- Email: [email protected]
Current advances in the development of therapies for neuromuscular disorders based on myostatin signalling, 3rd International Institute of Myology Workshop, Paris, September 12th, 2008
Neuromuscular Disorders 19 (2009) 797–799
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Workshop report
Current advances in the development of therapies for neuromuscular disorders based on myostatin signalling, 3rd International Institute of Myology Workshop, Paris, September 12th, 2008 Julie Dumonceaux, Helge Amthor * Faculté de Médecine Pierre et Marie Curie, Université Paris VI, UPMC INSERM UMR S 974/CNRS UMR 7215 - Institut de Myologie, 105 bd de l’Hôpital, 75013 Paris, France
1. Introduction 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.
2. Myostatin blockade and satellite cells activity Terence Partridge presented experiments strongly suggesting that myostatin blockade in fact drives satellite-cell-independent
* Corresponding author. E-mail address: [email protected] (H. Amthor). 0960-8966/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2009.08.001
muscle hypertrophy. First he showed that after 72 h of culture, the number of myoblasts derived from isolated myofibres of C57Bl6 mice, Mstn / mice and Mstn / mice treated J16-antibody was identical. He next demonstrated that when satellite cells from C57bl6 mice were exposed to recombinant myostatin, their proliferation was unaffected. Furthermore, over-expression of the myostatin propeptide in adult mice resulted muscle fibre hypertrophy, however, the number of myonuclei per muscle fibre and number of satellite cells remained unchanged. In addition, real time PCR revealed a strong decrease in the expression of the myostatin receptors AcvR 2A and 2B in satellite cells from prenatal to postnatal stages, and this could explain their unresponsiveness to myostatin stimulation. Thus, different mechanism than satellite cell proliferation are responsible for the myostatin-based control of postnatal muscle size, such as modulating synthesis and turnover of structural muscle fibre proteins. These results were recently published [4]. Finally, Terence Partridge pointed out that it remains difficult to determine the effect of myostatin blockade on mdx muscle, because fibre branching can reach up to 100% in 2 years old mdx mice and thus distorts the fibre counts. 3. Myostatin: a molecular rheostat for muscle mass Se-Jin Lee presented new data on the maturation of myostatin by BMP-1 mediated cleavage. He had previously shown that heating the latent complex (composed by the mature C-terminal portion of myostatin, which remains non-covalently bound to the Nterminal part) activates myostatin. He also demonstrated that the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases is responsible for the propeptide cleavage and this is essential for the activity of the complex [5]. When the mutated D76A propeptide is used, BMP-1 can no longer cleave the propeptide. Therefore, he created D76A-loxP mice and as expected, there was an increase in muscle mass, which was almost identical to the myostatin KO mouse. This result confirms the central role of BMP-1 for regulating myostatin activity. Because mice completely lacking BMP-1 die before birth, Se-Jin Lee created conditional knock out mice for BMP-1 and for TLL-2 (which can also cleave the propeptide). He observed that each of these conditional KO on their own did not induce an important increase in muscle mass, suggesting that other metalloproteinases can also cleave the propeptide [6]. However, he generated double
798
J. Dumonceaux, H. Amthor / Neuromuscular Disorders 19 (2009) 797–799
KO BMP-1+/ TLL-2 / mice, which resulted in a significant increase in muscle mass. Se-Jin Lee also reminded us that over-expressing follistatin (F66 transgenic mice) resulted in excessive muscle superior to myostatin knockout, suggesting the existence of other ligands in addition to myostatin [7]. He concluded that myostatin is not the exclusive regulator of muscle mass, which opens the possibility to develop novel therapeutic strategies in addition to myostatin blockade.
strategy) and myostatin blockade (using the sh-AcvRIIb strategy): the U7/sh-AcvRIIb vector. She demonstrated that such combined treatment of mdx tibialis anterior muscle not only stimulated muscle growth, moreover, maximal tetanic tension and specific force increased by 30% (Dumonceaux et al., submitted for publication). This is the proof of principle that a non-specific therapeutic approach (myostatin blockade) can increase the therapeutic benefit of a specific therapy (dystrophin exon skipping).
