; P<0.001). The force deficit induced by eccentric contractions was approximately twofold greater in myostatin-deficient than in normal EDL muscles (31% vs. 16% after five eccentric contractions; P=0.02). Myostatin-deficient mice ran 19% less distance (P<0.01) than control mice during the 12 wk following myostatin depletion, primarily because of fewer running bouts per night rather than diminished running speed or bout duration. Reduced specific tension (ratio of force to mass) and reduced running have been observed after muscle hypertrophy was induced by other means, suggesting that they are characteristics generally associated with abnormally large muscles rather than unique effects of myostatin deficiency.

Mechanisms involved in the enhancement of mammalian target of rapamycin signalling and hypertrophy in skeletal muscle of myostatin-deficient mice

Myostatin deficiency leads to both an increased rate of protein synthesis and skeletal muscle hypertrophy. However, the mechanisms involved in mediating these effects are not yet fully understood. Here, we demonstrate that genetic loss of myostatin leads to enhanced muscle expression of both protein kinase B and mammalian target of rapamycin/S6K signalling components, consistent with their elevated activity. This is associated with a reduction in the expression of PGC1α and COX IV, proteins which play important roles in maintaining mitochondrial function. Furthermore, we show that these changes in signalling and protein expression are largely independent of alterations in intramuscular amino acid content. Our findings, therefore, reveal potential new mechanisms and further contribute to our understanding of myostatin-regulated skeletal muscle growth and function.

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type-specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin-deficient double-muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse-transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC-2x and -2a and less -slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster-type muscles and on MyoD expression in slower-type muscles, suggesting a possible muscle type-specific effect of myostatin in skeletal muscle growth and maintenance.

Relation between extent of myostatin depletion and muscle growth in mature mice

Myostatin is a negative regulator of muscle growth and fiber size. Changes in myostatin expression might contribute to changes in muscle mass associated with various conditions, and reducing the amount of active myostatin is a potential strategy for preventing or reversing muscle atrophy. The present study was done to determine the extent to which myostatin levels must decline to induce growth of mature muscles. Myostatin expression was reduced by activating Cre recombinase in adult mice with floxed myostatin genes. The duration of Cre activation varied from 1 to 6 wk, and the residual myostatin mRNA expression after Cre activation varied from 3 to 63% of the normal level. Promyostatin levels declined in parallel with myostatin mRNA. There was no increase in muscle mass over the 3 mo following Cre activation if residual myostatin expression was >or=40% of normal. In mice with <40% of normal myostatin expression, muscle mass increased in proportion to the extent of myostatin depletion. In mice with <or=10% of normal myostatin expression, muscle mass increased approximately 25%. Myostatin depletion increased myonuclear domain volumes and the ratio of RNA to myonuclei probably by enhancing DNA transcription rather than by inhibiting RNA decay. There was no evidence that maintenance of the hypertrophy during chronic myostatin deficiency requires altered activity of Akt/mTOR or p38 MAPK signaling pathways. These data suggest that anabolic therapies based on reducing the concentration of active myostatin will be effective only if a very large proportion of the myostatin is removed or inactivated.

Skeletal muscle gene expression after myostatin knockout in mature mice

There is much interest in developing anti-myostatin agents to reverse or prevent muscle atrophy in adults, so it is important to characterize the effects of reducing myostatin activity after normal muscle development. For assessment of the effect of loss of myostatin signaling on gene expression in muscle, RNA from mice with postdevelopmental myostatin knockout was analyzed with oligonucleotide microarrays. Myostatin was undetectable in muscle within 2 wk after Cre recombinase activation in 4-month-old male mice with floxed myostatin genes. Three months after myostatin depletion, muscle mass had increased 26% (vs. 2% after induction of Cre activity in mice with normal myostatin genes), at which time the expression of several hundred genes differed in knockout and control mice at nominal P < 0.01. In contrast to previously reported effects of constitutive myostatin knockout, postdevelopmental knockout did not downregulate expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins involved in energy metabolism. Several collagen genes were expressed at 20-50% lower levels in the myostatin-deficient muscles, which had approximately 25% less collagen than normal muscles as reflected by hydroxyproline content. Most of the other genes affected by myostatin depletion have not been previously linked to myostatin signaling. Gene set enrichment analysis suggested that Smads are not the only transcription factors with reduced activity after myostatin depletion. These data reinforce other evidence that myostatin regulates collagen production in muscle and demonstrate that many of the previously reported effects of constitutive myostatin deficiency do not occur when myostatin is knocked out in mature muscles.

