Silencing Nfix rescues muscular dystrophy by delaying muscle regeneration

ARTICLE DOI: 10.1038/s41467-017-01098-y OPEN Silencing Nfix rescues muscular dystrophy by delaying muscle regeneration Giuliana Rossi 1, Chiara Bonfanti1, Stefania Antonini1, Mattia Bastoni1, Stefania Monteverde1, Anna Innocenzi2, Marielle Saclier1, Valentina Taglietti1 & Graziella Messina 1 Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration. 1 Department of Biosciences, University of Milan, via Celoria 26, 20133 Milan, Italy. 2 Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, via Olgettina 60, 20132 Milan, Italy. Correspondence and requests for materials should be addressed to G.M. (email: ) NATURE COMMUNICATIONS | 8: 1055 | DOI: 10.1038/s41467-017-01098-y | www.nature.com/naturecommunications 1 ARTICLE M NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01098-y uscular dystrophies (MDs) are inherited skeletal muscle disorders characterized by progressive muscle damage and weakness of variable distribution and severity, leading to wheelchair dependency and, in the most severe cases, to patient’s death1, 2. MDs are due to mutations in genes encoding for proteins of the structural dystrophin-glycoprotein complex, which induce sarcolemmal instability and muscle necrosis. The most common form is Duchenne muscular dystrophy (DMD), an X-linked autosomal recessive disorder due to mutations in the dystrophin gene, which encodes a protein anchoring the sarcolemmal membrane to the cytoskeleton, thus protecting the fibers from contraction-induced damage3. Another form is limb girdle muscular dystrophy 2D, an autosomal recessive disorder caused by mutations in the α-sarcoglycan gene4 and part of a group of MDs, with a prevalence ranging from 1 in 14,500 to 1 in 123,0005, 6. Unfortunately, there is no effective therapy, and corticosteroids represent the most widely used treatment to counteract chronic inflammation7. Although many attempts have been done to design cell and gene therapies, these approaches are limited by technical issues, related to difficulties in finding the appropriate cell type or vector, and are therefore still far to be curative8, 9. A shared knowledge in the field is that to be really successful, any therapeutic approach has to rely on good muscle quality, therefore restricting the number of patients eligible for clinical trials. In fact, there is no available approach able to rescue muscle damage when the muscle tissue has been completely lost and substituted by fibrotic deposits, thus restricting the cohort of patients eligible for clinical trials to the youngest and less compromised individuals. Therefore, many groups proposed drugs and genetic constructs to counteract skeletal muscle degeneration by promoting regeneration by endogenous satellite cells10, 11. Nevertheless, none of these strategies demonstrated to be efficacious when translated to humans12, and there is still the need for alternative approaches and new targets to be identified. MDs are indeed characterized by continuous cycles of degeneration and regeneration, as a consequence of the attempt to repair damage by satellite stem cells that unfortunately, sharing the same mutation of the myofibers, are not able to successfully repair damage, leading to the loss of muscle tissue and establishment of fibrosis. Interestingly, it has been shown that slow-twitch, oxidative fibers are more protected from damage-induced oxidative stress and degeneration13–16. In light of our recent observation that mice lacking Nfix are characterized by a delayed regeneration after injury and a switch toward a slow-twitch phenotype17, 18, we hypothesized that targeting Nfix in a dystrophic context may exert a protective effect. Nfix is part of a family of four closely related transcription factors (Nfia, b, c, and x) with a role in activating/repressing transcription of genes expressed in various organs19–25. We previously demonstrated that Nfix is responsible for the transcriptional switch from embryonic to fetal myogenesis, a crucial checkpoint during muscle development. In particular, fetuses lacking Nfix are characterized by a slow-twitch musculature, typical of the embryonic period, while embryos overexpressing Nfix switch to a more mature fetal-like phenotype17. In addition, we recently observed that, postnatally, Nfix is crucial for the maintenance of the correct timing of skeletal muscle regeneration upon injury18. Here, we show that silencing Nfix in both α-sarcoglycan (Sgca null)- and dystrophin (mdx)-deficient dystrophic mice strikingly protects from the degenerative process by promoting a more oxidative musculature and by slowing down muscle regeneration, in contrast to previous attempts that aimed to promote regeneration. These data are supportive of a new role for Nfix in the progression of MD and suggest Nfix as a novel target to treat this 2 severe disease. More in general, we provide proof of principle for an innovative therapeutic approach based on the idea that slowing down the degeneration–regeneration cycles, instead of increasing regeneration, delays the progression of the pathology. Results Absence of Nfix improves dystrophic signs of Sgca null mice. To verify whether the muscle phenotype observed in the Nfix null mouse17, 18 would be beneficial in a dystrophic context, we generated dystrophic mice lacking Nfix. The Sgca null mouse model26 was chosen for our analysis because of its very severe phenotype, resembling the human pathology. Muscle histology was analyzed at different weeks of age, to monitor the progression of the disease. At 3 weeks, the first signs of aberrant muscle structure were already present in Sgca null mice (Fig. 1a), characterized by few regenerative fibers (Supplementary Fig. 1A) and presence of inflammatory infiltrates, necrotic areas, and varying fiber calibre. On the contrary, Sgca null:Nfix null mice were characterized by a compact structure with less interstitial space between myofibers and absence of large degenerative areas. At 5 weeks, Sgca null mice showed increased central nucleation (Fig. 1b and Supplementary Fig. 1B), a typical sign of ongoing regenerat (...truncated)