4. AAV based myostatin blockade
5. Oxidative metabolism and exercise capacity
Keith Foster presented the work he has carried out using an AAV-8 vector over-expressing the myostatin propeptide fused to an Fc domain. This vector has been intravenously injected into MF-1 mice. He found an increase in muscle mass in both the soleus muscle (+41%) and the EDL muscle (+18%), however, the tetanic force was only increased in the soleus muscle. The specific force remained unchanged. In addition this increase in muscle mass was not accompanied by an increase in satellite cell activity, since there was no change in the nuclear number nor in the number of Pax7/ MyoD positive cells. However, the ratio of cytoplasm/nuclei was increased and there was a shift from oxidative to glycolytic fibres. These results were also recently published [8]. Keith Foster also combined myostatin blockade (systemic administration of the AAV propeptide) with a rescue of dystrophin expression using an AAV micro-dystrophin that was injected into the tibialis anterior muscle of mdx mouse. This combined approach did not change significantly the weight of the TA muscles. This may be explained by the fact that micro-dystrophin on one side prevents muscle hypertrophy, because it stops the degeneration and regeneration cycles, whereas on the other side over-expression of the myostatin propeptide induces a muscle mass increase. Interestingly, overexpression of the propeptide increases the force drop during a series of eccentric contractions compared to untreated mdx, whereas the combined treatment with micro-dystrophin and propeptide restored the eccentric contraction force drop almost to wild-type values. Julie Dumonceaux presented data showing that over-expression of the myostatin propeptide fused to the murine serum alkaline phosphatase (muSeAP) induces an increase in muscle mass in the C57Bl6 mice, which resulted from fibre hypertrophy. The same result was obtained when using the D76A mutated propeptide. Additionally, she developed new strategies for blocking the myostatin signalling pathway, which are based on the RNA interference technique. She constructed AAV based vectors encoding shRNA directed either against myostatin (sh-GDF8) or against the activin receptor IIb (sh-AcvRIIb). Intramuscular injection of these AAV vectors into adult mdx mice resulted in an efficient down-regulation of the target mRNA. However, the sh-GDF8 did not stimulate muscle growth, possibly because a local down-regulation of myostatin synthesis is insufficient to overcome the effect of circulating myostatin. Differently to sh-GDF8, AAV sh-AcvRIIb stimulated muscle growth when injected into tibialis anterior muscle of mdx mouse. Remarkably, fibre counts revealed that the muscle mass increase resulted from hyperplasia and not from hypertrophy. Such increase in fibre number could be explained by an increased muscle regeneration, increased fibre branching or by a failure of the fusion of small fibre regenerates as suggested by Julie Dumonceaux and Luis Garcia. They proposed a model to explain this phenomen and reasoned that during muscle regeneration in mdx mouse, signalling via the AcvRIIb promotes fusion of small fibre regenerates. Following AcvRIIb receptor knockdown, such fusion could be inhibited, which leads to an accumulation of small newly regenerated fibres. Julie Dumonceaux also presented a novel therapeutic strategy which combines dystrophin rescue (using the U7 exon skipping
Lack of myostatin results in a profound fibre type conversion towards a glycolytic phenotype, suggesting a deficit in the oxidative metabolism of skeletal muscle [9]. Christophe Hourdé presented his project on muscle metabolism following myostatin blockade and therapeutic strategies, such as AAV U7/sh-AcvRIIb. He demonstrated a shift from fast oxidative myosin heavy chains (IIa/IIx) to fast glycolytic myosin heavy chains (IIb) following treatment with U7/sh-AcvRIIb using SDS page technique. This shift was associated with a faster contraction time and half relaxation time. In ongoing work, he compares the activities of the mitochondrial enzymes (complex II, complex IV and citrate synthase) and the mitochondrial respiration following treatment with AAV U7/sh-AcvRIIb. Expected results will increase our understanding of how myostatin blockade modifies the muscle metabolism, especially if used as a therapeutic approach. Additional investigations have been initiated to identify transcription factors regulating muscle metabolism. Etienne Mouisel concentrated his presentation on the exercise capacity and force generation in the Mstn / mice. This work was performed in collaboration with Ketan Patel (University of Reading, UK) who was unfortunately hindered to attend this meeting. He compared the exercise capacity of Mstn / and wt mice using a swimming time limit test (exhaustion test). He showed that the wt mice were always able to swim for a longer time than the Mstn / mice. This result can be partially explained by the fact that Mstn / mice heavier than the wt mice, that they possess an increased lean muscle mass and have much less fat tissue. However, the ability of the Mstn / mice to swim was increased by a 5 weeks training protocol which was correlated with an increase in the maximum tetanic force (EDL muscle). Interestingly, the muscle weight of the Mstn / mice was not increased after exercise, resulting in an increase in the specific force compared to sedentary mice. Several analyses such as fibre typing or mitochondrial oxidative metabolism are in progress to better understand this phenomenon. Moreover, measures of running capacity on treadmill are in progress. In a different project, Etienne Mouisel studied whether myostatin blockade can prevent muscle atrophy following denervation. He sciatomised hindlimb muscles of C57Bl6 mice and then intramuscularly injected AAV propeptide or the muSeAP reporter gene. No difference on muscle size was observed which demonstrates that myostatin blockade is only effective on innervated muscle.
