Abstract
Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear:cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice.
Introduction
Skeletal muscle is a highly adaptable tissue that responds to various environmental and physiological stimuli by changing its phenotype with regard to its size and composition. Mechanical loading is considered a major positive regulator of muscle mass that significantly contributes to disease prevention, athletic performance and a high quality of life [1], [2]. Conversely, loss of skeletal muscle mass through either inadequate energy intake, cachexia or ageing has a major impact on public health [3], [4].
Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily that has emerged as a potent negative regulator of skeletal muscle mass [5], [6]. Several naturally occurring gene inactivating mutations have been reported to elicit a remarkable hyper muscular phenotype through both myofibre hypertrophy and hyperplasia accompanied by a reduction in body fat content in numerous species including cattle, mice, sheep, dogs and in one case in man [6], [7], [8], [9], [10]. Myostatin signals through a combination of activin type II receptors and activin receptor-like kinases 4 and 5. A number of differing intracellular signalling pathways have been proposed to explain the myostatin null phenotype. The activation of phosphorylated Smad2/3, which further associates with Smad4 and translocates into the nucleus to activate gene transcription has been proposed to control cell proliferation [11], [12]. Myostatin has been shown to control both muscle fibre number by blocking the proliferation and differentiation of myoblasts and muscle fibre size by maintaining the satellite cells in a quiescent state and inhibiting protein synthesis [13]. In addition, a novel role has recently been attributed to myostatin in the regulation of muscle growth and proliferation whereby myostatin functions to directly inhibit Akt phosphorylation, acting independently of the typical Smad2/3 pathway [14]. Elevated myostatin expression has been observed in some pathological states involving muscle tissue such as muscle atrophy and unloading, myocardial infarct, HIV associated muscle loss and muscle damage reviewed by [15]. Over expression of myostatin has been proposed by some to be a key feature in muscle wasting disorders like cachexia [16] and sarcopenia [17]. However, this issue is controversial given that myostatin null mice are more susceptible to muscle wastage [18].
Myostatin is synthesized as a precursor protein, which upon proteolytic processing gives rise to a N-terminal propeptide and a C-terminal biologically active peptide [11]. The N-terminal propeptide is capable of inhibiting the biological activity of the C-terminal peptide by binding to it directly and maintaining it in a latent state [19], [20], [21], [22]. In addition to its own propeptide, other myostatin inhibitors that interact with circulating myostatin sustaining it in an inactive state have also been identified. These include the naturally occurring hormone follistatin, the follistatin related gene product and growth and differentiation factor-associated serum protein-1 (GASP-1) [19], [20], [23]. Thus, bearing in mind the biological effect of myostatin on skeletal muscle growth, inhibition of myostatin signalling or preventing its biological activity have emerged as promising therapeutic approaches against muscle wasting and degenerative diseases such as muscular dystrophies [24], [25]. For instance, the generation of a dominant negative form of myostatin in transgenic mice which expressed myostatin mutated at its cleavage site showed a 20–35% increase in muscle mass [26], while over expression of a missense mutant myostatin in mouse was followed by hyperplasia [27]. In addition, over expression of the myostatin pro-domain in transgenic mice and a muscle-specific inactivation of the myostatin gene, have both been shown to lead to a hyper muscular phenotype [28], [29]. Pharmacological treatment of mice with a myostatin neutralizing antibody lead to increased skeletal muscle mass accompanied by increased grip strength [25]. On the contrary, both a muscle specific over expression of myostatin in transgenic mice and a systemic administration of myostatin were followed by reduced skeletal muscle mass and induction of cachexia [22], [30].
Myostatin inhibition through several strategies has proved beneficial for dystrophic muscle. More recently, Bogdanovich et al. [31] demonstrated that myostatin pharmacological blockage using a myostatin propeptide improved the dystrophic characteristics of the mdx mouse. Similarly, transgenic expression of a myostatin inhibitor derived from follistatin or one-time gene administration of myostatin inhibitor proteins have been reported to increase skeletal muscle mass and to ameliorate the dystrophic pathology observed in the mdx mouse, further supporting its therapeutic potential [32], [33]. Similar findings were obtained by independent studies when an adeno-associated virus (AAV)-mediated myostatin propeptide systemic delivery enhanced muscle growth in normal mice. This methodology also ameliorated dystrophic phenotypes both in mdx mice as well as in limb-girdle muscular dystrophy in calpain-3 deficient mice, although no improvement was seen in a different model of recessive limb-girdle muscular dystrophy with -sarcoglycan deficiency [34], [35]. Most recently, the muscle growth effect of myostatin blockade was demonstrated in normal dogs by gene transfer of an AAV8 canine myostatin propeptide vector, which lead to enhanced muscle growth in some but not all muscles [36]. However, clinical trials exploring the systemic administration of MYO-029, a neutralizing antibody to myostatin, did not show any muscle strength or function benefits in humans with muscle dystrophy, despite improvements on the cellular level [37], [38].
Given that myostatin is naturally inhibited by its own propeptide, in this study we generated recombinant AAV8 vectors expressing a mutated propeptide fused to a mouse immunoglobulin Fc molecule [39], in order to assess the histological and physiological properties of healthy muscle following systemic administration of myostatin propeptide and to evaluate its potential use in muscle wasting diseases and disorders.
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Section snippets
Vector generation
cDNA encoding a modified myostatin propeptide fused to mouse IgG2a moiety [39] was cloned into a pDD derived AAV plasmid under the control of the CAGGs promoter [40], generating the plasmid pProMyo. AAV8ProMyo vectors were produced and viral genome titred by Nantes Vector Core, France. The IgG2a moiety prolongs the half life of recombinant protein but does not affect muscle growth [39].
Recombinant AAV administration
This study only used Outbred MF-1 male mice that were housed in minimal disease facilities (Royal Holloway,
Systemic post-natal injection of adeno-associated virus 8 (AAV8) expressing modified myostatin propeptide results in muscle specific increases in mass
Western blots to detect myostatin propeptide demonstrated plasma expression in MF-1 AAV8ProMyo treated mice at weeks 1, 8 and 17, with no evidence that the level of expression altered with time (Fig. 1A). An extra band, migrating lower on the gel was noted on the Western blot that corresponds to IgG2a. The mean body weights at each measured time point were analysed using a two-way (treatment × time) ANOVA with repeated measures on time revealing that mice treated with AAV8ProMyo showed an overall
Discussion
Epigenetic protocols to interfere with myostatin function have been proposed as potential therapeutic strategies to ameliorate features of muscle wasting conditions. In this study, we evaluated antagonising myostatin activity through its ability to re-associate with the propeptide molecule thus rendering it inactive. We provide evidence that a single injection of the propeptide results in muscle-specific enlargement with a time related increase in satellite cell number.
A single intravenous
Acknowledgements
We would like to thank the Wellcome Trust (078649) and University of Reading for generous funding permitting the execution of this work.
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Myostatin: A novel insight into its role in metabolism, signal pathways, and expression regulation
2011, Cellular Signalling
Wild-type myostatin propeptide is unstable in vivo, but its mutated form at the cleavage site of metalloproteinase of BMP-1/TLD family has stronger effects on increasing muscle mass after it was injected into normal mice or mdx mice [66,67]. In addition, a single intravenous administration of a recombinant AAV8 vector expressing a mutated myostatin propeptide leads to increases in mice muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection [68]. Interestingly, a significant increase in skeletal muscle mass was observed after a single intramuscular injection of naked plasmid DNA encoding a mutant myostatin propeptide into mice [69]. View all citing articles on Scopus
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These authors have equally contributed to this study.View full text
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