Testosterone protects against dexamethasone-induced muscle atrophy, protein degradation and MAFbx upregulation

Administration of glucocorticoids in pharmacological amounts results in muscle atrophy due, in part, to accelerated degradation of muscle proteins by the ubiquitin-proteasome pathway. The ubiquitin ligase MAFbx is upregulated during muscle loss including that caused by glucocorticoids and has been implicated in accelerated muscle protein catabolism during such loss. Testosterone has been found to reverse glucocorticoid-induced muscle loss due to prolonged glucocorticoid administration. Here, we tested the possibility that testosterone would block muscle loss, upregulation of MAFbx, and protein catabolism when begun at the time of glucocorticoid administration. Coadministration of testosterone to male rats blocked dexamethasone-induced reduction in gastrocnemius muscle mass and upregulation of MAFbx mRNA levels. Administration of testosterone together with dexamethasone also prevented glucocorticoid-induced upregulation of MAFbx mRNA levels and protein catabolism in C2C12 myotube expressing the androgen receptor. Half-life of MAFbx was not altered by testosterone, dexamethasone or the combination. Testosterone blocked dexamethasone-induced increases in activity of the human MAFbx promotor. The findings indicate that administration testosterone prevents glucocorticoid-induced muscle atrophy and suggest that this results, in part at least, from reductions in muscle protein catabolism and expression of MAFbx.     

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.     

Upregulation of myostatin gene expression in streptozotocin-induced type 1 diabetes mice is attenuated by insulin

Myostatin is a strong inhibitor of muscle growth, and its expression is increased in several types of muscle atrophy. However, whether or not myostatin expression is altered in muscle atrophy associated with type 1 diabetes (T1D) remains uncertain. In this study, we provided experimental evidence to show that myostatin mRNA increased in the early stage of T1D but came back to control levels later on. This expression pattern was closely correlated with the loss of body weight and atrogin-1 expression. Furthermore, induction of myostatin expression could be attenuated by insulin in T1D mice. Taken together, our findings indicate that the upregulation of myostatin expression most likely contributes to the muscle atrophy process during insulin deficiency.     

Acute antibody-directed myostatin inhibition attenuates disuse muscle atrophy and weakness in mice

Counteracting the atrophy of skeletal muscle associated with disuse has significant implications for minimizing the wasting and weakness in plaster casting, joint immobilization, and other forms of limb unloading, with relevance to orthopedics, sports medicine, and plastic and reconstructive surgery. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the loss of muscle mass and functional capacity in mice during 14 or 21 days of unilateral hindlimb casting. Twelve-week-old C57BL/10 mice were subjected to unilateral hindlimb plaster casting or served as controls. Mice received subcutaneous injections of saline or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg; n = 6-9) on days 0 and 7 and were tested for muscle function on day 14, or were treated on days 0, 7, and 14 and tested for muscle function on day 21. Hindlimb casting reduced muscle mass, fiber size, and function of isolated soleus and extensor digitorum longus (EDL) muscles (P < 0.05). PF-354 attenuated the loss of muscle mass, fiber size, and function with greater effects after 14 days than after 21 days of casting, when wasting and weakness had plateaued (P < 0.05). Antibody-directed myostatin inhibition therefore attenuated the atrophy and loss of functional capacity in muscles from mice subjected to unilateral hindlimb casting with reductions in muscle size and strength being most apparent during the first 14 days of disuse. These findings highlight the therapeutic potential of antibody-directed myostatin inhibition for disuse atrophy especially within the first 2 wk of disuse.     

Antibody-directed myostatin inhibition enhances muscle mass and function in tumor-bearing mice

Cancer cachexia describes the progressive skeletal muscle wasting and weakness in many cancer patients and accounts for >20% of cancer-related deaths. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the atrophy and loss of function in muscles of tumor-bearing mice. Twelve-week-old C57BL/6 mice received a subcutaneous injection of saline (control) or Lewis lung carcinoma (LLC) tumor cells. One week later, mice received either once weekly injections of saline (control, n = 12; LLC, n = 9) or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg·kg?1·wk?1, LLC+PF-354, n = 11) for 5 wk. Injection of LLC cells reduced muscle mass and maximum force of tibialis anterior (TA) muscles by 8-10% (P < 0.05), but the muscle atrophy and weakness were prevented with PF-354 treatment (P > 0.05). Maximum specific (normalized) force of diaphragm muscle strips was reduced with LLC injection (P < 0.05) but was not improved with PF-354 treatment (P > 0.05). PF-354 enhanced activity of oxidative enzymes in TA and diaphragm muscles of tumor-bearing mice by 118% and 89%, respectively (P < 0.05). Compared with controls, apoptosis that was not of myofibrillar or satellite cell origin was 140% higher in TA muscle cross sections from saline-treated LLC tumor-bearing mice (P < 0.05) but was not different in PF-354-treated tumor-bearing mice (P > 0.05). Antibody-directed myostatin inhibition attenuated the skeletal muscle atrophy and loss of muscle force-producing capacity in a murine model of cancer cachexia, in part by reducing apoptosis. The improvements in limb muscle mass and function highlight the therapeutic potential of antibody-directed myostatin inhibition for cancer cachexia.     

