Effects of 2-week HMB-FA supplementation with or without eccentric resistance exercise on expression of some genes related to muscle protein turnover and serum irisin and IGF-1 concentrations.

Protein turnover is a process that is regulated by several factors and can lead to muscle hypertrophy or atrophy. The purpose of the present study was to determine the effects of ß-hydroxy-ß-methylbutyrate free acid (HMB-FA) and eccentric resistance exercise on variables related to protein turnover in rats. Thirty-two male rats were randomly assigned into four groups of eight, including control, control-HMB, exercise, and exercise-HMB. Animals in HMB groups received 340 mg/kg/day for two weeks. Animals in the exercise groups performed one session of eccentric resistance exercise consisting of eight repetitions descending from a ladder with a slope of 80 degree, with an extra load of two times body weight (100% 1RM). Twenty-four hours after the exercise session, triceps brachii muscle and serum were collected for further analysis. Exercise and HMB-FA induced lower muscle myostatin and higher muscle Fibronectin type III domain containing 5 (FNDC5), P70-S6 kinase 1 gene expression, as well as higher serum irisin and IGF-1 concentrations. Exercise alone induced higher caspase-3 and caspase-8 gene expression while HMB-FA alone induced lower caspase 3 gene expression. HMB-FA supplement increased the effect of exercise on muscle FNDC5, myostatin, and P70-S6 kinase 1 gene expression. The interaction of exercise and HMBFA resulted in an additive effect, increasing serum irisin and IGF-1 concentrations. In conclusion, a 2-week HMB-FA supplementation paired with acute eccentric resistance exercise can positively affect some genes related to muscle protein turnover.

Glucocorticoids counteract hypertrophic effects of myostatin inhibition in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a devastating genetic muscle disease resulting in progressive muscle degeneration and wasting. Glucocorticoids, specifically prednisone/prednisolone and deflazacort, are commonly used by DMD patients. Emerging DMD therapeutics include those targeting the muscle-wasting factor, myostatin (Mstn). The aim of this study was to investigate how chronic glucocorticoid treatment impacts the efficacy of Mstn inhibition in the D2.mdx mouse model of DMD. We report that chronic treatment of dystrophic mice with prednisolone (Pred) causes significant muscle wasting, entailing both activation of the ubiquitin-proteasome degradation pathway and inhibition of muscle protein synthesis. Combining Pred with Mstn inhibition, using a modified Mstn propeptide (dnMstn), completely abrogates the muscle hypertrophic effects of Mstn inhibition independently of Mstn expression or SMAD3 activation. Transcriptomic analysis identified that combining Pred with dnMstn treatment affects gene expression profiles associated with inflammation, metabolism, and fibrosis. Additionally, we demonstrate that Pred-induced muscle atrophy is not prevented by Mstn ablation. Therefore, glucocorticoids interfere with potential muscle mass benefits associated with targeting Mstn, and the ramifications of glucocorticoid use should be a consideration during clinical trial design for DMD therapeutics. These results have significant implications for past and future Mstn inhibition trials in DMD.

Inactivation of myostatin by photo-oxygenation using catalyst-functionalized peptides.

Inhibition of myostatin is an attractive treatment for muscular dystrophy and other amyotrophic diseases. A myostatin-binding peptide was functionalized by equipped with an on/off switchable photo-oxygenation catalyst. This peptide induces a selective oxygenation of myostatin under near-infrared light, resulting in inactivation of myostatin. This peptide shows several orders of magnitude greater inhibitory effect than the original peptide.

Low-Intensity Exercise Suppresses CCAAT/Enhancer-Binding Protein δ/Myostatin Pathway Through Androgen Receptor in Muscle Cells.

