Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.     

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.     

Molecular analysis of fiber type-specific expression of murine myostatin promoter

Myostatin is a negative regulator of muscle growth, and absence of the functional myostatin protein leads to the heavy muscle phenotype in both mouse and cattle. Although the role of myostatin in controlling muscle mass is established, little is known of the mechanisms regulating the expression of the myostatin gene. In this study, we have characterized the murine myostatin promoter in vivo. Various constructs of the murine myostatin promoter were injected into the quadriceps muscle of mice, and the reporter luciferase activity was analyzed. The results indicate that of the seven E-boxes present in the 2.5-kb fragment of the murine myostatin promoter, the E5 E-box plays an important role in the regulation of promoter activity in vivo. Furthermore, the in vitro studies demonstrated that MyoD preferentially binds and upregulates the murine myostatin promoter activity. We also analyzed the activity of the bovine and murine promoters in murine skeletal muscle and showed that, despite displaying comparable levels of activity in murine myoblast cultures, bovine myostatin promoter activity is much weaker than murine myostatin promoter in mice. Finally, we demonstrate that in vivo, the 2.5-kb region of the murine myostatin promoter is sufficient to drive the activity of the reporter gene in a fiber type-specific manner. myogenic regulatory factor; E-box; naked DNA     

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.     

Transgenic expression of myostatin propeptide prevents diet-induced obesity and insulin resistance

Obesity and insulin resistance cause serious consequences to human health. To study effects of skeletal muscle growth on obesity prevention, we focused on a key gene of skeletal muscle named myostatin, which plays an inhibitory role in muscle growth and development. We generated transgenic mice through muscle-specific expression of the cDNA sequence (5'-region 886 nucleotides) encoding for the propeptide of myostatin. The transgene effectively depressed myostatin function. Transgenic mice showed dramatic growth and muscle mass by 9 weeks of age. Here we reported that individual major muscles of transgenic mice were 45-115% heavier than those of wild-type mice, maintained normal blood glucose, insulin sensitivity, and fat mass after a 2-month regimen with a high-fat diet (45% kcal fat). In contrast, high-fat diet induced wild-type mice with 170-214% more fat mass than transgenic mice and developed impaired glucose tolerance and insulin resistance. Insulin signaling, measured by Akt phosphorylation, was significantly elevated by 144% in transgenic mice over wild-type mice fed a high-fat diet. Interestingly, high-fat diet significantly increased adiponectin secretion while blood insulin, resistin, and leptin levels remained normal in the transgenic mice. The results suggest that disruption of myostatin function by its propeptide favours dietary fat utilization for muscle growth and maintenance. An increased secretion of adiponectin may promote energy partition toward skeletal muscles, suggesting that a beneficial interaction between muscle and adipose tissue play a role in preventing obesity and insulin resistance.     

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.     

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer–promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.     

Relationships between transforming growth factor-beta1, myostatin, and decorin: implications for skeletal muscle fibrosis

Recent studies have shown that myostatin, first identified as a negative regulator of skeletal muscle growth, may also be involved in the formation of fibrosis within skeletal muscle. In this study, we further explored the potential role of myostatin in skeletal muscle fibrosis, as well as its interaction with both transforming growth factor-beta1 and decorin. We discovered that myostatin stimulated fibroblast proliferation in vitro and induced its differentiation into myofibroblasts. We further found that transforming growth factor-beta1 stimulated myostatin expression, and conversely, myostatin stimulated transforming growth factor-beta1 secretion in C2C12 myoblasts. Decorin, a small leucine-rich proteoglycan, was found to neutralize the effects of myostatin in both fibroblasts and myoblasts. Moreover, decorin up-regulated the expression of follistatin, an antagonist of myostatin. The results of in vivo experiments showed that myostatin knock-out mice developed significantly less fibrosis and displayed better skeletal muscle regeneration when compared with wild-type mice at 2 and 4 weeks following gastrocnemius muscle laceration injury. In wild-type mice, we found that transforming growth factor-beta1 and myostatin co-localize in myofibers in the early stages of injury. Recombinant myostatin protein stimulated myofibers to express transforming growth factor-beta1 in skeletal muscles at early time points following injection. In summary, these findings define a fibrogenic property of myostatin and suggest the existence of co-regulatory relationships between transforming growth factor-beta1, myostatin, and decorin.     

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.     

