Serum myokines as potential biomarkers of myostatin inhibition in sport doping: a preliminary study on their baseline levels in elite athletes.

We considered in this study the possibility of developing an indirect procedure for detecting myostatin inhibition/suppression, a practice that is prohibited as doping in sport. We have specifically considered the potential diagnostic utility of human serum myokines as indirect markers of myostatin inhibition. Myostatin, its main antagonist follistatin, and other myokines (follistatin-like 1, musclin, oncostatin, osteonectin, irisin, brain derived neurotrophic factor, and insulin-like growth factor-1) were selected as a panel of potential biomarkers whose levels may be altered following myostatine suppression. The serum levels of myostatin and of the nine myokines were measured in elite athletes of different age, sex, and sport discipline, and their cross correlation assessed by multivariate analysis. All myokines resulted to be measurable in human serum, except for musclin and irisine, whose levels were below the limits of quantitation in a reduced number of samples. Serum concentrations varied of different orders in magnitude (musclin and osteonectin < 1 ng/mL; follistatin, myostatine and irisine 1-5 ng/mL; brainderived neurotrophic factor, follistatin-like 1 and iinsulin-like growth factor-1 > 10 ng/mL), while no significant differences were found between female and male subjects, with the exceptions of follistatin-like 1 and musclin, showing a higher concentrations in females (p < 0.05). Levels of insulin-like growth factor 1 and brain derived neurotrophic factor were significantly higher in power athletes than in endurance ones. Multivariate statistics showed that musclin, follistatin-like 1 and oncostatin are more clustered and correlated to myostatin than other myokines, suggesting they could be considered as potential biomarkers of doping by myostatin inhibitors.

Inhibitor Design Strategy for Myostatin: Dynamics and Interaction Networks Define the Affinity and Release Mechanisms of the Inhibited Complexes.

Myostatin, an important negative regulator of muscle mass, is a therapeutic target for muscle atrophic disorders such as muscular dystrophy. Thus, the inhibition of myostatin presents a strategy to treat these disorders. It has long been established that the myostatin prodomain is a strong inhibitor of the mature myostatin, and the minimum peptide of the prodomain-corresponding to the α1-helix of its lasso-region-responsible for the inhibitory efficiency was defined and characterized as well. Here we show that the minimum peptide segment based on the growth differentiation factor 11 (GDF11), which we found to be more helical in its stand-alone solvated stfate than the similar segment of myostatin, is a promising new base scaffold for inhibitor design. The proposed inhibitory peptides in their solvated state and in complex with the mature myostatin were analyzed by in silico molecule modeling supplemented with the electronic circular dichroism spectroscopy measurements. We defined the Gaussian-Mahalanobis mean score to measure the fraction of dihedral angle-pairs close to the desired helical region of the Ramachandran-plot, carried out RING analysis of the peptide-protein interaction networks and characterized the internal motions of the complexes using our rigid-body segmentation protocol. We identified a variant-11m2-that is sufficiently ordered both in solvent and within the inhibitory complex, forms a high number of contacts with the binding-pocket and induces such changes in its internal dynamics that lead to a rigidified, permanently locked conformation that traps this peptide in the binding site. We also showed that the naturally evolved α1-helix has been optimized to simultaneously fulfill two very different roles: to function as a strong binder as well as a good leaving group. It forms an outstanding number of non-covalent interactions with the mature core of myostatin and maintains the most ordered conformation within the complex, while it induces independent movement of the gate-keeper ß-hairpin segment assisting the dissociation and also results in the least-ordered solvated form which provides extra stability for the dissociated state and discourages rebinding.

A phase Ib/IIa, open-label, multiple ascending-dose trial of domagrozumab in fukutin-related protein limb-girdle muscular dystrophy.

INTRODUCTION/AIMS: In this study we report the results of a phase Ib/IIa, open-label, multiple ascending-dose trial of domagrozumab, a myostatin inhibitor, in patients with fukutin-related protein (FKRP)-associated limb-girdle muscular dystrophy. METHODS: Nineteen patients were enrolled and assigned to one of three dosing arms (5, 20, or 40 mg/kg every 4 weeks). After 32 weeks of treatment, participants receiving the lowest dose were switched to the highest dose (40 mg/kg) for an additional 32 weeks. An extension study was also conducted. The primary endpoints were safety and tolerability. Secondary endpoints included muscle strength, timed function testing, pulmonary function, lean body mass, pharmacokinetics, and pharmacodynamics. As an exploratory outcome, muscle fat fractions were derived from whole-body magnetic resonance images. RESULTS: Serum concentrations of domagrozumab increased in a dose-dependent manner and modest levels of myostatin inhibition were observed in both serum and muscle tissue. The most frequently occurring adverse events were injuries secondary to falls. There were no significant between-group differences in the strength, functional, or imaging outcomes studied. DISCUSSION: We conclude that, although domagrozumab was safe in patients in limb-girdle muscular dystrophy type 2I/R9, there was no clear evidence supporting its efficacy in improving muscle strength or function.