6. Influence of myostatin on rhabdomyosarcoma growth and differentiation Rhabdomyosarcoma (RMS) is one of the most frequent sarcomas in childhood. Yet the prognosis remains dismal. Two cell lines originally derived from embryonic (RD) and alveolar (Rh30) RMS have been shown to express myostatin mRNA and protein. Addition of high doses of recombinant myostatin induced growth arrest in RD and Rh30 cell lines, whereas inhibition of myostatin signalling enhanced terminal differentiation in RD cells.
J. Dumonceaux, H. Amthor / Neuromuscular Disorders 19 (2009) 797–799
799
In the experiments presented by Kristina Juelich and Markus Schuelke, they show a 30% reduction in cell number 72 h after exposure of RD cells to myostatin. This was due to a cell cycle exit as confirmed by BrdU incorporation. There was no significant difference in the expression of terminal differentiation markers. However, the alveolar RMS cell line Rh30 did not respond at all to myostatin in their assays. Their opinion is that growth arrest of Rh30 cells in previous publications of other groups might be due to un-physiologically high myostatin concentrations (500-fold above physiological serum concentrations in humans). In addition, recombinant myostatin produced in bacteria might have not folded properly and might have acted differently on these cells. Additionally, they have produced monoclonal antibodies against the mature domain of human myostatin. Immunization of the Mstn / mice was done with recombinant protein produced in Escherichia coli comprising all amino acids of the mature portion of myostatin. Because this peptide is most probably not accurately folded and dimerized, these antibodies work very well in Western blot on denatured protein, but not as neutralizing antibodies that could be used for experimental therapies or for immunocapture of the myostatin complex. Additional immunizations are presently being performed with accurately folded bioactive myostatin.
Workshop participants
7. Summary
This workshop was sponsored by the Association Française contre les Myopathies. We thank Catherine Champseix for helping to organise this meeting.
In summary, there was a general agreement that myostatin blockade could be a promising therapeutic strategy to counteract muscle loss in the course of neuromuscular disorders, especially of muscular dystrophies. It was concluded that pharmacological approaches, such as soluble activin IIb receptors, are closer to clinical practise than gene vector mediated myostatin blockade. However, this should not discourage research on gene vectors, especially the development of AAV mediated muscle specific myostatin blockade (e.g. muscle targeted activin receptor knockdown), thus circumventing possible adverse effects following systemic myostatin depletion. Importantly, myostatin blockade can only be a non-specific treatment and the underlying defect, such as lack of dystrophin for DMD, will remain. On the other hand, the efficiency of specific therapy approaches, such as the restoration of dystrophin expression following exon skipping or over-expression of minidystrophin, is likely to be low if the disease has already progressed into substantial muscle wasting and fibrosis. Thus, a combination of myostatin blockade and specific therapy approaches might overcome the limitations of each and herein presented first results are very encouraging.