Myostatin (GDF-8) deficiency increases fracture callus size, Sox-5 expression, and callus bone volume

Myostatin (GDF-8) is a negative regulator of skeletal muscle growth and mice lacking myostatin show increased muscle mass. We have previously shown that myostatin deficiency increases bone strength and biomineralization throughout the skeleton, and others have demonstrated that myostatin is expressed during the earliest phase of fracture repair. In order to determine the role of myostatin in fracture callus morphogenesis, we studied fracture healing in mice lacking myostatin. Adult wild-type mice (+/+), mice heterozygous for the myostatin mutation (+/−), and mice homozygous for the disrupted myostatin sequence (−/−) were included for study at two and four weeks following osteotomy of the fibula. Expression of Sox-5 and BMP-2 were significantly upregulated in the fracture callus of myostatin-deficient (−/−) mice compared to wild-type (+/+) mice at two weeks following osteotomy. Fracture callus size was significantly increased in mice lacking myostatin at both two and four weeks following osteotomy, and total osseous tissue area and callus strength in three-point bending were significantly greater in myostatin −/− mice compared to myostatin +/+ mice at four weeks post-osteotomy. Our data suggest that myostatin functions to regulate fracture callus size by inhibiting the recruitment and proliferation of progenitor cells in the fracture blastema. Myostatin deficiency increases blastema size during the early inflammatory phase of fracture repair, ultimately producing an ossified callus having greater bone volume and greater callus strength. While myostatin is most well known for its effects on muscle development, it is also clear that myostatin plays a significant, direct role in bone formation and regeneration.

Muscle and nerve pathology in Dunnigan familial partial lipodystrophy

To characterize muscle and nerve pathology in Dunnigan familial partial lipodystrophy (FPLD). We used conventional histology, immunohistochemistry, messenger RNA (mRNA) expression, gene sequencing, and clinical studies of 13 patients with neuromuscular involvement. The clinical findings consisted of muscle hypertrophy (12/13), severe myalgias (9/13), and multiple nerve entrapment syndromes (8/13). Skeletal muscle histology demonstrated marked Type 1 and 2 muscle fiber hypertrophy and nonspecific myopathic changes, whereas numerous paranodal myelin swellings (tomacula) were found in sural nerve biopsies. We found that myostatin mRNA expression was reduced in patients with FPLD vs controls. We sequenced the myostatin gene in our subjects, but found no mutations. We then investigated whether or not SMAD, the intracellular mediator of myostatin signaling, might be impaired in patients with FPLD. We found that in FPLD muscle, a large number of SMAD molecules adhered to the nuclear membrane and were not found within the nucleus, compared with normal muscle or muscle from a patient with a non-FPLD lamin A/C disease. The myopathy and neuropathy associated with Dunnigan familial partial lipodystrophy are distinct from other lamin A/C disorders. We hypothesize that the lipodystrophy-associated mutation interferes with SMAD signaling, linking this type of lipodystrophy to the phenotypically similar myostatin deficiency.

Loss of myostatin (GDF8) function increases osteogenic differentiation of bone marrow-derived mesenchymal stem cells but the osteogenic effect is ablated with unloading

Myostatin (GDF8) is a negative regulator of skeletal muscle growth and mice lacking myostatin show a significant increase in muscle mass and bone density compared to normal mice. In order to further define the role of myostatin in regulating bone mass we sought to determine if loss of myostatin function significantly altered the potential for osteogenic differentiation in bone marrow-derived mesenchymal stem cells in vitro and in vivo. We first examined expression of the myostatin receptor, the type IIB activin receptor (AcvrIIB), in bone marrow-derived mesenchymal stem cells (BMSCs) isolated from mouse long bones. This receptor was found to be expressed at high levels in BMSCs, and we were also able to detect AcvrIIB protein in BMSCs in situ using immunofluorescence. BMSCs isolated from myostatin-deficient mice showed increased osteogenic differentiation compared to wild-type mice; however, treatment of BMSCs from myostatin-deficient mice with recombinant myostatin did not attenuate the osteogenic differentiation of these cells. Loading of BMSCs in vitro increased the expression of osteogenic factors such as BMP-2 and IGF-1, but treatment of BMSCs with recombinant myostatin was found to decrease the expression of these factors. We investigated the effects of myostatin loss-of-function on the differentiation of BMSCs in vivo using hindlimb unloading (7-day tail suspension). Unloading caused a greater increase in marrow adipocyte number, and a greater decrease in osteoblast number, in myostatin-deficient mice than in normal mice. These data suggest that the increased osteogenic differentiation of BMSCs from mice lacking myostatin is load-dependent, and that myostatin may alter the mechanosensitivity of BMSCs by suppressing the expression of osteogenic factors during mechanical stimulation. Furthermore, although myostatin deficiency increases muscle mass and bone strength, it does not prevent muscle and bone catabolism with unloading.