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.

Enzymatic Stability of Myostatin Inhibitory 16-mer Peptides.

Inhibition of myostatin is a promising strategy for treatment of muscle atrophic disorders. A 16-mer myostatin inhibitory linear peptide, MIPE-1686, administered intramuscularly, significantly increases muscle mass and hindlimb grip strength in Duchenne muscular dystrophic model mice. In this paper, we describe our examination of the enzymatic stabilities of this peptide with recombinant human proteases, aminopeptidase N, chymotrypsin C, and trypsin 3. MIPE-1686 was found to be stable in the presence of these enzymes, in contrast to a peptide (1), from which MIPE-1686 was developed. Modification of the peptides at a position distant from the protease cleavage site altered their enzymatic stability. These results suggest the possibility that the stability to proteases of 16-mer myostatin inhibitory peptides is associated with an increase in their known ß-sheet formation properties. This study suggests that MIPE-1686 has a potential to serve as a long-lasting agent in vivo.

Use Of Ankle Immobilization In Evaluating Treatments To Promote Longitudinal Muscle Growth In Mice.

INTRODUCTION: Difficulty in modeling congenital contractures (deformities of muscle-tendon unit development that include shortened muscles and lengthened tendons) has limited research of new treatments. METHODS: Early immobilization of the ankle in prepuberal mice was used to produce deformities similar to congenital contractures. Stretch treatment, electrostimulation, and local intramuscular injection of a follistatin analog (FST-288) were assessed as therapeutic interventions for these deformities. RESULTS: Ankle immobilization at full plantarflexion and 90 ° created tendon lengthening and muscle shortening in the tibialis anterior and soleus. Stretch treatment produced minimal evidence for longitudinal muscle growth and electrostimulation provided no additional benefit. Stretch treatment with FST-288 produced greater longitudinal muscle growth and less tendon lengthening, constituting the best treatment response. DISCUSSION: Ankle immobilization recapitulates key morphologic features of congenital contracture, and these features can be mitigated by a combination of stretch and pharmacological approaches that may be useful in patients. Muscle Nerve 58: 718-725, 2018.

ActRIIB blockade increases force-generating capacity and preserves energy supply in exercising mdx mouse muscle in vivo.

Postnatal blockade of the activin type IIB receptor (ActRIIB) represents a promising therapeutic strategy for counteracting dystrophic muscle wasting. However, its impact on muscle function and bioenergetics remains poorly documented in physiologic conditions. We have investigated totally noninvasively the effect of 8-wk administration of either soluble ActRIIB signaling inhibitor (sActRIIB-Fc) or vehicle PBS (control) on gastrocnemius muscle force-generating capacity, energy metabolism, and anatomy in dystrophic mdx mice using magnetic resonance (MR) imaging and dynamic [31P]-MR spectroscopy ([31P]-MRS) in vivo ActRIIB inhibition increased muscle volume (+33%) without changing fiber-type distribution, and increased basal animal oxygen consumption (+22%) and energy expenditure (+23%). During an in vivo standardized fatiguing exercise, maximum and total absolute contractile forces were larger (+40 and 24%, respectively) in sActRIIB-Fc treated animals, whereas specific force-generating capacity and fatigue resistance remained unaffected. Furthermore, sActRIIB-Fc administration did not alter metabolic fluxes, ATP homeostasis, or contractile efficiency during the fatiguing bout of exercise, although it dramatically reduced the intrinsic mitochondrial capacity for producing ATP. Overall, sActRIIB-Fc treatment increased muscle mass and strength without altering the fundamental weakness characteristic of dystrophic mdx muscle. These data support the clinical interest of ActRIIB blockade for reversing dystrophic muscle wasting.-Béchir, N., Pecchi, E., Vilmen, C., Le Fur, Y., Amthor, H., Bernard, M., Bendahan, D., Giannesini, B. ActRIIB blockade increases force-generating capacity and preserves energy supply in exercising mdx mouse muscle in vivo.

Transglutaminase-catalyzed covalent anti-myostatin peptide depots.

