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

Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies

Altres ajuts: Association Française contre les Myopathies (22525)Altres ajuts: Fundación Isabel GemioSkeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss

Identification of serum microRNAs as potential biomarkers in Pompe disease

Altres ajuts: This study was supported by a grant from Sanofi-Genzyme (GZ-2015-11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042).This study was supported by a grant from Sanofi-Genzyme (GZ-2015-11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042).To analyze the microRNA profile in serum of patients with Adult Onset Pompe disease (AOPD). We analyzed the expression of 185 microRNAs in serum of 15 AOPD patients and five controls using microRNA PCR Panels. The expression levels of microRNAs that were deregulated were further studied in 35 AOPD patients and 10 controls using Real-Time PCR. Additionally, the skeletal muscle expression of microRNAs which showed significant increase levels in serum samples was also studied. Correlations between microRNA serum levels and muscle function test, spirometry, and quantitative muscle MRI were performed (these data correspond to the study NCT01914536 at ClinicalTrials.gov). We identified 14 microRNAs that showed different expression levels in serum samples of AOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of three microRNAs, the so called dystromirs: miR-1-3p, miR-133a-3p, and miR-206. These microRNAs are involved in muscle regeneration and the expression of these was increased in patients' muscle biopsies. Significant correlations between microRNA levels and muscle function test were found. Serum expression levels of dystromirs may represent additional biomarkers for the follow-up of AOPD patients

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

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

PDGF-BB serum levels are decreased in adult onset Pompe patients

Adult onset Pompe disease is a genetic disorder characterized by slowly progressive skeletal and respiratory muscle weakness. Symptomatic patients are treated with enzymatic replacement therapy with human recombinant alfa glucosidase. Motor functional tests and spirometry are commonly used to follow patients up. However, a serological biomarker that correlates with the progression of the disease could improve follow-up. We studied serum concentrations of TGFβ, PDGF-BB, PDGF-AA and CTGF growth factors in 37 adult onset Pompe patients and 45 controls. Moreover, all patients performed several muscle function tests, conventional spirometry, and quantitative muscle MRI using 3-point Dixon. We observed a statistically significant change in the serum concentration of each growth factor in patients compared to controls. However, only PDGF-BB levels were able to differentiate between asymptomatic and symptomatic patients, suggesting its potential role in the follow-up of asymptomatic patients. Moreover, our results point to a dysregulation of muscle regeneration as an additional pathomechanism of Pompe disease

Búsqueda de biomarcadores y estudio de mecanismos fisiopatológicos en la enfermedad de Pompe

