RESUMEN
Duchenne muscular dystrophy is caused by the loss of functional dystrophin that secondarily causes systemic metabolic impairment in skeletal muscles and cardiomyocytes. The nutraceutical approach is considered as a possible complementary therapy for this pathology. In this work, we have studied the effect of pyrimidine nucleoside uridine (30 mg/kg/day for 28 days, i.p.), which plays an important role in cellular metabolism, on the development of DMD in the skeletal muscles of dystrophin deficient mdx mice, as well as its effect on the mitochondrial dysfunction that accompanies this pathology. We found that chronic uridine administration reduced fibrosis in the skeletal muscles of mdx mice, but it had no effect on the intensity of degeneration/regeneration cycles and inflammation, pseudohypetrophy, and muscle strength of the animals. Analysis of TEM micrographs showed that uridine also had no effect on the impaired mitochondrial ultrastructure of mdx mouse skeletal muscle. The administration of uridine was found to lead to an increase in the expression of the Drp1 and Parkin genes, which may indicate an increase in the intensity of organelle fission and the normalization of mitophagy. Uridine had little effect on OXPHOS dysfunction in mdx mouse mitochondria, and moreover, it was suppressed in the mitochondria of wild type animals. At the same time, uridine restored the transport of potassium ions and reduced the production of reactive oxygen species; however, this had no effect on the impaired calcium retention capacity of mdx mouse mitochondria. The obtained results demonstrate that the used dose of uridine only partially prevents mitochondrial dysfunction in skeletal muscles during Duchenne dystrophy, though it mitigates the development of destructive processes in skeletal muscles.
Asunto(s)
Distrofia Muscular de Duchenne , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Distrofina/metabolismo , Ratones , Ratones Endogámicos mdx , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Uridina/metabolismo , Uridina/farmacologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.
Asunto(s)
Distrofina , Metformina , Animales , Distrofina/genética , Terapia Genética/métodos , Glicina/uso terapéutico , Humanos , Metformina/uso terapéutico , Ratones , Ratones Endogámicos mdx , Morfolinos/genética , Morfolinos/uso terapéutico , Músculo Esquelético , Utrofina/genéticaRESUMEN
BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.
Asunto(s)
Sobrecarga de Hierro , Distrofia Muscular de Duchenne , Animales , Deferiprona , Distrofina/genética , Ferritinas , Fibrosis , Hemo/metabolismo , Hierro/metabolismo , Quelantes del Hierro , Sobrecarga de Hierro/etiología , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Protein lysine acetylation is a post-translational modification that regulates protein structure and function. It is targeted to proteins by lysine acetyltransferases (KATs) or removed by lysine deacetylases. This work identifies a role for the KAT enzyme general control of amino acid synthesis protein 5 (GCN5; KAT2A) in regulating muscle integrity by inhibiting DNA binding of the transcription factor/repressor Yin Yang 1 (YY1). Here we report that a muscle-specific mouse knockout of GCN5 (Gcn5skm-/-) reduces the expression of key structural muscle proteins, including dystrophin, resulting in myopathy. GCN5 was found to acetylate YY1 at two residues (K392 and K393), disrupting the interaction between the YY1 zinc finger region and DNA. These findings were supported by human data, including an observed negative correlation between YY1 gene expression and muscle fiber diameter. Collectively, GCN5 positively regulates muscle integrity through maintenance of structural protein expression via acetylation-dependent inhibition of YY1. This work implicates the role of protein acetylation in the regulation of muscle health and for consideration in the design of novel therapeutic strategies to support healthy muscle during myopathy or aging.
Asunto(s)
Distrofina/genética , Músculos/metabolismo , Factor de Transcripción YY1/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Envejecimiento/metabolismo , Animales , ADN/metabolismo , Distrofina/metabolismo , Regulación de la Expresión Génica , Humanos , Lisina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Músculos/patología , Músculos/ultraestructura , Atrofia Muscular/patología , Distrofias Musculares/patología , Transcriptoma/genética , Factores de Transcripción p300-CBP/deficienciaRESUMEN
Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited neuromuscular disorder that causes loss of muscle mass and motor skills. In the era of genomic medicine, there is still no known cure for DMD. In clinical practice, there is a growing awareness of the possible importance of nutrition in neuromuscular diseases. This is mostly the result of patients' or caregivers' empirical reports of how active substances derived from food have led to improved muscle strength and, thus, better quality of life. In this report, we investigate several nutraceutical principles in the sapje strain of zebrafish, a validated model of DMD, in order to identify possible natural products that, if supplemented in the diet, might improve the quality of life of DMD patients. Gingerol, a constituent of fresh ginger, statistically increased the locomotion of mutant larvae and upregulated the expression of heme oxygenase 1, a target gene for therapy aimed at improving dystrophic symptoms. Although three other compounds showed a partial positive effect on locomotor and muscle structure phenotypes, our nutraceutical screening study lent preliminary support to the efficacy and safety only of gingerol. Gingerol could easily be proposed as a dietary supplement in DMD.