Helge Amthor, Paris, France Serge Braun, Evry, France Gillian Butler-Browne, Paris, France Catherine Champseix, Paris, France Julie Dumonceaux, Paris, France George Dickson, Egham, UK Arnaud Ferry, Paris, France Keith Foster, Egham, UK Luis Garcia, Paris, France Christophe Hourdé, Paris, France Kristina Jülich, Berlin, Germany Se-Jin Lee, Baltimore, USA François Leterrier, Paris, France Etienne Mouisel, Paris, France Terence Partridge, Washington, USA France Pietri-Rouxel, Paris, France Markus Schuelke, Berlin, Germany Capucine Trollet, Paris, France Thomas Voit, Paris, France
Acknowledgements
References [1] Trollet C, Athanasopoulos T, Popplewell L, Malerba A, Dickson G. Gene therapy for muscular dystrophy: current progress and future prospects. Expert Opin Biol Ther 2009;9:1–20. [2] Magee TR, Artaza JN, Ferrini MG, et al. Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass. J Gene Med 2006;8:1171–81. [3] Qiao C, Li J, Zheng H, et al. Hydrodynamic limb vein injection of AAV8 canine myostatin propeptide gene in normal dogs enhances muscle growth. Hum Gene Ther 2009;20:1–10. [4] Amthor H, Otto A, Vulin A, et al. Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity. Proc Natl Acad Sci USA 2009;106:7479–84. [5] Wolfman NM, McPherron AC, Pappano WN, et al. Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases. Proc Natl Acad Sci USA 2003;100:15842–6. [6] Lee SJ. Genetic analysis of the role of proteolysis in the activation of latent myostatin. PLoS ONE 2008;3:e1628. [7] Lee SJ. Quadrupling muscle mass in mice by targeting TGF-beta signaling pathways. PLoS ONE 2007;2:e789. [8] Foster K, Graham IR, Otto A, et al. Adeno-associated virus-8-mediated intravenous transfer of myostatin propeptide leads to systemic functional improvements of slow but not fast muscle. Rejuvenation Res 2009;12:85–94. [9] Amthor H, Macharia R, Navarrete R, et al. Lack of myostatin results in excessive muscle growth but impaired force generation. Proc Natl Acad Sci USA 2007;104:1835–40.
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Workshop report
Current advances in the development of therapies for neuromuscular disorders based on myostatin signalling, 3rd International Institute of Myology Workshop, Paris, September 12th, 2008 Julie Dumonceaux, Helge Amthor * Faculté de Médecine Pierre et Marie Curie, Université Paris VI, UPMC INSERM UMR S 974/CNRS UMR 7215 - Institut de Myologie, 105 bd de l’Hôpital, 75013 Paris, France
1. Introduction 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.
2. Myostatin blockade and satellite cells activity Terence Partridge presented experiments strongly suggesting that myostatin blockade in fact drives satellite-cell-independent
* Corresponding author. E-mail address: [email protected] (H. Amthor). 0960-8966/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2009.08.001
muscle hypertrophy. First he showed that after 72 h of culture, the number of myoblasts derived from isolated myofibres of C57Bl6 mice, Mstn / mice and Mstn / mice treated J16-antibody was identical. He next demonstrated that when satellite cells from C57bl6 mice were exposed to recombinant myostatin, their proliferation was unaffected. Furthermore, over-expression of the myostatin propeptide in adult mice resulted muscle fibre hypertrophy, however, the number of myonuclei per muscle fibre and number of satellite cells remained unchanged. In addition, real time PCR revealed a strong decrease in the expression of the myostatin receptors AcvR 2A and 2B in satellite cells from prenatal to postnatal stages, and this could explain their unresponsiveness to myostatin stimulation. Thus, different mechanism than satellite cell proliferation are responsible for the myostatin-based control of postnatal muscle size, such as modulating synthesis and turnover of structural muscle fibre proteins. These results were recently published [4]. Finally, Terence Partridge pointed out that it remains difficult to determine the effect of myostatin blockade on mdx muscle, because fibre branching can reach up to 100% in 2 years old mdx mice and thus distorts the fibre counts. 