Myostatin Blockade Research Articles

Enhanced Muscle Growth by Plasmid-Mediated Delivery of Myostatin Propeptide

Myostatin is a member of the transforming growth factor beta (TGF-β) superfamily that functions as a negative regulator of skeletal muscle development and growth. Myostatin blockade therefore offers a strategy for promoting muscle growth in livestock production without resorting to genetic manipulation. In this report, we examined the effect of myostatin inhibition by plasmid-mediated delivery of a mutant myostatin propeptide (MProD76A), a natural inhibitor of myostatin, on the growth performance of mice. A significant increase in skeletal muscle mass was observed after a single intramuscular injection of naked plasmid DNA encoding MProD76A into mice. Enhanced muscle growth occurred because of myofiber hypertrophy, but no cardiac muscle hypertrophy and organomegaly was observed in the mice after myostatin inhibition by plasmid-mediated MProD76A delivery. These results demonstrate a promising approach to enhancing muscle growth that warrants further investigation in domestic animals.

Current advances in the development of therapies for neuromuscular disorders based on myostatin signalling, 3rd International Institute of Myology Workshop, Paris, September 12th, 2008

This one day workshop was organized by Helge Amthor and Thomas Voit and brought together 17 researchers from 4 different countries (Germany, France, UK, USA) with complimentary backgrounds and a particular expertise associated with myostatin. The aim of this workshop was to evaluate the prospective therapies based on myostatin blockade and to strengthen the collaboration between these different laboratories. Such a collective effort seemed particularly timely since many laboratories developed an increasing number of different strategies for myostatin blockade as well as an increasing number of different animal models. Some groups are using a recombinant protein to block the circulating myostatin, some viral gene delivery, while others are using nonviral gene delivery based strategies (for review see [1]). Most of the studies on the effect of myostatin blockade focused on wild type mice and on dystrophic mdx mice, however, a number of other mouse models for neuromuscular disorders have been explored. Several studies have used other species such as rats [2] and also dogs [3]. In addition, a number of different application routes were used and different outcome measures studied, therefore, comparison of data from different laboratories remains difficult. Helge Amthor pointed out that myostatin is highly conserved across species and is functional in healthy individuals. Thus, the hypothesis that deleting or blocking myostatin is beneficial for skeletal muscle can be misleading and may result in a negligence to explore its proper physiological function in skeletal muscle, such as the way it modifies the oxidative metabolism. He reminded us to be cautious about a possible experimentation and publication bias resulting from such hypothesis and the loss of scientific independence due to industrial partnership.

Myostatin Inhibition Enhances the Effects of Exercise on Performance and Metabolic Outcomes in Aged Mice

The objective of this study was to examine the effects of short-term exercise training, myostatin inhibition (PF-354), and exercise+PF-354, all relative to a vehicle control, on performance and metabolic measures in 24-month-old mice. At study termination, PF-354-treated mice exhibited significantly greater muscle weights. Performance measures revealed that exercise+PF-354 increased treadmill running time and distance to exhaustion (more than twofold) and increased habitual activity. Measures of strength were not different; however, all treatment groups demonstrated more than 30% reductions in muscle fatigue. Metabolic measures showed that basal metabolic rates were higher in PF-354- and exercise+PF-354-treated mice, and exercise and exercise+PF-354 groups exhibited significantly greater insulin sensitivity. PF-354 was associated with decreased Smad3 phosphorylation and increased peroxisome proliferator-activated receptor gamma coactivator-1alpha expression and, similar to exercise, decreased MuRF-1. The data suggest that the combination of exercise training and myostatin blockade may significantly improve physical function and whole-body metabolism in older individuals.

Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity

Myostatin, a member of the TGF-beta family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells.