Asunto(s)
Catecoles/administración & dosificación , Suplementos Dietéticos , Alcoholes Grasos/administración & dosificación , Distrofia Muscular de Duchenne/dietoterapia , Animales , Fibras de la Dieta , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Femenino , Hemo-Oxigenasa 1 , Larva , Locomoción , Masculino , Fuerza Muscular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Calidad de Vida , Pez CebraRESUMEN
BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). METHODS: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. RESULTS: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. CONCLUSIONS: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Metaboloma , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , NAD/metabolismo , Niacinamida/análogos & derivados , Compuestos de Piridinio/farmacología , ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , Animales , Distrofina/deficiencia , Inhibidores Enzimáticos/uso terapéutico , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Contracción Muscular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Niacinamida/farmacología , Niacinamida/uso terapéutico , Compuestos de Piridinio/uso terapéuticoRESUMEN
OBJECTIVE: To compare the effects of photobiomodulation therapy (PBMT) and pharmacological therapy (glucocorticoids and non-steroidal anti-inflammatory drugs) applied alone and in different combinations in mdx mice. METHODS: The animals were randomized and divided into seven experimental groups treated with placebo, PBMT, prednisone, non-steroidal anti-inflammatory drug (NSAIDs), PBMT plus prednisone and PBMT plus NSAID. Wild type animals were used as control. All treatments were performed during 14 consecutive weeks. Muscular morphology, protein expression of dystrophin and functional performance were assessed at the end of the last treatment. RESULTS: Both treatments with prednisone and PBMT applied alone or combined, were effective in preserving muscular morphology. In addition, the treatments with PBMT (p = 0.0005), PBMT plus prednisone (p = 0.0048) and PBMT plus NSAID (p = 0.0021) increased dystrophin gene expression compared to placebo-control group. However, in the functional performance the PBMT presented better results compared to glucocorticoids (p<0.0001). In contrast, the use of NSAIDs did not appear to add benefits to skeletal muscle tissue in mdx mice. CONCLUSION: We believe that the promising and optimistic results about the PBMT in skeletal muscle of mdx mice may in the future contribute to this therapy to be considered a safe alternative for patients with Duchenne Muscular Dystrophy (DMD) in a washout period (between treatment periods with glucocorticoids), allowing them to remain receiving effective and safe treatment in this period, avoiding at this way periods without administration of any treatment.
Asunto(s)
Distrofina/genética , Terapia por Luz de Baja Intensidad , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/efectos de la radiación , Distrofia Muscular de Duchenne/terapia , Animales , Antiinflamatorios no Esteroideos/farmacología , Terapia Combinada , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Glucocorticoides/farmacología , Humanos , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Prednisona/farmacologíaRESUMEN
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
: Duchenne muscular dystrophy (DMD) is one of the most severe forms of inherited muscular dystrophies. The disease is caused by the lack of dystrophin, a structurally essential protein; hence, a definitive cure would necessarily have to pass through some form of gene and/or cell therapy. Cell- and genetic-based therapeutics for DMD have been explored since the 1990s and recently, two of the latter have been approved for clinical use, but their efficacy is still very low. In parallel, there have been great ongoing efforts aimed at targeting the downstream pathogenic effects of dystrophin deficiency using classical pharmacological approaches, with synthetic or biological molecules. However, as it is always the case with rare diseases, R&D costs for new drugs can represent a major hurdle for researchers and patients alike. This problem can be greatly alleviated by experimenting the use of molecules that had originally been developed for different conditions, a process known as drug repurposing or drug repositioning. In this review, we will describe the state of the art of such an approach for DMD, both in the context of clinical trials and pre-clinical studies.