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.

Sexual dimorphism in the response of muscle and bone to myostatin (GDF8) deficiency

Males and females show marked differences in the deposition and modeling of bone during growth. Increased levels of testosterone in males are associated with increased muscle mass and periosteal expansion, whereas increased levels of estrogen in females are associated with endosteal contraction. Myostatin-deficiency increases muscle mass and bone density, and we proposed that the response of muscle and bone to loss of myostatin function may be affected by sex hormone status. We tested this hypothesis by measuring body weight, triceps brachii mass, and bone biomechanical properties of the radius in male and female wild-type and myostatin-deficient mice, 4 months of age (n=12/group). Two-factor ANOVA with sex and genotype as the two factors was used to test for significant interactions between sex and genotype for all variables. Significant sex-genotype interactions were detected for body mass, muscle mass, bone diameter, cortical thickness, area moment of inertia, and ultimate bone stress and strain, with males showing a greater response to myostatin deficiency than females for each variable. These data indicate that downregulation of myostatin produces a greater anabolic response in male skeletal muscle than in female muscle. Moreover, sex-specific differences in the response of muscle to myostatin deficiency in turn yield sex-specific differences in bone shape and biomechanical properties. These findings suggest that molecular therapies aimed at increasing muscle and bone strength via myostatin blockade may be more effective in men than in women. Funding for this research was provided by the National Institutes of Health (NIAMS AR049717-01A2).

Increased Muscle Mass With Myostatin Deficiency Improves Gains in Bone Strength With Exercise

We tested the hypothesis that increased muscle mass augments increases in bone strength normally observed with exercise. Myostatin-deficient mice, which show increased muscle mass, were exercised along with wildtype mice. Results indicate that increases in bone strength with exercise are greater in myostatin-deficient mice than in wildtype mice, suggesting that the combination of increased muscle mass and physical activity has a greater effect on bone strength than either increased muscle mass or intense exercise alone. Muscle (lean) mass is known to be a significant predictor of peak BMD in young people, and exercise is also found to increase bone mass in growing humans and laboratory animals. We sought to determine if increased muscle mass resulting from myostatin deficiency would enhance gains in bone strength that usually accompany exercise. Male mice lacking myostatin (GDF-8) were used as an animal model showing increased muscle mass. Wildtype and myostatin-deficient mice (n = 10-12 per genotype) were exercised on a treadmill for 30 minutes/day, 5 days/week, for 4 weeks starting at 12 weeks of age. Caged wildtype and myostatin-deficient mice (n = 10-12 per genotype) were included as sedentary controls. Structural and biomechanical parameters were measured from the radius. Ultimate force (F(u)), displacement (D(u)), toughness (energy-to-fracture; U), and ultimate strain (epsilon(u)) increased significantly with exercise in myostatin-deficient mice but not in normal mice. When F(u) is normalized by body mass, exercised myostatin-deficient mice show an increase in relative bone strength of 30% compared with caged controls, whereas exercised wildtype mice do not show a significant increase in ultimate force relative to caged controls. Relative to body weight, the radii of exercised myostatin-deficient mice are >25% stronger than those of exercised normal mice. Increased muscle mass resulting from inhibition of myostatin function improves the positive effects of exercise on bone strength.