Myostatin blockade with a form of the ActRIIB receptor has potent anti‐obesity and insulin sensitizing actions

Myostatin (GDF‐8), a member of the Growth and Differentiation Factor (GDF) family, inhibits muscle growth. The cellular action of myostatin is mediated by the activin receptor type IIB (ActRIIB). Loss‐of‐function mutations of the myostatin gene or disruption of myostatin signaling increases muscle mass. Skeletal muscle is the major organ for glucose disposal, thus we hypothesized that blockade of myostatin would enhance glucose metabolism. We administered a decoy form of the activin type IIB receptor [RAP‐031 10 mg/kg] or vehicle intraperitoneally twice weekly for 10 weeks in C57BL/6J mice. RAP‐031 increased body weight and lean tissue and decreased fat content in mice fed regular chow or high‐fat (45%) diet. RAP‐031 increased the weights of soleus, gastrocnemius and tibialis anterior muscles, as well muscle fiber diameters. We assessed glucose homeostasis using hyperinsulinemic‐euglycemic clamp and radioisotopic tracer kinetics. RAP‐031 increased the glucose infusion and disposal rates by 119% and 45% respectively, and suppressed hepatic glucose production by 66%. Together, these results demonstrate that blockade of myostatin signaling by treatment with an ActRIIB‐based therapeutic is a viable approach toward increasing muscle mass, decreasing fat and improving insulin sensitivity. [This work was supported by National Institutes of Health grant R01DK062348 and an Acceleron Pharma research gift].

Hydrodynamic Limb Vein Injection of Adeno-Associated Virus Serotype 8 Vector Carrying Canine Myostatin Propeptide Gene into Normal Dogs Enhances Muscle Growth

Inhibition or blockade of myostatin, a negative growth factor of skeletal muscle, enhances muscle growth and therefore is considered a promising strategy for the treatment of muscle-wasting diseases such as the muscular dystrophies. Previously, we showed that myostatin blockade in both normal and dystrophin-deficient mdx mice by systemic delivery of the myostatin propeptide (MPRO) gene by an adeno-associated virus serotype 8 (AAV8) vector could enhance muscle growth and ameliorate dystrophic lesions. Here, we further investigate whether the muscle growth effect of myostatin blockade can be achieved in dogs by gene transfer. First, we cloned the canine MPRO gene, packaged it in the AAV8 vector, and showed robust muscle-enhancing effects after systemic delivery into neonatal mice. This vector was then further tested in two 3-month-old normal dogs (weighing 9.7 and 6.3 kg). The vector was delivered to one limb by hydrodynamic vein injection, and the contralateral limb served as a control. The delivery procedure was safe, without discernible adverse effects. AAV vector DNA and MPRO gene expression were detected by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining of muscle biopsies. Overexpression of MPRO resulted in enhanced muscle growth without a cytotoxic T lymphocytic immune response, as evidenced by larger myofibers in multiple muscles, increased muscle volume determined by magnetic resonance imaging, and the lack of CD4+ and CD8+ T cell infiltration in the vector-injected limbs. Our preliminary study thus supports further investigation of this therapeutic strategy in the dystrophin-deficient golden retriever muscular dystrophy dog model.

G.P.8.07 Increased oxidative metabolism in mdx muscle treated by a combination of exon skipping and myostatin blockade

Myostatin blockade stimulates growth of skeletal muscle and has been suggested as a potential therapy for muscular dystrophies. Previously we observed a fibre type conversion towards glycolytic fibres in myostatin deficient mice, which was associated with a decreased number of mitochondria and reduction of oxidative enzymes SDH and COX, implicating a reduced oxidative metabolism of skeletal muscle as a possible adverse effect following lack of myostatin. Here we investigated the effect on muscle metabolism when myostatin blockade was used as a supportive therapy combined with skipping of dystrophin exon 23 in mdx mouse, the animal model for Duchenne muscular dystrophy. We used a AAV construct harboring a double vector which blocks myostatin signalling by RNA interference of the activin receptor IIb (sh-AcvRIIb-3) and which rescues dystrophin expression by using a modified exon skipping strategy (U7 snRNA targeting exon 23). Remarkably, here we demonstrate that combined exon skipping and myostatin blockade increased oxidative properties of treated mdx muscle. Intramuscular injection of U7/sh-AcvRIIb-inv vector into tibialis anterior muscles of mdx mice increased the activity of COX by 78% and of respiratory chain complexes II by 104% compared to the treatment with a control vector harboring scramble sh-RNA. Furthermore, we found an increase in citrate synthase activity of 13% compared to the controls. From these results we hypothesize that myostatin blockade has a different effect on muscle metabolism and subsequently on muscle function when used in combination with dystrophin rescue than myostatin blockade on its own. Therefore, side effects resulting from myostatin blockade may be reduced if applied as a supportive therapy. However, above demonstrated data do not permit to differentiate whether observed effects resulted from myostatin blockade or from exon skipping. Experiments to compare the effect of each strategy are on course.