Quercetin glycosides prevent dexamethasone-induced muscle atrophy in mice

Although quercetin has numerous biological benefits, including preventing muscle atrophy due to disuse, no reports have been published to date about the preventive effects and molecular mechanisms underlying drug-induced muscle atrophy. Highly soluble and bioavailable quercetin glycosides (QGs) were used to examine the inhibition of dexamethasone (DEX)-induced muscle atrophy in vivo. Male BALB/cCrSlc mice were treated with or without QGs for 7 days ad libitum, followed by addition of DEX to their drinking water for a further 7 days. The weight of gastrocnemius (GM) adjusted by body weight was significantly decreased on day 7 after DEX treatment. DEX-induced decrease of GM weight was improved by QG co-administration on day 7. The mRNA levels of muscle atrophy-related genes in the gastrocnemius were significantly lowered by QGs on day 1. In particular, the expression of myostatin, a master regulator of muscle mass homeostasis, was suppressed to that of the control level. In murine C2C12 myotubes, quercetin elevated the phosphorylation of Akt, which are downstream of the myostatin pathway, as well as expression of atrogenes. We demonstrated the protective effect of QGs in DEX-induced muscle atrophy, which might depend on the suppression of myostatin signaling.     

Effect of insulin on dexamethasone-induced ultrastructural changes in skeletal and cardiac muscle

Glucocorticoids help animals respond to stressors but excessive glucocorticoids cause muscle atrophy, while insulin can promote anabolism and growth. In order to compare the glucocorticoids-induced ultrastructural changes between skeletal muscle and cardiac muscle, and investigate the preventive effects of insulin on the changes, eighteen male chicks with similar initial weight were randomly divided into three groups. The two test groups were respectively treated with high-dose dexamethasone alone or together with low-dose insulin by intraperitoneal injection, and the control group was treated with an equal volume of saline solution. The experiment lasted for ten days, and then the body weight, muscle size and ultrastructure in skeletal and cardiac muscles of twelve chicks were qualitatively or quantitatively analyzed. The results showed that high-dose dexamethasone induced obvious skeletal and cardiac muscle atrophy. The differences of ultrastructural changes between skeletal muscle and cardiac muscle (such as for the former or the latter, the intermyofibrillar-and-interfilamentary spaces reducing or enlarging, the mitochondria swelling seriously or enlarging lightly, the myofibril filaments compacting or loosing) suggested that dexamethasone induced skeletal and cardiac muscle atrophy by different mechanisms. Low-dose insulin did not affect the dexamethasone-induced decreases of body weight and skeletal muscle size, but alleviated lightly the dexamethasone-induced ultrastructural changes in skeletal muscle. Different from skeletal muscle, low-dose insulin almost resisted the dexamethasone-induced ultrastructural changes in cardiac muscle.     

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.     

Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production.     

Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 μg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.     

Eccentric exercise training as a countermeasure to non-weight-bearing soleus muscle atrophy.

Although various exercise paradigms have been tested, none has completely prevented muscle atrophy during non-weight bearing. Because loaded eccentric contractions occur during normal daily activity but are absent during non-weight bearing, this investigation tested whether eccentric resistance training could prevent soleus muscle atrophy during non-weight bearing. Adult female rats were randomly assigned to either weight bearing +/- intramuscular electrodes or non-weight bearing +/- intramuscular electrodes groups. Electrically stimulated maximal eccentric contractions (4 sets of 6 repetitions at approximately 0.2 fiber lengths/s, 128 degrees range of motion) were performed on anesthetized animals at 48-h intervals during the 10-day experiment. Non-weight bearing significantly reduced soleus muscle wet weight (28-31%) and noncollagenous protein content (30-31%) compared with controls. Eccentric exercise training during non-weight bearing attenuated but did not prevent the loss of soleus muscle wet weight and noncollagenous protein by 77 and 44%, respectively. The potential of eccentric exercise training as an effective and highly efficient counter-measure to non-weight-bearing atrophy is demonstrated in the 44% attenuation of soleus muscle noncollagenous protein loss by eccentric exercise during only 0.035% of the total non-weight-bearing time period.     

