Identification and Evaluation of Traditional Chinese Medicine Natural Compounds as Potential Myostatin Inhibitors: An In Silico Approach.

Myostatin (MSTN), a negative regulator of muscle mass, is reported to be increased in conditions linked with muscle atrophy, sarcopenia, and other muscle-related diseases. Most pharmacologic approaches that treat muscle disorders are ineffective, emphasizing the emergence of MSTN inhibition. In this study, we used computational screening to uncover natural small bioactive inhibitors from the Traditional Chinese Medicine database (~38,000 compounds) for the MSTN protein. Potential ligands were screened, based on binding affinity (150), physicochemical (53) and ADMET properties (17). We found two hits (ZINC85592908 and ZINC85511481) with high binding affinity and specificity, and their binding patterns with MSTN protein. In addition, molecular dynamic simulations were run on each complex to better understand the interaction mechanism of MSTN with the control (curcumin) and the hit compounds (ZINC85592908 and ZINC85511481). We determined that the hits bind to the active pocket site (Helix region) and trigger conformational changes in the MSTN protein. Since the stability of the ZINC85592908 compound was greater than the MSTN control, we believe that ZINC85592908 has therapeutic potential against the MSTN protein and may hinder downstream singling by inhibiting the MSTN protein and increasing myogenesis in the skeletal muscle tissues.

Computational Identification of Dithymoquinone as a Potential Inhibitor of Myostatin and Regulator of Muscle Mass.

The skeletal muscle (SM) is the largest organ in the body and has tremendous regenerative power due to its myogenic stem cell population. Myostatin (MSTN), a protein produced by SM, is released into the bloodstream and is responsible for age-related reduced muscle fiber development. The objective of this study was to identify the natural compounds that inhibit MSTN with therapeutic potential for the management of age-related disorders, specifically muscle atrophy and sarcopenia. Sequential screening of 2000 natural compounds was performed, and dithymoquinone (DTQ) was found to inhibit MSTN with a binding free energy of -7.40 kcal/mol. Furthermore, the docking results showed that DTQ reduced the binding interaction between MSTN and its receptor, activin receptor type-2B (ActR2B). The global energy of MSTN-ActR2B was found to be reduced from -47.75 to -40.45 by DTQ. The stability of the DTQ-MSTN complex was subjected to a molecular dynamics analysis for up to 100 ns to check the stability of the complex using RMSD, RMSF, Rg, SASA, and H-bond number. The complex was found to be stable after 10 ns to the end of the simulation. These results suggest that DTQ blocks MSTN signaling through ActR2B and that it has potential use as a muscle growth-promoting agent during the aging process.

Identification of Molecules from Coffee Silverskin That Suppresses Myostatin Activity and Improves Muscle Mass and Strength in Mice.

Coffee has been shown to attenuate sarcopenia, the age-associated muscle atrophy. Myostatin (MSTN), a member of the TGF-ß growth/differentiation factor superfamily, is a potent negative regulator of skeletal muscle mass, and MSTN-inhibition increases muscle mass or prevents muscle atrophy. This study, thus, investigated the presence of MSTN-inhibitory capacity in coffee extracts. The ethanol-extract of coffee silverskin (CSE) but not other extracts demonstrated anti-MSTN activity in a pGL3-(CAGA)12-luciferase reporter gene assay. CSE also blocked Smad3 phosphorylation induced by MSTN but not by GDF11 or Activin A in Western blot analysis, demonstrating its capacity to block the binding of MSTN to its receptor. Oral administration of CSE significantly increased forelimb muscle mass and grip strength in mice. Using solvent partitioning, solid-phase chromatography, and reverse-phase HPLC, two peaks having MSTN-inhibitory capacity were purified from CSE. The two peaks were identified as ßN-arachinoyl-5-hydroxytryptamide (C20-5HT) and ßN-behenoyl-5-hydroxytryptamide (C22-5HT) using mass spectrometry and NMR analysis. In summary, the results show that CSE has the MSTN-inhibitory capacity, and C20-5HT and C22-5HT are active components of CSE-suppressing MSTN activity, suggesting the potential of CSE, C20-5HT, and C22-5HT being developed as agents to combat muscle atrophy and metabolic syndrome.