Asunto(s)
Reposicionamiento de Medicamentos/métodos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Fármacos Neuromusculares/uso terapéutico , Prednisona/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Distrofina/deficiencia , Distrofina/genética , Gentamicinas/uso terapéutico , Humanos , Metformina/uso terapéutico , Ratones Transgénicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Pregnenodionas/uso terapéutico , Simvastatina/uso terapéutico , Tadalafilo/uso terapéutico , Tamoxifeno/uso terapéuticoRESUMEN
Although the lack of dystrophin expression in muscle myofibers is the central cause of Duchenne muscular dystrophy (DMD), accumulating evidence suggests that DMD may also be a stem cell disease. Recent studies have revealed dystrophin expression in satellite cells and demonstrated that dystrophin deficiency is directly related to abnormalities in satellite cell polarity, asymmetric division, and epigenetic regulation, thus contributing to the manifestation of the DMD phenotype. Although metabolic and mitochondrial dysfunctions have also been associated with the DMD pathophysiology profile, interestingly, the role of dystrophin with respect to stem cells dysfunction has not been elucidated. In the past few years, editing of the gene that encodes dystrophin has emerged as a promising therapeutic approach for DMD, although the effects of dystrophin restoration in stem cells have not been addressed. Herein, we describe our use of a clustered regularly interspaced short palindromic repeats/Cas9-based system to correct the dystrophin mutation in dystrophic (mdx) muscle progenitor cells (MPCs) and show that the expression of dystrophin significantly improved cellular properties of the mdx MPCs in vitro. Our findings reveal that dystrophin-restored mdx MPCs demonstrated improvements in cell proliferation, differentiation, bioenergetics, and resistance to oxidative and endoplasmic reticulum stress. Furthermore, our in vivo studies demonstrated improved transplantation efficiency of the corrected MPCs in the muscles of mdx mice. Our results indicate that changes in cellular energetics and stress resistance via dystrophin restoration enhance muscle progenitor cell function, further validating that dystrophin plays a role in stem cell function and demonstrating the potential for new therapeutic approaches for DMD. Stem Cells 2019;37:1615-1628.
Asunto(s)
Distrofina/genética , Terapia Genética/métodos , Fibras Musculares Esqueléticas/patología , Distrofia Muscular de Duchenne/terapia , Células Satélite del Músculo Esquelético/patología , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Polaridad Celular/fisiología , Proliferación Celular/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Estrés del Retículo Endoplásmico/genética , Metabolismo Energético/genética , Epigénesis Genética , Edición Génica , Ratones , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Estrés Oxidativo/genética , Células Madre/fisiologíaRESUMEN
Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD+-dependent deacetylase, the depleted NAD+ in dystrophic skeletal muscle may limit quercetin efficacy; hence, supplementation with the NAD+ donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice; therefore, it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR, or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-mo-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed after treatment with Q, NR, and/or Lis for 7 mo. To mimic typical pharmacology of patients with Duchenne muscular dystrophy, a group was treated with prednisolone (Pred) in combination with Q, NR, and Lis. At 11 mo of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, whereas fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest that treatment with Q, NR, Lis, and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological damage.NEW & NOTEWORTHY Despite a compelling rationale and previous evidence to the contrary in short-term investigations, quercetin, nicotinamide riboside, or Lisinopril, alone or in combination, failed to restore muscle function or decrease histological injury in dystrophic limb muscle from D2-mdx mice after long-term administration. Importantly, we also found that in the D2-mdx model, an emerging and relatively understudied model of Duchenne muscular dystrophy dystrophin deficiency caused profound muscle dysfunction and histopathology in skeletal muscle.
Asunto(s)
Músculo Esquelético/efectos de los fármacos , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Preparaciones Farmacéuticas/administración & dosificación , Animales , Suplementos Dietéticos , Modelos Animales de Enfermedad , Distrofina/farmacología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos mdx , Contracción Muscular/efectos de los fármacos , Quercetina/farmacologíaRESUMEN
Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2'-Fluoro (2'-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2'-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2'-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2'-O-methyl (2'-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2'-F nucleotides induced efficient exon-23 skipping, with LNA/2'-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2'-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2'-OMe/2'-F chimeras. Overall, our findings certainly expand the scope of constructing 2'-F modified AOs in splice modulation by incorporating 2'-OMe and LNA modifications.