3. Myostatin: a molecular rheostat for muscle mass Se-Jin Lee presented new data on the maturation of myostatin by BMP-1 mediated cleavage. He had previously shown that heating the latent complex (composed by the mature C-terminal portion of myostatin, which remains non-covalently bound to the Nterminal part) activates myostatin. He also demonstrated that the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases is responsible for the propeptide cleavage and this is essential for the activity of the complex [5]. When the mutated D76A propeptide is used, BMP-1 can no longer cleave the propeptide. Therefore, he created D76A-loxP mice and as expected, there was an increase in muscle mass, which was almost identical to the myostatin KO mouse. This result confirms the central role of BMP-1 for regulating myostatin activity. Because mice completely lacking BMP-1 die before birth, Se-Jin Lee created conditional knock out mice for BMP-1 and for TLL-2 (which can also cleave the propeptide). He observed that each of these conditional KO on their own did not induce an important increase in muscle mass, suggesting that other metalloproteinases can also cleave the propeptide [6]. However, he generated double
798
J. Dumonceaux, H. Amthor / Neuromuscular Disorders 19 (2009) 797–799
KO BMP-1+/ TLL-2 / mice, which resulted in a significant increase in muscle mass. Se-Jin Lee also reminded us that over-expressing follistatin (F66 transgenic mice) resulted in excessive muscle superior to myostatin knockout, suggesting the existence of other ligands in addition to myostatin [7]. He concluded that myostatin is not the exclusive regulator of muscle mass, which opens the possibility to develop novel therapeutic strategies in addition to myostatin blockade.
strategy) and myostatin blockade (using the sh-AcvRIIb strategy): the U7/sh-AcvRIIb vector. She demonstrated that such combined treatment of mdx tibialis anterior muscle not only stimulated muscle growth, moreover, maximal tetanic tension and specific force increased by 30% (Dumonceaux et al., submitted for publication). This is the proof of principle that a non-specific therapeutic approach (myostatin blockade) can increase the therapeutic benefit of a specific therapy (dystrophin exon skipping).
4. AAV based myostatin blockade
5. Oxidative metabolism and exercise capacity
Keith Foster presented the work he has carried out using an AAV-8 vector over-expressing the myostatin propeptide fused to an Fc domain. This vector has been intravenously injected into MF-1 mice. He found an increase in muscle mass in both the soleus muscle (+41%) and the EDL muscle (+18%), however, the tetanic force was only increased in the soleus muscle. The specific force remained unchanged. In addition this increase in muscle mass was not accompanied by an increase in satellite cell activity, since there was no change in the nuclear number nor in the number of Pax7/ MyoD positive cells. However, the ratio of cytoplasm/nuclei was increased and there was a shift from oxidative to glycolytic fibres. These results were also recently published [8]. Keith Foster also combined myostatin blockade (systemic administration of the AAV propeptide) with a rescue of dystrophin expression using an AAV micro-dystrophin that was injected into the tibialis anterior muscle of mdx mouse. This combined approach did not change significantly the weight of the TA muscles. This may be explained by the fact that micro-dystrophin on one side prevents muscle hypertrophy, because it stops the degeneration and regeneration cycles, whereas on the other side over-expression of the myostatin propeptide induces a muscle mass increase. Interestingly, overexpression of the propeptide increases the force drop during a series of eccentric contractions compared to untreated mdx, whereas the combined treatment with micro-dystrophin and propeptide restored the eccentric contraction force drop almost to wild-type values. Julie Dumonceaux presented data showing that over-expression of the myostatin propeptide fused to the murine serum alkaline phosphatase (muSeAP) induces an increase in muscle mass in the C57Bl6 mice, which resulted from fibre hypertrophy. The same result was obtained when using the D76A mutated propeptide. Additionally, she developed new strategies for blocking the myostatin signalling pathway, which are based on the RNA interference technique. She constructed AAV based vectors encoding shRNA directed either against myostatin (sh-GDF8) or against