Fortetropin inhibits disuse muscle atrophy in dogs after tibial plateau leveling osteotomy.

OBJECTIVE: To determine if a commercial myostatin reducer (Fortetropin®) would inhibit disuse muscle atrophy in dogs after a tibial plateau leveling osteotomy. DESIGN: A prospective randomized, double-blinded, placebo-controlled clinical trial. ANIMALS: One hundred client-owned dogs presenting for surgical correction of cranial cruciate ligament rupture by tibial plateau leveling osteotomy. PROCEDURES: Patients were randomly assigned into the Fortetropin® or placebo group and clients were instructed to add the assigned supplement to the dog's normal diet once daily for twelve weeks. Enrolled patients had ultrasound measurements of muscle thickness, tape measure measurements of thigh circumference, serum myostatin level assays, and static stance analysis evaluated at weeks 0, 8, and 12. RESULTS: From week 0 to week 8, there was no change for thigh circumference in the Fortetropin® group for the affected limb (-0.54cm, P = 0.31), but a significant decrease in thigh circumference for the placebo group (-1.21cm, P = 0.03). There was no significant change in serum myostatin levels of dogs in the Fortetropin® group at any time point (P>0.05), while there was a significant rise of serum myostatin levels of dogs in placebo group during the period of forced exercise restriction (week 0 to week 8; +2,892 pg/ml, P = 0.02). The percent of body weight supported by the affected limb increased in dogs treated with Fortetropin® (+7.0%, P<0.01) and the placebo group (+4.9%, P<0.01) at the end of the period of forced exercise restriction. The difference in weight bearing between the Fortetropin® and placebo groups was not statistically significant (P = 0.10). CONCLUSION: Dogs receiving Fortetropin® had a similar increase in stance force on the affected limb, no significant increase in serum myostatin levels, and no significant reduction in thigh circumference at the end of the period of forced exercise restriction compared to the placebo. These findings support the feeding of Fortetropin® to prevent disuse muscle atrophy in canine patients undergoing a tibial plateau leveling osteotomy.

Therapeutic considerations of sarcopenia in heart failure patients.

INTRODUCTION: Sarcopenia is a common feature, and affects 20-47% of patients with heart failure (HF). Sarcopenia is also an independent predictor of impaired functional capacity, even after adjusting for clinical relevant variables, which is associated with adverse outcome in patients with HF. Areas covered: Several different pathophysiological pathways are involved in sarcopenic processes including altered nutrient intake and absorption, hormonal factor, inflammatory processes, oxidative stress, cellular proteolysis, and unhealthy lifestyle. Nutritional therapy, physical activity and/or exercise training have been associated with improved muscle mass or physical performance in HF. Few studies reported beneficial effects for muscle mass and physical performance, in those who received angiotensin-converting enzyme (ACE) inhibitors, or/and beta-blocker. In addition, testosterone, selective androgen receptor modulators, ghrelin agonist and myostatin inhibitors are currently under study as possible future therapeutic options. Expert commentary: Regular and adequate level of physical activity and/or exercise training, and sufficient nutritional intake or special nutritional supplementation may represent the best strategy for prevention or delay of sarcopenia and worsening physical performance in patients with HF. Maximal tolerated dosages of standard therapies for HF such as ACE-inhibitors or beta-blockers are first-line strategy, however it is difficult to recommend other pharmacological agents as part of routine treatment of sarcopenia.

Pharmacological inhibition of myostatin protects against skeletal muscle atrophy and weakness after anterior cruciate ligament tear.