Myostatin blockade improves function but not histopathology in a murine model of limb‐girdle muscular dystrophy 2C

Myostatin is a negative regulator of skeletal muscle growth. Myostatin mutations and pharmacological strategies increase muscle mass in vivo, suggesting that myostatin blockade may prove useful in diseases characterized by muscle wasting, such as the muscular dystrophies. We subjected the gamma-sarcoglycan-deficient (Sgcg(-/-)) mouse model of limb-girdle muscular dystrophy (LGMD) 2C to antibody-mediated myostatin blockade in vivo. Myostatin inhibition led to increased fiber size, muscle mass, and absolute force. However, no clear improvement in muscle histopathology was evident, demonstrating discordance between physiological and histological improvement. These results and previous studies on the dyw/dyw mouse model of congenital muscular dystrophy and in the late-stage delta-sarcoglycan-deficient (Sgcd(-/-)) mouse model of LGMD2F document disease-specific limitations to therapeutic strategies based on myostatin blockade in the more severe mouse models of different muscular dystrophies.

Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology inmdxmice

Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin-related proteins, and myostatin antibodies. Although follistatin shows potent myostatin-inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (K(d)) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (K(d)) of a follistatin-derived myostatin inhibitor, designated FS I-I, to activin and myostatin are 64.3 microM and 46.8 nM, respectively. Transgenic mice expressing FS I-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I-I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I-I mice. These results indicate that myostatin blockade by FS I-I has a therapeutic potential for muscular dystrophy.

G.P.3.14 No evidence for increased muscle regeneration in myostatin deficient mdx mice ( mstn-/- mdx)

At the 11th WMS congress in Brugge we reported that dystrophic muscle from mdx mice that lack myostatin (mstn−/−mdx) contained no increased number of satellite cells. Preliminary histological analysis furthermore revealed no increase in number of revertant fibres. These results did not support the current view that blockade of myostatin can stimulate regeneration of dystrophic muscle.

Last Word on Point:Counterpoint: Satellite cell addition is/is not obligatory for skeletal muscle hypertrophy

To the Editor : The authors of the Point:Counterpoint debate ([1][1], [2][2]) collectively thank our colleagues for their thoughtful responses to the topic “Satellite cell addition is/is not obligatory for skeletal muscle hypertrophy.” While the goal of our initial position piece and rebuttal

Myostatin Blockade: A New Way to Enhance Skeletal Muscle Repair in Old Age?

Age-related muscle deterioration, called sarcopenia, is characterized by a decline in skeletal muscle mass, strength, and endurance. Sarcopenia is a major health issue in aging humans, because it reduces their capacity to carry out basic activities of daily life and puts the elderly at increased risk of injury.1 Repair of myofibers, the functional blocks of skeletal muscle, is made possible by satellite cells, myogenic stem cells residing beneath the myofiber basal lamina.2 Even without overt muscle trauma, subtle muscle injuries that take place during routine activity raise a continuous demand for myofiber repair throughout life.