the activin receptor IIb (sh-AcvRIIb). Intramuscular injection of these AAV vectors into adult mdx mice resulted in an efficient down-regulation of the target mRNA. However, the sh-GDF8 did not stimulate muscle growth, possibly because a local down-regulation of myostatin synthesis is insufficient to overcome the effect of circulating myostatin. Differently to sh-GDF8, AAV sh-AcvRIIb stimulated muscle growth when injected into tibialis anterior muscle of mdx mouse. Remarkably, fibre counts revealed that the muscle mass increase resulted from hyperplasia and not from hypertrophy. Such increase in fibre number could be explained by an increased muscle regeneration, increased fibre branching or by a failure of the fusion of small fibre regenerates as suggested by Julie Dumonceaux and Luis Garcia. They proposed a model to explain this phenomen and reasoned that during muscle regeneration in mdx mouse, signalling via the AcvRIIb promotes fusion of small fibre regenerates. Following AcvRIIb receptor knockdown, such fusion could be inhibited, which leads to an accumulation of small newly regenerated fibres. Julie Dumonceaux also presented a novel therapeutic strategy which combines dystrophin rescue (using the U7 exon skipping
Lack of myostatin results in a profound fibre type conversion towards a glycolytic phenotype, suggesting a deficit in the oxidative metabolism of skeletal muscle [9]. Christophe Hourdé presented his project on muscle metabolism following myostatin blockade and therapeutic strategies, such as AAV U7/sh-AcvRIIb. He demonstrated a shift from fast oxidative myosin heavy chains (IIa/IIx) to fast glycolytic myosin heavy chains (IIb) following treatment with U7/sh-AcvRIIb using SDS page technique. This shift was associated with a faster contraction time and half relaxation time. In ongoing work, he compares the activities of the mitochondrial enzymes (complex II, complex IV and citrate synthase) and the mitochondrial respiration following treatment with AAV U7/sh-AcvRIIb. Expected results will increase our understanding of how myostatin blockade modifies the muscle metabolism, especially if used as a therapeutic approach. Additional investigations have been initiated to identify transcription factors regulating muscle metabolism. Etienne Mouisel concentrated his presentation on the exercise capacity and force generation in the Mstn / mice. This work was performed in collaboration with Ketan Patel (University of Reading, UK) who was unfortunately hindered to attend this meeting. He compared the exercise capacity of Mstn / and wt mice using a swimming time limit test (exhaustion test). He showed that the wt mice were always able to swim for a longer time than the Mstn / mice. This result can be partially explained by the fact that Mstn / mice heavier than the wt mice, that they possess an increased lean muscle mass and have much less fat tissue. However, the ability of the Mstn / mice to swim was increased by a 5 weeks training protocol which was correlated with an increase in the maximum tetanic force (EDL muscle). Interestingly, the muscle weight of the Mstn / mice was not increased after exercise, resulting in an increase in the specific force compared to sedentary mice. Several analyses such as fibre typing or mitochondrial oxidative metabolism are in progress to better understand this phenomenon. Moreover, measures of running capacity on treadmill are in progress. In a different project, Etienne Mouisel studied whether myostatin blockade can prevent muscle atrophy following denervation. He sciatomised hindlimb muscles of C57Bl6 mice and then intramuscularly injected AAV propeptide or the muSeAP reporter gene. No difference on muscle size was observed which demonstrates that myostatin blockade is only effective on innervated muscle.
6. Influence of myostatin on rhabdomyosarcoma growth and differentiation Rhabdomyosarcoma (RMS) is one of the most frequent sarcomas in childhood. Yet the prognosis remains dismal. Two cell lines originally derived from embryonic (RD) and alveolar (Rh30) RMS have been shown to express myostatin mRNA and protein. Addition of high doses of recombinant myostatin induced growth arrest in RD and Rh30 cell lines, whereas inhibition of myostatin signalling enhanced terminal differentiation in RD cells.