Dependence of dexamethasone-induced Akt/FOXO1 signaling,upregulation of MAFbx,and protein catabolism upon the glucocorticoid receptor

The muscle ubiquitin ligases MAFbx and MuRF1 are upregulated in and promote muscle atrophy. Upregulation of MAFbx and MuRF1 by glucocorticoids has been linked to activation of FOXO1 and FOXO3A resulting from reduced Akt activity. We determined the requirements for the glucocorticoid receptor (GR) in these biological responses in C2C12 cells in which GR expression was knocked down by stable expression of an shRNA. Loss of GR prevented dexamethasone-induced increases in protein catabolism. Loss of GR, or inhibition of ligand binding to GR with RU486, prevented upregulation of MAFbx and MuRF1 by dexamethasone. Loss of GR also prevented dexamethasone-induced decreases in Akt phosphorylation, and increases in the fraction of FOXO1 that was unphosphorylated. The findings establish a requirement for the GR in activating molecular signals that promote muscle protein catabolism.     

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.     

Skeletal muscle atrophy is associated with an increased expression of myostatin and impaired satellite cell function in the portacaval anastamosis rat

Proliferation and differentiation of satellite cells are critical in the regeneration of atrophied muscle following immobilization and aging. We hypothesized that impaired satellite cell function is responsible for the atrophy of skeletal muscle also seen in cirrhosis. Myostatin and insulin-like growth factor 1 (IGF1) have been identified to be positive and negative regulators, respectively, of satellite cell function. Using a rat model of cirrhosis [portacaval anastamosis (PCA)] and sham-operated controls, we examined the expression of myostatin, its receptor activinR2b, and its downstream messenger cyclin-dependent kinase inhibitor p21 (CDKI p21) as well as IGF1 and its receptor in the gastrocnemius muscle. Expression of PCNA, a marker of proliferation, and myogenic regulatory factors (myoD, myf5, and myogenin), markers of differentiation of satellite cells, were also measured. Real- time PCR for mRNA and Western blot assay for protein quantification were performed. PCA rats had lower body weight and gastrocnemius weight compared with sham animals (P < 0.05). PCNA and myogenic regulatory factors were lower in PCA rats (P < 0.05). Myostatin, activinR2b, and CDKI p21 were higher in the PCA animals (P < 0.05). The expression of IGF1 and its receptor was lower in liver and skeletal muscle of PCA animals (P < 0.05). These data suggest that skeletal muscle atrophy seen in the portacaval shunted rats is a consequence of impaired satellite cell proliferation and differentiation mediated, in part, by higher myostatin and lower IGF1 expression.     

Modulation of myostatin expression during modified muscle use.

Previous findings have provided strong evidence that myostatin functions as a negative regulator of muscle mass during development and growth. In the present study, we test the hypothesis that myostatin may serve a similar function in fully differentiated muscle experiencing modified loading. Our findings show that myostatin expression can be modulated in fully differentiated, nonpathological skeletal muscle in a manner that is inversely related to changes in muscle mass. Atrophy of rat hind limb muscles induced by 10 days of unloading resulted in a 16% decrease in plantaris mass, a 110% increase in myostatin mRNA, and a 37% increase in myostatin protein. Immunohistochemical observations showed a detectable increase in myostatin concentration at myotendinous junctions during muscle unloading. The concentration of myostatin mRNA and protein returned to values not significantly different from ambulatory controls after 4 days of reloading, during which time plantaris mass also returned to control values. However, the results also show that periods of 30 min of daily muscle loading during the unloading period were sufficient to prevent significant losses of muscle mass caused by unloading, although myostatin mRNA still showed a 55% increase in concentration. Thus, significant increases in myostatin expression are not sufficient for muscle mass loss, although muscle mass loss during unloading is accompanied by increases in myostatin.     

Effect of weight loss on muscle lipid content in morbidly obese subjects

The purpose of this study was to test the hypothesis that weight loss results in a reduction in intramuscular lipid (IMCL) content that is concomitant with enhanced insulin action. Muscle biopsies were obtained from morbidly obese individuals [body mass index (BMI) 52.2 +/- 2.5 kg/m(2); n = 6] before and after gastric bypass surgery, an intervention that improves insulin action. With intervention, there was a 47% reduction (P < 0.01) in BMI and a 93% decrease in homeostasis model assessment, or HOMA (7.0 +/- 1.9 vs. 0.5 +/- 0.1). Histochemically determined IMCL content decreased (P < 0.05) by approximately 30%. In relation to fiber type, IMCL was significantly higher in type I vs. type II fibers. In both fiber types, there were reductions in IMCL and trends for muscle atrophy. Despite these two negating factors, the IMCL-to-fiber area ratio still decreased by approximately 44% with weight loss. In conclusion, despite differing initial levels and possible atrophy, weight loss appears to decrease IMCL deposition to a similar relative extent in type I and II muscle fibers. This reduction in intramuscular triglyceride may contribute to enhanced insulin action seen with weight loss.     

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.