Extracellular matrix remodelling is associated with muscle force increase in overloaded mouse plantaris muscle

International audienceExtracellular matrix remodelling is associated with muscle force increase in overloaded mouse plantaris muscle Aims: Transforming growth factor-b (TGF-b) signalling is thought to contribute to the remodelling of extracellular matrix (ECM) of skeletal muscle and to functional decline in patients with muscular dystrophies. We wanted to determine the role of TGF-b-induced ECM remodelling in dystrophic muscle. Methods: We experimentally induced the pathological hallmarks of severe muscular dystrophy by mechanically overloading the plantaris muscle in mice. Furthermore, we determined the role of TGF-b signalling on dystrophic tissue modulation and on muscle function by (i) overloading myostatin knockout (Mstn À/À) mice and (ii) by additional pharmacological TGF-b inhibition via halofuginone. Results: Transcriptome analysis of overloaded muscles revealed upregulation predominantly of genes associated with ECM, inflammation and metalloproteinase activity. Histology revealed in wild-type mice signs of severe muscular dystrophy including myofibres with large variation in size and internalized myonuclei, as well as increased ECM deposition. At the same time, muscle weight had increased by 208% and muscle force by 234%. Myostatin deficiency blunted the effect of overload on muscle mass (59% increase) and force (76% increase), while having no effect on ECM deposition. Concomitant treatment with halofuginone blunted overload-induced muscle hypertro-phy and muscle force increase, while reducing ECM depo-sition and increasing myofibre size. Conclusions: ECM remodelling is associated with an increase in muscle mass and force in overload-modelled dystrophic muscle. Lack of myostatin is not advantageous and inhibition of ECM deposition by halofuginone is disadvantageous for muscle plasticity in response to stimuli that induce dystrophic muscle

Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models

INTRODUCTION: The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. METHODS: We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KO(Lee) ) and (ii) the Grobet model (KO(Grobet) ), and measured the contraction of tibialis anterior muscle in situ. RESULTS: Absolute maximal isometric force was increased in 6-month-old KO(Lee) and KO(Grobet) mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KO(Lee) mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KO(Grobet) mice, whereas specific maximal power was reduced only in male KO(Lee) mice. CONCLUSIONS: Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017

Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy

International audienceBackground: Dmdmdx, harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. Methods: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. Results: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. Conclusion: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo

Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle

International audienceDystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called “basal sarcolemmal dystrophin units (BSDUs).” These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber—with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle–tendon junction

BMP signaling controls muscle mass.

Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate myostatin lead to excessive muscle growth in animals and humans, but the signals and pathways responsible for this hypertrophy remain largely unknown. Here we show that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is the fundamental hypertrophic signal in mice. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin-deficient mice and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 signaling negatively regulates a gene (Fbxo30) that encodes a ubiquitin ligase required for muscle loss, which we named muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1). Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy

Past, present, and future perspective of targeting myostatin and related signaling pathways to counteract muscle atrophy

Myostatin was identified more than 20 years ago as a negative regulator of muscle mass in mice and cattle. Since then, a wealth of studies have uncovered the potential involvement of myostatin in muscle atrophy and sparked interest in myostatin as a promising therapeutic target to counteract decline of muscle mass in patients afflicted with different muscle-wasting conditions. Insight in the molecular mechanism of myostatin signaling and regulation of myostatin activity has resulted in the identification of specific treatments to inhibit myostatin signaling and related signaling pathways. Currently, several treatments that target myostatin and related proteins have been evaluated in preclinical animal models of muscle wasting, and some potential therapies have progressed to clinical trials. However, studies also revealed potential downsides of myostatin targeting in skeletal muscle and other tissues, which raises the question if myostatin is indeed a valuable target to counteract muscle atrophy. In this review we provide an updated overview of the molecular mechanisms of myostatin signaling, the preclinical evidence supporting a role for myostatin and related proteins in muscle atrophy, and the potential issues that arise when targeting myostatin. In addition, we evaluate the current clinical status of different treatments aimed at inhibiting myostatin and discuss future perspectives of targeting myostatin to counteract muscle atrophy