Anterior cruciate ligament (ACL) tears are among the most frequent knee injuries in sports medicine, with tear rates in the US up to 250,000 per year. Many patients who suffer from ACL tears have persistent atrophy and weakness even after considerable rehabilitation. Myostatin is a cytokine that directly induces muscle atrophy, and previous studies rodent models and patients have demonstrated an upregulation of myostatin after ACL tear. Using a preclinical rat model, our objective was to determine if the use of a bioneutralizing antibody against myostatin could prevent muscle atrophy and weakness after ACL tear. Rats underwent a surgically induced ACL tear and were treated with either a bioneutralizing antibody against myostatin (10B3, GlaxoSmithKline) or a sham antibody (E1-82.15, GlaxoSmithKline). Muscles were harvested at either 7 or 21 days after induction of a tear to measure changes in contractile function, fiber size, and genes involved in muscle atrophy and hypertrophy. These time points were selected to evaluate early and later changes in muscle structure and function. Compared to the sham antibody group, 7 days after ACL tear, myostatin inhibition reduced the expression of proteolytic genes and induced the expression of hypertrophy genes. These early changes in gene expression lead to a 22% increase in muscle fiber cross-sectional area and a 10% improvement in maximum isometric force production that were observed 21 days after ACL tear. Overall, myostatin inhibition lead to several favorable, although modest, changes in molecular biomarkers of muscle regeneration and reduced muscle atrophy and weakness following ACL tear. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2499-2505, 2017.

A myostatin inhibitor (propeptide-Fc) increases muscle mass and muscle fiber size in aged mice but does not increase bone density or bone strength.

Loss of muscle and bone mass with age are significant contributors to falls and fractures among the elderly. Myostatin deficiency is associated with increased muscle mass in mice, dogs, cows, sheep and humans, and mice lacking myostatin have been observed to show increased bone density in the limb, spine, and jaw. Transgenic overexpression of myostatin propeptide, which binds to and inhibits the active myostatin ligand, also increases muscle mass and bone density in mice. We therefore sought to test the hypothesis that in vivo inhibition of myostatin using an injectable myostatin propeptide (GDF8 propeptide-Fc) would increase both muscle mass and bone density in aged (24 mo) mice. Male mice were injected weekly (20 mg/kg body weight) with recombinant myostatin propeptide-Fc (PRO) or vehicle (VEH; saline) for four weeks. There was no difference in body weight between the two groups at the end of the treatment period, but PRO treatment significantly increased mass of the tibialis anterior muscle (+ 7%) and increased muscle fiber diameter of the extensor digitorum longus (+ 16%) and soleus (+ 6%) muscles compared to VEH treatment. Bone volume relative to total volume (BV/TV) of the femur calculated by microCT did not differ significantly between PRO- and VEH-treated mice, and ultimate force (Fu), stiffness (S), toughness (U) measured from three-point bending tests also did not differ significantly between groups. Histomorphometric assays also revealed no differences in bone formation or resorption in response to PRO treatment. These data suggest that while developmental perturbation of myostatin signaling through either gene knockout or transgenic inhibition may alter both muscle and bone mass in mice, pharmacological inhibition of myostatin in aged mice has a more pronounced effect on skeletal muscle than on bone.

Molecular mechanisms in aging and current strategies to counteract sarcopenia.

Sarcopenia, the progressive loss of muscle mass with age, is characterized by a deterioration of muscle quantity and quality leading to a gradual slowing of movement and a decline in strength and power. Sarcopenia is a highly significant public health problem. Since these age-related changes in skeletal muscle are largely attributed to various molecular mediators affecting fiber size, mitochondrial homeostatis, and apoptosis, the mechanisms responsible for these deleterious changes present numerous therapeutic targets for drug discovery. We and other researchers demonstrated that a disruption of Akt-mTOR and RhoA-SRF signaling but not Atrogin-1 or MuRF1 contributes to sarcopenia. In addition, sarcopenia seems to include a marked loss of fibers attributable to apoptosis. This review deals with molecular mechanisms of muscle atrophy and provides an update on current strategies (resistance training, myostatin inhibition, treatment with amino acids or testosterone, calorie restriction, etc) for counteracting this loss. Resistance training in combination with amino acid-containing nutrition would be the best candidate to attenuate, prevent, or ultimately reverse age-related muscle wasting and weakness.