BACKGROUND: Androgen production following exercise has been suggested to contribute anabolic actions of muscle. However, the underlying mechanisms of the androgen receptor (AR) in androgen's action are still unclear. OBJECTIVE: In the present study, we examined androgen/AR-mediated action in exercise, especially for the suppression of myostatin, a potent negative regulator of muscle mass. METHODS: To examine the effects of exercise, we employed low-intensity exercise in mice and electric pulse stimulation (EPS) in C2C12 myotubes. Androgen production by C2C12 myotubes was measured by enzyme-linked immunosorbent assay. To block the action of AR, we pretreated C2C12 myotubes with flutamide. Quantitative real-time polymerase chain reaction was used to determine the expression levels of proteolytic genes including CCAAT/enhancer-binding protein delta (C/EBPδ), myostatin and muscle E3 ubiquitin ligases, as well as myogenic genes such as myogenin and PGC1α. The activation of 5'-adenosine-activated protein kinase and STAT3 was determined by Western blot analysis. RESULTS: Both mRNA and protein levels of AR significantly increased in skeletal muscle of low-intensity exercised mice and C2C12 myotubes exposed to EPS. Production of testosterone and dihydrotestosterone from EPS-treated C2C12 myotubes was markedly increased. Of interest, we found that myostatin was clearly inhibited by EPS, and its inhibition was significantly abrogated when AR was blocked by flutamide. To test how AR suppresses myostatin, we examined the effects of EPS on C/EBPδ because the promoter region of myostatin has several C/EBP recognition sites. C/EBPδ expression was decreased by EPS, and this decrease was negated by flutamide. IL-6 and phospho-STAT3 (pSTAT3) expression, the downstream pathway of myostatin, were decreased by EPS and this was also reversed by flutamide. Similar downregulation of C/EBPδ, myostatin, and IL-6 was seen in skeletal muscle of low-intensity exercised mice. CONCLUSIONS: Muscle AR expression and androgen production were increased by exercise and EPS treatment. As a mechanistical insight, it is suggested that AR inhibited myostatin expression transcriptionally by C/EBPδ suppression, which negatively influences IL-6/pSTAT3 expression and consequently contributes to the prevention of muscle proteolysis during exercise.

Sarcopenia.

Sarcopenia is defined as a combination of low muscle mass with low muscle function. The term was first used to designate the loss of muscle mass and performance associated with aging. Now, recognized causes of sarcopenia also include chronic disease, a physically inactive lifestyle, loss of mobility, and malnutrition. Sarcopenia should be differentiated from cachexia, which is characterized not only by low muscle mass but also by weight loss and anorexia. Sarcopenia results from complex and interdependent pathophysiological mechanisms that include aging, physical inactivity, neuromuscular compromise, resistance to postprandial anabolism, insulin resistance, lipotoxicity, endocrine factors, oxidative stress, mitochondrial dysfunction, and inflammation. The prevalence of sarcopenia ranges from 3% to 24% depending on the diagnostic criteria used and increases with age. Among patients with rheumatoid arthritis 20% to 30% have sarcopenia, which correlates with disease severity. Sarcopenia exacts a heavy toll of functional impairment, metabolic disorders, morbidity, mortality, and healthcare costs. Thus, the consequences of sarcopenia include disability, quality of life impairments, falls, osteoporosis, dyslipidemia, an increased cardiovascular risk, metabolic syndrome, and immunosuppression. The adverse effects of sarcopenia are particularly great in patients with a high fat mass, a condition known as sarcopenic obesity. The diagnosis of sarcopenia rests on muscle mass measurements and on functional tests that evaluate either muscle strength or physical performance (walking, balance). No specific biomarkers have been identified to date. The management of sarcopenia requires a multimodal approach combining a sufficient intake of high-quality protein and fatty acids, physical exercise, and antiinflammatory medications. Selective androgen receptor modulators and anti-myostatin antibodies are being evaluated as potential stimulators of muscle anabolism.

A Soluble Activin Receptor IIB Fails to Prevent Muscle Atrophy in a Mouse Model of Spinal Cord Injury.

Myostatin (MST) is a potent regulator of muscle growth and size. Spinal cord injury (SCI) results in marked atrophy of muscle below the level of injury. Currently, there is no effective pharmaceutical treatment available to prevent sublesional muscle atrophy post-SCI. To determine whether inhibition of MST with a soluble activin IIB receptor (RAP-031) prevents sublesional SCI-induced muscle atrophy, mice were randomly assigned to the following groups: Sham-SCI; SCI+Vehicle group (SCI-VEH); and SCI+RAP-031 (SCI-RAP-031). SCI was induced by complete transection at thoracic level 10. Animals were euthanized at 56 days post-surgery. RAP-031 reduced, but did not prevent, body weight loss post-SCI. RAP-031 increased total lean tissue mass compared to SCI-VEH (14.8%). RAP-031 increased forelimb muscle mass post-SCI by 38% and 19% for biceps and triceps, respectively (p < 0.001). There were no differences in hindlimb muscle weights between the RAP-031 and SCI-VEH groups. In the gastrocnemius, messenger RNA (mRNA) expression was elevated for interleukin (IL)-6 (8-fold), IL-1ß (3-fold), and tumor necrosis factor alpha (8-fold) in the SCI-VEH, compared to the Sham group. Muscle RING finger protein 1 mRNA was 2-fold greater in the RAP-031 group, compared to Sham-SCI. RAP-031 did not influence cytokine expression. Bone mineral density of the distal femur and proximal tibia were decreased post-SCI (-26% and -28%, respectively) and were not altered by RAP-031. In conclusion, MST inhibition increased supralesional muscle mass, but did not prevent sublesional muscle or bone loss, or the inflammation in paralyzed muscle.

