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.

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.

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.