La malaltia de Pompe d'inici tardà (EPIT) és un trastorn genètic rar produït per mutacions al gen GAA i es caracteritza per una debilitat muscular progressiva. Les biòpsies musculars de pacients amb EPIT mostren una acumulació de glucogen que s'associa a la presència de vacuoles autofàgiques que indueixen una atròfia de les fibres musculars en fases més avançades. Aquesta tesi doctoral es focalitza en dues investigacions principals. D'una banda, es va realitzar un estudi sobre els nivells de miRNA en sèrum de pacients amb EPIT per tal de trobar possibles biomarcadors de la progressió de la malaltia. D'altra banda, es van estudiar diferents mecanismes fisiopatològics als pacients amb EPIT, centrant-se en el procés d'atròfia muscular. Al primer estudi, identifiquem 14 miRNAs que van mostrar diferents nivells d'expressió en mostres de sèrum de pacients amb EPIT en comparació amb els controls. Vam validar aquests resultats en una cohort més gran de pacients i vam trobar nivells elevats de 3 miRNAs, els anomenats dystromiRs: miR-1-3p, miR-133a-3p i miR-206. Aquests miRNAs estan involucrats en la regeneració muscular i l'expressió es va trobar incrementada en biòpsies musculars de pacients amb EPIT. Es van trobar correlacions significatives entre els nivells de miRNA i la prova de funció muscular. En conclusió, els nivells d'expressió sèrica de dystromiRs podrien representar biomarcadors potencials per al seguiment de pacients amb EPIT. Al segon estudi, es va realitzar l'anàlisi de l'expressió de molècules relacionades amb l'atròfia de fibres musculars en biòpsies de pacients amb EPIT mitjançant tècniques d'immunotinció i RT-PCR. BNIP3, un atrogen conegut, es va identificar com un mediador potencial de l'atròfia en pacients amb EPIT. Observem que les fibres vacuolades en biòpsies musculars de pacients amb EPIT eren més petites que les fibres no vacuolades i que, a més, expressaven BNIP3. Les nostres dades suggereixen que l'expressió de BNIP3 es troba regulada per la inhibició de la via AKT-mTOR, fet que porta a la fosforilació de ULK1 a Ser317 per AMPK. Vam estudiar mioblasts i miotubs obtinguts de pacients amb EPIT i controls per confirmar aquests resultats utilitzant diferents tècniques moleculars. Els miotubs derivats de pacients amb EPIT també eren més petits i expressaven BNIP3. Addicionalment, la transfecció de BNIP3 a miotubs controls va conduir a l'atròfia dels miotubs. En conclusió, tots aquests resultats suggereixen una cascada que comença amb la inhibició de la via AKT-mTOR i l'activació de l'expressió de BNIP3 que, alhora, condueix a l'atròfia de les fibres musculars. Els nostres resultats obren la porta a nous tractaments potencials dirigits a BNIP3 per reduir els seus efectes nocius sobre l'atròfia de les fibres musculars a la malaltia de Pompe.La enfermedad de Pompe de inicio tardío (EPIT) es un trastorno genético raro producido por mutaciones en el gen GAA y se caracteriza por una debilidad muscular progresiva. Las biopsias musculares de pacientes con EPIT muestran una acumulación de glucógeno que se asocia a la presencia de vacuolas autofágicas induciendo en fases avanzadas una atrofia de las fibras musculares. Esta tesis doctoral se centra en dos investigaciones principales. Por un lado, se realizó un estudio sobre los niveles de miRNAs en suero de pacientes con EPIT con el fin de encontrar posibles biomarcadores de la progresión de la enfermedad. Por otro lado, se estudiaron diferentes mecanismos fisiopatológicos en los pacientes con EPIT, centrándose en el proceso de atrofia muscular. En el primer estudio, identificamos 14 miRNAs que mostraron diferentes niveles de expresión en muestras de suero de pacientes con EPIT en comparación con los controles. Validamos estos resultados en una cohorte más grande de pacientes y encontramos niveles elevados de 3 miRNAs, los llamados dystromiRs: miR-1-3p, miR-133a-3p y miR-206. Estos miRNAs están involucrados en la regeneración muscular y la expresión de éstos se encontró incrementada en biopsias musculares de pacientes con EPIT. Se encontraron correlaciones significativas entre los niveles de miRNAs y la prueba de función muscular. En conclusión, los niveles de expresión sérica de dystromiRs pueden representar biomarcadores potenciales para el seguimiento de pacientes con EPIT. En el segundo estudio, se realizó el análisis de la expresión de moléculas relacionadas con la atrofia de fibras musculares en biopsias de pacientes con EPIT mediante técnicas de inmunotinción y RT-PCR. BNIP3, un atrogén bien conocido, se identificó como un mediador potencial de la atrofia en pacientes con EPIT. Observamos que las fibras vacuoladas en biopsias musculares de pacientes con EPIT eran más pequeñas que las fibras no vacuoladas y que, además, expresaban BNIP3. Nuestros datos sugieren que la expresión de BNIP3 está regulada por la inhibición de la vía AKT-mTOR, lo que lleva a la fosforilación de ULK1 en Ser317 por AMPK. Estudiamos mioblastos y miotubos obtenidos de pacientes con EPIT y controles para confirmar estos resultados utilizando diferentes técnicas moleculares. Los miotubos derivados de pacientes con EPIT también eran más pequeños y expresaban BNIP3. Adicionalmente, la transfección de BNIP3 en miotubos de control condujo a la atrofia de los miotubos. En conclusión, todos estos hallazgos sugieren una cascada que comienza con la inhibición de la vía AKT-mTOR y la activación de la expresión de BNIP3 que, a su vez, conduce a la atrofia de las fibras musculares. Nuestros resultados abren la puerta a nuevos tratamientos potenciales dirigidos a BNIP3 para reducir sus efectos nocivos sobre la atrofia de las fibras musculares en la enfermedad de Pompe.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 a progressive accumulation of glycogen and autophagic vacuoles eventually leading to the atrophy of muscle fibers. This doctoral thesis focuses on two main investigations. A first set of experiments was performed with the aim to identify miRNAs in the serum of LOPD patients that could be biomarkers of the disease progression. A second group of experiments was performed to study the different pathophysiological mechanisms of the disease leading to muscle atrophy. In the first study, we identified 14 miRNAs that showed different expression levels in serum samples of LOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of 3 miRNAs, the so-called dystromiRs: miR-1-3p, miR-133a-3p and miR-206. These miRNAs are involved in muscle regeneration and the expression was increased in muscle biopsies from patients. Significant correlations between miRNA levels and muscle function tests were found. In conclusion, serum expression levels of dystromiRs may represent potential biomarkers for the follow-up of LOPD patients. In the second study, the expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was analyzed using immunostaining techniques and RT-PCR. BNIP3, a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. We observed that vacuolated fibers in LOPD patients' muscle biopsies were smaller than non-vacuolated fibers and expressed BNIP3. Our data suggested that BNIP3 expression is regulated by inhibition of the AKT-mTOR pathway, leading to phosphorylation of ULK1 at Ser317 by AMPK. We studied myoblasts and myotubes obtained from LOPD patients and controls 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 leads to myotube atrophy. In conclusion, all these findings suggest a cascade which starts with the inhibition of the AKT-mTOR pathway and activation of BNIP3 expression leading to progressive muscle fiber atrophy. Our results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.Universitat Autònoma de Barcelona. Programa de Doctorat en Medicin

