Estrogen effects on skeletal muscle insulin-like growth factor 1 and myostatin in ovariectomized rats

Previous work showed that estrogen replacement attenuates muscle growth in immature rats. The present study examined muscle insulin-like growth factor-1 (IGF-1) and myostatin expression to determine whether these growth regulators might be involved in mediating estrogen's effects on muscle growth. IGF-1 and myostatin message and protein expression in selected skeletal muscles from 7-week-old sham-ovariectomized (SHAM) and ovariectomized rats that received continuous estrogen (OVX/E2) or solvent vehicle (OVX/CO) from an implant for 1 week or 5 weeks was measured. In the 1-week study, ovariectomy increased IGF-1 mRNA expression in fast extensor digitorum longus and gastrocnemius muscles; the increase was reversed by estrogen replacement. A similar trend was observed in the slow soleus muscle, although the change was not statistically significant. In contrast to mRNA, muscle IGF-1 protein expression was not different between SHAM and OVX/ CO animals in the 1-week study. One week of estrogen replacement significantly decreased IGF-1 protein level in all muscles examined. Myostatin mRNA expression was not different among the 1-week treatment groups. One week of estrogen replacement significantly increased myostatin protein in the slow soleus muscle but not the fast extensor digitorum longus and gastrocnemius muscles. There was no treatment effect on IGF-1 and myostatin expression in the 5-week study; this finding suggested a transient estrogen effect or upregulation of a compensatory mechanism to counteract the estrogen effect observed at the earlier time point. This investigation is the first to explore ovariectomy and estrogen effects on skeletal muscle IGF-1 and myostatin expression. Results suggest that reduced levels of muscle IGF-1 protein may mediate estrogen's effect on growth in immature, ovariectomized rats. Increased levels of muscle myostatin protein may also have a role in mediating estrogen's effects on growth in slow but not fast skeletal muscle.     

Sepsis downregulates myostatin mRNA levels without altering myostatin protein levels in skeletal muscle

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis‐induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham‐operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of activin A, follistatin, and total and phosphorylated Smad2 (p‐Smad2) were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Interestingly, total and p‐Smad3 levels were increased in septic muscle, possibly reflecting altered signaling through pathways other than myostatin. Similar to sepsis, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. Fasting, an additional condition characterized by muscle wasting, reduced myostatin mRNA and activin A protein levels, increased myostatin protein, and did not influence follistatin and p‐Smad2 levels. Of note, total and p‐Smad3 levels were reduced in muscle during fasting. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting. Downstream signaling through Smad2 and 3 is probably regulated not only by myostatin but by other mechanisms as well. J. Cell. Biochem. 111: 1059–1073, 2010. © 2010 Wiley‐Liss, Inc.     

Myostatin expression is regulated by underfeeding and neonatal programming in rats

Confusing results have been reported regarding the influence of nutritional status on myostatin levels. Some studies indicate that short-term fasting results in increased myostatin mRNA levels in skeletal muscle, evident in several species. In contrast, other studies have demonstrated either a decrease or no change in myostatin levels during fasting. In the present study, we investigated the effect of different patterns of food deprivation on muscle myostatin expression in both newborn and adult rats. Adjustment of litter size in neonatal rats is a well-established model to study the effect of early overfeeding or underfeeding on body composition and in this study resulted in modifications in the pattern of muscle myostatin expression. Rat pups growing in large litters (22–24 newborns) showed a decrease in muscle myostatin mRNA and protein levels at 24 days of age. Interestingly, these effects were maintained at 60 days of age despite rats having free access to food since weaning, thus suggesting that changes in myostatin expression induced by neonatal reduction of food intake are long-lasting. In contrast, no changes in myostatin mRNA levels were observed in adult rats when food intake was decreased during 7 days by either food restriction or central leptin treatment. Similar results were obtained when food restriction was maintained in adult rats for a longer period (7 weeks), despite significant muscle loss. Overall, these data suggest that myostatin gene expression is programmed by nutritional status in neonatal life.     

Myostatin promotes the wasting of human myoblast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins

Myostatin is a negative regulator of skeletal muscle growth and in fact acts as a potent inducer of "cachectic-like" muscle wasting in mice. The mechanism of action of myostatin in promoting muscle wasting has been predominantly studied in murine models. Despite numerous reports linking elevated levels of myostatin to human skeletal muscle wasting conditions, little is currently known about the signaling mechanism(s) through which myostatin promotes human skeletal muscle wasting. Therefore, in this present study we describe in further detail the mechanisms behind myostatin regulation of human skeletal muscle wasting using an in vitro human primary myotube atrophy model. Treatment of human myotube populations with myostatin promoted dramatic myotubular atrophy. Mechanistically, myostatin-induced myotube atrophy resulted in reduced p-AKT concomitant with the accumulation of active dephosphorylated Forkhead Box-O (FOXO1) and FOXO3. We further show that addition of myostatin results in enhanced activation of atrogin-1 and muscle-specific RING finger protein 1 (MURF1) and reduced expression of both myosin light chain (MYL) and myosin heavy chain (MYH). In addition, we found that myostatin-induced loss of MYL and MYH proteins is dependent on the activity of the proteasome and mediated via SMAD3-dependent regulation of FOXO1 and atrogin-1. Therefore, these data suggest that the mechanism through which myostatin promotes muscle wasting is very well conserved between species, and that myostatin-induced human myotube atrophy is mediated through inhibition of insulin-like growth factor (IGF)/phosphoinositide 3-kinase (PI3-K)/AKT signaling and enhanced activation of the ubiquitin-proteasome pathway and elevated protein degradation.     

Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting

A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-β (TGF-β) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future.     

Interactions between the consumption of a high-fat diet and fasting in the regulation of fatty acid oxidation enzyme gene expression: an evaluation of potential mechanisms

The consumption of high-fat diets (HFDs) and fasting are known to increase the expression of enzymes involved in fatty acid oxidation (FAO). However, it has been reported that the ability of physiological stressors to induce enzymes of FAO in skeletal muscle is blunted with obesity. In this regard, we sought to explore the effects and potential mechanisms of an HFD on the expression of FAO enzymes in the fed and fasted state. The consumption of an HFD increased the mRNA expression or protein content of medium-chain acyl-CoA dehydrogenase (MCAD), uncoupling protein-3 (UCP3), and pyruvate dehydrogenase kinase 4 (PDK4) in the fed state. Fasting increased the mRNA expression of PDK4, MCAD, and UCP-3, and the protein content of UCP-3 in chow but not HFD rats. HFDs did not increase carnitine palmitoyl transfer-1 (CPT-1) mRNA levels in the fed state and the effects of fasting were markedly reduced compared with chow-fed rats. The expression of peroxisome-proliferator-activated receptor-γ coactivator-1β (PGC-1β) was increased in muscle from HFD rats in the fed state, while PGC-1-related coactivator (PRC) was increased with fasting in chow-fed but not HFD rats. Plasma fatty acid levels were elevated in the fed state from HFD rats but not increased further with fasting, whereas fasting increased plasma fatty acids in chow-fed animals. Fasting-mediated increases in plasma epinephrine, and the activation of PKA and AMPK in skeletal muscle were similar between chow and HFD rats. p38 MAPK phosphorylation was increased with fasting in chow-fed but not HFD rats. Our findings suggest that a blunted effect of fasting on the induction of PDK4, MCAD, and UCP3 in skeletal muscle from HFD rats is likely a result of already elevated levels of these enzymes, the induction of which is associated with increases in plasma fatty acid and PGC-1β. On the other hand, a blunted induction of PRC and CPT-1 mRNA may be explained by decreases in p38 MAPK signaling.     

IGF and myostatin pathways are respectively induced during the earlier and the later stages of skeletal muscle hypertrophy induced by clenbuterol,a β2‐adrenergic agonist

Clenbuterol, a β₂‐adrenergic agonist, increases the hypertrophy of skeletal muscle. Insulin‐like growth factor (IGF) is reported to work as a potent positive regulator in the clenbuterol‐induced hypertrophy of skeletal muscles. However, the precise regulatory mechanism for the hypertrophy of skeletal muscle induced by clenbuterol is unknown. Myostatin, a member of the TGFβ super family, is a negative regulator of muscle growth. The aim of the present study is to elucidate the function of myostatin and IGF in the hypertrophy of rat masseter muscle induced by clenbuterol. To investigate the function of myostatin and IGF in regulatory mechanism for the clenbuterol‐induced hypertrophy of skeletal muscles, we analysed the expression of myostatin and phosphorylation levels of myostatin and IGF signaling components in the masseter muscle of rat to which clenbuterol was orally administered for 21 days. Hypertrophy of the rat masseter muscle was induced between 3 and 14 days of oral administration of clenbuterol and was terminated at 21 days. The expression of myostatin and the phosphorylation of smad2/3 were elevated at 21 days. The phosphorylation of IGF receptor 1 (IGFR1) and akt1 was elevated at 3 and 7 days. These results suggest that myostatin functions as a negative regulator in the later stages in the hypertrophy of rat masseter muscle induced by clenbuterol, whereas IGF works as a positive regulator in the earlier stages. Copyright © 2012 John Wiley & Sons, Ltd.     

Androgens negatively regulate myostatin expression in an androgen-dependent skeletal muscle

Myostatin is an important negative regulator of skeletal muscle growth, while androgens are strong positive effectors. In order to investigate the possible interaction between myostatin and androgen pathways, we followed myostatin expression in the androgen-dependent levator ani (LA) muscle of the rat as a function of androgen status. By testosterone deprivation (castration), we induced LA growth arrest in young male rats, whilst atrophy in adult ones, however, both processes could be reversed by testosterone supplementation. After castration, a significant up-regulation of active myostatin protein (and its propeptide) was found, whereas the subsequent testosterone treatment reduced myostatin protein levels to normal values in both young and adult rats. Similarly, a testosterone-induced suppression of myostatin mRNA levels was observed in castrated adult but not in young animals. Altogether, androgens seem to have strong negative impact on myostatin expression, which might be a key factor in the weight regulation of LA muscle.     

Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts

TGF-β and myostatin are the two most important regulators of muscle growth. Both growth factors have been shown to signal through a Smad3-dependent pathway. However to date, the role of Smad3 in muscle growth and differentiation is not investigated. Here, we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy. Consistent with this, we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice. Loss of Smad3 also led to defective satellite cell (SC) functionality. Smad3-null SCs showed reduced propensity for self-renewal, which may lead to a progressive loss of SC number. Indeed, decreased SC number was observed in skeletal muscle from Smad3-null mice showing signs of severe muscle wasting. Further in vitro analysis of primary myoblast cultures identified that Smad3-null myoblasts exhibit impaired proliferation, differentiation and fusion, resulting in the formation of atrophied myotubes. A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts. Given that myostatin is a negative regulator, we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice. Consistent with this theory, inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.     

Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization

The expression of myostatin mRNA was examined in regenerating skeletal muscle of the rat. Skeletal muscle regeneration was induced by injecting bupivacaine or hypertonic saline solution into the femoral muscle, and the tissues were collected 48 h after the treatment. In situ hybridization analysis revealed that the cells positive for myostatin message were localized in the regenerating area of the bupivacaine-treated tissues, where a numerous number of mononucleated cells were present. The myostatin-positive mononucleated cells contained both myogenic and nonmyogenic cells, as revealed by immunohistochemical staining for desmin and vimentin. Bupivacaine treatment to the testes resulted in no myostatin message expression in the testicular vimentin-positive cells, suggesting that the expression of myostatin message in vimentin-positive cells is a skeletal muscle-specific phenomenon. Furthermore, crushed muscle extract prepared from regenerating skeletal muscle had induced myostatin mRNA expression in skeletal muscle-derived fibroblasts in a dose-dependent manner. These results indicated that myostatin is expressed during skeletal muscle regeneration both in myogenic and nonmyogenic cells, and suggested that some factor(s) capable of inducing myostatin expression in fibroblasts are present in regenerating skeletal muscle.     

Inbred strains of zebrafish exhibit variation in growth performance and myostatin expression following fasting

Although the zebrafish (Danio rerio) has been widely utilized as a model organism for several decades, there is little information available on physiological variation underlying genetic variation among the most commonly used inbred strains. This study evaluated growth performance using physiological and molecular markers of growth in response to fasting in six commonly used zebrafish strains [AB, TU, TL, SJA, WIK, and petstore (PET) zebrafish]. Fasting resulted in a standard decrease in whole blood glucose levels, a typical vertebrate glucose metabolism pattern, in AB, PET, TL, and TU zebrafish strains. Alternatively, fasting did not affect glucose levels in SJA and WIK zebrafish strains. Similarly, fasting had no effect on myostatin mRNA levels in AB, PET, TU, and WIK zebrafish strains, but decreased myostatin-1 and ? 2 mRNA levels in SJA zebrafish. Consistent with previous work, fasting increased myostatin-2 mRNA levels in TL zebrafish. These data demonstrate that variation is present in growth performance between commonly used inbred strains of zebrafish. These data can help future research endeavors by highlighting the attributes of each strain with regard to growth performance so that the most fitting strain may be utilized.     

Effects of fasting and refeeding on expression of atrogin-1 and Akt/FOXO signaling pathway in skeletal muscle of chicks

This experiment was conducted to study the effects of fasting and refeeding on expression of the atrogin-1 and Akt/FOXO signaling pathway in skeletal muscle of chicks. Chicks were fasted for 24 h and refed for 2 h. Atrogin-1 mRNA expression was increased by fasting, and their increment was reduced by refeeding. Phosphorylations of Akt and FOXO1 were not decreased by fasting, but, they were increased by refeeding. These results indicate that refeeding stimulates phosphorylation of Akt/FOXO, resulting in a decrease in atrogin-1 expression in skeletal muscle of chicks.     

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish

Myostatin belongs to the transforming growth factor (TGF)-β superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.     

A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the ¹²⁵I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis.     

The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.     

Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study

In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata(sea bream), Solea solea(sole) and Brachydanio rerio(zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).     

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-)M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated.     

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-κB

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro-oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor-β (TGF-β) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor-α (TNF-α) signaling via NF-κB and NADPH oxidase. Aged Mstn null (Mstn(-/-) ) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF-κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF-α and hydrogen peroxide (H(2) O(2) ) are potent inducers of Mstn and require NF-κB signaling for Mstn induction. These results demonstrate that Mstn and TNF-α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF-α and NADPH oxidase, and the elevated TNF-α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal-mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.     

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.