J. Dumonceaux, H. Amthor / Neuromuscular Disorders 19 (2009) 797–799
799
In the experiments presented by Kristina Juelich and Markus Schuelke, they show a 30% reduction in cell number 72 h after exposure of RD cells to myostatin. This was due to a cell cycle exit as confirmed by BrdU incorporation. There was no significant difference in the expression of terminal differentiation markers. However, the alveolar RMS cell line Rh30 did not respond at all to myostatin in their assays. Their opinion is that growth arrest of Rh30 cells in previous publications of other groups might be due to un-physiologically high myostatin concentrations (500-fold above physiological serum concentrations in humans). In addition, recombinant myostatin produced in bacteria might have not folded properly and might have acted differently on these cells. Additionally, they have produced monoclonal antibodies against the mature domain of human myostatin. Immunization of the Mstn / mice was done with recombinant protein produced in Escherichia coli comprising all amino acids of the mature portion of myostatin. Because this peptide is most probably not accurately folded and dimerized, these antibodies work very well in Western blot on denatured protein, but not as neutralizing antibodies that could be used for experimental therapies or for immunocapture of the myostatin complex. Additional immunizations are presently being performed with accurately folded bioactive myostatin.
Workshop participants
7. Summary
This workshop was sponsored by the Association Française contre les Myopathies. We thank Catherine Champseix for helping to organise this meeting.
In summary, there was a general agreement that myostatin blockade could be a promising therapeutic strategy to counteract muscle loss in the course of neuromuscular disorders, especially of muscular dystrophies. It was concluded that pharmacological approaches, such as soluble activin IIb receptors, are closer to clinical practise than gene vector mediated myostatin blockade. However, this should not discourage research on gene vectors, especially the development of AAV mediated muscle specific myostatin blockade (e.g. muscle targeted activin receptor knockdown), thus circumventing possible adverse effects following systemic myostatin depletion. Importantly, myostatin blockade can only be a non-specific treatment and the underlying defect, such as lack of dystrophin for DMD, will remain. On the other hand, the efficiency of specific therapy approaches, such as the restoration of dystrophin expression following exon skipping or over-expression of minidystrophin, is likely to be low if the disease has already progressed into substantial muscle wasting and fibrosis. Thus, a combination of myostatin blockade and specific therapy approaches might overcome the limitations of each and herein presented first results are very encouraging.
Helge Amthor, Paris, France Serge Braun, Evry, France Gillian Butler-Browne, Paris, France Catherine Champseix, Paris, France Julie Dumonceaux, Paris, France George Dickson, Egham, UK Arnaud Ferry, Paris, France Keith Foster, Egham, UK Luis Garcia, Paris, France Christophe Hourdé, Paris, France Kristina Jülich, Berlin, Germany Se-Jin Lee, Baltimore, USA François Leterrier, Paris, France Etienne Mouisel, Paris, France Terence Partridge, Washington, USA France Pietri-Rouxel, Paris, France Markus Schuelke, Berlin, Germany Capucine Trollet, Paris, France Thomas Voit, Paris, France
Acknowledgements
References [1] Trollet C, Athanasopoulos T, Popplewell L, Malerba A, Dickson G. Gene therapy for muscular dystrophy: current progress and future prospects. Expert Opin Biol Ther 2009;9:1–20. [2] Magee TR, Artaza JN, Ferrini MG, et al. Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass. J Gene Med 2006;8:1171–81. [3] Qiao C, Li J, Zheng H, et al. Hydrodynamic limb vein injection of AAV8 canine myostatin propeptide gene in normal dogs enhances muscle growth. Hum Gene Ther 2009;20:1–10. [4] Amthor H, Otto A, Vulin A, et al. Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity. Proc Natl Acad Sci USA 2009;106:7479–84. [5] Wolfman NM, McPherron AC, Pappano WN, et al. Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases. Proc Natl Acad Sci USA 2003;100:15842–6. [6] Lee SJ. Genetic analysis of the role of proteolysis in the activation of latent myostatin. PLoS ONE 2008;3:e1628. [7] Lee SJ. Quadrupling muscle mass in mice by targeting TGF-beta signaling pathways. PLoS ONE 2007;2:e789. [8] Foster K, Graham IR, Otto A, et al. Adeno-associated virus-8-mediated intravenous transfer of myostatin propeptide leads to systemic functional improvements of slow but not fast muscle. Rejuvenation Res 2009;12:85–94. [9] Amthor H, Macharia R, Navarrete R, et al. Lack of myostatin results in excessive muscle growth but impaired force generation. Proc Natl Acad Sci USA 2007;104:1835–40.