PDGF-BB serum levels are decreased in adult onset Pompe patients

Spanish Pompe Study Group.Adult onset Pompe disease is a genetic disorder characterized by slowly progressive skeletal and respiratory muscle weakness. Symptomatic patients are treated with enzymatic replacement therapy with human recombinant alfa glucosidase. Motor functional tests and spirometry are commonly used to follow patients up. However, a serological biomarker that correlates with the progression of the disease could improve follow-up. We studied serum concentrations of TGFβ, PDGF-BB, PDGF-AA and CTGF growth factors in 37 adult onset Pompe patients and 45 controls. Moreover, all patients performed several muscle function tests, conventional spirometry, and quantitative muscle MRI using 3-point Dixon. We observed a statistically significant change in the serum concentration of each growth factor in patients compared to controls. However, only PDGF-BB levels were able to differentiate between asymptomatic and symptomatic patients, suggesting its potential role in the follow-up of asymptomatic patients. Moreover, our results point to a dysregulation of muscle regeneration as an additional pathomechanism of Pompe disease.We would like to thank the Spanish Association of Patients with Glycogenosis (www.glucogenosis.org) for their support to our investigation. This investigation was sponsored by a grant from the Spanish Ministry of Health, Fondos FEDER-ISCIII PI15/01822 to Dr. Jordi Díaz-Manera and funds from Fundacion Isabel Gemio.Peer reviewe

Identification of serum microRNAs as potential biomarkers in Pompe disease

Coinvestigators – The Spanish Pompe Study Group: Miguel Angel Barba-Romero; Joseba Barcena; María Rosario Carzorla; Carlota Creus; Jaume Coll-Canti; Noemí de Luna; Manuel Díaz; Cristina Domínguez; Roberto Fernández Torrón; María José García Antelo; Josep María Grau; María Teresa Gómez Caravaca; Juan Carlos León Hernández; Adolfo López de Munain; Francisco Antonio Martinez-García; Yolanda Morgado; Antonio Moreno; Germán Morís; Miguel Angel Muñoz-Blanco; Andres Nascimento; Carmen Paradas; José Luis Parajua Pozo; Luis Querol; Arturo Robledo-Strauss; Ricard Rojas García, Ricard; Íñigo Rojas-Marcoso; José Antonio Salazar; Mercedes Usón[Objective] To analyze the microRNA profile in serum of patients with Adult Onset Pompe disease (AOPD). [Methods] We analyzed the expression of 185 microRNAs in serum of 15 AOPD patients and five controls using microRNA PCR Panels. The expression levels of microRNAs that were deregulated were further studied in 35 AOPD patients and 10 controls using Real‐Time PCR. Additionally, the skeletal muscle expression of microRNAs which showed significant increase levels in serum samples was also studied. Correlations between microRNA serum levels and muscle function test, spirometry, and quantitative muscle MRI were performed (these data correspond to the study NCT01914536 at ClinicalTrials.gov). [Results] We identified 14 microRNAs that showed different expression levels in serum samples of AOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of three microRNAs, the so called dystromirs: miR‐1‐3p, miR‐133a‐3p, and miR‐206. These microRNAs are involved in muscle regeneration and the expression of these was increased in patients' muscle biopsies. Significant correlations between microRNA levels and muscle function test were found. [Interpretation] Serum expression levels of dystromirs may represent additional biomarkers for the follow‐up of AOPD patients.This study was supported by a grant from Sanofi‐Genzyme (GZ‐2015‐11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042)