Expression of myostatin pro domain results in muscular transgenic mice

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Depression of myostatin activity leads to increased muscle growth and carcass lean yield. In an attempt to down-regulate myostatin, transgenic mice were produced with a ribozyme-based construct or a myostatin pro domain construct. Though the expression of the ribozyme was detected, muscle development was not altered by the ribozyme transgene. However, a dramatic muscling phenotype was observed in transgenic mice carrying the myostatin pro domain gene. Expression of the pro domain transgene at 5% of beta-actin mRNA levels resulted in a 17-30% increase in body weight (P < 0.001). The carcass weight of the transgenic mice showed a 22-44% increase compared with nontransgenic littermates at 9 weeks of age (16.05 +/- 0.67 vs. 11.16 +/- 0.28 g in males; 9.99 +/- 0.38 vs. 8.19 +/- 0.19 g in females, P < 0.001). Extreme muscling was present throughout the whole carcass of transgenic mice as hind and fore limbs and trunk weights, all increased significantly (P < 0.001). Epididymal fat pad weight, an indicator of body fat, was significantly decreased in pro domain transgenic mice (P < 0.001). Analysis of muscle morphology indicated that cross-sectional areas of fast-glycolytic fibers (gastrocnemius) and fast-oxidative glycolytic fibers (tibialis) were larger in pro domain transgenic mice than in their controls (P < 0.01), whereas fiber number (gastrocnemius) was not different (P > 0.05). Thus, the muscular phenotype is attributable to myofiber hypertrophy rather than hyperplasia. The results of this study suggest that the over-expression of myostatin pro domain may provide an alternative to myostatin knockouts as a means of increasing muscle mass in other mammals.     

Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.     

Administration of a mutated myostatin propeptide to neonatal mice significantly enhances skeletal muscle growth

Myostatin is a dominant inhibitor of skeletal muscle development and growth. As transgenic over‐expression of myostatin propeptide dramatically enhanced muscle mass, we hypothesized that administration of myostatin propeptide will increase muscle growth. In this study, the wild‐type form of porcine myostatin propeptide and its mutated form at the cleavage site of metalloproteinases of BMP‐1/TLD family were produced from insect cells. In vitro A204 cells reporter assays showed that both wild‐type and the mutated propeptides depressed myostatin activity. The recombinant propeptides at four‐fold myostatin concentration can effectively block myostatin function during co‐incubation with A204 cells. In particular, the mutated propeptide appeared much more effective than wild‐type propeptide over a long period during the in vitro co‐incubation. Administration of the mutated propeptide to neonatal mice at the age of 11 and 18 days was tested and showed significant increase in growth performance by 11–15% from the age of 25 to 57 days (P < 0.05). The major skeletal muscles of mice that were injected with mutated propeptide were 13.5–24.8% heavier than the control group (P < 0.05) as a result of muscle fiber hypertrophy. In conclusion, administration of the mutated myostatin propeptide during the neonatal period is an effective way for promoting muscle growth. Mol. Reprod. Dev. 77: 76–82, 2010. © 2009 Wiley‐Liss, Inc.     

Follistatin-derived peptide expression in muscle decreases adipose tissue mass and prevents hepatic steatosis

Myostatin, a member of the transforming growth factor (TGF)-β superfamily, plays a potent inhibitory role in regulating skeletal muscle mass. Inhibition of myostatin by gene disruption, transgenic (Tg) expression of myostatin propeptide, or injection of propeptide or myostatin antibodies causes a widespread increase in skeletal muscle mass. Several peptides, in addition to myostatin propeptide and myostatin antibodies, can bind directly to and neutralize the activity of myostatin. These include follistatin and follistatin-related gene. Overexpression of follistatin or follistatin-related gene in mice increased the muscle mass as in myostatin knockout mice. Follistatin binds to myostatin but also binds to and inhibits other members of the TGF-β superfamily, notably activins. Therefore, follistatin regulates both myostatin and activins in vivo. We previously reported the development and characterization of several follistatin-derived peptides, including FS I-I (Nakatani M, Takehara Y, Sugino H, Matsumoto M, Hashimoto O, Hasegawa Y, Murakami T, Uezumi A, Takeda S, Noji S, Sunada Y, Tsuchida K. FASEB J 22: 477-487, 2008). FS I-I retained myostatin-inhibitory activity without affecting the bioactivity of activins. Here, we found that inhibition of myostatin increases skeletal muscle mass and decreases fat accumulation in FS I-I Tg mice. FS I-I Tg mice also showed decreased fat accumulation even on a control diet. Interestingly, the adipocytes in FS I-I Tg mice were much smaller than those of wild-type mice. Furthermore, FS I-I Tg mice were resistant to high-fat diet-induced obesity and hepatic steatosis and had lower hepatic fatty acid levels and altered fatty acid composition compared with control mice. FS I-I Tg mice have improved glucose tolerance when placed on a high-fat diet. These data indicate that inhibiting myostatin with a follistatin-derived peptide provides a novel therapeutic option to decrease adipocyte size, prevent obesity and hepatic steatosis, and improve glucose tolerance.     