Asunto(s)
Fibras Musculares Esqueléticas/efectos de los fármacos , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/farmacología , Empalme del ARN/efectos de los fármacos , Animales , Células Cultivadas , Técnicas de Química Sintética/economía , Técnicas de Química Sintética/métodos , Química Farmacéutica/economía , Química Farmacéutica/métodos , Evaluación Preclínica de Medicamentos , Distrofina/genética , Distrofina/metabolismo , Exones/efectos de los fármacos , Exones/genética , Terapia Genética/economía , Terapia Genética/métodos , Humanos , Ratones , Ratones Endogámicos mdx , Morfolinos/economía , Morfolinos/uso terapéutico , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Oligonucleótidos/química , Oligonucleótidos/economía , Oligonucleótidos/farmacología , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/economía , Oligonucleótidos Antisentido/uso terapéuticoRESUMEN
Dp71 is the major form of dystrophins (Dp) in the supraoptic nucleus (SON) and in the neural lobe of hypophysis (NL/HP). Dp71-null mice exhibit a hypo-osmolar status attributed to an altered osmosensitivity of the SON and to a perturbed vasopressinergic axis. Because oxytocin (OT) is implicated in osmoregulation via natriuresis, this study explored the oxytocinergic axis in Dp71-null mice after salt-loading (SL). Under normosmolar conditions, OT-mRNA expression was higher in the Dp71-null SON compared to wild-type (wt) and the OT peptide level has not changed. Dp-immunostaining was localized in astrocytes end-feet surrounding vessels in wt SON. This distribution changed in Dp71-null SON, Dp being detected in OT-soma of MCNs. nNOS and NADPH-diaphorase levels increased in the OT area of the Dp71-null SON compared to wt. In the NL/HP, OT level reduced in Dp71-null mice and Dp localization changed from pituicytes end-feet in wt SON to OT terminals in Dp71-null SON. Salt-Loading resulted in an increase of OT-mRNA and peptide levels in wt SON but had no effect in Dp71-null SON. In the NL/HP, OT content was reduced after SL. For Dp71-null mice, OT level, already low in control, was not modified by SL. Dp level was not affected by SL in the SON nor in the NL/HP. Our data confirmed the importance of Dp71 for the SON functionality in osmoregulation. The localization of Dp71 at the glial-vascular interface could be associated with SON osmosensitivity, leading to an adequate OT synthesis in the SON and release from the NL/HP upon plasmatic hyperosmolality.
Asunto(s)
Distrofina/deficiencia , Hipotálamo/metabolismo , Osmorregulación/fisiología , Oxitocina/metabolismo , Animales , Distrofina/metabolismo , Ratones Noqueados , NADPH Deshidrogenasa/metabolismo , Neuronas/metabolismo , Oxitocina/genética , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Supraóptico/metabolismoRESUMEN
BACKGROUND: Duchenne muscular dystrophy (Duchenne) is caused by pathogenic variants in the DMD gene. Antisense oligonucleotides (AONs) are one emerging precision medicine treatment for Duchenne. DMD molecular genetic testing results guide precision-therapy molecular eligibility, requiring healthcare providers to perform analyses currently uncommon in clinical laboratory and medical practices. Clear DMD variant notation and interpretation are key components of clinical care with the availability of precision medicine. METHODS: The DMD Open-access Variant Explorer (DOVE) is a web-based aid for DMD variant interpretation which additionally reports variant-specific predicted molecular eligibility for therapy. DOVE was developed in Python and adapted to the Django Web framework, integrates existing open-access tools, and does not rely on previous variant report/classification. RESULTS: DOVE [www.dmd.nl/DOVE] interprets colloquial and HGMD inputs of DMD variants to output HGMD variant nomenclature, theoretical molecular eligibility for therapy, and any predicted deleterious molecular consequences of therapy. DOVE relies on holistic in silico prediction of molecular eligibility for therapy in lieu of reference to an empirically defined, "variant-eligible" list. Examples illustrate the advantage and necessity for holistic variant interpretation. CONCLUSION: DOVE may prove useful for variant interpretation both at patient-level and in large-scale programs such as newborn screening and has broad application in concept to molecular genetic test result interpretation.