Nature realizes protein and peptide depots by catalyzing covalent bonds with the extracellular matrix (ECM) of tissues. We are translating this natural blueprint for the sustained delivery of a myostatin-inhibiting peptide (Anti-Myo), resulting in an enzyme depot established from injectable solutions. For that, we fused Anti-Myo to the D-domain of insulin-like growth factor I, a transglutaminase (TG) substrate. TG catalyzed the covalent binding of the D-domain to ECM proteins, such as laminin and fibronectin, on bioengineered ECM and in mice. ECM decorated with Anti-Myo suppressed myostatin activity and pathway activation and reduced the differentiation of preconditioned bone marrow-derived macrophages into osteoclasts in vitro.

Insight on sarcopenic obesity and epicatechin as a promising treatment option.

BACKGROUND AND AIM: Sarcopenic Obesity (SO) in the elderly population is a complex and multifactorial condition which refers to the loss of skeletal muscle mass, strength, and function associated with aging, while obesity involves excessive adipose tissue accumulation. The simultaneous occurrence of these two conditions presents a unique set of challenges to public health and clinical management. This narrative review aims to provide an overview of the use of epicatechin (EC) in the treatment of SO and its related complications. METHOD: A survey of studies related to preclinical and clinical evidence of Epicatechin in sarcopenic obesity and its complications was performed in the following database Medline, Scopus, ProQuest, Embase, Web of Science, and Google scholar. Followed by structural activity relationship and pharmacokinetic profile of Epicatechin was discussed in this paper. RESULTS: The main pharmacological effect of Epicatechin is myostatin inhibition activity which has been described by both in vitro and in vivo studies earlier. The SO is directly correlated with the alteration of Myostatin. The pre-clinical and clinical studies suggest that epicatechin can be a potential candidate in the management of SO and its related complication. CONCLUSION: The present review describes the pharmacokinetic profile and structural activity of epicatechin respective to SO and its related complications. The goal of this review is to update the scientific community on the therapeutic potential of epicatechin in SO and age-related factors. Conduction of clinical and pre-clinical trials, also drug dosage optimization may provide with insights on the use of epicatechin in SO.

Myostatin: Basic biology to clinical application.

Myostatin is a member of the transforming growth factor (TGF)-ß superfamily. It is expressed by animal and human skeletal muscle cells where it limits muscle growth and promotes protein breakdown. Its effects are influenced by complex mechanisms including transcriptional and epigenetic regulation and modulation by extracellular binding proteins. Due to its actions in promoting muscle atrophy and cachexia, myostatin has been investigated as a promising therapeutic target to counteract muscle mass loss in experimental models and patients affected by different muscle-wasting conditions. Moreover, growing evidence indicates that myostatin, beyond to regulate skeletal muscle growth, may have a role in many physiologic and pathologic processes, such as obesity, insulin resistance, cardiovascular and chronic kidney disease. In this chapter, we review myostatin biology, including intracellular and extracellular regulatory pathways, and the role of myostatin in modulating physiologic processes, such as muscle growth and aging. Moreover, we discuss the most relevant experimental and clinical evidence supporting the extra-muscle effects of myostatin. Finally, we consider the main strategies developed and tested to inhibit myostatin in clinical trials and discuss the limits and future perspectives of the research on myostatin.

Lessons Learned from Discontinued Clinical Developments in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked condition caused by a deficiency of functional dystrophin protein. Patients experience progressive muscle weakness, cardiomyopathy and have a decreased life expectancy. Standards of care, including treatment with steroids, and multidisciplinary approaches have extended the life expectancy and improved the quality of life of patients. In the last 30 years, several compounds have been assessed in preclinical and clinical studies for their ability to restore functional dystrophin levels or to modify pathways involved in DMD pathophysiology. However, there is still an unmet need with regards to a disease-modifying treatment for DMD and the attrition rate between early-phase and late-phase clinical development remains high. Currently, there are 40 compounds in clinical development for DMD, including gene therapy and antisense oligonucleotides for exon skipping. Only five of them have received conditional approval in one jurisdiction subject to further proof of efficacy. In this review, we present data of another 16 compounds that failed to complete clinical development, despite positive results in early phases of development in some cases. We examine the reasons for the high attrition rate and we suggest solutions to avoid similar mistakes in the future.

The Failed Clinical Story of Myostatin Inhibitors against Duchenne Muscular Dystrophy: Exploring the Biology behind the Battle.