Contractile properties of skeletal muscles from myostatin deficient mice

Myostatin (GDF-8) is a negative regulator of skeletal muscle mass and has a profound impact on the structure of muscles. Consequently, the impact of myostatin deficiency on the contractile properties of muscle was determined. Measurements were made of the physiological cross sectional area (CSA), in vitro maximum isometric tetanic isometric force (Fo), and the force-deficit (decrease in Fo expressed as a percentage of initial Fo) following a two-stretch lengthening contraction protocol designed to produce severe injury to the whole EDL and soleus muscles of wild type (MSTN+/+), MSTN heterozygous null (MSTN+/-) and homozygous null (MSTN-/-) adult male mice. For most models of mammalian muscle hypertrophy, Fo increases, but the specific Fo (sFo), Fo normalized by CSA, decreases. Compared with MSTN+/+ mice, EDL muscles of MSTN+/- mice had a 13% increase in Fo, but no difference in sFo. MSTN-/- mice had a 34% increase in Fo and an 18% decrease in sFo. Compared with MSTN+/+ mice, the Fo of soleus muscles were 10% greater in MSTN+/- mice and 30% greater in MSTN-/- mice. Despite this increase in Fo, sFo was not different. Following the injury protocol, the force deficit of EDL muscles from MSTN-/- mice was 15% greater than that of MSTN+/+ and MSTN+/- mice. No difference in force deficits was observed for soleus muscles. Our results support a significant role for myostatin in the determination of the contractile properties of skeletal muscles.

Resistance to body fat gain in ‘double-muscled’ mice fed a high-fat diet

To determine if myostatin deficiency attenuates body fat gain with increased dietary fat intake. Normal and myostatin-deficient mice were fed control (8-10 kcal %fat) and high-fat (HF) (45 kcal %fat) diets for a period of 8 weeks, starting at 2 months of age. Body composition, including percent body fat, lean mass, and fat mass, were measured using DXA. Serum adipokines were measured using a Beadlyte assay. Two-factor ANOVA revealed significant treatment x genotype interactions for body fat (g), percent body fat, and serum leptin. The HF diet significantly increased body fat, percent body fat, and serum leptin in normal mice but not in myostatin-deficient mice. Loss of myostatin function not only increases muscle mass in animal models but also attenuates the body fat accumulation that usually accompanies an HF diet.

Myofibrillar protein synthesis in myostatin-deficient mice

Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn(DeltaE3/DeltaE3)) or normal myostatin expression (Mstn(+/+)) by measuring tracer incorporation after a systemic flooding dose of l-[ring-(2)H(5)]phenylalanine. At 5-6 wk of age, Mstn(DeltaE3/DeltaE3) mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn(+/+) mice. With maturation, fractional rates of synthesis declined >50% in parallel with decreased DNA and RNA [total, 28S rRNA, and poly(A) RNA] concentrations in muscle. At 6 mo of age, Mstn(DeltaE3/DeltaE3) mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn(+/+) mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn(DeltaE3/DeltaE3) mice, whereas RNA concentrations were normal, so the ratio of RNA to DNA was approximately 30% greater than normal in Mstn(DeltaE3/DeltaE3) mice. Thus the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.

Marker-assisted introgression of the Compact mutant myostatin allele MstnCmpt-dl1Abc into a mouse line with extreme growth effects on body composition and muscularity

Myostatin is a negative regulator of muscle growth and mutations in its gene lead to muscular hypertrophy and reduced fat. In cattle, this is seen in 'double muscled' breeds. We have used marker-assisted introgression to introduce a murine myostatin mutation, MstnCmpt-dl1Abc [Compact (C)], into an inbred line of mice (DUHi) that had been selected on body weight and had exceptional growth. Compared with homozygous wild-type mice, homozygous (C/C) mice of this line were approximately 4-5 % lighter, had approximately 7-8 % shorter tails, substantially increased muscle weights (e.g. quadriceps muscle in males was 59 % heavier) and an increased 'dressing percentage' (approximately 49 % vs 39 %), an indicator of overall muscularity. The weights of several organs (e.g. liver, kidney, heart and digestive tract) were significantly reduced, by 12-20 %. Myostatin deficiency also resulted in drastic reductions of total body fat and of various fat depots, total body fat proportion falling from approximately 17.5 % in wild-type animals of both sexes to 9.5 % and 11.6% in homozygous (C/C) females and males, respectively. Males with a deficiency in myostatin had higher gains in muscle traits than females. Additionally, there was a strong distortion of the segregation ratio on the DUHi background. Of 838 genotyped pups from inter se matings 29 %, 63 % and 8 % were homozygous wild type (+/+), heterozygous (C/+) and homozygous (C/C), respectively, showing that MstnCmpt-dl1Abc has lower fitness on this background. This line, when congenic, will be a useful resource in gene expression studies and for finding modifying genes.