NOX4 inhibition promotes the remodeling of dystrophic muscle

The muscular dystrophies (MDs) are genetic muscle diseases that result in progressive muscle degeneration followed by the fibrotic replacement of affected muscles as regenerative processes fail. Therapeutics that specifically address the fibrosis and failed regeneration associated with MDs represent a major unmet clinical need for MD patients, particularly those with advanced stage disease progression. The current study investigates targeting NAD(P)H oxidase (NOX) 4 as a potential strategy to reduce fibrosis and promote regeneration in disease-burdened muscle that models Duchenne muscular dystrophy (DMD). NOX4 is elevated in the muscles of dystrophic mice and DMD patients, localizing primarily to interstitial cells located between muscle fibers. Genetic and pharmacological targeting of NOX4 significantly reduces fibrosis in dystrophic respiratory and limb muscles. Mechanistically, NOX4 targeting decreases the number of fibrosis-depositing cells (myofibroblasts) and restores the number of muscle-specific stem cells (satellite cells) to their physiological niche, thereby, rejuvenating muscle regeneration. Furthermore, acute inhibition of NOX4 is sufficient to induce apoptotic clearing of myofibroblasts within dystrophic muscle. These data indicate that targeting NOX4 is an effective strategy to promote the beneficial remodeling of disease-burdened muscle representative of DMD and, potentially, other MDs and muscle pathologies.

Dlk1-Dio3 cluster miRNAs regulate mitochondrial functions in Duchenne muscular dystrophy

Background: Duchenne Muscular Dystrophy (DMD) is a severe muscle disease caused by impaired expression of dystrophin. While mitochondrial dysfunction is thought to play an important role in DMD, the mechanism of this dysfunction remains to be clarified. We recently identified in DMD and in other muscular dystrophies the upregulation of a large number of the Dlk1-Dio3 clustered miRNAs (DD-miRNAs), in both the muscle and the serum. The objective of the present study was to define the biological functions of DD-miRNAs in skeletal muscle, particularly in the context of muscular dystrophy. Methods: DD-miRNAs expression pattern was characterized in vitro and in vivo, in normal and dystrophic situations. Epigenomic characterization was performed, to elucidate the molecular control of DD-miRNAs dysregulation. The biological effect of muscle DD-miRNAs dysregulation was investigated by an in vivo simultaneous overexpression of 14 DD-miRNAs in the wild-type muscle, together with CRISPR-Cas9-based knockdown of the entire DD-miRNA cluster in an iPS-derived myotubes. Omics data and bioinformatics tools were used for the prediction of DD-miRNAs biological functions, and functional characterization of mitochondrial pathways was performed. Results: We found that DD-miRNAs dysregulation is not specific to DMD since observed in mouse models for other muscular dystrophies. We showed that DD-miRNAs expression in mdx, is reduced in satellite cells, but highly upregulated in regenerating myofibers, suggesting a myofibers origin of DD6miRNA upregulation in muscular dystrophy in both muscles and serum. We demonstrated that upregulation of DD-miRNAs in the dystrophic muscle is controlled epigenetically by DNA and histone methylation (p<0.0001 and p=0.001, respectively) at the Intergenic Differentially Methylated Region (IG-DMR) of Dlk1-Dio3 locus. Transcriptomic analysis revealed a substantial overlap between the dystrophic muscle of the mdx mouse and the normal muscle that overexpressed 14 DD-miRNAs. Bioinformatics analysis predicted that DD-miRNAs could regulate mitochondrial functions. The ectopic overexpression of 14 DD-miRNAs, in the healthy muscle, resulted in a drastic downregulation of mitochondrial oxidative phosphorylation (OxPhos) (NES=-2.8, p=8.7E-17), similarly to the level in dystrophic muscles of mdx mice and DMD patients (NES=-2.88, p=7.7E-28). Knocking down the entire DD-miRNA cluster in iPS-derived myotubes resulted in increased mitochondrial OxPhos expression and activities. Conclusions: The present study provides evidence for the modulation of mitochondrial activity in the dystrophic muscle by the upregulated DD-miRNAs and supports an updated model for mitochondrial dysfunction in DMD. The regulation of mitochondrial OxPhos by DD-miRNAs may have a broader impact beyond DMD in physiological and pathological situations of muscle adaptation and regeneration.

A revised model for mitochondrial dysfunction in Duchenne muscular dystrophy

We recently identified a signaling pathway that links the upregulation of miR-379 with a mitochondrial response in dystrophic muscle. In the present commentary, we explain the significance that this pathway may have in mitochondrial dysfunction in Duchenne muscular dystrophy (DMD). We identified the upregulation of miR-379 in the serum and muscles of DMD animal models and patients. We found that miR-379 is one of very few miRNAs whose expression was normalized in DMD patients treated with glucocorticoid. We identified EIF4G2 as a miR-379 target, which may promote mitochondrial oxidative phosphorylation (OxPhos) in the skeletal muscle. We found enriched EIF4G2 expression in oxidative fibers, and identified the mitochondrial ATP synthase subunit DAPIT as a translational target of EIF4G2. The identified signaling cascade, which comprises miR-379, EIF4G2 and DAPIT, may link the glucocorticoid treatment in DMD to a recovered mitochondrial ATP synthesis rate. We propose an updated model of mitochondrial dysfunction in DMD.

Interplay between myofibers and pro-inflammatory macrophages controls muscle damage in mdx mice

ABSTRACT Duchenne muscular dystrophy is a genetic muscle disease characterized by chronic inflammation and fibrosis mediated by a pro-fibrotic macrophage population expressing pro-inflammatory markers. Our aim was to characterize cellular events leading to the alteration of macrophage properties and to modulate macrophage inflammatory status using the gaseous mediator hydrogen sulfide (H2S). Using co-culture experiments, we first showed that myofibers derived from mdx mice strongly skewed the polarization of resting macrophages towards a pro-inflammatory phenotype. Treatment of mdx mice with NaHS, an H2S donor, reduced the number of pro-inflammatory macrophages in skeletal muscle, which was associated with a decreased number of nuclei per fiber, as well as reduced myofiber branching and fibrosis. Finally, we established the metabolic sensor AMP-activated protein kinase (AMPK) as a critical NaHS target in muscle macrophages. These results identify an interplay between myofibers and macrophages where dystrophic myofibers contribute to the maintenance of a highly inflammatory environment sustaining a pro-inflammatory macrophage status, which in turn favors myofiber damage, myofiber branching and establishment of fibrosis. Our results also highlight the use of H2S donors as a potential therapeutic strategy to improve the dystrophic muscle phenotype by dampening chronic inflammation. This article has an associated First Person interview with the first author of the paper.

Mislocalisation of Pax7 and Its Interaction with Importin-α1 (KPNA2) in Dystrophin-Deficient Myoblasts

Background: Pax 7 is one of the key factors in the development of tissues and organs during embryogenesis. It has been suggested that Pax7 may play a major role during myogenesis. Our previous study has shown that Pax7 cell is attenuated in the mdx embryo during gestation as well as in dystrophic muscle indicating that an absence of dystrophin in muscle affects pax7 regulation in Duchene Muscular Dystrophy (DMD). Therefore, we aimed to investigate the Pax7 expression pattern as well as their specific transport protein in dystrophin-deficient myoblasts at postnatal/juvenile stage. Methods: In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured under normal conditions prior to further analyse its expression pattern at proliferating stage via western blot and immunofluorescence analysis. Protein prediction and protein interaction study was done via in silico and co-immunoprecipitation analyses, respectively.Results: It was found that Pax7 localised in the cytoplasm of dystrophin-deficient myoblasts and high expression retained during differentiation. Co-localisation analysis of Pax7 with subcellular markers indicated that Pax7 is highly synthesised at their proliferative state. Interestingly, it is shown that Pax7 possess a nuclear location signal and KPNA2 was suggested as an escort protein for Pax7 translocation into the nucleus. Conclusion: For the first time, our study showed that Pax7 is mislocalised in dystrophin-deficient myoblasts and it is postulated that KPNA2 is the karyopherin-α which might be responsible for Pax7 translocation into the nucleus.

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice

Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Anti-inflammatory nanoparticles significantly improve muscle function in a murine model of advanced muscular dystrophy

Chronic inflammation contributes to the pathogenesis of all muscular dystrophies. Inflammatory T cells damage muscle, while regulatory T cells (Tregs) promote regeneration. We hypothesized that providing anti-inflammatory cytokines in dystrophic muscle would promote proregenerative immune phenotypes and improve function. Primary T cells from dystrophic (mdx) mice responded appropriately to inflammatory or suppressive cytokines. Subsequently, interleukin-4 (IL-4)– or IL-10–conjugated gold nanoparticles (PA4, PA10) were injected into chronically injured, aged, mdx muscle. PA4 and PA10 increased T cell recruitment, with PA4 doubling CD4+/CD8− T cells versus controls. Further, 50% of CD4+/CD8− T cells were immunosuppressive Tregs following PA4, versus 20% in controls. Concomitant with Treg recruitment, muscles exhibited increased fiber area and fourfold increases in contraction force and velocity versus controls. The ability of PA4 to shift immune responses, and improve dystrophic muscle function, suggests that immunomodulatory treatment may benefit many genetically diverse muscular dystrophies, all of which share inflammatory pathology.

Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21 and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence associated secretory phenotype which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne Muscular Dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-week-old) and D2-mdx mice (4-week and 8-week-old), two mouse models of DMD, that cells displaying canonical markers of senescence are found within skeletal muscle. 8-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation which were mostly Cdkn1a-positive macrophages while in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wildtype (WT) muscle which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes (Cdkn1a (p21), Cdkn2a (p16INK4A), Trp53 (p53)) which correlated with the quantity of SA-b-Gal-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.