Effects of myostatin deletion in aging mice

Inhibitors of myostatin, a negative regulator of skeletal muscle mass, are being developed to mitigate aging-related muscle loss. Knock-out (KO) mouse studies suggest myostatin also affects adiposity, glucose handling and cardiac growth. However, the cardiac consequences of inhibiting myostatin remain unclear. Myostatin inhibition can potentiate cardiac growth in specific settings ( Morissette et al., 2006) , a concern because of cardiac hypertrophy is associated with adverse clinical outcomes. Therefore, we examined the systemic and cardiac effects of myostatin deletion in aged mice (27–30 months old). Heart mass increased comparably in both wild-type (WT) and KO mice. Aged KO mice maintained twice as much quadriceps mass as aged WT; however, both groups lost the same percentage (36%) of adult muscle mass. Dual-energy X-ray absorptiometry revealed increased bone density, mineral content, and area in aged KO vs. aged WT mice. Serum insulin and glucose levels were lower in KO mice. Echocardiography showed preserved cardiac function with better fractional shortening (58.1% vs. 49.4%, P  = 0.002) and smaller left ventricular diastolic diameters (3.41 vs. 2.71, P  = 0.012) in KO vs. WT mice. Phospholamban phosphorylation was increased 3.3-fold in KO hearts ( P  < 0.05), without changes in total phospholamban, sarco(endo)plasmic reticulum calcium ATPase 2a or calsequestrin. Aged KO hearts showed less fibrosis by Masson's Trichrome staining. Thus, myostatin deletion does not affect aging-related increases in cardiac mass and appears beneficial for bone density, insulin sensitivity and heart function in senescent mice. These results suggest that clinical interventions designed to inhibit skeletal muscle mass loss with aging could have beneficial effects on other organ systems as well.     

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.     

Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.     

Genetic variability affects the response of skeletal muscle to disuse

Objective:To examine whether genetic variability plays a role in skeletal muscle response to disuse.Methods:We examined skeletal muscle response to disuse in five different strains of mice: CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, 129S1/SvImJ and A/J. Mice had one limb immobilized by a cast for three weeks.Results:Response to immobilization was dependent on the strain of mice. Skeletal muscle mass/body weight was decreased by immobilization in all strains except 1291/SvImJ. Immobilization decreased absolute skeletal muscle mass in quadriceps and gastrocnemius in NOD/ShiltJ and NZO/HILtJ mice. Three weeks of immobilization resulted in an increase in quadriceps levels of atrogenes in CAST/EiJ. Immobilization resulted in an increase in quadriceps and gastrocnemius levels of Myh4 in CAST/EiJ. A similar trend was observed for Myh7 in gastrocnemius muscle. Immobilization resulted in a decrease of the p-p70S6K1/total p706SK1 ratio in quadriceps of NOD/ShiLtJ mice and the gastrocnemius of A/J mice. Immobilization did not affect the p-4EBP1/total 4EBP1 ratio in quadriceps of any of the strains examined. However, the p-4EBP1/total 4EBP1 ratio in gastrocnemius was greater in immobilized, relative to control, limbs in CAST/EiJ mice.Conclusion:Genetic variability affects the response of skeletal muscle to disuse.     

Myostatin expression in age and denervation-induced skeletal muscle atrophy

Myostatin is hypothesized to regulate skeletal muscle mass and to be a potential target for therapeutic intervention in sarcopenia. To clarify whether myostatin is invariably associated with sarcopenia, this study examined the levels of expression of myostatin mRNA and protein in Sprague Dawley rats during aging- and denervation-induced sarcopenia. The level of myostatin mRNA in the gastrocnemius decreased progressively with age being 9, 34 and 56% lower at 6, 12 and 27 months, respectively, compared with mRNA levels at 1.5 months. In contrast, two low molecular mass isoforms of myostatin protein identified by Western blotting increased progressively with age. With denervation, myostatin mRNA was 31% higher on day 1 but by 14 days after sciatic neurectomy when the muscle had atrophied 50%, myostatin expression decreased 34% relative to the sham operated limb. Western analysis of the denervated gastrocnemius showed that myostatin protein levels varied in parallel with mRNA. These disparate patterns of expression of myostatin during age- and denervation-induced atrophy suggest that the regulation of myostatin is complex and variable depending on whether the atrophy is slowly or rapidly progressive.     

Muscle-specific transgenic expression of porcine myostatin propeptide enhances muscle growth in mice

Myostatin is a well-known negative regulator of skeletal muscle growth. Inhibition of myostatin activity results in increased muscle mass. Myostatin propeptide, as a myostatin antagonist, could be applied to promote meat production in livestock such as pigs. In this study, we generated a transgenic mouse model expressing porcine myostatin propeptide under the control of muscle-specific regulatory elements. The mean body weight of transgenic mice from a line expressing the highest level of porcine myostatin propeptide was increased by 5.4 % (P = 0.023) and 3.2 % (P = 0.031) in males and females, respectively, at 8 weeks of age. Weight of carcass, fore limb and hind limb was respectively increased by 6.0 % (P = 0.038), 9.0 % (P = 0.014), 8.7 % (P = 0.036) in transgenic male mice, compared to wild-type male controls at the age of 9 weeks. Similarly, carcass, fore limb and hind limb of transgenic female mice was 11.4 % (P = 0.002), 14.5 % (P = 0.006) and 14.5 % (P = 0.03) respectively heavier than that of wild-type female mice. The mean cross-section area of muscle fiber was increased by 17 % (P = 0.002) in transgenic mice, in comparison with wild-type controls. These results demonstrated that porcine myostatin propeptide is effective in enhancement of muscle growth. The present study provided useful information for future study on generation of transgenic pigs overexpressing porcine myostatin propeptide for improvement of muscle mass.