Role of IGF-I in follistatin-induced skeletal muscle hypertrophy.

Follistatin, a physiological inhibitor of myostatin, induces a dramatic increase in skeletal muscle mass, requiring the type 1 IGF-I receptor/Akt/mTOR pathway. The aim of the present study was to investigate the role of IGF-I and insulin, two ligands of the IGF-I receptor, in the follistatin hypertrophic action on skeletal muscle. In a first step, we showed that follistatin increases muscle mass while being associated with a downregulation of muscle IGF-I expression. In addition, follistatin retained its full hypertrophic effect toward muscle in hypophysectomized animals despite very low concentrations of circulating and muscle IGF-I. Furthermore, follistatin did not increase muscle sensitivity to IGF-I in stimulating phosphorylation of Akt but, surprisingly, decreased it once hypertrophy was present. Taken together, these observations indicate that increased muscle IGF-I production or sensitivity does not contribute to the muscle hypertrophy caused by follistatin. Unlike low IGF-I, low insulin, as obtained by streptozotocin injection, attenuated the hypertrophic action of follistatin on skeletal muscle. Moreover, the full anabolic response to follistatin was restored in this condition by insulin but also by IGF-I infusion. Therefore, follistatin-induced muscle hypertrophy requires the activation of the insulin/IGF-I pathway by either insulin or IGF-I. When insulin or IGF-I alone is missing, follistatin retains its full anabolic effect, but when both are deficient, as in streptozotocin-treated animals, follistatin fails to stimulate muscle growth.

The effects of exogenous cortisol on myostatin transcription in rainbow trout, Oncorhynchus mykiss.

Glucocorticoids (GCs) strongly regulate myostatin expression in mammals via glucocorticoid response elements (GREs), and bioinformatics methods suggest that this regulatory mechanism is conserved among many vertebrates. However, the multiple myostatin genes found in some fishes may be an exception. In silico promoter analyses of the three putative rainbow trout (Oncorhynchus mykiss) myostatin promoters have failed to identify putative GREs, suggesting a divergence in myostatin function. Therefore, we hypothesized that myostatin mRNA expression is not regulated by glucocorticoids in rainbow trout. In this study, both juvenile rainbow trout and primary trout myoblasts were treated with cortisol to examine the effects on myostatin mRNA expression. Results suggest that exogenous cortisol does not regulate myostatin-1a and -1b expression in vivo, as myostatin mRNA levels were not significantly affected by cortisol treatment in either red or white muscle tissue. In red muscle, myostatin-2a levels were significantly elevated in the cortisol treatment group relative to the control, but not the vehicle control, at both 12 h and 24 h post-injection. As such, it is unclear if cortisol was acting alone or in combination with the vehicle. Cortisol increased myostatin-1b expression in a dose-dependent manner in vitro. Further work is needed to determine if this response is the direct result of cortisol acting on the myostatin-1b promoter or through an alternative mechanism. These results suggest that regulation of myostatin by cortisol may not be as highly conserved as previously thought and support previous work that describes potential functional divergence of the multiple myostatin genes in fishes.

Myostatin as a therapeutic target in Amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis is a devastating neurological disease that is inevitably fatal after 3-5years duration. Treatment options are minimal and as such new therapeutic modalities are required. In this review, we discuss the role of the myostatin pathway as a modulator of skeletal muscle mass and therapeutic approaches using biological based therapies. Both monoclonal antibodies to myostatin and a soluble receptor decoy to its high affinity receptor have been used in clinical trials of neuromuscular diseases and while there have been efficacy signals with the latter approach there have also been safety issues. Our approach is to target the high affinity receptor-binding site on myostatin and to develop a next generation set of therapeutic reagents built on a novel protein scaffold. This is the natural single domain VNAR found in sharks which is extremely versatile and has the ability to develop products with superior properties compared to existing therapeutics.