Antagonism of Myostatin Enhances Muscle Regeneration During Sarcopenia

A reduction in muscle mass and strength is often observed with aging, and this phenomenon is known as sarcopenia. This age-related atrophy frequently correlates with insufficient levels of muscle regeneration resulting from impairment of satellite cell involvement and myogenesis brought about by the aged environment. Using myostatin-null mice, we recently showed that negative regulators of muscle mass such as myostatin play an active role in the regulation of myogenesis during aging. The present study specifically tests the therapeutic value of a myostatin antagonist in sarcopenia. We report here that a short-term blockade of myostatin, through stage-specific administration of a myostatin antagonist, significantly enhanced muscle regeneration in aged mice after injury and during sarcopenia. Antagonism of myostatin led to satellite cell activation, increased Pax7 and MyoD protein levels, and greater myoblast and macrophage cell migration, resulting in enhanced muscle regeneration after notexin injury in aged mice. In addition, the antagonist demonstrated a high degree of efficacy, as only minimal doses during the critical period of regeneration after injury were sufficient to restore the myogenic and inflammatory responses in the aged environment. Thus, we propose that the antagonism of myostatin has significant therapeutic potential in the alleviation of sarcopenia.

Translating Mighty Mice into Neuromuscular Therapeutics: Is Bigger Muscle Better?

There is increasing clinical research interest in skeletal muscle due in part to the rising incidence of morbidities associated with muscle, particularly insulin resistance and type 2 diabetes (inactive and over-fed muscle) as well as aging and frailty (sarcopenia). A promising avenue of drug development is focused on muscle hypertrophy: the rationale being that a larger muscle is healthier and stronger. Molecular targets for drug development include the myostatin pathway and the IGF1/AKT1 pathway, with both preclinical and clinical testing underway in the inherited muscular dystrophies and in other muscle disorders. As research intensifies, the complexity of these networks in both normal physiology and pathophysiology is becoming evident. As described by Parsons et al1 in this issue of The American Journal of Pathology, myostatin blockade in mouse models of inherited muscle disease is highly variable in its effectiveness, depending on the disease model used, the specific muscle groups studied, and even the age of the animal. Here, we discuss the underlying rationale and current data concerning modulation of muscle size in clinical disorders of muscle.

Age-Dependent Effect of Myostatin Blockade on Disease Severity in a Murine Model of Limb-Girdle Muscular Dystrophy

Myostatin (MSTN) is a muscle-specific secreted peptide that functions to limit muscle growth through an autocrine regulatory feedback loop. Loss of MSTN activity in cattle, mice, and humans leads to a profound phenotype of muscle overgrowth, associated with more and larger fibers and enhanced regenerative capacity. Deletion of MSTN in the mdx mouse model of Duchenne muscular dystrophy enhances muscle mass and reduces disease severity. In contrast, loss of MSTN activity in the dyW/dyW mouse model of laminin-deficient congenital muscular dystrophy, a much more severe and lethal disease model, does not improve all aspects of muscle pathology. Here we examined disease severity associated with myostatin (mstn-/-) deletion in mice nullizygous for delta-sarcoglycan (scgd-/-), a model of limb-girdle muscular dystrophy. Early loss of MSTN activity achieved either by monoclonal antibody administration or by gene deletion each improved muscle mass, regeneration, and reduced fibrosis in scgd-/- mice. However, antibody-mediated inhibition of MSTN in late-stage dystrophic scgd-/- mice did not improve disease. These findings suggest that MSTN inhibition may benefit muscular dystrophy when instituted early or if disease is relatively mild but that MSTN inhibition in severely affected or late-stage disease may be ineffective.

Myostatin Expression in Muscular Dystrophies and Mitochondrial Encephalomyopathies

The demonstration that myostatin may negatively regulate muscle mass in adult individuals has raised the possibility of targeting the myostatin pathway in order to increase muscle growth in a variety of muscle degenerative and wasting conditions. In this regard, blockade of endogenous myostatin results in anatomic, biochemical, and physiologic improvement in the dystrophic phenotype in the mdx mouse. Moreover, myostatin messenger ribonucleic acid levels are decreased in the regenerated muscle of these mice, suggesting that myostatin may also be involved in the pathogenesis of the disease. To gain further insight into the possible role of myostatin in muscle degenerative diseases, the present work investigates the expression of muscle myostatin in children with muscular dystrophies and mitochondrial encephalomyopathies. No differences in the pattern of myostatin expression were evident in any case, even in those patients with prominent muscular atrophy. These findings suggest that muscle loss that can be observed in muscle degenerative diseases does not depend on changes in myostatin expression.

Sexual dimorphism in the response of muscle and bone to myostatin (GDF8) deficiency

Males and females show marked differences in the deposition and modeling of bone during growth. Increased levels of testosterone in males are associated with increased muscle mass and periosteal expansion, whereas increased levels of estrogen in females are associated with endosteal contraction. Myostatin-deficiency increases muscle mass and bone density, and we proposed that the response of muscle and bone to loss of myostatin function may be affected by sex hormone status. We tested this hypothesis by measuring body weight, triceps brachii mass, and bone biomechanical properties of the radius in male and female wild-type and myostatin-deficient mice, 4 months of age (n=12/group). Two-factor ANOVA with sex and genotype as the two factors was used to test for significant interactions between sex and genotype for all variables. Significant sex-genotype interactions were detected for body mass, muscle mass, bone diameter, cortical thickness, area moment of inertia, and ultimate bone stress and strain, with males showing a greater response to myostatin deficiency than females for each variable. These data indicate that downregulation of myostatin produces a greater anabolic response in male skeletal muscle than in female muscle. Moreover, sex-specific differences in the response of muscle to myostatin deficiency in turn yield sex-specific differences in bone shape and biomechanical properties. These findings suggest that molecular therapies aimed at increasing muscle and bone strength via myostatin blockade may be more effective in men than in women. Funding for this research was provided by the National Institutes of Health (NIAMS AR049717-01A2).

1125. AAV-Mediated Myostatin Propeptide Gene Delivery Results in Growth and Hypertrophy of Skeletal but Not Cardiac Muscles

Muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness. Because of the lack of effective treatment with traditional pharmaceutical agents, gene therapy and stem-cell therapy have been vigorously explored. Besides gene replacement therapy, a different form of gene therapy, i.e., expression of |[ldquo]|booster genes|[rdquo]|, has been investigated, aiming at alleviating the secondary deficiencies in muscular dystrophies rather than the primary ones. Blockade of myostatin is one of such strategies. Myostatin is a member of the TGF-beta family and a negative regulator of skeletal muscle growth. Studies using transgenic mice or antagonist antibody against myostatin showed promotion of muscle growth and amelioration of muscular dystrophy. However, gene delivery of myostatin inhibitors has not been reported.

Myostatin propeptide‐mediated amelioration of dystrophic pathophysiology

Mutations in myostatin (GDF8) cause marked increases in muscle mass, suggesting that this transforming growth factor-beta (TGF-beta) superfamily member negatively regulates muscle growth. Myostatin blockade therefore offers a strategy for reversing muscle wasting in Duchenne's muscular dystrophy (DMD) without resorting to genetic manipulation. Here, we demonstrate that pharmacological blockade using a myostatin propeptide stabilized by fusion to IgG-Fc improved pathophysiology of the mdx mouse model of DMD. Functional benefits evidenced by specific force improvement, exceeded those reported previously using myostatin antibody-mediated blockade. More importantly, use of a propeptide blockade strategy obviates possibilities of anti-idiotypic responses that could potentially limit the effectiveness of antibody-mediated myostatin blockade strategies over time. This study provides a novel pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD and since it uses an endogenous inhibitor of myostatin should help circumvent technical hurdles and toxicity associated with conventional gene or cell based therapies.

Muscle regeneration in the prolonged absence of myostatin.

Myostatin is an endogenous inhibitor of muscle conserved across diverse species. In the absence of myostatin, there is massive muscle growth in mice, cattle, and humans. Previous studies in the mdx mouse model of muscular dystrophy demonstrate that inhibiting myostatin attenuates several features of dystrophic muscle. These findings have encouraged the development of human therapies to block myostatin. However, little is known of the long-term effects on muscle of myostatin blockade. To evaluate potential sequelae from the prolonged absence of myostatin, senescent myostatin null (mstn-/-) mice were studied. Senescent mstn-/- mice continue to have normal muscle with increased mass and strength relative to controls. Muscles of senescent mstn-/- mice regenerate robustly from both chronic and acute injury. Early markers of regeneration are enhanced in the absence of myostatin, suggesting a mechanism for the attenuation of dystrophic features found in mdx mice lacking myostatin.