Recovery from skeletal muscle injury is often incomplete because of the formation of fibrosis and inadequate myofiber regeneration; therefore, injured muscle could benefit significantly from therapies that both stimulate muscle regeneration and inhibit fibrosis. To this end, we focused on blocking myostatin, a member of the transforming growth factor-β superfamily and a negative regulator of muscle regeneration, with the myostatin antagonist follistatin. In vivo, follistatin-overexpressing transgenic mice underwent significantly greater myofiber regeneration and had less fibrosis formation compared with wild-type mice after skeletal muscle injury. Follistatin's mode of action is likely due to its ability to block myostatin and enhance neovacularization. Furthermore, muscle progenitor cells isolated from follistatin-overexpressing mice were significantly superior to muscle progenitors isolated from wild-type mice at regenerating dystrophin-positive myofibers when transplanted into the skeletal muscle of dystrophic mdx/severe combined immunodeficiency mice. In vitro, follistatin stimulated myoblasts to express MyoD, Myf5, and myogenin, which are myogenic transcription factors that promote myogenic differentiation. Moreover, follistatin's ability to enhance muscle differentiation is at least partially due to its ability to block myostatin, activin A, and transforming growth factor-β1, all of which are negative regulators of muscle cell differentiation. The findings of this study suggest that follistatin is a promising agent for improving skeletal muscle healing after injury and muscle diseases, such as the muscular dystrophies.
Although skeletal muscle injuries are extremely common, accounting for up to 35% to 55% of all sports-related injuries, the treatments that are currently available have not progressed during the last few decades and are often ineffective. Unfortunately, significant morbidity is associated with these injuries, such as the development of painful contractures, loss of muscle extensibility and strength, and the increased risk for repeated injury, which is largely due to extensive fibrosis formation. In response to traumas and disease, the local secretion of transforming growth factor (TGF)-β1, a potent fibrotic cytokine, induces the formation of fibrosis in various tissues and organs, including skeletal muscles.1-9 Various agents, including suramin,10,11 interferon-γ,12 decorin,5,8,13-15 relaxin,16,17 and losartan,9,18 have been shown to significantly enhance skeletal muscle regeneration, reduce fibrosis in injured muscles, and, in a broad spectrum of myopathic diseases, partially block TGF-β1. Although much of the pathogenesis after skeletal muscle injury has been attributed to TGF-β1 expression, it has become clear that myostatin, a member of the TGF-β superfamily, can also be implicated in the formation of muscle fibrosis.19-24 Myostatin was initially known as a primary negative regulator of the growth and development of fetal and postnatal skeletal muscle.25,26 A variety of approaches to block myostatin function have been developed during the past few years, including i) the creation of a myostatin gene knockout animal model, ii) the use of a myostatin neutralizing antibody, and iii) the delivery of the myostatin propeptide (MPRO) gene via an adenoassociated virus (AAV). These different methods of myostatin blockade have unequivocally shown that the inhibition of myostatin reduces fibrosis and enhances muscle regeneration in both injured and dystrophic murine skeletal muscles.19-24 Myostatin directly stimulates the formation of skeletal muscle fibrosis by stimulating muscle fibroblasts, whose excessive activities are responsible for the development of fibrosis in injured muscle.23,27 Muscle fibroblasts express the myostatin protein23,27 and its receptor, ACVR2B.27 Myostatin increases the proliferation and secretion of extracellular matrix products by muscle fibroblasts.23,27 These effects may be due to the activation of the canonical TGF-β signaling pathway, as well as the PI3K/Akt/mTOR pathway in muscle fibroblasts, as evidenced by increased phosphorylation of SMAD2/3 and Akt/mTOR, respectively.27 Injection of myostatin-coated beads into skeletal muscle leads to the formation of fibrosis around the injected beads, which could be reversed with the addition of follistatin, an antagonist of myostatin.27 Myostatin and TGF-β1 have been observed to reciprocally induce the expression of one another.23,28 The blockade of TGF-β1 signaling impairs myostatin's biological activity and vice versa, which suggests that TGF-β1 acts synergistically with myostatin to induce fibrosis in injured skeletal muscle.23
In addition to impairing skeletal muscle healing by promoting fibrosis, myostatin also inhibits myofiber regeneration in mouse models that mimic diseases such as Duchenne muscular dystrophy (DMD)22 and amyotrophic lateral sclerosis-associated muscular atrophy,29 as well as after experiencing a traumatic injury.20,23 Specifically, the diaphragm muscles of mdx mice, an animal model of DMD, were noted to undergo significantly more myofiber damage and less myofiber regeneration when compared with transgenic mdx mice that also had their myostatin gene knocked out.22 Similarly, in two acute muscle injury models, the first where the tibialis anterior muscle was injured by the injection of notexin and the other where the gastrocnemius muscle (GM) was injured via laceration, there was significantly greater regeneration and significantly less fibrosis formation in the injured myostatin knockout mice than the injured wild-type (WT) controls.20,23 Given the promising benefits of blocking myostatin in skeletal muscle, a safety trial using MYO-29, a neutralizing antibody of myostatin, was conducted in adult patients with various forms of muscular dystrophy, including Becker muscular dystrophy, facioscapulohumeral dystrophy, and limb-girdle muscular dystrophy.30 The results of this trial demonstrated that the patients could tolerate MYO-29 very well when it was administrated systemically.30
Research into the development of therapies to antagonize myostatin has led to the discovery of several new functions exhibited by follistatin. Follistatin was originally found to antagonize activin A in reproductive tissues and was also observed to neutralize several other proteins within the TGF-β superfamily.31-33 Follistatin is also well known as a potent myostatin antagonist in skeletal muscle.34,35 Follistatin-overexpressing transgenic mice exhibit a significant increase in muscle mass, much as is seen to occur in myostatin knockout mice.35 Several in vivo studies on follistatin have shown that the systemic administration of this agent directly inhibits myostatin and also reduces myostatin-induced muscle wasting.26,34,36 Moreover, a single injection of AAV-mediated follistatin into the quadriceps and tibialis anterior muscles, of both young and aged WT C57BL/6J or dystrophic mice, increased the muscles weight, and more interestingly, it also promoted an increase in the weight of noninjected muscles located remotely (eg, triceps muscles). This increase in muscular weight was accompanied by an increase in hind limb grip strength. It is also noteworthy that increased follistatin levels were not detrimental to the reproductive capacity of the treated animals.37 Apart from these animal model findings, it has also been reported that follistatin plays a beneficial role in human myoblast transplantation. Human myoblasts-overexpressing follistatin outperformed normal human myoblast controls in both proliferation and differentiation capacities in vitro and regenerated much larger engraftment areas when injected into the tibialis anterior muscles of severe combined immunodeficiency (SCID) mice.38 The safety and effectiveness of follistatin treatment have been evaluated in nonhuman primates.39 The long-term expression of the AAV-mediated follistatin in the quadriceps muscles of cynomolgus macaque monkeys increased the monkeys' muscle mass and strength without having any deleterious effects on any of their critical organ systems.39 This minimal off-target effect makes this molecule a promising potential therapeutic agent to treat muscles injured acutely and injured by degenerative muscle disorders; however, before translating follistatin-based therapies from the bench to the bedside, clear mechanisms of how follistatin promotes muscle regeneration requires extensive investigation.