The anabolic steroid methandienone targets the hypothalamic-pituitary-testicular axis and myostatin signaling in a rat training model.

There is increasing evidence that the biological activity of myostatin (MSTN), a negative regulator of muscle growth, is affected by training but also anabolic steroids. In this study, we analyzed the effects of the frequently abused anabolic steroid methandienone (Md) on the hypothalamic-pituitary-testicular axis and androgen-sensitive tissues in intact rats performing a treadmill training to simulate the situation of abusing athletes. The anabolic effects were correlated with the expression of members of the MSTN signaling cascade. Md treatment resulted in a significant stimulation of anabolic activity of the levator ani muscle, which was further increased by training, while prostate and seminal vesicle weights decreased in conformance with hormone concentrations of LH and testosterone. In gastrocnemius muscle, mRNA expression of genes of the MSTN signaling cascade (MSTN, Smad7 and MyoD) was reduced by training but not after Md treatment, in soleus muscle MSTN and its inhibitors, follistatin (FLST) and Smad-7 were only affected after training in combination with Md treatment. In summary, our data demonstrate that Md treatment of intact rats results in anabolic effects which are enhanced in combination with physical activity. Interestingly, the anabolic activity on the levator ani was increased in combination with training, although the levator ani muscle was not specifically stimulated by our training protocol. In the m. gastrocnemius and soleus, the anabolic effects correlate with changes in the expression patterns of genes involved in MSTN signaling. Our data provide evidence that the decrease in the weight of androgen-sensitive sexual glands, observed after Md treatment, is caused by a suppression of endogenous testosterone synthesis. These observations provide new insights into the molecular mechanisms of the interaction between anabolic steroids, training and MSTN signaling during skeletal muscle adaptation.

Targeting the myostatin signaling pathway to treat muscle wasting diseases.

PURPOSE OF REVIEW: To understand the mechanisms of muscle wasting and how inhibiting myostatin signaling affects them. RECENT FINDINGS: Myostatin signaling is critical for the understanding of the pathogenesis of muscle wasting as blocking signaling mitigates muscle losses in rodent models of catabolic diseases including cancer, chronic kidney, or heart failure. SUMMARY: Muscle wasting increases the risks of morbidity and mortality. But, the reliability of estimates of the degree of muscle wasting is controversial as are definitions of terms like cachexia. Much information has been learnt about the pathophysiology of muscle wasting, including the major role of the ubiquitin-proteasome system (UPS) which along with other proteases degrades protein and limits protein synthesis. In contrast, few successful strategies for reversing muscle loss have been tested. Several catabolic conditions are characterized by inflammation, increased glucocorticoid production, and impaired intracellular signaling in response to insulin and IGF-1. These characteristics lead to activation of the UPS and other proteases producing muscle wasting. Another potential initiator of muscle wasting is myostatin and its expression is increased in muscles of animal models and patients with certain catabolic conditions. Myostatin is a member of the TGF-ß family; it suppresses muscle growth and its absence stimulates muscle growth substantially. Recently, pharmacologic suppression of myostatin was found to counteract inflammation, increased glucocorticoids and impaired insulin/IGF-1 signaling and most importantly, prevents muscle wasting in rodent models of cancer and kidney failure. Myostatin antagonism as a therapy for patients with muscle wasting should become a topic of clinical investigation.

Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor.

Inhibition of the myostatin signaling pathway is emerging as a promising therapeutic means to treat muscle wasting and degenerative disorders. Activin type IIB receptor (ActRIIB) is the putative myostatin receptor, and a soluble activin receptor (ActRIIB-Fc) has been demonstrated to potently inhibit a subset of transforming growth factor (TGF)-ß family members including myostatin. To determine reliable and valid biomarkers for ActRIIB-Fc treatment, we assessed gene expression profiles for quadriceps muscles from mice treated with ActRIIB-Fc compared with mice genetically lacking myostatin and control mice. Expression of 134 genes was significantly altered in mice treated with ActRIIB-Fc over a 2-wk period relative to control mice (fold change > 1.5, P < 0.001), whereas the number of significantly altered genes in mice treated for 2 days was 38, demonstrating a time-dependent response to ActRIIB-Fc in overall muscle gene expression. The number of significantly altered genes in Mstn(-/-) mice relative to control mice was substantially higher (360), but for most of these genes the expression levels in the 2-wk treated mice were closer to the levels in the Mstn(-/-) mice than in control mice (P < 10⁻³°). Expression levels of 30 selected genes were further validated with quantitative real-time polymerase chain reaction (qPCR), and a correlation of ≥ 0.89 was observed between the fold changes from the microarray analysis and the qPCR analysis. These data suggest that treatment with ActRIIB-Fc results in overlapping but distinct gene expression signatures compared with myostatin genetic mutation. Differentially expressed genes identified in this study can be used as potential biomarkers for ActRIIB-Fc treatment, which is currently in clinical trials as a therapeutic agent for muscle wasting and degenerative disorders.