Asunto(s)
Distrofina/genética , Pruebas Genéticas/métodos , Estudio de Asociación del Genoma Completo/métodos , Distrofia Muscular de Duchenne/genética , Polimorfismo de Nucleótido Simple , Programas Informáticos , Humanos , Distrofia Muscular de Duchenne/patologíaRESUMEN
BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, results in chronic inflammation and irreversible skeletal muscle degeneration. Moreover, the associated impairment of autophagy greatly contributes to the aggravation of muscle damage. We explored the possibility of using non-euphoric compounds present in Cannabis sativa, cannabidiol (CBD), cannabidivarin (CBDV) and tetrahydrocannabidivarin (THCV), to reduce inflammation, restore functional autophagy and positively enhance muscle function in vivo. EXPERIMENTAL APPROACH: Using quantitative PCR, western blots and [Ca2+ ]i measurements, we explored the effects of CBD and CBDV on the differentiation of both murine and human skeletal muscle cells as well as their potential interaction with TRP channels. Male dystrophic mdx mice were injected i.p. with CBD or CBDV at different stages of the disease. After treatment, locomotor tests and biochemical analyses were used to evaluate their effects on inflammation and autophagy. KEY RESULTS: CBD and CBDV promoted the differentiation of murine C2C12 myoblast cells into myotubes by increasing [Ca2+ ]i mostly via TRPV1 activation, an effect that undergoes rapid desensitization. In primary satellite cells and myoblasts isolated from healthy and/or DMD donors, not only CBD and CBDV but also THCV promoted myotube formation, in this case, mostly via TRPA1 activation. In mdx mice, CBD (60 mg·kg-1 ) and CBDV (60 mg·kg-1 ) prevented the loss of locomotor activity, reduced inflammation and restored autophagy. CONCLUSION AND IMPLICATIONS: We provide new insights into plant cannabinoid interactions with TRP channels in skeletal muscle, highlighting a potential opportunity for novel co-adjuvant therapies to prevent muscle degeneration in DMD patients. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.
Asunto(s)
Cannabidiol/farmacología , Cannabinoides/farmacología , Cannabis/química , Dronabinol/análogos & derivados , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Mioblastos/efectos de los fármacos , Animales , Calcio/metabolismo , Cannabidiol/aislamiento & purificación , Cannabinoides/aislamiento & purificación , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Dronabinol/aislamiento & purificación , Dronabinol/farmacología , Distrofina/genética , Endocannabinoides/metabolismo , Humanos , Masculino , Ratones , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
The smallest product of the Duchenne muscular dystrophy gene, dystrophin (Dp)71, is ubiquitously expressed in nonmuscle tissues. We previously showed that Dp71 expression in hepatic cells is modulated in part by stimulating factor 1 (Sp1), stimulating protein 3 (Sp3), and yin yang 1 (YY1) transcription factors, and that the polyaromatic hydrocarbon, ß-naphthoflavone (ßNF), downregulates Dp71 expression. The aim of the present study was to determine whether ßNF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcriptionquantitative polymerase chain reaction was used to measure halflife mRNA levels in ßNFtreated cells exposed to actinomycin D, an inhibitor of transcription, for 0, 4, 8, 12 and 16 h. Transient transfections with a plasmid carrying the Dp71 basal promoter fused to luciferase reporter gene were carried out in control and ßNFtreated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and ßNFtreated cells. To the best of our knowledge, the results demonstrated for the first time that this negative regulation takes place at the promoter level rather than the mRNA stability level. Interestingly, using EMSAs, ßNF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that ßNF may modulate the expression of other genes regulated by these transcription factors. In conclusion, ßNF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.
Asunto(s)
Distrofina/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción YY1/metabolismo , beta-naftoflavona/farmacología , Células Hep G2 , Humanos , Unión ProteicaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
Asunto(s)
Secuencia de Bases , Distrofina , Exones , Oligodesoxirribonucleótidos Antisentido , Eliminación de Secuencia , Animales , Evaluación Preclínica de Medicamentos , Distrofina/genética , Distrofina/metabolismo , Humanos , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacologíaRESUMEN
This study aimed to analyze the protective effects of photobiomodulation therapy (PBMT) with combination of low-level laser therapy (LLLT) and light emitting diode therapy (LEDT) on skeletal muscle tissue to delay dystrophy progression in mdx mice (DMD mdx ). To this aim, mice were randomly divided into five different experimental groups: wild type (WT), placebo-control (DMD mdx ), PBMT with doses of 1 J (DMD mdx ), 3 J (DMD mdx ), and 10 J (DMD mdx ). PBMT was performed employing a cluster probe with 9 diodes (1 x 905nm super-pulsed laser diode; 4 x 875nm infrared LEDs; and 4 x 640nm red LEDs, manufactured by Multi Radiance Medical®, Solon - OH, USA), 3 times a week for 14 weeks. PBMT was applied on a single point (tibialis anterior muscle-bilaterally). We analyzed functional performance, muscle morphology, and gene and protein expression of dystrophin. PBMT with a 10 J dose significantly improved (p < 0.001) functional performance compared to all other experimental groups. Muscle morphology was improved by all PBMT doses, with better outcomes with the 3 and 10 J doses. Gene expression of dystrophin was significantly increased with 3 J (p < 0.01) and 10 J (p < 0.01) doses when compared to placebo-control group. Regarding protein expression of dystrophin, 3 J (p < 0.001) and 10 J (p < 0.05) doses also significantly showed increase compared to placebo-control group. We conclude that PBMT can mainly preserve muscle morphology and improve muscular function of mdx mice through modulation of gene and protein expression of dystrophin. Furthermore, since PBMT is a non-pharmacological treatment which does not present side effects and is easy to handle, it can be seen as a promising tool for treating Duchenne's muscular dystrophy.