Myostatin inhibition therapy has held much promise for the treatment of muscle wasting disorders. This is particularly true for the fatal myopathy, Duchenne Muscular Dystrophy (DMD). Following on from promising pre-clinical data in dystrophin-deficient mice and dogs, several clinical trials were initiated in DMD patients using different modality myostatin inhibition therapies. All failed to show modification of disease course as dictated by the primary and secondary outcome measures selected: the myostatin inhibition story, thus far, is a failed clinical story. These trials have recently been extensively reviewed and reasons why pre-clinical data collected in animal models have failed to translate into clinical benefit to patients have been purported. However, the biological mechanisms underlying translational failure need to be examined to ensure future myostatin inhibitor development endeavors do not meet with the same fate. Here, we explore the biology which could explain the failed translation of myostatin inhibitors in the treatment of DMD.

Myostatin inhibition in combination with antisense oligonucleotide therapy improves outcomes in spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN-restoring therapies substantially prolong survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle-enhancing approach as a novel therapeutic strategy for SMA. METHODS: The experiments were performed in a mouse model of severe SMA. A previously reported 25-mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno-associated virus-mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 µg/g (therapeutic dose) or 10 µg/g (low-dose) PMO25 on its own or together with systemic delivery of a single dose of adeno-associated virus-mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated. RESULTS: We show that myostatin inhibition acts synergistically with SMN-restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 µg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two-fold increase in time score) and treadmill exercise test (two-fold increase in running distance). In SMA mice treated with low-dose PMO25 (10 µg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase). CONCLUSIONS: These data suggest that myostatin inhibition, in addition to the well-known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN-enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN-restoring drugs.

Effect of myostatin inhibitor (myostatin pro-peptide) microinjection on in vitro maturation and subsequent early developmental stages of buffalo embryo.

Expression of myostatin (MSTN, also known as growth differentiation factor 8, GDF8) was recently detected in cumulus-oocytes complexes (COCs), however little is known about its role in in vitro maturation (IVM) and fertilization (IVF) in large animals. Therefore, this study was designed to investigate the effect of MSTN inhibition on IVM of buffalo oocytes through investigation of IVM efficiency and expression of some specific genes in COCs from IVM till subsequent developmental stages following IVF. To reach this goal, we prepared a construct of adeno-associated virus (AAV) carrying MSTN pro-peptides (AAV-MSTNP) to inhibit MSTN. Over-expression of MSTNP was verified by upregulated expression of MSTNP and downregulated expression of the TGFß receptor ActRIIb, the TGFß signal transducer SMAD2 in COCs using qPCR. Microinjection of AAV-MSTNP to oocytes before IVM yielded a significant decrease in maturation rate as revealed by less cumulus cells expansion, fewer oocytes reaching metaphase II, and downregulation of cumulus expansion-related genes pentraxin 3 (Ptx3) and prostaglandin-endoperoxide synthase 2 (Ptgs2) as compared to the control and vehicle groups. These changes were also accompanied by elevated intracellular reactive oxygen species (ROS), upregulated expression of the apoptotic Bax gene, reduced antioxidant enzymes (SOD, CAT, GPX) activities, and downregulated expression of the antioxidant gene nuclear factor erythroid 2 like 2 (Nrf2), and the anti-apoptotic gene Bcl2 in COCs after IVM. Overexpression of MSTN inhibitor, MSTNP, also inhibited GDF9 and BMP15 genes expression in COCs. Additionally, both the fertilization efficiency and cleavage and blastocyst rates were significantly lower in MSTNP group than in the control and vehicle groups. The obtained data suggest an important role for MSTN during IVM and the subsequent developmental stages probably through, at least in part, inhibition of ROS production and apoptosis and modulation of IVM-related gene expression in COCs.

Electrical impedance myography as a biomarker of myostatin inhibition with ActRIIB-mFc: a study in wild-type mice.

AIM: We sought to determine the sensitivity of electrical impedance myography (EIM) to myofiber hypertrophy induced by treatment with various doses of ActRIIB-mFc, an inhibitor of myostatin signaling. METHODS: Wild-type C57BL/6 J mice (n = 40, male) were treated with three different doses of ActRIIB-mFc (i.e., RAP-031) or vehicle twice weekly for 5 weeks. End point assessments included gastrocnemius EIM, force measurements, muscle mass and myofiber size quantification. RESULTS: ActRIIB-mFc increased body mass, muscle mass and myofiber size across all doses. Alterations in EIM 50 kHz phase and center frequency (fc) were also present, with trends in a dose-dependent fashion. Significant correlations between EIM parameters and myofiber/functional data were identified. CONCLUSION: EIM outcomes can serve as effective biomarkers of myostatin signaling inhibition, demonstrating a dose sensitivity and correlation to standard assessments.

Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

BACKGROUND: Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. METHODS: To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn (+/-)) whippets. A total of four GRippets (dystrophic and Mstn (+/-)), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. RESULTS: Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age. CONCLUSIONS: Partial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors.