Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

Duchenne muscle dystrophy (DMD) is a genetic disorder characterized by progressive skeletal muscle weakness. Dystrophin deficiency induces instability of the sarcolemma during muscle contraction that leads to muscle necrosis and replacement of muscle by fibro-adipose tissue. Several therapies have been developed to counteract the fibrotic process. We report the effects of nintedanib, a tyrosine kinase inhibitor, in the mdx murine model of DMD. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1, and PDGFA. We treated seven mdx mice with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, nintedanib reduced the fibrotic process in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential use as a therapeutic drug in DMD patients.España, Ministerio de Economía y Competitividad BFU2016-74975-PEspaña, Instituto Ramón y Cajal PI13/0134

Hypoxia triggers IFN-I production in muscle: Implications in dermatomyositis

Dermatomyositis is an inflammatory myopathy characterized by symmetrical proximal muscle weakness and skin changes. Muscle biopsy hallmarks include perifascicular atrophy, loss of intramuscular capillaries, perivascular and perimysial inflammation and the overexpression of IFN-inducible genes. Among them, the retinoic-acid inducible gene 1 (RIG-I) is specifically overexpressed in perifascicular areas of dermatomyositis muscle. The aim of this work was to study if RIG-I expression may be modulated by hypoxia using an in vitro approach. We identified putative hypoxia response elements (HRE) in RIG-I regulatory regions and luciferase assays confirmed that RIG-I is a new HIF-inducible gene. We observed an increase expression of RIG-I both by Real time PCR and Western blot in hypoxic conditions in human muscle cells. Cell transfection with a constitutive RIG-I expression vector increased levels of phospho-IRF-3, indicating that RIG-I promotes binding of transcription factors to the enhancer sequence of IFN. Moreover, release of IFN-beta was observed in hypoxic conditions. Finally, HIF-1 alpha overexpression was confirmed in the muscle biopsies and in some RIG-I positive perifascicular muscle fibres but not in controls. Our results indicate that hypoxia triggers the production of IFN-I in vitro, and may contribute to the pathogenesis of DM together with other inflammatory factors

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy

Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Dystrophinopathies

Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked muscular disorders produced by mutations in the DMD gene which encodes the protein dystrophin. Both diseases are characterized by progressive involvement of skeletal, cardiac, and respiratory muscles. As new treatment strategies become available, reliable biomarkers and outcome measures that can monitor disease progression are needed for clinical trials. Methods: We collected clinical and functional data and blood samples from 19 DMD patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the serum concentration of 4 growth factors involved in the process of muscle fibrosis. We correlated the serum concentration of these growth factors with several muscle function tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI. Results: We found significant differences in the serum concentration of Platelet Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, and in and Transforming Growth Factor- β1 (TGF-β1) between BMD and DYSF patients. PDGF-AA showed a good correlation with several muscle function tests for both DMD and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies. Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases

RIG-I expression in perifascicular myofibers is a reliable biomarker of dermatomyositis

Background: Dermatomyositis (DM) is inflammatory myopathy or myositis characterized by muscle weakness and skin manifestations. In the differential diagnosis of DM the evaluation of the muscle biopsy is of importance among other parameters. Perifascicular atrophy in the muscle biopsy is considered a hallmark of DM. However, perifascicular atrophy is not observed in all patients with DM and, conversely, perifascicular atrophy can be observed in other myositis such as antisynthetase syndrome (ASS), complicating DM diagnosis. Retinoic acid inducible-gene I (RIG-I), a receptor of innate immunity that promotes type I interferon, was observed in perifascicular areas in DM. We compared the value of RIG-I expression with perifascicular atrophy as a biomarker of DM. Methods: We studied by immunohistochemical analysis the expression of RIG-I and the presence of perifascicular atrophy in 115 coded muscle biopsies: 44 patients with DM, 18 with myositis with overlap, 8 with ASS, 27 with non-DM inflammatory myopathy (16 with polymyositis, 6 with inclusion body myositis, 5 with immune-mediated necrotizing myopathy), 8 with muscular dystrophy (4 with dysferlinopathy, 4 with fascioscapulohumeral muscle dystrophy) and 10 healthy controls. Results: We found RIG-I-positive fibers in 50% of DM samples vs 11% in non-DM samples (p < 0.001). Interestingly, RIG-I staining identified 32% of DM patients without perifascicular atrophy (p = 0.007). RIG-I sensitivity was higher than perifascicular atrophy (p < 0.001). No differences in specificity between perifascicular atrophy and RIG-I staining were found (92% vs 88%). RIG-I staining was more reproducible than perifascicular atrophy (κ coefficient 0.52 vs 0.37). Conclusions: The perifascicular pattern of RIG-I expression supports the diagnosis of DM. Of importance for clinical and therapeutic studies, the inclusion of RIG-I in the routine pathological staining of samples in inflammatory myopathy will allow us to gather more homogeneous subgroups of patients in terms of immunopathogenesis

BNIP3 Is Involved in Muscle Fiber Atrophy in Late-Onset Pompe Disease Patients

Late-onset Pompe disease (LOPD) is a rare genetic disorder produced by mutations in the GAA gene and is characterized by progressive muscle weakness. LOPD muscle biopsies show accumulation of glycogen along with the autophagic vacuoles associated with atrophic muscle fibers. The expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was studied using immunofluorescence and real-time PCR. BCL2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. Vacuolated fibers in LOPD patient muscle biopsies were smaller than nonvacuolated fibers and expressed BNIP3. The current data suggested that BNIP3 expression is regulated by inhibition of the AKT-mammalian target of rapamycin pathway, leading to phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) at Ser317 by AMP-activated protein kinase. Myoblasts and myotubes obtained from LOPD patients and age-matched controls were studied to confirm these results using different molecular techniques. Myotubes derived from LOPD patients were likewise smaller and expressed BNIP3. Conclusively, transfection of BNIP3 into control myotubes led to myotube atrophy. These findings suggest a cascade that starts with the inhibition of the AKT-mammalian target of rapamycin pathway and activation of BNIP3 expression, leading to progressive muscle fiber atrophy. These results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.Peer reviewe

Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Dystrophinopathies

Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked muscular disorders produced by mutations in the DMD gene which encodes the protein dystrophin. Both diseases are characterized by progressive involvement of skeletal, cardiac, and respiratory muscles. As new treatment strategies become available, reliable biomarkers and outcome measures that can monitor disease progression are needed for clinical trials.Methods: We collected clinical and functional data and blood samples from 19 DMD patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the serum concentration of 4 growth factors involved in the process of muscle fibrosis. We correlated the serum concentration of these growth factors with several muscle function tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI.Results: We found significant differences in the serum concentration of Platelet Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, and in and Transforming Growth Factor- β1 (TGF-β1) between BMD and DYSF patients. PDGF-AA showed a good correlation with several muscle function tests for both DMD and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies.Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases

Autoantibody screening in Guillain-Barré syndrome

Background: Guillain-Barré syndrome (GBS) is an acute inflammatory neuropathy with a heterogeneous presentation. Although some evidences support the role of autoantibodies in its pathogenesis, the target antigens remain unknown in a substantial proportion of GBS patients. The objective of this study is to screen for autoantibodies targeting peripheral nerve components in Guillain-Barré syndrome. Methods: Autoantibody screening was performed in serum samples from all GBS patients included in the International GBS Outcome study by 11 different Spanish centres. The screening included testing for anti-ganglioside antibodies, anti-nodo/paranodal antibodies, immunocytochemistry on neuroblastoma-derived human motor neurons and murine dorsal root ganglia (DRG) neurons, and immunohistochemistry on monkey peripheral nerve sections. We analysed the staining patterns of patients and controls. The prognostic value of anti-ganglioside antibodies was also analysed. Results: None of the GBS patients (n = 100) reacted against the nodo/paranodal proteins tested, and 61 (61%) were positive for, at least, one anti-ganglioside antibody. GBS sera reacted strongly against DRG neurons more frequently than controls both with IgG (6% vs 0%; p = 0.03) and IgM (11% vs 2.2%; p = 0.02) immunodetection. No differences were observed in the proportion of patients reacting against neuroblastoma-derived human motor neurons. Reactivity against monkey nerve tissue was frequently detected both in patients and controls, but specific patterns were only detected in GBS patients: IgG from 13 (13%) patients reacted strongly against Schwann cells. Finally, we confirmed that IgG anti-GM1 antibodies are associated with poorer outcomes independently of other known prognostic factors. Conclusion: Our study confirms that (1) GBS patients display a heterogeneous repertoire of autoantibodies targeting nerve cells and structures; (2) gangliosides are the most frequent antigens in GBS patients and have a prognostic value; (3) further antigen-discovery experiments may elucidate other potential antigens in GBS

A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb-girdle muscular dystrophy, we identified a missense mutation in 1 (protein O -glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O -glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle-specific α-dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent 7 + cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch-dependent loss of satellite cells

Nous aspectes fisiopatogènics en la dermatomiositis

Les miopaties inflamatòries són un grup de malalties neuromusculars molt heterogeni que es caracteritzen clínicament per la presència de debilitat muscular que pot arribar a ser invalidant. Aquest grup de malalties inclou la dermatomiositis (DM), la polimiositis, la miopatia per cossos d’inclusió i recentment s’hi ha inclòs la miopatia necrotitzant autoimmune. Per arribar a un diagnòstic precís és necessari l’estudi de la biòpsia muscular entre d’altres paràmetres clínics. La biòpsia muscular d’aquest grup de pacients es caracteritza per la presència d’infiltrats inflamatoris que fenotípicament i quantitativament varia a cada entitat. A més, s’observa la sobreexpressió del complexe major d’histocompatibilitat de classe I (MHC-I) a les fibres musculars havent-li atribuït un paper patogènic. Fins ara no hi ha evidències de l’etiologia de la DM, havent-hi dues hipòtesis principals: la primera considera que la malaltia comença amb un atac autoimmune contra un antigen desconegut de l’endoteli muscular provocant la destrucció dels capil•lars i una hipòxia tissular. De fet a les biòpsies de pacients amb DM és característica una disminució dels capil·lars que, com a conseqüència, es pensa que provoca la típica atròfia perifascicular. La segona atribueix un paper principal a l’ interferó (IFN) ja que múltiples gens induïts per IFN s’han trobat sobreregulats a la DM. L’origen de l’IFN s’ha atribuït als infiltrats inflamatoris. Per estudiar específicament la contribució del múscul a la patogènia d’aquestes malalties, evitant l’efecte confusor dels infiltrats inflamatoris, vam aïllar fibres musculars patològiques mitjançant microdissecció làser a partir de biòpsies musculars d’aquests pacients. L’anàlisi transcriptòmic de les fibres aïllades va demostrar una contribució específicament a la DM de la immunitat innata, de l’IFN de tipus I (IFN-I) i de la hipòxia. En particular, es va observar una sobreregulació de RIG-I a la DM, un receptor de la immunitat innata induïble per IFN i que fisiològicament reconeix àcids nucleics vírics per iniciar una resposta antivírica a través de IFN-I. Els estudis de immunofluorescència van demostrar que la sobreexpressió de RIG-I colocalitzava amb fibres MHC-I positives a la DM. Els estudis in vitro utilitzant miotubs humans va demostrar la capacitat de RIG-I d’induïr una resposta de IFNβ –un subtipus de IFN-I- i com a conseqüència es sobreexpressava MHC-I i altre vegada RIG-I suggerint un mecanisme inflamatori auto-sustentat a la DM. A partir d’aquest primer resultat l’estudi va evolucionar al paper de la hipòxia en relació a la immunitat innata. La hipòxia està modulada principalment per un factor de transcripció clau anomenat HIF1α que indueix l’expressió de gens que continguin al seu promotor unes seqüències específiques. Els estudis in silico i in vitro van demostrar que RIG-I és un gen induït per HIF1α. Els resultats experimentals conclouen que la hipòxia és un fenomen primerenc en la seqüència d’esdeveniment fisiopatogènics en la DM ja que la sobreexpressió de RIG-I era capaç d’induir la seva activació i per tant, de promoure l’expressió d’IFN-I i en canvi, l’expressió de HIF1α no es modificava per l’estimulació amb interferons. Finalment es va avaluar la utilitat de RIG-I com a biomarcador histològic per la DM. A partir de 115 biòpsies musculars incloent malalties neuromusculars que podien confondre’s clinicament o histologicament amb la DM, vam demostrar que RIG-I és un biomarcador mes sensible que la atrofia perifascicular per la DM (50% a la DM vs 11% a la resta). De fet, en els pacients amb DM on a la biòpsia l’atròfia perifascicular no era evident, un 32% dels pacients eren RIG-I positius. Analitzant la reproductibilitat de la interpretació de l’atròfia perifascicular i RIG-I, vam trobar que RIG-I era un biomarcador més fàcilment interpretable que la atrofia perifascicular. Com a conclusió, aquesta tesi unifica les dues hipòtesis patogèniques actuals sobre la DM on la hipòxia es el primer esdeveniment que a través de RIG-I explica la signatura de IFN-I.Inflammatory myopathies are an heterogeneous group of neuromuscular diseases that are clinically characterized by the presence of muscle weakness that leads to disability. This group of diseases includes dermatomyositis (DM), polymyositis, inclusion body myositis and recently, necrotizing autoimmune myopathies have also been included. The study of the muscle biopsy, among other parameters, is necessary to obtain an accurate diagnosis. The muscle biopsy of these patients is characterized by the presence of inflammatory infiltrates that vary phenotypically and quantitatively in each entity. However, overexpression of the major histocompatibility complex class I (MHC-I) is shared by all these entities and it is believed that it has a pathogenic role. Until now, the etiology of DM is a matter of debate but there are two main hypotheses: The first hypothesis considers that the disease starts with an autoimmune attack against unknown antigens in the endothelium leading to the destruction of capillaries promoting, eventually, tissular hypoxia and muscle fiber atrophy. In fact, in the muscle biopsies of DM patients there is a characteristic reduction in the number of capillaries and perifascicular atrophy. The second hypothesis is based on the role of type I interferons (IFN-I). Experiments of gene expression analysis have demonstrated upregulation of multiple IFN-I-induced genes in DM. The origin of this IFN-I has been attributed to perimysial inflammatory infiltrates. To study the pathological mechanisms that occur specifically in the muscle fibers we isolated MHC-I positive muscle fibers using laser-microdissection from the muscle biopsies of these patients. This technology allowed us to exclude inflammatory infiltrates that could interfere with the results. The transcriptomic profile of the isolated fibers demonstrated a significant contribution of innate immunity, IFN-I and hypoxia specifically in DM. In particular, we observed the upregulation of RIG-I in DM, a receptor of the innate immunity that recognizes nucleic acids derived from virus to initiate an antiviral response and promoting IFN-I. Immunofluorescence studies demonstrated that RIG-I is overexpressed in MHC-I-expressing fibers in DM. In vitro studies using human myotubes showed the ability of RIG-I to induce the secretion of IFNβ – an IFN-I subtype – and as a consequence, induced the expression of MHC-I and RIG-I itself, suggesting a self sustained autoimmune mechanism in DM. These results prompted us to evaluate the relation between hypoxia and innate immunity. Hypoxia is mainly modulated by a key transcription factor named HIF1α that induces the expression of genes that contain specific sequences in its promoter. Our studies in silico and in vitro demonstrated that RIG-I is an HIF-inducible gene. From these studies, we concluded that hypoxia is a primary event in the pathogenic sequence of events in DM because the overexpression of RIG-Iwas able to induce its own activation promoting the expression of IFN-I-stimulated genes. In contrast, the expression of HIF1α was not modified by the stimulation of IFNs. Finally we evaluated the utility of RIG-I as a histological biomarker for DM. We studied 115 muscle biopsies using immunohistochemistry including diseases that may be confused clinically or histologically with DM. We demonstrated that RIG-I has a higher sensitivity than perifascicular atrophy for DM diagnosis (50% in DM vs 11 % in non-DM). Interestingly, 32% of patients with DM in whom perifascicular atrophy was not evident, showed expression of RIG-I at the perifascicular areas. We analyzed the reproducibility of perifascicular atrophy and RIG-I staining and we found that RIG-I has a higher reproducibility and therefore constitutes a more easily interpretable biomarker than perifascicular atrophy. We conclude that this thesis unifies both current hypotheses on DM pathogenesis and shows that hypoxia contributes to the pathology of DM by activating the RIG-I signaling pathway and consequently inducing IFN-I expression