Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy-dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here, we show in mice, that 4-month pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling downregulates porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phosphorus Magnetic Resonance Spectroscopy ((31)P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn(-/-) mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn(-/-) mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn(-/-) mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn(-/-) mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.

Antisense oligonucleotides (AONs) hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA) is considered very promising for the treatment of Duchenne muscular dystrophy (DMD), a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients.

Adeno-associated virus (AAV)-U7-mediated skipping of dystrophin-exon-23 restores dystrophin expression and muscle function in the mdx mouse model of Duchenne muscular dystrophy. Soluble activin receptor IIB (sActRIIB-Fc) inhibits signaling of myostatin and homologous molecules and increases muscle mass and function of wild-type and mdx mice. We hypothesized that combined treatment with AAV-U7 and sActRIIB-Fc may synergistically improve mdx muscle function. Bioactivity of sActRIIB-Fc on skeletal muscle was first demonstrated in wild-type mice. In mdx mice we show that AAV-U7-mediated dystrophin restoration improved specific muscle force and resistance to eccentric contractions when applied alone. Treatment of mdx mice with sActRIIB-Fc increased body weight, muscle mass and myofiber size, but had little effect on muscle function. Combined treatment stimulated muscle growth comparable to the effect of sActRIIB-Fc alone and dystrophin rescue was similar to AAV-U7 alone. Moreover, combined treatment improved maximal tetanic force and the resistance to eccentric contraction to similar extent as AAV-U7 alone. In conclusion, combination of dystrophin exon skipping with sActRIIB-Fc brings together benefits of each treatment; however, we failed to evidence a clear synergistic effect on mdx muscle function.

Christensenellaceae is a family of subdominant commensal bacteria found in humans. It is thought to play an important role in gut health by maintaining microbial symbiosis. Indeed, these bacteria occur at significantly lower levels or are absent in individuals suffering from inflammatory bowel diseases (IBDs). Here, we explored if type species Christensenella minuta (strain: DSM 22607) could have the potential to help treat IBDs. We assessed key properties displayed by the bacterium using a combination of in vitro and in vivo assays. We found that while C. minuta is a strict anaerobe, it is also oxygen tolerant. Additionally, we observed that the species produces high levels of acetate and moderate levels of butyrate. We performed deep phenotyping using Biolog microarrays. Using human intestinal cell lines, we discovered that C. minuta demonstrated strong anti-inflammatory activity, resulting in reduced levels of proinflammatory IL-8 cytokines via the inhibition of the NF-κB signaling pathway. Furthermore, C. minuta protected intestinal epithelial integrity in vitro. Finally, in two distinct animal models of acute colitis, C. minuta prevented intestinal damage, reduced colonic inflammation, and promoted mucosal healing. Together, these results indicate that C. minuta has potent immunomodulatory properties, underscoring its potential use in innovative microbiome-based IBD biotherapies.

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.

No abstract

Antisense oligonucleotides (ASOs) hold promise for therapeutic splice switching correction for genetic diseases, in particular for Duchenne muscular dystrophy (DMD), for which ASO-exon skipping represents one of the most advanced therapeutic strategies. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting the unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. TcDNA-ASOs demonstrate an encouraging safety profile and no particular class-related toxicity, however, when administered in high doses for several months, mild renal toxicity is observed secondary to predictable phosphorothioate (PS)-ASO accumulation in kidneys. In this study, we investigate the influence of the relative content of PS linkages in tcDNA-ASOs on exon skipping efficacy. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0%, 25%, 33%, 50%, 67%, 83%, and 100%). The results indicate that levels of exon-23 skipping and dystrophin rescue increase with the number of PS linkages in most skeletal muscles except in the heart. As expected, plasma coagulation times are shortened with decreasing PS content, and tcDNA-protein binding in serum directly correlates with the number of PS linkages on the tcDNA backbone. Altogether, these data contribute in establishing the appropriate sulfur content within the tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.