Transcription activation of myostatin by trichostatin A in differentiated C2C12 myocytes via ASK1-MKK3/4/6-JNK and p38 mitogen-activated protein kinase pathways.

Myostatin is a negative regulator of skeletal muscle mass. The pathways employed in modulating myostatin gene expression are scarcely known. We aimed to determine the signaling pathway of myostatin induction by a histone deacetylase (HDAC) inhibitor-trichostatin A (TSA) in differentiated C(2)C(12) myocytes. TSA increased myostatin mRNA expression up to 40-fold after treatment for 24 h, and induced myostatin promoter activity up to 3.8-fold. Pretreatment with actinomycin D reduced the TSA-induced myostatin mRNA by 93%, suggesting TSA-induced myostatin expression mainly at the transcriptional level. Pretreatment with p38 MAPK (SB203580) and JNK (SP600125) inhibitors, but not ERK (PD98059) inhibitor, blocked TSA-induced myostatin expression, respectively, by 72% and 43%. Knockdown of p38 MAPK by RNAi inhibited the TSA-induced myostatin expression by 77% in C(2)C(12) myoblasts. The protein levels of phosphorylated p38 MAPK, JNK, but not ERK, increased with TSA treatment in differentiated C(2)C(12) cells. Direct activation of p38 MAPK and JNK by anisomycin in the absence of TSA increased myostatin mRNA by fourfold. The phosphorylated form of the kinase MKK3/4/6 and ASK1, upstream cascades of p38 MAPK and JNK, also increased with TSA treatment. We concluded that the induction of myostatin by TSA treatment in differentiated C(2)C(12) cells is in part through ASK1-MKK3/6-p38 MAPK and ASK1-MKK4-JNK signaling pathways. Activation of p38 MAPK and JNK axis is necessary, but not sufficient for TSA-induced myostatin expression.

Changes in insulin like growth factors, myostatin and vascular endothelial growth factor in rat musculus latissimus dorsi by poly-3-hydroxybutyrate implants.

The present study aimed at researching the synergistic effect between an ectopic bone substitute and surrounding muscle tissue. To describe this effect, changes of insulin like growth factors (IGF1, IGF2), myostatin (GDF8) and vascular endothelial growth factor (VEGF) mRNA content of 12 Wistar-King rats musculus latissimus dorsi with implanted poly-3-hydroxybutyrate (PHB) scaffold were examined after 6 and 12 weeks. At each time interval six rats were killed and implants and surrounding tissues prepared for genetic evaluation. Eight rats without any implants served as controls. RNA was extracted from homogenized muscle tissue and reverse transcribed. Changes in mRNA content were measured by Real-Time PCR using specific primers for IGF1, IGF2, GDF8 and VEGF. Comparing the level of VEGF mRNA in muscle after 6 and 12 weeks to the controls, we could assess a significant increase of VEGF gene expression (p<0.05) whereas the level of mRNA expression was higher after 6 than after 12 weeks of treatment. Expression of IGF1 gene was also significantly increased as compared to the controls over the observed period of time (p<0.05). In the case of the IGF2 gene, the expression was significantly elevated after 6 weeks (p<0.05), but not significantly increased after 12 weeks (p>0.05). We observed a significantly decreased GDF8 gene expression (p<0.05) both after retrieval of implants after 6 as well as after 12 weeks. Moreover, mRNA level of GDF8 after 6 and 12 weeks were comparable the same. Our results show that PHB implants in rat musculus latissimus dorsi interact with the surrounding muscle tissue. This interaction works itself on growth potential of the muscle.