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Distrofina/metabolismo , Terapia por Luz de Baja Intensidad/métodos , Músculo Esquelético/fisiopatología , Músculo Esquelético/efectos de la radiación , Distrofia Muscular de Duchenne/fisiopatología , Distrofia Muscular de Duchenne/radioterapia , Animales , Relación Dosis-Respuesta en la Radiación , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Placebos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In Duchenne muscular dystrophy (DMD), lack of dystrophin leads to progressive muscle degeneration, with DMD patients suffering from cardiorespiratory failure. Cell therapy is an alternative to life-long corticoid therapy. Satellite cells, the stem cells of skeletal muscles, do not completely compensate for the muscle damage in dystrophic muscles. Elevated levels of proinflammatory and profibrotic factors, such as metalloproteinase 9 (MMP-9), impair muscle regeneration, leading to extensive fibrosis and poor results with myoblast transplantation therapies. Omega-3 is an anti-inflammatory drug that protects against muscle degeneration in the mdx mouse model of DMD. In the present study, we test our hypothesis that omega-3 affects MMP-9 and thereby benefits muscle regeneration and myoblast transplantation in the mdx mouse. We observe that omega-3 reduces MMP-9 gene expression and improves myoblast engraftment, satellite cell activation, and muscle regeneration by mechanisms involving, at least in part, the regulation of macrophages, as shown here with the fluorescence-activated cell sorting technique. The present study demonstrates the benefits of omega-3 on satellite cell survival and muscle regeneration, further supporting its use in clinical trials and cell therapies in DMD.
Asunto(s)
Distrofina/deficiencia , Ácidos Grasos Omega-3/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Fibras Musculares Esqueléticas/patología , Mioblastos/enzimología , Mioblastos/trasplante , Células Satélite del Músculo Esquelético/patología , Animales , Biomarcadores/metabolismo , Distrofina/metabolismo , Femenino , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/efectos de los fármacos , Atrofia Muscular/patología , Mioblastos/efectos de los fármacos , Necrosis , Receptores Notch/metabolismo , Regeneración/efectos de los fármacos , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Satélite del Músculo Esquelético/metabolismo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Duchenne muscular dystrophy (DMD) is an X chromosome-linked lethal muscular disorder with progressing muscle wasting and weakness caused by mutations in the gene encoding a subsarcolemmal protein dystrophin. For a long time, there was no effective cure; however, advances in molecular biology have allowed the development of radical treatment approaches. Among them, exon-skipping therapy using antisense oligonucleotides is very promising, because it corrects the reading frame of the dystrophin-encoding gene and restores protein expression, resulting in the conversion of DMD to a clinically milder form, Becker muscular dystrophy (BMD). However, clinical trials in exon-skipping therapy did not provide satisfactory results, which may be attributed to inefficient exon skipping, low expression level of restored dystrophin and inadequate methods of muscle function evaluation. To date, exon-skipping approaches have particularly focused on the correction of the gene-reading frame. However, the problem is that the relationship between the resultant and expected phenotypes in terms of definite symptomatic improvement has not yet been elucidated. In other words, previously conducted clinical trials have not been planned based on the comprehensive assessment of genotype-phenotype relationship in BMD, which demonstrates a broad range of symptom severity depending on the functional activity of the truncated dystrophin. The analysis I present in this review strongly suggests that the development of exon-skipping therapy and its clinical trials should be based on large-cohort studies of BMD.