RESUMEN
Viruses and multinucleated cells rely on fusogens to facilitate the fusion of their membranes. In this issue of Cell, Millay and colleagues demonstrate that replacing viral fusogens with mammalian skeletal muscle fusogens leads to the specific transduction of skeletal muscle and the ability to deliver gene therapy constructs in a therapeutically relevant muscle disease.
Asunto(s)
Terapia Genética , Músculo Esquelético , Virus , Animales , Fusión Celular , MamíferosRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease that is caused by the loss of functional dystrophin protein in cardiac and skeletal muscles. DMD patient muscles become weakened, leading to eventual myofiber breakdown and replacement with fibrotic and adipose tissues. Inflammation drives the pathogenic processes through releasing inflammatory cytokines and other factors that promote skeletal muscle degeneration and contributing to the loss of motor function. Selective inhibitors of nuclear export (SINEs) are a class of compounds that function by inhibiting the nuclear export protein exportin 1 (XPO1). The XPO1 protein is an important regulator of key inflammatory and neurological factors that drive inflammation and neurotoxicity in various neurological and neuromuscular diseases. Here, we demonstrate that SINE compound KPT-350 can ameliorate dystrophic-associated pathologies in the muscles of DMD models of zebrafish and mice. Thus, SINE compounds are a promising novel strategy for blocking dystrophic symptoms and could be used in combinatorial treatments for DMD.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Carioferinas/antagonistas & inhibidores , Distrofia Muscular de Duchenne/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Pez Cebra/genética , Administración Oral , Animales , Biomarcadores/sangre , Citocinas/antagonistas & inhibidores , Citocinas/sangre , Modelos Animales de Enfermedad , Locomoción/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos mdx , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación , Proteínas de Pez Cebra/genética , Proteína Exportina 1RESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.
Asunto(s)
Distrofia Muscular de Duchenne/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/fisiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Perros , Distrofina/genética , Distrofina/metabolismo , Humanos , Células Musculares/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/fisiopatología , Mutación , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Pez Cebra/metabolismoRESUMEN
Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening.
Asunto(s)
Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Pez Cebra/fisiología , Animales , Modelos Animales de Enfermedad , Cinética , Contracción Muscular , Fibras Musculares de Contracción Rápida/patología , Fibras Musculares de Contracción Lenta/patología , Análisis de Regresión , Sarcómeros/metabolismo , Tetania/fisiopatologíaRESUMEN
The role of the cellular microenvironment has recently gained attention in the context of muscle health, adaption, and disease. Emerging evidence supports major roles for the extracellular matrix (ECM) in regeneration and the dynamic regulation of the satellite cell niche. Satellite cells normally reside in a quiescent state in healthy muscle, but upon muscle injury, they activate, proliferate, and fuse to the damaged fibers to restore muscle function and architecture. This chapter reviews the composition and mechanical properties of skeletal muscle ECM and the role of these factors in contributing to the satellite cell niche that impact muscle regeneration. In addition, the chapter details the effects of satellite cell-matrix interactions and provides evidence that there is bidirectional regulation affecting both the cellular and extracellular microenvironment within skeletal muscle. Lastly, emerging methods to investigate satellite cell-matrix interactions will be presented.
Asunto(s)
Microambiente Celular , Matriz Extracelular , Músculo Esquelético , Células Satélite del Músculo Esquelético , Humanos , Animales , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/fisiología , Células Satélite del Músculo Esquelético/metabolismo , Matriz Extracelular/metabolismo , Músculo Esquelético/fisiología , Músculo Esquelético/citología , Adaptación Fisiológica , Nicho de Células Madre/fisiología , Regeneración/fisiología , Enfermedades Musculares/patología , Enfermedades Musculares/fisiopatología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Duchenne muscular dystrophy (DMD) is caused by an out-of-frame mutation in the DMD gene that results in the absence of a functional dystrophin protein, leading to a devastating progressive lethal muscle-wasting disease. Muscle stem cell-based therapy is a promising avenue for improving muscle regeneration. However, despite the efforts to deliver the optimal cell population to multiple muscles most efforts have failed. Here we describe a detailed optimized method of for the delivery of human skeletal muscle progenitor cells (SMPCs) to multiple hindlimb muscles in healthy, dystrophic and severely dystrophic mouse models. We show that systemic delivery is inefficient and is affected by the microenvironment. We found that significantly less human SMPCs were detected in healthy gastrocnemius muscle cross-sections, compared to both dystrophic and severely dystrophic gastrocnemius muscle. Human SMPCs were found to be detected inside blood vessels distinctly in healthy, dystrophic and severely dystrophic muscles, with prominent clotting identified in severely dystrophic muscles after intra arterial (IA) systemic cell delivery. We propose that muscle microenvironment and the severity of muscular dystrophy to an extent impacts the systemic delivery of SMPCs and that overall systemic stem cell delivery is not currently efficient or safe to be used in cell based therapies for DMD. This work extends our understanding of the severe nature of DMD, which should be taken into account when considering stem cell-based systemic delivery platforms.
RESUMEN
Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human-mouse myofibres in vivo. However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches. We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment. Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1. Similarly, ACTC1+ human myofibres supporting PAX7+ SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches. To demonstrate ACTC1+ myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1+ myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls. We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1+ myofibres and PAX7+ SMPCs in vivo. This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.
Asunto(s)
Músculo Esquelético , Factor de Transcripción PAX7 , Regeneración , Células Satélite del Músculo Esquelético , Animales , Humanos , Ratones , Músculo Esquelético/fisiología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Células Madre Pluripotentes , Células Satélite del Músculo Esquelético/fisiologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the dystrophin gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient sapje and sapje-like zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.
Asunto(s)
Distrofina/deficiencia , Serotonina/metabolismo , Pez Cebra/metabolismo , Animales , Birrefringencia , Evaluación Preclínica de Medicamentos , Distrofina/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Receptores de Serotonina , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Análisis de SupervivenciaRESUMEN
Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s-1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18-24% faster Umax of adult males vs. females, and a 14-20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.
Asunto(s)
Modelos Biológicos , Programas Informáticos , Natación/fisiología , Pez Cebra/fisiología , AnimalesRESUMEN
Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.
Asunto(s)
Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Distrofia Muscular de Cinturas/tratamiento farmacológico , Distrofia Muscular de Cinturas/fisiopatología , Distrofias Musculares/tratamiento farmacológico , Distrofias Musculares/fisiopatología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Técnicas de Inactivación de Genes , Humanos , Locomoción , Movimiento , Músculo Esquelético/fisiopatología , Distrofias Musculares/genética , Distrofia Muscular de Cinturas/genética , Mutación , Pentosiltransferasa , Fenotipo , Proteínas , Transcriptoma , Síndrome de Walker-Warburg , Pez CebraRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-a (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.